ENTERIC GLIAL MODULATION OF IMMUNE ACTIVATION DURING 

INFLAMMATORY STRESS 

By  

Aaron Kin Yeung Chow 

 

 

 

 

 

A DISSERTATION 

Michigan State University 

in partial fulfillment of the requirements 

Submitted to 

for the degree of 

Physiology–Doctor of Philosophy  

2020 

 

 

ABSTRACT 

ENTERIC GLIAL MODULATION OF IMMUNE ACTIVATION DURING 

INFLAMMATORY STRESS 

By 

Aaron Kin Yeung Chow 

Gastrointestinal (GI) disorders such as inflammatory bowel disease (IBD), 

irritable bowel syndrome, and other functional GI disorders are major health concerns 

and account for about $26.4 billion in yearly costs in the United States alone. Many of 

these GI disorders manifest symptoms such as GI dysmotility, intestinal secretion and 

absorption dysfunction, and abdominal pain. Inflammation plays a major role in the 

pathogenesis of these diseases, and current therapies for many GI disorders aim to 

regulate the activation and progression of the inflammatory cascade. However, due to a 

gap in knowledge in how the immune system is regulated within the GI tract, there 

remains a lack of effective treatments for these common GI disorders. Interactions 

between the nervous system and immune system point to neurons having important 

roles in immune modulation, but the mechanisms of neuro-immune regulation in the gut 

is not completely understood. 

The enteric nervous system (ENS) consists of enteric neurons and enteric glia 

arranged in plexuses embedded in the gut wall. This neural network is responsible for 

the normal secretomotor functioning of the GI tract, and the disruption of the ENS 

network alters GI functioning and underlies pathological GI symptoms. As part of the 

ENS, enteric glia work in tandem with enteric neurons to coordinate GI functions. In 

addition to their contributions to maintain normal secretomotor functioning of the GI 

 

tract, enteric glia are activated by immunomodulatory signals, they can secrete and 

respond to cytokines, can exert immunosuppressive effects, and share characteristics 

with antigen presenting cells. Therefore, we hypothesize that enteric glia play an active 

role in immune regulation in the ENS. 

In this dissertation, we specifically examine the role enteric glial cells play as an 

antigen presenting cell to regulate immune activation.  Our results show that enteric glia 

have the machinery necessary to act as an antigen presenting cell and can express 

major histocompatibility complex (MHC) type II molecules during inflammatory stress to 

interact with T-lymphocytes. Enteric glial MHC II expression has functional relevance, 

as it modulates the activation in Th17 and Treg subtypes, but not Th1 or Th2 T-

lymphocyte subtypes. Although MHC II molecules are typically associated with the 

expression of phagocytosed extracellular antigens, our results show that enteric glia do 

not readily phagocytose extracellular antigens. Instead, MHC II expression in enteric 

glia is mediated by autophagy. The activation of autophagic pathways is necessary, but 

not sufficient in eliciting enteric glial MHC II expression. Finally, although enteric glia 

regulate T-lymphocyte activation, cytokine levels at the whole organism or regional 

tissue levels remain unchanged, suggesting that enteric glial cytokine effects primarily 

operate at the local microenvironment level. 

Our findings provide support for enteric glial cells having an active role as an 

immunomodulator. Specifically, we show that enteric glia modulate T-lymphocyte 

activation via autophagy-mediated MHC II expression and propose a novel mechanism 

of neuro-immune modulation in the gut.

 

ACKNOWLEDGEMENTS 

 

I would like to first thank my mentor Dr. Brian Gulbransen for the guidance he 

has given me in this journey. His support and mentorship on how to become an efficient 

and successful scientist has brought me to where I am today in my research career. His 

positive outlook on any given situation has encouraged me to never give up even when 

faced with discouraging experimental results. Brian always had time to sit down to 

discuss any concerns I had whether it was science-related or otherwise, and he was 

never too busy to talk about data. I am especially grateful for the patience and 

understanding Brian has for the DO-PhD program – the sheer length of the program, 

the extra obligations I have that pull me away from experiments, and the times during 

medical school where I spent studying and wasn’t very productive in lab. 

 

I would also like to thank the past and present members of the Gulbransen Lab 

who have made being part of the lab such an enjoyable experience. Jonathan, David, 

Vlado, Siomara, Isola, Ninotchska, Wilmarie, Luisa, Christine, Mustafa, RJ, Becca, and 

Erika, thank you for all the help in lab and experiments when I overestimate how much I 

can manage in one day. Thank you for your insights and and helping me develop as a 

scientist, and our time spent hanging out outside of lab as well. Your help and friendship 

have made my experiences here very enjoyable. 

 

I would also like to thank the DO-PhD program who made my graduate career 

here at MSU possible. Thank you, Dr. Justin McCormick and Ms. Bethany Heinlen, for 

admitting me to the DO-PhD program and allowing me on this wonderful journey. Thank 

you to Dr. Brian Schutte and Ms. Michelle Volker who are the current DO-PhD 

 
 

 iv 

administrators who continue to run the DO-PhD program and who are supportive of my 

journey. I would also like to thank the BMS program, especially Becky and Jasmine, 

who have been extremely patient and helpful as I navigate the intricacies of graduate 

school. 

 

It is true that time flies when you’re having fun, and I would like to thank the 

friends that I have made along the way here in Michigan that have made it feel like I 

moved to Michigan only a few short weeks ago. I would like to especially thank Briana, 

Laura, Sean, Scott, and Yajing – the times that we have shared together and friendship 

that we have makes this long journey so much fun.  

 

I would like to thank my friends and family back in Canada as well, who have 

been ever supportive of my studies and studies here in the United States, no matter the 

physical distance that separate us, or the sheer number of years that I have spent as a 

student. They have never ceased supporting my endeavors here and have been my 

biggest supporters, reminding me during stressful times that it will all be worth it in the 

end. 

 

I would also like to thank my committee members Dr. Julia Busik, Dr. James 

Galligan, Dr. Linda Mansfield, Dr. Adam Moeser, and Dr. Narayanan Parameswaran 

who have helped me in refining my project, giving me valuable feedback, and your 

attention during the countless hours of committee meetings. Your support has helped 

guide my development as a scientist. 

 
 

 v 

 

I could not have been able to accomplish this journey without the help and 

support of family, friends, staff, faculty, collaborators, and countless others that are not 

mentioned here. Thank you from the bottom of my heart. 

 

 
 

 vi 

TABLE OF CONTENTS 

 

LIST OF TABLES ............................................................................................................ x 

LIST OF FIGURES ..........................................................................................................xi 

KEY TO ABBREVIATIONS ........................................................................................... xiii 

CHAPTER 1: Introduction ............................................................................................... 1 
Overview .............................................................................................................. 2 
The Enteric Nervous System ................................................................................ 3 
Intrinsic ENS innervation ............................................................................ 3 
Extrinsic inputs into the ENS ...................................................................... 7 
ENS damage and disease ......................................................................... 9 
Enteric Glia .............................................................................................. 13 
The Immune System .......................................................................................... 17 
Inflammation ............................................................................................ 19 
The Innate Immune System ..................................................................... 22 
Antigen Presenting Cells .......................................................................... 24 
Type II Major Histocompatibility Complex ................................................ 26 
The Adaptive Immune System ................................................................. 31 
T-lymphocytes ......................................................................................... 33 
Current models of intestinal inflammation ................................................ 35 
Summary and Aims of Dissertation .......................................................... 37 
REFERENCES ................................................................................................... 39 

CHAPTER 2: Potential roles of enteric glia in bridging neuroimmune communication in 
the gut ........................................................................................................................... 58 
Abstract .............................................................................................................. 59 
Introduction ......................................................................................................... 60 
Contribution of enteric neurons to inflammation ....................................... 61 
Glia in the healthy intestine ...................................................................... 66 
Glial cells are activated by cytokines and immunomodulatory signals ..... 68 
Glia express and secrete cytokines and pro-inflammatory signals .......... 72 
Glial cells act as antigen presenting cells ................................................ 74 
Enteric glia exert immunosuppressive and anti-inflammatory effects ...... 75 
Conclusions ........................................................................................................ 77 
REFERENCES ................................................................................................... 79 

 
 

 vii 

CHAPTER 3: Enteric glial cells induce Th17 and Treg activation via autophagy-mediated 
MHC II expression ......................................................................................................... 86 
Abstract .............................................................................................................. 87 
Background and Aims .............................................................................. 87 
Methods ................................................................................................... 87 
Results ..................................................................................................... 87 
Conclusions ............................................................................................. 88 
Introduction ......................................................................................................... 89 
Materials and Methods ....................................................................................... 91 
Animals .................................................................................................... 91 
Induction of glial MHC II ........................................................................... 92 
Phagocytosis assay ................................................................................. 92 
Whole-Mount Immunofluorescence ......................................................... 93 
H&E Staining ........................................................................................... 94 
Frozen section Immunofluorescence ....................................................... 94 
Flow cytometry ......................................................................................... 95 
Gene expression ...................................................................................... 96 
Cytokine quantification ............................................................................. 98 
Autophagy induction and detection .......................................................... 99 
Data Analysis ........................................................................................... 99 
Statistics ................................................................................................ 101 
Results ............................................................................................................. 104 
Conditional ablation of glial MHC II in Sox10CreERT2;IABfl/fl mice and IFNγ + 
LPS challenge ........................................................................................ 104 
Enteric glia express mature MHC II in response to IFNγ + LPS ............ 109 
Enteric glial MHC II expression contributes to CD4+ T-cell activation in 
mesenteric lymph nodes ........................................................................ 112 
Enteric glia affect local, but not serum or colonic cytokine levels ........... 120 
Pro-inflammatory stimuli IFNγ and LPS do not trigger phagocytosis in 
enteric glia ............................................................................................. 132 
Glial autophagy regulates MHC II expression ........................................ 134 
Discussion ........................................................................................................ 139 
REFERENCES ................................................................................................. 147 

CHAPTER 4: Summary, Key Findings and Future Directions ..................................... 157 
Summary .......................................................................................................... 158 
Key Findings ..................................................................................................... 158 

Enteric glial cells express MHC II in response to low dose IFNγ and LPS
 ............................................................................................................... 158 
Enteric glial MHC II alter T-lymphocyte activation .................................. 159 
Enteric glial MHC II expression is derived from autophagy pathways .... 160 
Study Limitations .............................................................................................. 161 
Pharmacological modulators .................................................................. 161 

 
 

viii 

Sex of experimental animals .................................................................. 161 
MHC II knockout animals ....................................................................... 161 
Future Directions .............................................................................................. 161 
Determination of antigens presented on enteric glial MHC II ................. 161 
Enteric glial autophagy effects in modulating T-lymphocyte activation .. 162 
Glial MHC II contributions in overt inflammation .................................... 162 
Sympathetic effects on enteric glial-mediated T-lymphocyte activation . 163 
Local renin angiotensin effects .............................................................. 165 
REFERENCES ................................................................................................. 169 

 ix 

 

 
 

LIST OF TABLES 

 

Table 3.1: Antibodies used in Chapter 3 ..................................................................... 102 

 
 

 
 

 x 

LIST OF FIGURES 

 

Figure 1.1: Arrangement of the ENS within the GI tract .................................................. 6 

Figure 1.2: Enteric glial proximity to enteric neurons ..................................................... 14 

Figure 1.3: Overview of enteric glial functions ............................................................... 16 

Figure 1.4: Overview of typical GI immunity .................................................................. 18 

Figure 1.5: Murine MHC II. ............................................................................................ 27 

Figure 1.6: Maturation of MHC II molecules .................................................................. 29 

Figure 2.1: Overview of enteric neuron responses to select signals ............................. 64 

Figure 2.2: Overview of enteric glial cell responses to select signals ............................ 70 

Figure 3.1: Enteric glial cells express MHC II when stimulated with IFNγ and LPS .... 105 

Figure 3.2: Exposure to IFNγ and LPS causes weight loss but does not cause 
significant tissue damage or observable inflammation ................................................ 107 

Figure 3.3: Pattern and level of MHC II expression in macrophages does not change 
when treated with IFNγ and LPS ................................................................................. 111 

Figure 3.4: CD4 expressing T-lymphocytes are in close proximity with GFAP expressing 
enteric glia in the myenteric plexus ............................................................................. 115 

Figure 3.5: Enteric glial MHC II expression affects T-lymphocyte activation ............... 116 

Figure 3.6: Activation of CD8+ T-lymphocytes decreased when MHC II ablated in 
enteric glia ................................................................................................................... 118 

Figure 3.7: Enteric glial MHC II expression affect FoxP3+ and IL-17+ cell populations to 
a greater degree than IFNγ+ and IL-4+ cell populations ............................................. 119 

Figure 3.8: Enteric glial gene expression .................................................................... 122 

Figure 3.9: Enteric glial MHC II expression has limited effects on serum and colonic 
tissue cytokine levels ................................................................................................... 124 

Figure 3.10: MHC II ablation in enteric glial cells do not show changes in colonic tissue 
level of select cytokines ............................................................................................... 126 

 
 

 xi 

Figure 3.11: MHC II ablation in enteric glial cells do not show changes in serum level of 
select cytokines ........................................................................................................... 128 

Figure 3.12: Sox10 expressing cells are present in sparse numbers in spleen tissue and 
express MHC II ........................................................................................................... 130 

Figure 3.13: Enteric glia do not exhibit phagocytic activity when stimulated with IFNγ 
and LPS ...................................................................................................................... 133 

Figure 3.14: Enteric glia express MHC II via autophagy pathways ............................. 136 

Figure 3.15: Schematic mechanism of enteric glial MHC II expression ....................... 146 

Figure 4.1: Autonomic innervation onto enteric glia may modulate MHC II expression
 .................................................................................................................................... 164 

Figure 4.2: The renin-angiotensin pathway neuronal damage and inflammation ........ 167 

Figure 4.3: Enteric glia increase expression of ACE2 during high fat diet conditions .. 168 

 

 

 
 

 xii 

KEY TO ABBREVIATIONS 

 
3-MA – 3-methyladenine 

6-OHDA – 6-hydroxydopamine 

CD – Cluster of differentiation 

CNS – Central nervous system 

DMEM – Dulbecco’s Modified Eagle Medium 

EDNRB – Endothelin B receptor 

ENS – Enteric nervous system  

FACS – Fluorescence-activated cell sorting 

GDNF – Glial derived neurotrophic factor 

GFAP – Glial fibrillary acidic protein 

GI – Gastrointestinal 

GSNO – Glial derived S-nitrosoglutatione 

IBD – Inflammatory bowel disease 

IBS – Irritable bowel syndrome 

IFN-γ – Interferon gamma 

IL – Interleukin  

IP – Intraperitoneal 

 
 

xiii 

KO – Knockout 

LMMP – Longitudinal muscle-myenteric plexus 

LPS – Lipopolysaccharide 

MAPK – Mitogen-activated protein kinase 

MHC – Major histocompatibility complex 

NF-κB – Nuclear factor kappa-light-chain-enhancer of activated B cells 

PBS – Phosphate buffered saline 

TGF-β – Transforming growth factor beta 

TLR – Toll-like receptor 

TNBS – Trinitrobenzene sulfonic acid 

TNF – Tumor necrosis factor 

VIP – Vasoactive intestinal peptide  

WT – Wildtype 

 
 

xiv 

CHAPTER 1: 
Introduction 

 

 1 

 

 

 
 

Overview 

The gastrointestinal (GI) tract is an important organ system responsible for the 

movement, digestion, absorption, excretion, and barrier defense against ingested 

particles (60, 128). The proper functioning of the GI tract is coordinated primarily by the 

enteric nervous system (ENS) (60), with modulating inputs from the central nervous 

system (CNS) (60). The ENS is a part of the autonomic nervous system and is 

embedded within the walls of the GI tract, and dysfunction in the ENS is a large 

component to the development of varying GI disorders (57). The loss of neuronal inputs 

into the GI tract leads to uncoordinated GI function, dysmotility, improper GI secretion, 

absorption, and improper regulation in GI blood flow (56). ENS neuropathies can be due 

to congenital causes, effects from the surrounding environment or disease, or a 

combination of factors (56, 57), leading pathological GI findings in diseases such as in 

Hirschsprung’s disease, Parkinson’s disease, Inflammatory bowel disease (IBD), 

Diabetes mellitus, Chronic intestinal pseudo-obstruction, Chagas disease, among 

others (56, 57, 148, 202).  

Inflammation plays a major role in neuronal damage and death in the ENS, and 

is the hallmark finding in IBD. Given that the lumen of the GI tract is continuous with the 

outside environment, the constant exposure to bacteria, viruses, parasites, and other 

foreign particles are major sources for potential inflammatory stimuli (60, 192). 

Therefore, the GI tract plays a critical role in pathogen defense and is a major site for 

immune regulation (60, 143, 149). Although components such as the microbiome has 

been the focus of many studies examining GI immune regulation, there is growing 

support that the ENS has a major role in the modulating immune activity (114, 219). 

 
 

 2 

Increasing evidence show that both enteric neurons and enteric glia, which are non-

myelinating peripheral glia in the ENS, play substantial roles in immune regulation as 

well (22, 114). Specifically, enteric glia have the prerequisite cellular machinery to 

directly interact with the immune system as an antigen presenting cell and modulate its 

activity (4, 53, 90, 201). However, many of the studies have been performed in vitro, or 

have not shown functional effects on the immune system. Therefore, the specific roles 

that enteric glia play in immune regulation is not well understood. Here, this dissertation 

examines enteric glial function and responses in inflammation and its role in immune 

modulation in the ENS. 

Intrinsic ENS innervation 

The Enteric Nervous System 

The ENS, also referred to the “brain of the gut”, is part of the autonomic nervous 

system (48) and is embedded along the entire length of the GI tract. The ENS receives 

inputs from the CNS but is functionally independent (48, 97). In fact, the GI tract 

maintains coordinated activity and does not cease to function when completely 

separated from the CNS (6, 7). The ENS is composed primarily of two ganglionated 

plexuses, termed the submucosal, or Meissner’s plexus, and the myenteric, or 

Auerbach’s plexus (82, 185, 194). These two plexuses regulate major functions in the 

GI tract. Anatomically, the submucosal plexus is located between the muscularis 

mucosae and circular muscle layers in the GI tract (Fig.1.1). The submucosal plexus is 

the smaller of the two plexuses and consists of fewer neurons (82). It is primarily 

involved in controlling absorption, secretion, and local blow flood to the gut (174, 198, 

 
 

 3 

206). In contrast, the myenteric plexus, the larger of the two plexuses, is located 

between the circular and longitudinal muscle layers of the GI tract (185)(Fig.1.1). The 

role of the myenteric plexus is primarily motility focused, as it is responsible for the 

coordinated contraction and relaxation within GI tract (89, 97, 182). This is an important 

process, as it allows for the proper transit of ingested food particles through the GI tract, 

a process known as peristalsis.  

The ENS develops from neural crest progenitors expressing gene markers 

Sox10, FoxD3, p75 neurotrophin receptor, Phox2b, RET, and endothelin B receptor 

(EDNRB) (32). As these progenitors differentiate into enteric neurons that populate the 

GI tract, they retain expression of Phox2b and RET, but lose expression of other 

progenitor cell markers. Enteric neurons also gain the expression of PGP9.5, β3-tubulin, 

and HuC-D (32). As they mature, enteric neurons further differentiate into different types 

of neurons and can be further classified by immunohistochemical labeling of neuronal 

markers and neurotransmitters they express. Classification of these enteric neurons 

falls into three major types: motor neurons (both excitatory and inhibitory), interneurons, 

and sensory neurons (49). Excitatory motor neurons elicit their excitatory effects 

primarily via release of acetylcholine, and to a lesser degree, takykinin proteins such as 

substance P (49, 61). These neurons can also be identified by their expression of 

calretinin and γ-aminobutyric acid. In contrast, inhibitory motor neurons primarily utilize 

nitric oxide as a neurotransmitter, along with vasoactive intestinal peptide (VIP), ATP, 

and carbon monoxide (48, 214). Interneurons (ascending and descending) present 

along the length of the GI tract responsible for reflex pathways utilize acetylcholine as a 

neurotransmitter, along with a combination of other markers such as serotonin, 

 
 

 4 

somatostatin, nitric oxide, VIP, and calretinin (48). Finally, intrinsic sensory neurons 

utilize acetylcholine, calcitonin gene-related peptide, and tachykinins as 

neurotransmitters, and can be further identified via labeling for calbindin, calretinin, and 

isolectin B4 (48).  

 

 
 

 

 5 

 

Figure 1.1: Arrangement of the ENS within the GI tract. The ENS is distributed within 

the walls of the GI tract in primarily two plexuses, the myenteric plexus and submucosal 

plexus. The submucosal plexus is located between the muscularis mucosae and 

circular muscle, while the myenteric plexus is located between the circular and 

longitudinal muscle layers.  

 

 
 

 

 6 

Extrinsic inputs into the ENS 

 

Despite extensive intrinsic neuronal inputs into the ENS to control GI function, 

the ENS also receives extrinsic inputs from the CNS to modulate GI activity (128, 150). 

CNS inputs into the ENS consist of sympathetic, parasympathetic, and sensory 

innervation arranged in vagal, thoracolumbar, and craniosacral pathways (60). The 

sympathetic and parasympathetic inputs to the ENS can modulate GI function by 

stimulating GI motility and digestion with parasympathetic stimuli, and inhibiting GI 

motility and digestion with the sympathetic pathway (73, 128, 150). GI motility is 

modulated by parasympathetic efferent motoneurons that innervate the GI tract and are 

derived primarily from the dorsal motor nucleus of the vagal nerve for the upper regions 

of the GI tract, and preganglionic fibers from lumbosacral regions of the spinal cord in 

the distal regions of the colon (19). These preganglionic efferent motoneurons release 

acetylcholine to bind nicotinic receptors and activate interstitial cells of Cajal and 

neurons in the myenteric plexus to elicit muscle contraction in the GI tract (19, 168). 

However, vagal activation of non-adrenergic, non-cholinergic postganglionic pathways 

also allows for release of nitric oxide and vasoactive intestinal polypeptide in order for 

the relaxation of intestinal smooth muscle (19). In contrast, sympathetic modulation of 

GI activity originate from thoracolumbar regions of the spinal cord to several 

sympathetic ganglia in order to supply sympathetic innervation to the GI tract. 

Postganglionic fibers from the celiac ganglion supply sympathetic innervation to the 

proximal GI tract such as the stomach, while neurons from the superior mesenteric 

ganglion and inferior mesenteric ganglion provide sympathetic innervation to the small 

and large intestines, respectively (19). Unlike parasympathetic nerves, the preganglionic 

 
 

 7 

sympathetic neurons primarily utilize norepinephrine as a neurotransmitter (19). 

Sympathetic modulation of intrinsic ENS neurons contributes to decreased motility in 

the GI tract, and increased tone in intestinal sphincters (106).  

In addition to modulating GI motility, sympathetic and parasympathetic nerves 

are distributed to lymphoid organs and immune tissues in the GI tract and have 

immunomodulatory effects on the GI system (24, 118, 186).  Broadly speaking, 

parasympathetic activation is associated with anti-inflammatory effects in the gut, while 

sympathetic activation can lead to either pro- or anti- inflammatory effects, depending 

on levels of released neurotransmitters (16, 119, 186). Sympathetic signaling through α-

adrenergic receptors can induce release of TNF-α from macrophages, promote Th2 T-

lymphocyte activation, and increase Th17 T-lymphocyte activation through increased 

expression of IL-23 by antigen presenting cells (3, 91). In contrast, cholinergic signaling 

from parasympathetic pathways can act directly onto enteric neurons that interact with 

nearby macrophages. Release of acetylcholine by these enteric neurons can bind α-7-

nicotinic acetylcholine receptors present on macrophages to inhibit the release of pro-

inflammatory cytokine TNF-α (16). Interestingly, vagal efferent nerves can also interact 

with splenic sympathetic nerves within the celiac ganglion to elicit anti-inflammatory 

effects via the splenic nerve. Activation here leads to acetylcholine release in T-

lymphocytes and thereby bind α-7-nicotinic acetylcholine receptors on splenic 

macrophages to inhibit release of TNF-α (16).  

In addition to parasympathetic and sympathetic nerves, even sensory neurons 

have immunomodulatory properties and can inhibit immune activation in neutrophils, 

monocytes, and macrophages (28). Therefore, the contributions from the CNS in 

 
 

 8 

immune modulation play important roles in homeostasis and can contribute to intestinal 

inflammation, ENS damage, and the pathogenesis of IBD. 

ENS damage and disease 

Damage to the ENS can arise from a multitude of sources and can lead to 

neuronal death. Since the ENS regulates GI secretomotor function, injury to the ENS 

can manifest as symptoms such as constipation, diarrhea, nutrient malabsorption, or 

abdominal pain (60, 108). ENS dysfunction can be due to malformations congenitally, 

direct modifications to neuronal structure and function, inflammatory effects, exposure 

to chemicals, changes in microbiota, or as a part of normal aging (57, 71, 120, 166).  

In congenital ENS malformation, dysfunctions in the ENS arises due to 

inappropriate development of enteric neurons. For example, Hirschsprung’s disease is 

due to the improper migration of neural crest cells during gestation and leads to 

aganglionsis in the distal portions of the GI tract, primarily affecting the colon (191). 

Genetic abnormalities in multiple genes such as RET, endothelin receptor type B, and 

Sox10 contribute to the pathogenesis of Hirschsprung’s disease (17, 21, 148). Without 

the development of a proper ENS in sections of the gut, aganglionic regions lack enteric 

neurons and enteric glia. These regions do not have proper motility functions and 

typically cannot relax, leading to fecal stasis, bacterial overgrowth in the GI tract, and 

potentially lead to sepsis (120, 195).  

Diseases causing neuropathies, such as diabetes, can lead to ENS damage by 

modifying neurons and neuronal function. In diabetes, elevated glucose levels in the 

body leads to the covalent linkage of glucose molecules, termed glycation, of neurons. 

 
 

 9 

This occurs through a process known as non-enzymatic glycation, which can hinder 

neuronal functions and trigger nerve degradation (93, 202). Furthermore, diabetes may 

also activate other pathways such as the polyol, protein kinase C, mitogen-activated 

protein kinase (MAPK), TNF-α, NF-κB, cyclooxygenase, interleukin (IL), Wnt, and 

autophagy pathways, among others, to elicit neuronal damage and pathogenesis (38). 

This can lead to a host of GI symptoms (42) and can contribute to motility disorders and 

gastroparesis seen in diabetes patients (38, 202). Enteric glial cells within the ENS can 

act to limit neuronal degradation (109), but diabetic changes in enteric glial cells, such 

as changes in glial morphology and number, can limit their ability to protect enteric 

neurons and further contribute to diabetes-associated ENS dysfunction (144, 153).  

Inflammation in the GI tract is a very common cause of enteric neuropathies, but 

the pathogenesis varies significantly between diseases. For example, inflammatory 

damage to the ENS can be caused by infection, such as in Chagas disease caused by 

Trypanasoma cruzi infection. The inflammatory response stimulated by this infection 

can lead to megacolon or megaesophagus due to destruction of ENS segments (81, 

180). In IBD, pathogenesis is thought to be due to the inappropriate immune activation 

against the microbiota in genetically susceptible patients (29, 179, 210, 222). This can 

lead to inflammation in the GI tract, a hallmark of IBD. Inflammatory stress on the ENS 

is distributed differently in the two forms of IBD: Crohn’s disease and ulcerative colitis. 

While tissue inflammation is limited to the superficial layers of the colon in ulcerative 

colitis (225), Crohn’s disease can elicit inflammation anywhere along the length of the 

GI tract. In addition, inflammation in Crohn’s disease can affect the entire thickness of 

the GI tract and not only the superficial layers as in ulcerative colitis (225). Inflammatory 

 
 

10 

damage in the GI tract can lead to the reorganization, damage, and death of both 

enteric neurons and enteric glial cells, thereby contributing to a loss of GI secretomotor 

functions and symptoms seen in IBD patients.  

Inflammatory stress causes neuronal death through the activation of caspase-

dependent apoptotic pathways in enteric neurons via activation of purinergic receptors 

and pannexin channels (63, 129). Neuronal death due to inflammation does not target 

specific subpopulations (129), but instead leads to a generalized decreased neuronal 

density in the ENS. However, inflammatory stress can also contribute to changes in 

neuronal behavior, such as the development of hyperactivity in intrinsic primary afferent 

neurons during, and after inflammation, thereby contributing to greater 

afterhyperpolarizing potential (104, 105, 129). The change in neuronal activity in these 

intrinsic primary afferent neurons suggest alterations in normal intestinal sensation and 

changes in motor reflexes that rely on these afferent nerves. In addition to alterations in 

neuron activity, IBD elicit changes in neurotransmitter expression in enteric neurons. 

For example, IBD patients have an increased proportion of neurons expressing 

substance P (134), neuropeptide Y (10), and changes in number of neurons expressing 

vasoactive intestinal polypeptide (10, 173). 

In addition to inflammatory stress, ENS exposure to certain chemicals in the body 

can lead to detrimental effects. For example, chemotherapy treatment has many effects 

on the GI tract, but chemotherapy-induced neuronal death in the ENS can lead to 

serious dysmotility disorders (2, 43, 121, 208). Furthermore, chemical-induced ENS 

damage and alterations in neuronal and glial function can persist for extended periods 

of time, such as in Gulf War veterans exposed to pyridostigmine bromide decades ago 

 
 

11 

(70, 71). In this way, chemical damage to the ENS can have lasting effects even after 

offending chemicals are removed. 

The interplay between the ENS and the microbiota in the GI tract also has 

significant implications on GI disease and secretomotor function. Many groups have 

shown interactions between the microbiota, the ENS, and the CNS (35, 171, 204). 

Changes in the microbiota composition has profound effects on GI function, and GI 

diseases such as IBD have been linked with intestinal dysbiosis and alterations in 

microbiota (1, 139). Due to the complex nature of microbiota interactions, the specific 

effects due to changes in microbe distribution are not completely known and is a topic of 

study. 

Finally, the process of normal aging causes increased neurodegeneration 

overall, but neurons in the ENS are especially susceptible (166). Despite the plasticity of 

the ENS and its capability of neuronal outgrowth, aging leads to the reduction in the 

number of neurons and glia in the ENS (166). Furthermore, aging causes alterations in 

neuronal morphology and arrangement, suggesting alterations in neuronal behavior and 

function, and may contribute to GI motility disorders seen in old age, such as fecal 

incontinence and constipation (26, 52). Taken together, ENS neuropathy can be due to 

numerous reasons ranging from congenital malformations, direct neuronal 

modifications, inflammation, the exposure to chemicals, changes in microbiota, just from 

normal aging, or a combination of any of these factors. ENS neuropathy, irrelevant of 

cause, leads to GI secretomotor dysfunction by causing damage to both enteric neurons 

and enteric glia, thereby disrupting the ENS network.  

 
 

12 

Enteric Glia 

 

Enteric glia, together with enteric neurons, form the ENS. They are a type of non-

myelinating peripheral glia that share similar characteristics as astrocytes of the CNS 

(50, 55, 84). They are in extremely close contact with each other (15) (Fig.1.2), and 

were historically thought of being merely a ‘support’ cell to enteric neurons by providing 

nutritional and structural support to the ENS. However, enteric glia have since been 

shown to have an important impact on enteric neurogenesis, activity, and survival (32, 

65, 135).  

Enteric glia are derived from neural crest cells and begin development and 

colonization of the intestinal tract within the first two weeks in utero (55, 88, 163). During 

development, enteric glia express markers such as Sox10, FoxD3, B-FABP, glial 

fibrillary acidic protein (GFAP), and S100β, making them a distinct population compared 

to developing enteric neurons or neural crest progenitor cells (32). Therefore, current 

identification of enteric glia within the ENS is achieved by the labeling of glial-specific 

markers S100β and GFAP (32, 135, 164), and by morphology, as they have distinct 

astrocyte-like star shaped morphology with long and highly branched processes (50, 

164, 165) (Fig.1.2). However, despite their similarities with glial populations in the CNS, 

enteric glia are a distinct population. This is evidenced by their embryonic origins from 

the neural crest, in opposed to astrocytes being derived from the neural tube (8, 55). 

Due to the differences in embryologic origin, enteric glia also express the Sox10 

transcription factor, unlike astrocytes (127).  

 

 
 

 

13 

Figure 1.2: Enteric glial proximity to enteric neurons. Immunofluorescent labeling of 

enteric neurons with HuC/D (red) and enteric glia with glial-specific marker GFAP 

(grey). Enlarged area further demonstrates the proximity of glial processes to enteric 

 

neurons. Scale bar represents 50µm. 

 

 
 

 

14 

 

In the established ENS, enteric glia communicate with enteric neurons and other 

cell types such as intestinal epithelium to play an active role in GI secretomotor function. 

To interact with neuronal signaling, enteric glia express receptors for numerous 

neurotransmitters such as cholinergic receptors (27, 36), adrenergic receptors (62, 

132), purinergic receptors (58, 64, 92), serotonin receptors (14), among others  (130, 

175, 224). This allows for enteric neurons to respond to nearby neuronal activity and be 

targets of innervation. In addition, enteric glia can also modulate neuronal signals in the 

ENS, thereby contributing to the control of GI motility (32, 65, 135) (Fig.1.3). In addition 

to interactions with enteric neurons, enteric glia can interact with non-neuronal cell types 

as well. Their interactions with intestinal epithelial cells play a role in the control of 

intestinal secretion and barrier function (110, 111, 170), and their ability to secrete and 

respond to cytokines can affect immune activity (90, 162) (Fig.1.3). A more in-depth 

examination of enteric glial cell characteristics that support their role in immune 

modulation is presented in chapter 2 of this dissertation. Taken together, enteric glia 

play an active role in normal ENS signaling as well as secretomotor and immune 

modulation activities in the GI tract.  

 

 
 

 

15 

 

Figure 1.3: Overview of enteric glial functions. Enteric glia play an active role in the 

communication and interaction between enteric neurons, enterocytes, and immune cells 

in the intestinal tract. These interactions are through neurotransmitters and soluble 

molecules such as nitric oxide or secreted cytokines in order to affect GI secretomotor 

function, barrier function, and immune activity. 

 

 

 
 

 

16 

 

 

In addition to their role in ENS signaling, enteric glia also play a role in 

neurogenesis and neuronal maintenance in the ENS. Studies demonstrate that enteric 

glia help maintain the health of the ENS and recover from injury via the expression of 

neurotrophic factors that can stimulate neuron outgrowth and regeneration (11, 66, 

181). However, during oxidative stress and neuronal damage, enteric glia can also play 

the role of executioner and contribute to neuronal death (18). In mice, there are roughly 

equal numbers of enteric glia and enteric neurons in the GI tract, but the ratio between 

glia and neurons increases in higher order animals, reaching roughly seven-to-one 

enteric glia-to-neuron ratios in humans (51, 74). This suggests that enteric glia can 

effectively monitor neuronal health and function, and that increasing numbers of enteric 

glia are necessary for the increasing complexity of GI functions in higher organisms. 

The Immune System 

The ENS in the walls of the GI tract does not exist in isolation. In fact, many 

immune cells are in the surrounding microenvironment of both the submucosal and 

myenteric plexus. Dendritic cells, muscularis macrophages and memory T-lymphocytes 

are all normally distributed within the mucosa, submucosa, and muscular layers of the 

GI tract (5, 33) to monitor the local microenvironment for any potential pathogens 

(Fig.1.4). The proximity of immune cells to the ENS lends to interactions between cells 

of the ENS and the immune system (138, 209). While having immune cells in close 

proximity to the ENS is beneficial in eliminating nearby pathogens, immune activation or 

dysregulation can theoretically have immediate effects on the ENS. 

 

 
 

17 

Figure 1.4: Overview of typical GI immunity. Spread within the mucosa, submucosa, 

and muscular layers of the GI tract are dendritic cells, lymphocytes, and macrophages. 

Nearby GI-associated lymphoid tissues (GALT) and regional mesenteric lymph nodes 

contain additional immune cells to respond to pathogens. In the submucosal and 

myenteric plexuses, muscularis macrophages are present in close proximity to the 

 

ganglia. 

 

 
 

 

18 

 

Broadly, the immune system is responsible for host defense and is involved in 

the recognition and removal of pathogens, foreign molecules, and damaged cells and 

tissues. It is divided into two main components, the innate and adaptive immune 

systems. The innate immune system is responsible for a rapid response to an invading 

pathogen by limiting its spread, phagocytosing and destroying pathogens, and 

communicate with the adaptive immune system to mount an additional response to a 

given pathogen (169). Innate immunity relies on molecular patterns common on many 

pathogens to generate a generic immune response (83, 145). 

In contrast, the adaptive immune system does not respond with the same speed 

as the innate immune system and relies on activity in the innate immune system to 

convey antigens to direct it to the proper target. In addition, the adaptive immune 

system has the ability to generate a ‘memory’ of a previous immunologic insult such that 

subsequent exposure to the same insult elicits a stronger and more rapid adaptive 

immune response (145, 169). Activation in either the innate and adaptive immune 

systems can lead to inflammation within the ENS, contributing to the pathogenesis of 

ENS disorders and IBD. Currently, numerous therapeutics have been developed to 

target specific steps in each arms of the immune system (75), and there continues to be 

rigorous study into immune regulation as a means to limit inflammatory damage to the 

ENS. 

Inflammation 

 

Inflammation has been recognized since ancient Roman times. The cardinal 

signs of inflammation were described by Aulus Cornelius Celsus as being rubor 

(redness), tumor (swelling), calor (heat), and dolor (pain) (193). The process of 

 
 

19 

inflammation represents a broad response to limit the spread of pathogens and deliver 

immune cells, among other products, to help eliminate threats. However, inflammation 

is also a major driver for neuroplastic changes in the ENS (207). Changes to neuron 

survival and the overall ENS network leads to functional alterations in the ENS and 

contributes to secretomotor symptoms in diseases such as IBD. 

 

The overall inflammatory process is accomplished by first increasing blood flow 

and vascular permeability to the site of infection or injury. During infection, the increase 

in fluid to the area helps to isolate a pathogen to prevent spread (122). Furthermore, the 

increase in blood and lymph flow to the area helps bring leukocytes to the site of injury 

or infection to help progress the immune reaction (122). Initial immune cells recruited to 

the inflamed area primarily consists of neutrophils and monocytes, but progressively 

expands to include macrophages and T-lymphocytes over time (122, 152). If the 

recruited immune cells are incapable of clearing the pathogen, granulomas may form to 

sequester and isolate the offending agent from spreading to other regions of the tissue 

or body (122). The inflammatory pathway resolves once the pathogen or inflammatory 

agent is cleared from the body, and tissue repair and regeneration is activated to 

reverse damage incurred by the inflammatory process. 

 

One of the ways immune cells carry about effector functions is through the 

expression of cytokine molecules. Cytokines, which are soluble chemicals in the 

bloodstream used for cell signaling, and chemokines, which are chemotactic cytokines, 

contribute either to the development or resolution of inflammation, depending on the 

molecule (41, 78). They are expressed by both the innate and adaptive arms of the 

immune system, and the expression and levels of different cytokines are relevant in the 

 
 

20 

pathogenesis of inflammatory diseases such as IBD. They cytokine landscape in IBD is 

very diverse and includes a multitude of pro-inflammatory and anti-inflammatory 

cytokines and chemokines in its pathogenesis (47, 136, 199). Among them, 

therapeutics have been developed and are being tested to neutralize a portion of those 

cytokines and chemokines, such as Fontolizumab targeting IFNγ, Infliximab (tumor 

necrosis factor), Tocilizumab (IL-6), Ustekinumab and briakinumab (IL-12 and IL-23), 

Anrukinzumab (IL-13), Secukinumab (IL-17), Vercirnon (chemokine receptor CCR9),  

Eldelumab (CXCL10), and Smad7 anti-sense oligonucleotides (CCL20) (47, 136, 199). 

Furthermore, the therapeutic administration of recombinant IFNβ, recombinant IL-10, or 

recombinant IL-11 is also being studied as a means to limit inflammatory burden in IBD 

(136). Therefore, the regulation of cytokine and chemokine levels is an important area 

to regulate inflammation and is the current strategy to limit inflammatory damage to the 

ENS and surrounding tissue in IBD. 

The inflammatory pathway represents a destructive process as recruited 

neutrophils and monocytes phagocytose and destroy and clear foreign pathogens and 

cellular debris. However, their destructive nature and release of reactive oxygen species 

can be non-discriminant and cause collateral damage to the surrounding tissue (124, 

142, 152). The prolonged activation of inflammation in a tissue can lead to fibrosis and 

scar formation, leading to the replacement of damaged tissue with non-functional scar 

tissue. In the context of GI inflammation, intestinal fibrosis presents a very serious 

complication in IBD patients. The combination of inflammation-induced neuron loss in 

the ENS and increased intestinal fibrosis leads to GI secretomotor dysfunction in IBD 

patients and leads to an increased morbidity and mortality (76, 99). 

 
 

21 

Despite its destructive properties, the process of inflammation can be beneficial 

to tissue repair and regeneration. Inflammatory recruitment of immune cells, especially 

macrophages, play a major role in wound healing and tissue regeneration. Specifically, 

M2-subtype macrophages function to remove neutrophils and apoptotic cellular debris 

from inflammatory regions, to promote angiogenesis, extracellular matrix synthesis, 

stimulate progenitor cell activation, and fibroblast proliferation (80, 94, 142). 

Furthermore, at the resolution of inflammatory processes, the release of mediators such 

as resolvins by endothelial cells inhibits neutrophil recruitment and extravasation to 

damaged tissue, and stimulates macrophages to clear apoptotic neutrophils and other 

cell debris from the inflammatory site (46, 176). Many of these inflammatory functions, 

especially during the early stages of inflammation, are carried out by the innate immune 

system, and this arm of the immune system represents a major player in the GI 

inflammation. 

The Innate Immune System 

 

The innate immune system represents one of the first lines of defense against 

infectious agents and pathogens, the first line of defense being epithelial barriers that 

separate our bodies from the external environment and potential pathogens. The innate 

immune system consists of fast responding immune cells such as dendritic cells, 

macrophages, polymorphonuclear cells (including neutrophils, basophils, and 

eosinophils), and mast cells (83). These cells are positioned in areas of close proximity 

with potential pathogens, such as the mucosal layer of the intestinal tract. Innate 

immune cells recognize pathogens by common molecular patterns expressed on their 

cell surface, termed pathogen-associated molecular patterns (67, 83). Binding of these 

 
 

22 

pathogen-associated molecular patterns onto ‘pattern recognition receptors’ expressed 

on immune cells activates the immune cell, leading to phagocytosis and clearing of the 

pathogen (67, 83). Activation of innate immune cells such as dendritic cells can also 

lead to the migration of the dendritic cell to nearby lymph nodes to stimulate and 

activate the adaptive immune system. Depending on the severity of pathogen invasion, 

tissue inflammation may also ensue. 

 

Pattern recognition receptors are divided into 3 major types: Toll-like receptors 

(TLR), NOD-like receptors, and RIG-I-like receptors (96). These pattern recognition 

receptors bind pathogen-associated molecular patterns typically consisting of bacterial, 

viral, or fungal components. However, pattern recognition receptors can also bind 

molecules that represent cellular damage, termed damage-associated molecular 

patterns (59). For example, TLR1&2 recognize bacterial cell wall components like 

peptidoglycan, and triacyl lipopeptides, TLR3 recognize dsRNA molecules, TLR4 

recognize lipopolysaccharide (LPS), TLR5 recognize flagellin molecules, TLR7&8 

recognize ssRNA molecules, and TLR9 recognize dsDNA (83, 96, 226). RIG-I-like 

receptors recognize different viral pathogens, while NOD-like receptors a variety of 

bacterial, viral, and fungal pathogen-associated molecular patterns, along with non-

microbial damage-associated molecular patterns (96, 226). However, binding onto 

pattern recognition receptors does not always elicit the destruction of the 

microorganism. Pattern recognition receptors have also been shown as a means to 

monitor and recognize commensal microorganisms in the microbiota (23, 95). Binding of 

TLR molecules by commensal organisms is necessary for intestinal homeostasis, is a 

way to modulate microbiota populations, can induce anti-inflammatory responses in the 

 
 

23 

intestines, and modulates intestinal epithelial metabolism (23, 30, 95, 154, 218). In the 

context of the ENS, enteric glial cells can express pattern recognition receptors such as 

TLR4 to detect LPS molecules, and can release nitric oxide via TLR4 binding (107, 

201). 

 

As described above, the activation of innate immune cells can trigger 

phagocytosis of potential pathogens. The process of phagocytosis represents an 

important pathway in innate immune cells to both clear pathogens and to prepare 

antigens for presentation to the adaptive immune system (67). Once recognized by 

innate immune cells, restructuring of the cellular cytoskeleton allows for the engulfment 

and internalization of foreign molecules into phagosomes (44). The maturation of the 

phagosome into a phagolysosome involves the fusion with lysosomes, the acidification 

of vesicle contents, and the accumulation of reactive oxygen species and hydrolases 

including various proteases, lipases, nucleases, glycosidases, and phosphatases to 

degrade internalized particles (44, 203). In the GI tract, dendritic cells resident 

macrophages, and recruited neutrophils and monocytes are the primary cell populations 

carrying out phagocytosis activities. 

Antigen Presenting Cells 

 

While phagocytosis in neutrophils is primarily to clear pathogens and not to 

present antigens to the adaptive immune system, dendritic cells and macrophages play 

an additional role as professional antigen presenting cells to load phagocytosed 

particles onto major histocompatibility complex (MHC) II molecules for presentation to 

the adaptive immune system (145, 183). Antigen presenting cells form an important 

transition between the innate and adaptive immune system and can direct an immune 

 
 

24 

response towards specific targets. In addition to their phagocytic ability and constitutive 

expression of MHC II molecules, professional antigen presenting cells are also capable 

binding and activating T-lymphocytes (145, 169). This is accomplished by the 

expression of T-lymphocyte co-stimulatory molecules such as CD40, CD80, and CD86, 

along with activating cytokines to stimulate T-lymphocyte proliferation and differentiation 

(169).  

Although antigen presentation to T-lymphocytes is primarily carried out by 

dendritic cells and macrophages as professional antigen presenting cells, growing 

evidence supports the presence of ‘atypical’ antigen presenting cells as well (87, 137, 

183).  These atypical antigen presenting cells are characterized by the ability to express 

MHC II molecules in an inducible manner, as opposed to constitutive expression of 

MHC II in professional antigen presenting cells (87, 183). Furthermore, although 

atypical antigen presenting cells can express co-stimulatory molecules, they are 

generally not as effective as professional antigen presenting cells at triggering T-

lymphocyte activation, differentiation, and proliferation (87, 177). In fact, there are 

conflicting studies debating whether atypical antigen presenting cells are functional at 

inducing T-lymphocyte activation (87, 157, 177). Currently, cells such as mast cells, 

basophils, eosinophils, and type 3 innate lymphoid cells have been classified as atypical 

antigen presenting cells (20, 40, 45, 69, 87, 112, 141, 151, 220). However, due to 

conflicting findings on their capabilities to effectively function as an antigen presenting 

cell, the true role of atypical antigen presenting cells remains to be elucidated. 

Within the ENS, enteric glial cells show promise to also act as an atypical antigen 

presenting cell. Enteric glia have been shown to express MHC II molecules, T-cell co-

 
 

25 

stimulatory molecules, and can interact activate T-lymphocytes in vitro (4, 53, 201). 

Moreover, astrocyte cell populations in the CNS that are related to enteric glia show 

phagocytic capabilities and can uptake (85). A more detailed examination into the 

characteristics of enteric glial cells that supports their potential role as an immune 

modulator is presented in chapter 2 of this dissertation. 

Type II Major Histocompatibility Complex 

As described above, the MHC II molecule is constitutively expressed on 

professional antigen and functions as a crucial component by which self-derived or 

phagocytosed antigens are presented on the cell surface to T-lymphocytes (145, 183). 

The identity of presented peptides activates specific T-lymphocytes, and therefore 

modulates immune activation. Slight changes in the peptides presented can drastically 

change the resultant immune reaction, as simple post-translational modification in 

presented peptides can change a non-existent immune response into a complete 

autoimmune cascade (13, 72). Inappropriate peptide loading onto MHC II molecules 

and dysfunctional antigen presentation are contributing factors in the development of 

multiple diseases, such as IBD, systemic lupus erythematosus, and multiple sclerosis 

(25, 113, 123, 211, 216). Therefore, the proper loading of peptides onto MHC II 

molecules is crucial in directing appropriate immune responses. In the mouse (the 

animal model used in this dissertation), the MHC II molecule is a heterodimer composed 

of α1, α2, β1 and β2 chains (77, 184, 189) (Fig.1.5a). These are expressed on two 

functional α loci (Aa and Ea), and two functional β loci (Ab and Eb) on chromosome 17 

(77, 189) (Fig.1.5b). Gene products from either the Aa and Ab pair or Ea and Eb pair 

combine to form functional MHC II molecules. 

 
 

26 

 
Figure 1.5: Murine MHC II. (A) The MHC II molecule is a heterodimer composed of the 

α1-α2 peptide dimerized with the β1-β2 peptide, forming a peptide binding groove to 

present peptide fragments to the adaptive immune system. (B) The MHC II molecule is 

expressed from two functional α loci (Aa and Ea) and two functional β loci (Ab and Eb). 

Gene products from either Aa and Ab or Ea and Eb combine to form the MHC II 

molecule. 

 
 

 
 

 

27 

Newly synthesized MHC II molecules from the endoplasmic reticulum are 

attached with CD74, also known as the invariant chain (158, 190). The attachment of 

the invariant chain serves two major purposes: 1) to occupy the peptide binding groove 

on MHC II molecules and prevent inappropriate peptide binding, and 2) to guide newly 

synthesized MHC II to low-pH endosome compartments (158, 190). As MHC II 

molecules mature, the invariant chain is degraded until only a small peptide, the class II-

associated invariant-chain peptide (CLIP), remains bound to the MHC II peptide binding 

groove. It is not until the fusion with phagolysosomes that CLIP molecules are displaced 

from the peptide binding groove via the activity of the H2-M enzyme (158), and MHC II 

molecules are free to load antigens (Fig.1.6). Loaded MHC II molecules are transported 

to the cell surface where it can then, in conjunction with other co-stimulatory molecules, 

bind and activate T-lymphocytes. Although peptide loading onto MHC II molecules are 

generally derived from extracellular antigens that have been phagocytosed and 

processed, atypical loading of MHC II molecules can also occur through the loading of 

intracellular peptides via the autophagy pathway (37, 187). Despite loading intracellular 

antigens onto MHC II molecules, autophagy-induced peptide loading onto MHC II can 

not only avoid the development of auto-immunity but can also elicit an anti-inflammatory 

effect by limiting T-lymphocyte activation (212). 

 

 
 

 

28 

 
Figure 1.6: Maturation of MHC II molecules. Newly synthesized MHC II molecules are 

bound with the invariant chain peptide and transported into phagolysosomes, where 

proteolytic cleavage processes the bound invariant chain into the CLIP peptide while 

maintaining MHC II binding. The association of MHC II with the H2-M molecule (the 

murine equivalent to the human DM molecule) allows for the release of the CLIP  

 

 
 

29 

Figure 1.6 (cont’d): 

peptide from the MHC II peptide binding groove in order to load endocytosed peptides 

for presentation on the cell surface. Processing of cytoplasmic proteins in 

autophagolysosomes can also merge into this pathway and load cytoplasmic peptides 

onto MHC II molecules for presentation. 

 

 
 

 

30 

The autophagy pathway is important during development, but it also represents a 

method for cells to degrade and recycle cellular components during times of stress or 

starvation (102, 146). The autophagy pathway also plays a role in pathogen defense by 

playing a role in the degradation and presentation of intracellular bacterial and viral 

pathogens (103, 131). Furthermore, in a normal resting state, basal activation of 

autophagy and loading of autophagy-derived intracellular peptides contributes to 

immune tolerance (156). In the context of GI homeostasis, autophagic pathways are 

important in the maintenance of proper microbiota composition (200, 217), as 

autophagy dysfunction is associated with intestinal dysbiosis (98). 

The regulation of the autophagy pathways is an important method of maintaining 

homeostasis, and defective autophagy pathways have far reaching effects in the body. 

For example, dysfunction in the autophagy pathway is associated with genetic instability 

and tumorigenesis (101, 117), neurodegenerative diseases such as Huntington’s 

disease and Parkinson’s disease (115, 156, 213), GI diseases such as IBD and liver 

dysfunction (79, 147), and myopathies in both cardiac and skeletal muscle (140, 196). 

In the context of antigen presentation, while basal activation of autophagy-mediated 

MHC II loading is beneficial, a dysregulated autophagy pathway could theoretically lead 

to the presentation of inappropriate antigens to the adaptive immune system, but this 

has yet to be established (34, 172, 187, 205).  

The Adaptive Immune System 

 

The adaptive immune system demonstrates fairly different characteristics 

compared to the innate immune system. While innate immunity occurs quickly after 

binding pattern recognition receptors, the activation of the adaptive immune system is 

 
 

31 

much slower and generally involves cellular proliferation of specific adaptive immune 

cells after activation (215). This represents a second ‘phase’ of immunity after the innate 

immune system. A factor contributing to response time is the fact that innate immune 

cells are positioned in areas near potential infection sites. However, the adaptive 

immune system is generally positioned in different lymphoid organs and require 

recruitment (145). As described above, the adaptive immune system also requires 

contributions from the adaptive immune system in the form of antigen presentation cells. 

The presentation of antigens on MHC II molecules is crucial in order to direct the 

adaptive immune response (145, 183). In contrast, the activation of the innate immune 

system by binding of pattern recognition receptors elicits a consistent innate response, 

and not a ‘tailored’ adaptive response to specific antigens. Finally, unlike the innate 

immune system which provides the same response at each exposure to a given 

antigen, the adaptive immune system provides a lasting ‘memory’ for a quicker and 

more robust reaction at subsequent exposures to the same antigen. 

 

The adaptive immune system is comprised of two major cell types: T-

lymphocytes and B-lymphocytes. While B-lymphocytes are associated with humoral 

immunity from antibodies secreted by activated B-lymphocytes, T-lymphocytes are 

associated with cell-mediated immunity via direct cell-to-cell interactions. These two cell 

types do not act independently from each other, as specific T-lymphocyte subsets are 

involved in the activation of B-lymphocytes as well (12, 145). Contributions from both B 

and T-lymphocytes leads to robust adaptive immune responses. 

 
 

32 

T-lymphocytes 

 

T-lymphocytes form in the thymus, and through rigorous selection processes, 

they develop into either CD4-expressing or CD8-expressing T-lymphocytes (54). Once 

developed, but prior to encountering an antigen or stimulation, they are in a naïve and 

inactive state stored in lymphoid organs (145). However, antigen presentation and 

stimulation from antigen presenting cells causes the activation and proliferation of T-

lymphocytes. The activation state of a T-lymphocyte can be determined not only by the 

cytokines it expresses and proliferation rate, but also by the cell surface markers that 

are present. For example, acute markers for T-lymphocyte activation include CD69, 

which becomes detectable on the cell surface within several hours, and CD25, which 

becomes detectable within several days (31, 178). Once activated, T-lymphocytes take 

on effector roles to bring about immune responses (145). 

CD8-expressing T-lymphocytes are also known as cytotoxic T-lymphocytes, and 

they carry out their cytotoxic effector functions via cell-to-cell contact and binding of 

‘non-self’ MHC I molecules (145). These T-lymphocytes are especially important in the 

detection of intracellular pathogens such as viruses and modified ‘self’, such as in 

cancer (159). CD8+ T-lymphocytes rely on the recognition and binding of cell surface 

receptors and perform cytotoxic effects by perforating the target cell wall with perforin. 

The subsequent introduction of granzyme molecules into the target cell cytoplasm 

activates the caspase cascade and triggers apoptosis in the target cell (145). 

 

Unlike their CD8-expressing counterparts, CD4+ T-lymphocytes do not generally 

carry out their effector functions via cell receptor binding. Instead, their effector 

functions are carried out by releasing soluble molecules and cytokines (145). CD4+ T-

 
 

33 

lymphocytes are also referred to as helper T-lymphocytes due to their function to aid in 

the activation of other T-lymphocyte subtypes or other immune cells. CD4+ T-

lymphocytes can be further subdivided into different cell types based on the primary 

cytokine or transcription factor expressed by each CD4+ T-lymphocyte subtype. T-

lymphocyte subtypes that are of interest in this dissertation include Th1, Th2, Th17, and 

Treg T-lymphocytes. 

Each T-lymphocyte subtype expresses a unique combination of cytokines and 

can drive different forms of immune reactions. For example, Th1 T-lymphocytes are 

identified by their expression of IFNγ, IL-2, and TNF-β to activate macrophages, B cells, 

and cell mediated immunity with a predominantly phagocytic-dependent inflammation 

(161). Crohn’s disease has traditionally been thought of as a Th1-mediated disease 

(161), but recent studies demonstrate a joint role for Th1 and Th17 in its pathogenesis 

(133). In contrast, ulcerative colitis is regarded as a Th2-mediated disease with distinct 

cytokine profiles, expressing IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 (133, 160, 161, 188). 

Th2 T-lymphocytes can also inhibit Th1 effects such as phagocyte-dependent 

inflammation, while inducing an antibody-mediated response (161). Depending on the 

surrounding environment, activated Th17 T-lymphocytes express multiple cytokines 

including IL-9, IL-10, IL-17A, IL-17F, IL-21, IL-22, granulocyte-macrophage colony 

stimulating factor, and IFNγ (9, 116). The physiologic role for Th17 T-lymphocytes is to 

regulate neutrophil recruitment and provide immunity against bacterial and fungal 

pathogens via cytokine and chemokine release (9). However, improper Th17 regulation 

contributes to IBD pathogenesis and is associated with auto-immune diseases such as 

type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus and multiple 

 
 

34 

sclerosis (9, 100, 197). Finally, the Treg subtype of T-lymphocytes act in an anti-

inflammatory manner and limits proliferation in the other T-lymphocyte subtypes to 

promote immune tolerance, limit auto-immunity, and maintain tissue homeostasis and 

wound repair (86, 167, 221). They can be identified through their expression of the 

FoxP3 transcription factor, and they carry out immunosuppressive activities through the 

release of anti-inflammatory cytokines IL-10 and transforming growth factor beta (TGF-

β), sequestering pro-inflammatory cytokines, and inducing the anergy and apoptosis of 

other pro-inflammatory T-lymphocyte subtypes (86, 100, 167). 

Current models of intestinal inflammation 

 

A variety of animal models have been developed in order to model intestinal 

inflammation and inflammatory bowel diseases. These models are primarily chemically 

induced, genetically induced, or cell-transfer induced colitis models. While many 

different genetic models have been developed that develop intestinal inflammation, 

these models generally also trigger inflammatory responses or pathology in other organ 

systems as well. One of the more widely used animal knockouts are IL-10 knockout 

animals that demonstrate a spontaneous development of colitis after roughly 3 months 

of age (125, 126). Other knockout models include IL-2 knockout, TGFβ knockout, Toll-

Like Receptor 5 knockout, signal transducer and activator of transcription 3 knockout, T-

lymphocyte receptor α knockout, among others (125, 126). Adoptive transfer of CD4+ or 

CD8+ T-lymphocytes is another technique to model intestinal inflammation. Naïve CD4+ 

T-lymphocytes transferred from the spleen of wildtype animals to immune-deficient mice 

cause a Th1-mediated response to elicit transmural colitis when in the absence of 

regulatory T-lymphocytes (125). Similarly, transfer of naïve CD8+ T-lymphocytes to 

 
 

35 

recombination activating gene-2 knockout animals elicits colitis via activated CD8+ T-

lymphocytes expressing IL-17. In addition to the transfer of naïve T-lymphocytes, the 

transfer of CD8+ T-lymphocytes that are specific for heat shock protein 60 or ovalbumin 

(in the presence of administered ovalbumin) also elicits intestinal inflammation (125). 

Despite the effectiveness of genetic models or adoptive transfer of T-lymphocytes in 

generating intestinal inflammation, the most common type of model utilized for IBD 

comes in the form of chemically induced intestinal inflammation and IBD, due to the low 

cost and the ease of these models (155). These chemicals include dextran sulfate 

sodium (DSS), oxazolone, dinitrobenzene sulfonic acid, trinitrobenzene sulfonic acid, 

and acetic acid. These models are beneficial in that it does not require specific genetic 

strains of animals and can be administered to form discrete regions of intestinal 

inflammation. For example, DSS-induced colitis utilizes 1-5% of the polymer DSS in 

drinking water to induce acute phase colitis, characterized by increased levels of TNF-α 

(125, 155). Since colitis is triggered by intestinal epithelial damage due to osmotic 

stress from DSS and not due to the activation of adaptive immune cells, colitis can be 

elicited in animals lacking T- and B- lymphocytes (39). In contrast, oxazolone-induced 

colitis utilizes an intrarectal administration of haptenizing agent oxazolone with ethanol 

to elicit an acute development of colonic inflammation. As oxazolone is a haptenizing 

agent, it requires an intact adaptive immune system, specifically working via natural 

killer T cells (68). Similarly, the administration of haptenizing agents dinitrobenzene 

sulfonic acid and trinitrobenzene sulfonic acid by enema with ethanol allows for the 

development of regional intestinal inflammation and favors a Th1-mediated inflammatory 

response (125, 155). Finally, acetic acid enema causes intestinal epithelial damage and 

 
 

36 

is characterized by neutrophil recruitment and ulceration and necrosis in the mucosal 

and submucosal layers (155). 

 

This dissertation aims to examine the contributions of enteric glia on the 

development of intestinal inflammation. Given the severity of inflammation in the 

developed IBD models described above, experiments in this dissertation utilize a 

different form of inflammatory stimuli. Based on findings from Zhang et al. (223), we 

utilize intraperitoneal (IP) injections of IFNγ and LPS as inflammatory stimuli. This is to 

ensure that inflammatory stress does not overwhelm our experimental model and to 

prevent inflammatory responses being dominated by other immune cell types, thereby 

masking contributions from enteric glia. Details of our model and injection of 

inflammatory stimuli are described in Chapter 3.  

Summary and Aims of Dissertation 

 

The ENS is composed of enteric neurons and enteric glia, and it is the primary 

regulator of GI function (48, 194). Inflammatory damage to the ENS can lead to the loss 

of enteric neurons and glia, contributing to secretomotor dysfunctions seen in the GI 

diseases (56). Therefore, immune modulation in the GI tract is extremely important in 

maintaining ENS health and function. Neuroimmune interactions are becoming 

increasingly recognized as important aspects to immune modulation. As a part of the 

ENS, enteric glia demonstrate similar characteristics to antigen presenting cells. This 

suggests that enteric glia may play a similar role as antigen presenting cells and interact 

with the adaptive immune system, but their exact role in immune modulation has yet to 

be studied. 

 
 

37 

 

During homeostasis, enteric glia play important roles in the maintenance of 

enteric neuronal health and can modulate neuronal activity to maintain normal GI 

secretomotor functioning (32, 65, 135). However, during inflammation, enteric glia can 

contribute to neuronal death and express MHC II and T-lymphocyte co-stimulatory 

molecules (4, 18, 53, 201). Co-culture experiments demonstrate the ability for enteric 

glia to also modulate T-lymphocyte activity (90). 

 

The objective of this dissertation is to understand the role enteric glia play in vivo 

in modulating immune activity during homeostasis and in pro-inflammatory 

environments. Chapter 2 takes a further look into recent studies that demonstrate the 

potential for enteric glia to function as an immunomodulator. Chapter 3 investigates the 

capabilities of enteric glia to act as an antigen presenting cell, their effects on T-

lymphocyte activation, and the contributions autophagy has on glial MHC II expression. 

Together, these chapters aim to improve the understanding of how enteric glia interact 

with T-lymphocytes, and their role in immunomodulation. 

 

 
 

 

38 

REFERENCES

 
 

39 

REFERENCES 

1.   Ananthakrishnan AN, Bernstein CN, Iliopoulos D, Macpherson A, Neurath 

MF, Ali RAR, Vavricka SR, Fiocchi C. Environmental triggers in IBD: A review of 
progress and evidence. Nat. Rev. Gastroenterol. Hepatol. 15 Nature Publishing 
Group: 39–49, 2018. 

2.   Appelbaum J, Wells D, Hiatt JB, Steinbach G, Stewart FM, Thomas H, 

Nghiem P, Kapur RP, Thompson JA, Bhatia S. Fatal enteric plexus neuropathy 
after one dose of ipilimumab plus nivolumab: A case report. J Immunother Cancer 
6, 2018. 

3.   Bai A, Lu N, Guo Y, Chen J, Liu Z. Modulation of inflammatory response via α2-
adrenoceptor blockade in acute murine colitis. Clin Exp Immunol 156: 353–362, 
2009. 

4.   Barcelos Morais Da Silveira A, De Oliveira EC, Neto SG, Luquetti AO, Toshio 

Fujiwara R, Correa Oliveira R, Brehmer A, Barcelos A, Oliveira EC De, Neto 
SG, Luquetti AO, Toshio R, Correa R, Brehmer A. Enteroglial cells act as 
antigen-presenting cells in chagasic megacolon. Hum Pathol 42: 522–532, 2011. 

5.   Bartolomé-Casado R, Landsverk OJB, Chauhan SK, Richter L, Phung D, 
Greiff V, Risnes LF, Yao Y, Neumann RS, Yaqub S, Øyen O, Horneland R, 
Aandahl EM, Paulsen V, Sollid LM, Qiao SW, Baekkevold ES, Jahnsen FL. 
Resident memory CD8 T cells persist for years in human small intestine. J Exp 
Med 216: 2412–2426, 2019. 

6.   Bayliss WM, Starling EH. The movements and innervation of the small intestine. 

J Physiol 24: 99–143, 1899. 

7.   Bayliss WM, Starling EH. The movements and the innervation of the large 

intestine. J Physiol 26: 107–18, 1900. 

8.   Bayraktar OA, Fuentealba LC, Alvarez-Buylla A, Rowitch DH. Astrocyte 

development and heterogeneity. Cold Spring Harb Perspect Biol 7, 2015. 

9.   Bedoya SK, Lam B, Lau K, Larkin J. Th17 cells in immunity and autoimmunity. 

Clin. Dev. Immunol. 2013 Hindawi Limited: 986789, 2013. 

10.   Belai A, Boulos PB, Robson T, Burnstock G. Neurochemical coding in the 

small intestine of patients with Crohn’s disease. Gut 40: 767–774, 1997. 

11.   Le Berre-Scoul C, Chevalier J, Oleynikova E, Cossais F, Talon S, Neunlist M, 
Boudin H. A novel enteric neuron-glia coculture system reveals the role of glia in 
neuronal development. J Physiol 1542: 33–36, 2016. 

12.   Biram A, Davidzohn N, Shulman Z. T cell interactions with B cells during 

 
 

40 

germinal center formation, a three‐step model. Immunol Rev 288: 37–48, 2019. 

13.   Birnboim HC, Lemay A-M, Lam DKY, Goldstein R, Webb JR. Cutting edge: 

MHC class II-restricted peptides containing the inflammation-associated marker 3-
nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune 
response. J Immunol 171: 528–32, 2003. 

14.   Boesmans W, Cirillo C, Van den Abbeel V, Van den Haute C, Depoortere I, 

Tack J, Vanden Berghe P. Neurotransmitters involved in fast excitatory 
neurotransmission directly activate enteric glial cells. Neurogastroenterol Motil 25, 
2013. 

15.   Boesmans W, Martens MA, Weltens N, Hao MM, Tack J, Cirillo C, Vanden 

Berghe P. Imaging neuron-glia interactions in the enteric nervous system. Front 
Cell Neurosci 7: 183, 2013. 

16.   Bonaz, Bruno; Sinniger, Valerie; Pellissier S. Anti-inflammatory properties of 
the vagus nerve: potential therapeuitc implications of vagus nerve stimulation. J 
Physiol 82: 1496–1514, 2011. 

17.   Brosens E, Burns AJ, Brooks AS, Matera I, Borrego S, Ceccherini I, Tam PK, 

García-Barceló M-M, Thapar N, Benninga MA, Hofstra RMW, Alves MM. 
Genetics of enteric neuropathies. Dev Biol 417: 198–208, 2016. 

18.   Brown IAM, McClain JL, Watson RE, Patel BA, Gulbransen BD. Enteric Glia 

Mediate Neuron Death in Colitis Through Purinergic Pathways That Require 
Connexin-43 and Nitric Oxide. C Cell Mol Gastroenterol Hepatol 2: 77–91, 2016. 

19.   Browning KN, Travagli RA. Central nervous system control of gastrointestinal 

motility and secretion and modulation of gastrointestinal functions. Compr Physiol 
4: 1339–1368, 2014. 

20.   Von Burg N, Chappaz S, Baerenwaldt A, Horvath E, Bose Dasgupta S, 

Ashok D, Pieters J, Tacchini-Cottier F, Rolink A, Acha-Orbea H, Finke D. 
Activated group 3 innate lymphoid cells promote T-cell-mediated immune 
responses. Proc Natl Acad Sci U S A 111: 12835–12840, 2014. 

21.   Cantrell VA, Owens SE, Chandler RL, Airey DC, Bradley KM, Smith JR, 

Southard-Smith EM. Interactions between Sox10 and EdnrB modulate 
penetrance and severity of aganglionosis in the Sox10Dom mouse model of 
Hirschsprung disease. Hum Mol Genet 13: 2289–2301, 2004. 

22.   Capoccia E, Cirillo C, Gigli S, Pesce M, D’Alessandro A, Cuomo R, Sarnelli 

G, Steardo L, Esposito G. Enteric glia: A new player in inflammatory bowel 
diseases. Int J Immunopathol Pharmacol 28: 443–451, 2015. 

23.   Cario E. Toll-like receptors in inflammatory bowel diseases: A decade later. 

Inflamm. Bowel Dis. 16 Oxford Academic: 1583–1597, 2010. 

 
 

41 

24.   Cervi AL, Lukewich MK, Lomax AE. Neural regulation of gastrointestinal 

inflammation: role of the sympathetic nervous system. Auton Neurosci 182: 83–8, 
2014. 

25.   Chastain EML, Duncan DS, Rodgers JM, Miller SD. The role of antigen 

presenting cells in multiple sclerosis. Biochim. Biophys. Acta - Mol. Basis Dis. 
1812 NIH Public Access: 265–274, 2011. 

26.   Chatoor DR, Taylor SJ, Cohen CRG, Emmanuel A V. Faecal incontinence. Br J 

Surg 94: 134–144, 2007. 

27.   Cheadle G a, Costantini TW, Bansal V, Eliceiri BP, Coimbra R. Cholinergic 

Signaling in the Gut: A Novel Mechanism of Barrier Protection through Activation 
of Enteric Glia Cells. Surg Infect (Larchmt) 15: 387–93, 2014. 

28.   Chiu IM, Heesters BA, Ghasemlou N, Von Hehn CA, Zhao F, Tran J, Wainger 

B, Strominger A, Muralidharan S, Horswill AR, Bubeck Wardenburg J, 
Hwang SW, Carroll MC, Woolf CJ. Bacteria activate sensory neurons that 
modulate pain and inflammation. Nature 501: 52–7, 2013. 

29.   Cho JH. The genetics and immunopathogenesis of inflammatory bowel disease. 

Nat. Rev. Immunol. 8 Nat Rev Immunol: 458–466, 2008. 

30.   Chu H, Mazmanian SK. Innate immune recognition of the microbiota promotes 
host-microbial symbiosis. Nat. Immunol. 14 NIH Public Access: 668–675, 2013. 

31.   Cibrián D, Sánchez-Madrid F. CD69: from activation marker to metabolic 

gatekeeper. Eur. J. Immunol. 47 Wiley-VCH Verlag: 946–953, 2017. 

32.   Coelho-Aguiar J de M, Bon-Frauches AC, Gomes ALT, Verissimo CP, Aguiar 

DP, Matias D, Thomasi BB de M, Gomes AS, Brito GA de C, Moura-Neto V. 
The enteric glia: Identity and functions. Glia 63: 921–935, 2015. 

33.   Coombes JL, Powrie F. Dendritic cells in intestinal immune regulation. Nat. Rev. 

Immunol. 8 Europe PMC Funders: 435–446, 2008. 

34.   Crotzer VL, Blum JS. Autophagy and Its Role in MHC-Mediated Antigen 

Presentation. J Immunol 182: 3335–3341, 2009. 

35.   Cryan JF, O’riordan KJ, Cowan CSM, Sandhu K V., Bastiaanssen TFS, 

Boehme M, Codagnone MG, Cussotto S, Fulling C, Golubeva A V., Guzzetta 
KE, Jaggar M, Long-Smith CM, Lyte JM, Martin JA, Molinero-Perez A, 
Moloney G, Morelli E, Morillas E, O’connor R, Cruz-Pereira JS, Peterson VL, 
Rea K, Ritz NL, Sherwin E, Spichak S, Teichman EM, van de Wouw M, 
Ventura-Silva AP, Wallace-Fitzsimons SE, Hyland N, Clarke G, Dinan TG. 
The microbiota-gut-brain axis. Physiol Rev 99: 1877–2013, 2019. 

36.   Delvalle NM, Fried DE, Rivera-Lopez G, Gaudette L, Gulbransen BD. 

 
 

42 

Cholinergic activation of enteric glia is a physiological mechanism that contributes 
to the regulation of gastrointestinal motility. Am J Physiol - Gastrointest Liver 
Physiol 315: G473–G483, 2018. 

37.   Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Müller M, Kreymborg K, 

Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen C, 
Rammensee H, Stevanovic S. Autophagy promotes MHC class II presentation of 
peptides from intracellular source proteins. Proc Natl Acad Sci U S A 102: 7922–
7, 2005. 

38.   Dewanjee S, Das S, Das AK, Bhattacharjee N, Dihingia A, Dua TK, Kalita J, 

Manna P. Molecular mechanism of diabetic neuropathy and its 
pharmacotherapeutic targets. Eur J Pharmacol 833: 472–523, 2018. 

39.   Dieleman LA, Ridwan BU, Tennyson GS, Beagley KW, Bucy RP, Elson CO. 

Dextran sulfate sodium-induced colitis occurs in severe combined 
immunodeficient mice. Gastroenterology 107: 1643–1652, 1994. 

40.   Dimitriadou V, Mécheri S, Koutsilieris M, Fraser W, Al-Daccak R, Mourad W. 

Expression of functional major histocompatility complex class II molecules on 
HMC-1 human mast cells. J Leukoc Biol 64: 791–799, 1998. 

41.   Dinarello CA. Proinflammatory cytokines. Chest 118: 503–508, 2000. 

42.   Du YT, Rayner CK, Jones KL, Talley NJ, Horowitz M. Gastrointestinal 

symptoms in diabetes: Prevalence, assessment, pathogenesis, and management. 
Diabetes Care 41: 627–637, 2018. 

43.   Escalante J, McQuade RM, Stojanovska V, Nurgali K. Impact of chemotherapy 

on gastrointestinal functions and the enteric nervous system. Maturitas 105 
Elsevier Ireland Ltd: 23–29, 2017. 

44.   Flannagan RS, Jaumouillé V, Grinstein S. The cell biology of phagocytosis. 

Annu Rev Pathol Mech Dis 7: 61–98, 2012. 

45.   Fox CC, Jewell SD, Whitacre CC. Rat peritoneal mast cells present antigen to a 

PPD-specific T cell line. Cell Immunol 158: 253–264, 1994. 

46.   Freire MO, Van Dyke TE. Natural resolution of inflammation. Periodontol 2000 

63: 149–164, 2013. 

47.   Friedrich M, Pohin M, Powrie F. Cytokine Networks in the Pathophysiology of 

Inflammatory Bowel Disease. Immunity 50: 992–1006, 2019. 

48.   Furness JB. The enteric nervous system and neurogastroenterology. Nat. Rev. 

Gastroenterol. Hepatol. 9: 286–294, 2012. 

49.   Furness JB, Costa M. Types of neurons in the enteric nervous system. J Auton 

 
 

43 

Nerv Syst 81: 87–96, 2000. 

50.   Gabella G. Ultrastructure of the nerve plexuses of the mammalian intestine: The 

enteric glial cells. Neuroscience 6: 425–436, 1981. 

51.   Gabella G, Trigg P. Size of neurons and glial cells in the enteric ganglia of mice, 

guinea-pigs, rabbits and sheep. J Neurocytol 13: 49–71, 1984. 

52.   Gallagher P, O’Mahony D. Constipation in old age. Best Pract Res Clin 

Gastroenterol 23: 875–887, 2009. 

53.   Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, Vantrappen G, 

Desmet VJ. Major histocompatibility class II expression on the small intestinal 
nervous system in Crohn’s disease. Gastroenterology 103: 439–447, 1992. 

54.   Germain RN. t-cell development and the CD4-CD8 lineage decision. Nat Rev 

Immunol 2: 309–322, 2002. 

55.   Gershon MD, Rothman TP. Enteric glia. Glia 4: 195–204, 1991. 

56.   De Giorgio R, Guerrini S, Barbara G, Stanghellini V, De Ponti F, Corinaldesi 
R, Moses PL, Sharkey KA, Mawe GM. Inflammatory neuropathies of the enteric 
nervous system. Gastroenterology 126: 1872–1883, 2004. 

57.   De Giorgio R, Stanghellini V, Barbara G, Corinaldesi R, De Ponti F, Tonini M, 
Bassotti G, Sternini C. Primary enteric neuropathies underlying gastrointestinal 
motor dysfunction. Scand J Gastroenterol 35: 114–122, 2000. 

58.   Gomes P, Chevalier J, Boesmans W, Roosen L, Van Den Abbeel V, Neunlist 

M, Tack J, Vanden Berghe P. ATP-dependent paracrine communication 
between enteric neurons and glia in a primary cell culture derived from embryonic 
mice. Neurogastroenterol Motil 21, 2009. 

59.   Gong T, Liu L, Jiang W, Zhou R. DAMP-sensing receptors in sterile 

inflammation and inflammatory diseases. Nat. Rev. Immunol. 20 Nature 
Research: 95–112, 2020. 

60.   Greenwood-Van Meerveld B, Johnson AC, Grundy D. Gastrointestinal 

Physiology and Function. Handb Exp Pharmacol 239: 1–16, 2017. 

61.   Grider JR. Neurotransmitters mediating the intestinal peristaltic reflex in the 

mouse. J Pharmacol Exp Ther 307: 460–7, 2003. 

62.   Gulbransen BD, Bains JS, Sharkey KA. Enteric Glia Are Targets of the 

Sympathetic Innervation of the Myenteric Plexus in the Guinea Pig Distal Colon. J 
Neurosci 30: 6801–6809, 2010. 

63.   Gulbransen BD, Bashashati M, Hirota SA, Gui X, Roberts JA, MacDonald JA, 

 
 

44 

Muruve DA, McKay DM, Beck PL, Mawe GM, Thompson RJ, Sharkey KA. 
Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric 
neurons during colitis. Nat Med 18: 600–604, 2012. 

64.   Gulbransen BD, Sharkey KA. Purinergic Neuron-to-Glia Signaling in the Enteric 

Nervous System. Gastroenterology 136: 1349–1358, 2009. 

65.   Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the 
gastrointestinal tract. Nat. Rev. Gastroenterol. Hepatol. 9 Nature Publishing 
Group: 625–632, 2012. 

66.   Hansebout CR, Su CX, Reddy K, Zhang D, Jiang C, Rathbone MP, Jiang S. 
Enteric glia mediate neuronal outgrowth through release of neurotrophic factors. 
Neural Regen Res 7: 2165–2175, 2012. 

67.   Hato T, Dagher PC. How the innate immune system senses trouble and causes 

trouble. Clin J Am Soc Nephrol 10: 1459–1469, 2015. 

68.   Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W. Oxazolone 
colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-
producing NK-T cells. Immunity 17: 629–638, 2002. 

69.   Hepworth MR, Monticelli LA, Fung TC, Ziegler CGK, Grunberg S, Sinha R, 

Mantegazza AR, Ma HL, Crawford A, Angelosanto JM, John Wherry E, Koni 
PA, Bushman FD, Elson CO, Eberl G, Artis D, Sonnenberg GF. Innate 
lymphoid cells regulate CD4 + T-cell responses to intestinal commensal bacteria. 
Nature 498: 113–117, 2013. 

70.   Hernandez S, Fried DE, Grubišić V, McClain JL, Gulbransen BD. 

Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness. 
FASEB J 33: 6168–6184, 2019. 

71.   Hernandez S, Morales-Soto W, Grubišić V, Fried D, Gulbransen BD. 

Pyridostigmine bromide exposure creates chronic, underlying neuroimmune 
disruption in the gastrointestinal tract and brain that alters responses to 
palmitoylethanolamide in a mouse model of Gulf War Illness. Neuropharmacology 
(2020). doi: 10.1016/j.neuropharm.2020.108264. 

72.   Herzog J, Maekawa Y, Cirrito TP, Illian BS, Unanue ER. Activated antigen-

presenting cells select and present chemically modified peptides recognized by 
unique CD4 T cells. Proc Natl Acad Sci U S A 102: 7928–7933, 2005. 

73.   Hirst GD, McKirdy HC. Presynaptic inhibition at mammalian peripheral synapse? 

Nature 250: 430–1, 1974. 

74.   Hoff S, Zeller F, Von Weyhern CWH, Wegner M, Schemann M, Michel K, Rühl 

A. Quantitative assessment of glial cells in the human and guinea pig enteric 
nervous system with an anti-Sox8/9/10 antibody. J Comp Neurol 509: 356–371, 

 
 

45 

2008. 

75.   Holleran G, Lopetuso L, Petito V, Graziani C, Ianiro G, McNamara D, 

Gasbarrini A, Scaldaferri F. The innate and adaptive immune system as targets 
for biologic therapies in inflammatory bowel disease. Int. J. Mol. Sci. 18 MDPI AG: 
2017. 

76.   Holvoet T, Devriese S, Castermans K, Boland S, Leysen D, Vandewynckel 

YP, Devisscher L, Van den Bossche L, Van Welden S, Dullaers M, 
Vandenbroucke RE, De Rycke R, Geboes K, Bourin A, Defert O, Hindryckx 
P, De Vos M, Laukens D. Treatment of Intestinal Fibrosis in Experimental 
Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase 
Inhibitor. Gastroenterology 153: 1054–1067, 2017. 

77.   Hood L, Steinmetz M, Malissen B. Genes of the major histocompatibility 

complex of the mouse. Annu Rev Immunol 1: 529–68, 1983. 

78.   Hughes CE, Nibbs RJB. A guide to chemokines and their receptors. FEBS J. 

285 Blackwell Publishing Ltd: 2944–2971, 2018. 

79.  

80.  

Iida T, Onodera K, Nakase H. Role of autophagy in the pathogenesis of 
inflammatory bowel disease. World J Gastroenterol 23: 1944–1953, 2017. 

Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: Phenotypical 
vs. functional differentiation. Front. Immunol. 5 Frontiers Research Foundation: 
2014. 

81.   Jabari S, De Oliveira EC, Brehmer A, Da Silveira ABM. Chagasic megacolon: 

Enteric neurons and related structures. Histochem. Cell Biol. 142 Springer Verlag: 
235–244, 2014. 

82.   Jabbur SJ, El-Kak FH, Nassar CF. The enteric nervous system--an overview. 

Med Res Rev 8: 459–69, 1988. 

83.   Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 20: 

197–216, 2002. 

84.   Jessen KR, Mirsky R. Astrocyte-like glia in the peripheral nervous system: an 

immunohistochemical study of enteric glia. J Neurosci 3: 2206–18, 1983. 

85.   Jones RS, Minogue AM, Connor TJ, Lynch MA. Amyloid-β-induced astrocytic 

phagocytosis is mediated by CD36, CD47 and RAGE. J Neuroimmune Pharmacol 
8: 301–11, 2013. 

86.   Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: Mechanisms of 

differentiation and function. Annu. Rev. Immunol. 30 NIH Public Access: 531–564, 
2012. 

 
 

46 

87.   Kambayashi T, Laufer TM. Atypical MHC class II-expressing antigen-presenting 
cells: can anything replace a dendritic cell? Nat Rev Immunol 14: 719–30, 2014. 

88.   Kapur RP, Yost C, Palmiter RD. A transgenic model for studying development of 

the enteric nervous system in normal and aganglionic mice. [Online]. 
Development 116: 167–75, 1992. https://pubmed.ncbi.nlm.nih.gov/1483385/ [4 
Oct. 2020]. 

89.   Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is 

not required for the generation or propagation of colonic migrating motor 
complexes. Gastroenterology 138: 659-70 670.e1–2, 2010. 

90.   Kermarrec L, Durand T, Neunlist M, Naveilhan P, Neveu I. Enteric glial cells 
have specific immunosuppressive properties. J Neuroimmunol 295–296: 79–83, 
2016. 

91.   Kim BJ, Jones HP. Epinephrine-primed murine bone marrow-derived dendritic 
cells facilitate production of IL-17A and IL-4 but not IFN-γ by CD4+ T cells. Brain 
Behav Immun 24: 1126–1136, 2010. 

92.   Kimball BC, Mulholland MW. Enteric glia exhibit P2U receptors that increase 

cytosolic calcium by a phospholipase C-dependent mechanism. J Neurochem 66: 
604–612, 1996. 

93.   King RHM. The role of glycation in the pathogenesis of diabetic polyneuropathy. 

J. Clin. Pathol. - Mol. Pathol. 54 BMJ Publishing Group: 400–408, 2001. 

94.   Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the 
macrophage. Expert Rev. Mol. Med. 13 NIH Public Access: e23, 2011. 

95.   Kubinak JL, Round JL. Toll-like receptors promote mutually beneficial 

commensal-host interactions. PLoS Pathog 8: 1, 2012. 

96.   Kumar H, Kawai T, Akira S. Pathogen recognition by the innate immune system. 

Int Rev Immunol 30: 16–34, 2011. 

97.   Kunze WA, Furness JB. The enteric nervous system and regulation of intestinal 

motility. Annu Rev Physiol 61: 117–42, 1999. 

98.   Larabi A, Barnich N, Nguyen HTT. New insights into the interplay between 

autophagy, gut microbiota and inflammatory responses in IBD. Autophagy 16: 38–
51, 2020. 

99.   Latella G, Di Gregorio J, Flati V, Rieder F, Lawrance IC. Mechanisms of 

initiation and progression of intestinal fibrosis in IBD. Scand J Gastroenterol 50: 
53–65, 2015. 

100.   Lee GR. The balance of th17 versus treg cells in autoimmunity. Int J Mol Sci 19: 

 
 

47 

1–14, 2018. 

101.   Levine B. Cell biology: Autophagy and cancer. Nature 446 Nature Publishing 

Group: 745–747, 2007. 

102.   Levine B, Kroemer G. Biological Functions of Autophagy Genes: A Disease 

Perspective. Cell 176 Cell Press: 11–42, 2019. 

103.   Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, Herman 

B, Levine B. Protection against Fatal Sindbis Virus Encephalitis by Beclin, a 
Novel Bcl-2-Interacting Protein. J Virol 72: 8586–8596, 1998. 

104.   Linden DR. Enhanced excitability of guinea pig ileum myenteric AH neurons 
during and following recovery from chemical colitis. Neurosci Lett 545: 91–95, 
2013. 

105.   Linden DR, Sharkey KA, Mawe GM. Enhanced excitability of myenteric AH 

neurones in the inflamed guinea-pig distal colon. J. Physiol. 547 J Physiol: 589–
601, 2003. 

106.   Lomax AE, Sharkey KA, Furness JB. The participation of the sympathetic 

innervation of the gastrointestinal tract in disease states. Neurogastroenterol Motil 
22: 7–18, 2010. 

107.   Lu Y, Li X, Liu S, Zhang Y, Zhang D. Toll-like receptors and inflammatory bowel 

disease. Front. Immunol. 9 Frontiers Media S.A.: 72, 2018. 

108.   Lundgren O, Svanvik J, Jivegård L. Enteric nervous system - I. Physiology and 

pathophysiology of the intestinal tract. Dig Dis Sci 34: 264–283, 1989. 

109.   Luo P, Liu D, Li C, He WX, Zhang CL, Chang MJ. Enteric glial cell activation 

protects enteric neurons from damage due to diabetes in part via the promotion of 
neurotrophic factor release. Neurogastroenterol Motil 30, 2018. 

110.   MacEachern SJ, Patel BA, Keenan CM, Dicay M, Chapman K, McCafferty D-
M, Savidge TC, Beck PL, MacNaughton WK, Sharkey KA. Inhibiting Inducible 
Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice 
With Colitis. Gastroenterology 149: 445–55.e3, 2015. 

111.   Maceachern SJ, Patel BA, Mckay DM, Sharkey KA. Nitric oxide regulation of 

colonic epithelial ion transport: A novel role for enteric glia in the myenteric 
plexus. J Physiol 589: 3333–3348, 2011. 

112.   MacKenzie JR, Mattes J, Dent LA, Foster PS.  Eosinophils Promote Allergic 

Disease of the Lung by Regulating CD4 + Th2 Lymphocyte Function . J Immunol 
167: 3146–3155, 2001. 

113.   Mahdi BM. Role of HLA typing on Crohn’s disease pathogenesis. Ann Med Surg 

 
 

48 

4: 248–253, 2015. 

114.   Margolis KG, Gershon MD. Enteric Neuronal Regulation of Intestinal 

Inflammation. Trends Neurosci xx: 1–11, 2016. 

115.   Martinez-Vicente M, Cuervo AM. Autophagy and neurodegeneration: when the 

cleaning crew goes on strike. Lancet Neurol. 6 Lancet Neurol: 352–361, 2007. 

116.   Marwaha AK, Leung NJ, McMurchy AN, Levings MK. TH17 cells in 

autoimmunity and immunodeficiency : Protective orpathogenic? Front. Immunol. 3 
Frontiers Media SA: 2012. 

117.   Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat. 

Rev. Cancer 7 Nat Rev Cancer: 961–967, 2007. 

118.   Matteoli G, Boeckxstaens GE. The vagal innervation of the gut and immune 

homeostasis. Gut 62: 1214–22, 2013. 

119.   Matteoli G, Gomez-Pinilla PJ, Nemethova A, Di Giovangiulio M, Cailotto C, 

van Bree SH, Michel K, Tracey KJ, Schemann M, Boesmans W, Vanden 
Berghe P, Boeckxstaens GE. A distinct vagal anti-inflammatory pathway 
modulates intestinal muscularis resident macrophages independent of the spleen. 
Gut (2013). doi: 10.1136/gutjnl-2013-304676. 

120.   Mckeown SJ, Stamp L, Hao MM, Young HM. Hirschsprung disease: A 

developmental disorder of the enteric nervous system. Wiley Interdiscip Rev Dev 
Biol 2: 113–129, 2013. 

121.   McQuade RM, Al Thaalibi M, Nurgali K. Impact of chemotherapy-induced 

enteric nervous system toxicity on gastrointestinal mucositis. Curr Opin Support 
Palliat Care 14: 293–300, 2020. 

122.   Medzhitov R. Origin and physiological roles of inflammation. Nature 454: 428–

435, 2008. 

123.   Middel P, Raddatz D, Gunawan B, Haller F, Radzun HJ. Increased number of 

mature dendritic cells in Crohn’s disease: Evidence for a chemokine mediated 
retention mechanism. Gut 55: 220–227, 2006. 

124.   Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB. Reactive oxygen species 
in inflammation and tissue injury. Antioxidants Redox Signal. 20 Antioxid Redox 
Signal: 1126–1167, 2014. 

125.   Mizoguchi A. Animal models of inflammatory bowel disease. Prog Mol Biol Transl 

Sci 105: 263–320, 2012. 

126.   Mizoguchi A, Mizoguchi E. Animal models of IBD: linkage to human disease. 

Curr Opin Pharmacol 10: 578–87, 2010. 

 
 

49 

127.   Mollaaghababa R, Pavan WJ. The importance of having your SOX on: Role of 

SOX10 in the development of neural crest-derived melanocytes and glia. 
Oncogene 22 Oncogene: 3024–3034, 2003. 

128.   Mourad FH, Saadé NE. Neural regulation of intestinal nutrient absorption. Prog 

Neurobiol 95: 149–162, 2011. 

129.   Moynes DM, Lucas GH, Beyak MJ, Lomax AE. Effects of inflammation on the 

innervation of the colon. Toxicol Pathol 42: 111–117, 2014. 

130.   Murakami M, Ohta T, Otsuguro KI, Ito S. Involvement of prostaglandin E2 

derived from enteric glial cells in the action of bradykinin in cultured rat myenteric 
neurons. Neuroscience 145: 642–653, 2007. 

131.   Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto 

T, Nara A, Funao J, Nakata M, Tsuda K, Hamada S, Yoshimori T. Autophagy 
defends cells against invading group A Streptococcus. Science (80- ) 306: 1037–
1040, 2004. 

132.   Nasser Y, Ho W, Sharkey KA. Distribution of adrenergic receptors in the enteric 
nervous system of the guinea pig, mouse, and rat. J Comp Neurol 495: 529–553, 
2006. 

133.   Nemeth ZH, Bogdanovski DA, Barratt-Stopper P, Paglinco SR, Antonioli L, 
Rolandelli RH. Crohn’s Disease and Ulcerative Colitis Show Unique Cytokine 
Profiles. Cureus 9, 2017. 

134.   Neunlist M, Aubert P, Toquet C, Oreshkova T, Barouk J, Lehur PA, 

Schemann M, Galmiche JP. Changes in chemical coding of myenteric neurones 
in ulcerative colitis. Gut 52: 84–90, 2003. 

135.   Neunlist M, Rolli-Derkinderen M, Latorre R, Van Landeghem L, Coron E, 
Derkinderen P, De Giorgio R. Enteric glial cells: Recent developments and 
future directions. Gastroenterology 147 W.B. Saunders: 1230–1237, 2014. 

136.   Neurath MF. Cytokines in inflammatory bowel disease. Nat Rev Immunol 14: 

329–42, 2014. 

137.   Nickoloff BJ, Turka LA. Immunological functions of non-professional antigen-

presenting cells: new insights from studies of T-cell interactions with 
keratinocytes. Immunol Today 15: 464–469, 1994. 

138.   Nijhuis LEJ, Olivier BJ, de Jonge WJ. Neurogenic regulation of dendritic cells in 

the intestine. Biochem Pharmacol 80: 2002–8, 2010. 

139.   Nishida A, Inoue R, Inatomi O, Bamba S, Naito Y, Andoh A. Gut microbiota in 

the pathogenesis of inflammatory bowel disease. Clin. J. Gastroenterol. 11 
Springer Tokyo: 2018. 

 
 

50 

140.   Nishino I. Autophagic Vacuolar Myopathy. Semin Pediatr Neurol 13: 90–95, 

2006. 

141.   Oh K, Shen T, Le Gros G, Min B. Induction of Th2 type immunity in a mouse 
system reveals a novel immunoregulatory role of basophils. Blood 109: 2921–
2927, 2007. 

142.   Oishi Y, Manabe I. Macrophages in inflammation, repair and regeneration. Int 

Immunol 30: 511–528, 2018. 

143.   Oshima T, Miwa H. Gastrointestinal mucosal barrier function and diseases. J 

Gastroenterol 51: 768–778, 2016. 

144.   Panizzon CP do NB, Zanoni JN, Hermes-Uliana C, Trevizan AR, Sehaber CC, 

Pereira RVF, Linden DR, Neto MH de M. Desired and side effects of the 
supplementation with L-glutamine and L-glutathione in enteric glia of diabetic rats. 
Acta Histochem 118: 625–631, 2016. 

145.   Parkin J, Cohen B. An overview of the immune system. Lancet (London, 

England) 357: 1777–89, 2001. 

146.   Parzych KR, Klionsky DJ. An overview of autophagy: morphology, mechanism, 

and regulation. Antioxid Redox Signal 20: 460–73, 2014. 

147.   Perlmutter DH. The role of autophagy in α-1-antitrypsin deficiency: A specific 

cellular response in genetic diseases associated with aggregation-prone proteins. 
Autophagy 2 Taylor and Francis Inc.: 258–263, 2006. 

148.   Pesce M, Borrelli O, Saliakellis E, Thapar N. Gastrointestinal Neuropathies: 

New Insights and Emerging Therapies. Gastroenterol Clin North Am 47: 877–894, 
2018. 

149.   Peterson LW, Artis D. Intestinal epithelial cells: Regulators of barrier function 

and immune homeostasis. Nat. Rev. Immunol. 14 Nature Publishing Group: 141–
153, 2014. 

150.   De Ponti F, Giaroni C, Cosentino M, Lecchini S, Frigo G. Adrenergic 

mechanisms in the control of gastrointestinal motility: from basic science to 
clinical applications. Pharmacol Ther 69: 59–78, 1996. 

Tramón P, Palomino P, Lahoz C, Leyva‐Cobian F. Eosinophil as antigen‐

151.   Pozo V Del, de Andrés B, Martín E, Cárdaba B, Fernández JC, Gallardo S, 

presenting cell: activation of T cell clones and T cell hybridoma by eosinophils 
after antigen processing. Eur J Immunol 22: 1919–1925, 1992. 

152.   Prame Kumar K, Nicholls AJ, Wong CHY. Partners in crime: neutrophils and 

monocytes/macrophages in inflammation and disease. Cell Tissue Res 371: 551–
565, 2018. 

 
 

51 

153.   Qi R, Yang W, Chen J. Role of enteric glial cells in gastric motility in diabetic rats 

at different stages. J Huazhong Univ Sci Technol - Med Sci 33: 496–500, 2013. 

154.   Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. 

Recognition of commensal microflora by toll-like receptors is required for intestinal 
homeostasis. Cell 118: 229–241, 2004. 

155.   Randhawa PK, Singh K, Singh N, Jaggi AS. A review on chemical-induced 

inflammatory bowel disease models in rodents. Korean J. Physiol. Pharmacol. 18 
Korean Physiological Soc. and Korean Soc. of Pharmacology: 279–288, 2014. 

156.   Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-

Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, 
Massey DCO, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, 
Underwood BR, Winslow And AR, Rubinsztein DC. Regulation of mammalian 
autophagy in physiology and pathophysiology. Physiol. Rev. 90  American 
Physiological Society Bethesda, MD: 1383–1435, 2010. 

157.   van Rijt LS, Vos N, Hijdra D, de Vries VC, Hoogsteden HC, Lambrecht BN. 

Airway Eosinophils Accumulate in the Mediastinal Lymph Nodes but Lack 
Antigen-Presenting Potential for Naive T Cells. J Immunol 171: 3372–3378, 2003. 

158.   Roche PA, Furuta K. The ins and outs of MHC class II-mediated antigen 

processing and presentation. Nat Rev Immunol 15: 203–16, 2015. 

159.   Rock KL, Reits E, Neefjes J. Present Yourself! By MHC Class I and MHC Class 

II Molecules. Trends Immunol 37: 724–737, 2016. 

160.   Romagnani S. Th1/Th2 cells. Inflamm Bowel Dis 5: 285–94, 1999. 

161.   Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy, Asthma Immunol 

85: 9–18, 2000. 

162.   Rosenbaum C, Schick MA, Wollborn J, Heider A, Scholz C-J, Cecil A, Niesler 

B, Hirrlinger J, Walles H, Metzger M. Activation of Myenteric Glia during Acute 
Inflammation In Vitro and In Vivo. PLoS One 11: e0151335, 2016. 

163.   Rothman TP, Tennyson VM, Gershon MD. Colonization of the bowel by the 

precursors of enteric glia: Studies of normal and congenitally aganglionic mutant 
mice. J Comp Neurol 252: 493–506, 1986. 

164.   Rühl A. Glial cells in the gut. Neurogastroenterol Motil 17: 777–790, 2005. 

165.   Rühl A, Nasser Y, Sharkey KA. Enteric glia. Neurogastroenterol Motil 16 Suppl 

1: 44–9, 2004. 

166.   Saffrey MJ. Cellular changes in the enteric nervous system during ageing. Dev 

Biol 382: 344–55, 2013. 

 
 

52 

167.   Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T Cells and Immune 

Tolerance. Cell 133 Cell Press: 775–787, 2008. 

168.   Sanders KM, Koh SD, Ro S, Ward SM. Regulation of gastrointestinal motility-

insights from smooth muscle biology. Nat. Rev. Gastroenterol. Hepatol. 9 Nat Rev 
Gastroenterol Hepatol: 633–645, 2012. 

169.   Santana MA, Esquivel-Guadarrama F. Cell biology of T cell activation and 

differentiation. Int Rev Cytol 250: 217–74, 2006. 

170.   Savidge TC, Newman P, Pothoulakis C, Ruhl A, Neunlist M, Bourreille A, 
Hurst R, Sofroniew M V. Enteric Glia Regulate Intestinal Barrier Function and 
Inflammation Via Release of S-Nitrosoglutathione. Gastroenterology 132: 1344–
1358, 2007. 

171.   Scheperjans F, Aho V, Pereira PAB, Koskinen K, Paulin L, Pekkonen E, 

Haapaniemi E, Kaakkola S, Eerola-Rautio J, Pohja M, Kinnunen E, Murros K, 
Auvinen P. Gut microbiota are related to Parkinson’s disease and clinical 
phenotype. Mov Disord 30: 350–358, 2015. 

172.   Schmid D, Pypaert M, Münz C. Antigen-loading compartments for major 

histocompatibility complex class II molecules continuously receive input from 
autophagosomes. Immunity 26: 79–92, 2007. 

173.   Schneider J, Jehle EC, Starlinger MJ, Neunlist M, Michel K, Hoppe S, 

Schemann M. Neurotransmitter coding of enteric neurones in the submucous 
plexus is changed in non-inflamed rectum of patients with Crohn’s disease. 
Neurogastroenterol Motil 13: 255–264, 2001. 

174.   Schulzke JD, Fromm M, Hegel U, Riecken EO. Ion transport and enteric 

nervous system (ENS) in rat rectal colon: mechanical stretch causes electrogenic 
Cl-secretion via plexus Meissner and amiloride-sensitive electrogenic Na-
absorption is not affected by intramural neurons. Pflugers Arch 414: 216–21, 
1989. 

175.   Segura BJ, Zhang W, Cowles RA, Xiao L, Lin TR, Logsdon C, Mulholland 

MW. Lysophosphatidic acid stimulates calcium transients in enteric glia. 
Neuroscience 123: 687–693, 2004. 

176.   Serhan CN, Levy BD. Resolvins in inflammation: Emergence of the pro-resolving 

superfamily of mediators. J. Clin. Invest. 128 American Society for Clinical 
Investigation: 2657–2669, 2018. 

177.   Sharma M, Hegde P, Aimanianda V, Beau R, Sénéchal H, Poncet P, Latgé JP, 

Kaveri S V., Bayry J. Circulating human basophils lack the features of 
professional antigen presenting cells. Sci Rep 3, 2013. 

178.   Shipkova M, Wieland E. Surface markers of lymphocyte activation and markers 

 
 

53 

of cell proliferation. Clin. Chim. Acta 413 Elsevier: 1338–1349, 2012. 

179.   Silva FAR, Rodrigues BL, Ayrizono MDLS, Leal RF. The Immunological Basis 

of Inflammatory Bowel Disease. Gastroenterol. Res. Pract. 2016 Hindawi 
Publishing Corporation: 2016. 

180.   da Silveira ABM, Lemos EM, Adad SJ, Correa-Oliveira R, Furness JB, 

D’Avila Reis D. Megacolon in Chagas disease: a study of inflammatory cells, 
enteric nerves, and glial cells. Hum Pathol 38: 1256–1264, 2007. 

181.   Soret R, Schneider S, Bernas G, Christophers B, Souchkova O, Charrier B, 
Righini-Grunder F, Aspirot A, Landry M, Kembel S, Faure C, Heuckeroth R, 
Pilon N. Glial Cell Derived Neurotrophic Factor Induces Enteric Neurogenesis 
and Improves Colon Structure and Function in Mouse Models of Hirschsprung 
Disease. Gastroenterology (2020). doi: 10.1053/j.gastro.2020.07.018. 

182.   Spencer NJ, Dinning PG, Brookes SJ, Costa M. Insights into the mechanisms 

underlying colonic motor patterns. J Physiol 594: 4099–116, 2016. 

183.   Sprent J. Antigen-Presenting Cells: Professionals and amateurs. Curr Biol 5: 

1095–1097, 1995. 

184.   Steinmetz M, Hood LE. Genes of the major histocompatibility complex in mouse 

and man. Biotechnol Biol Front : 303–317, 2019. 

185.   Sternini C. Structural and chemical organization of the myenteric plexus. Annu 

Rev Physiol 50: 81–93, 1988. 

186.   Straub RH, Wiest R, Strauch UG, Härle P, Schölmerich J. The role of the 

sympathetic nervous system in intestinal inflammation. Gut 55: 1640–9, 2006. 

187.   Strawbridge AB, Blum JS. Autophagy in MHC class II antigen processing. Curr 

Opin Immunol 19: 87–92, 2007. 

188.   Strober W, Fuss IJ. Proinflammatory cytokines in the pathogenesis of 
inflammatory bowel diseases. Gastroenterology 140: 1756–1767, 2011. 

189.   Stuart PM. Major Histocompatibility Complex (MHC): Mouse. eLS : 1–7, 2015. 

190.   Su H, Na N, Zhang X, Zhao Y. The biological function and significance of CD74 

in immune diseases. Inflamm Res 66: 209–216, 2017. 

191.   Sullivan PB. Hirschprung’s disease. Arch Dis Child 74: 5–7, 1996. 

192.   Takiishi T, Fenero CIM, Câmara NOS. Intestinal barrier and gut microbiota: 
Shaping our immune responses throughout life. Tissue barriers 5: e1373208, 
2017. 

 
 

54 

193.   Talamonti G, D’Aliberti G, Cenzato M. Aulus Cornelius Celsus and the Head 

Injuries. World Neurosurg. 133 Elsevier Inc.: 127–134, 2020. 

194.   Taylor GS, Bywater RA. Intrinsic control of the gut. Baillieres Clin Gastroenterol 

2: 1–22, 1988. 

195.   Teitelbaum DH, Caniano DA, Qualman SJ. The pathophysiology of 

Hirschsprung’s-associated enterocolitis: importance of histologic correlates. J 
Pediatr Surg 24: 1271–1277, 1989. 

196.   Terman A, Brunk UT. Autophagy in cardiac myocyte homeostasis, aging, and 

pathology. Cardiovasc. Res. 68 Cardiovasc Res: 355–365, 2005. 

197.   Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. 

Immunol. Rev. 223 NIH Public Access: 87–113, 2008. 

198.   Timmermans JP, Hens J, Adriaensen D. Outer submucous plexus: an intrinsic 
nerve network involved in both secretory and motility processes in the intestine of 
large mammals and humans. Anat Rec 262: 71–8, 2001. 

199.   Trivedi PJ, Adams DH. Chemokines and chemokine receptors as therapeutic 

targets in inflammatory bowel disease; Pitfalls and promise. J. Crohn’s Colitis 12 
Oxford University Press: S641–S652, 2018. 

200.   Tsuboi K, Nishitani M, Takakura A, Imai Y, Komatsu M, Kawashima H. 

Autophagy protects against colitis by the maintenance of normal gut microflora 
and secretion of mucus. J Biol Chem 290: 20511–20526, 2015. 

201.   Turco F, Sarnelli G, Cirillo C, Palumbo I, De Giorgi F, D’Alessandro A, 
Cammarota M, Giuliano M, Cuomo R. Enteroglial-derived S100B protein 
integrates bacteria-induced Toll-like receptor signalling in human enteric glial 
cells. Gut 63: 105–15, 2014. 

202.   Uranga-Ocio JA, Bastús-Díez S, Delkáder-Palacios D, García-Cristóbal N, 
Leal-García MÁ, Abalo-Delgado R. Enteric neuropathy associated to diabetes 
mellitus. [Online]. Rev Esp Enferm Dig 107: 366–73, 2015. 
http://www.ncbi.nlm.nih.gov/pubmed/26031865. 

203.   Uribe-Quero E, Rosales C. Control of phagocytosis by microbial pathogens. 

Front. Immunol. 8 Frontiers Media S.A.: 24, 2017. 

204.   De Vadder F, Grasset E, Holm LM, Karsenty G, Macpherson AJ, Olofsson 

LE, Bäckhed F. Gut microbiota regulates maturation of the adult enteric nervous 
system via enteric serotonin networks. Proc Natl Acad Sci U S A 115: 6458–6463, 
2018. 

205.   Valečka J, Almeida CR, Su B, Pierre P, Gatti E. Autophagy and MHC-restricted 

antigen presentation. Mol Immunol 99: 163–170, 2018. 

 
 

55 

206.   Vanner S, Surprenant A. Neural reflexes controlling intestinal microcirculation. 

Am J Physiol 271: G223-30, 1996. 

207.   Vasina V, Barbara G, Talamonti L, Stanghellini V, Corinaldesi R, Tonini M, 

De Ponti F, De Giorgio R. Enteric neuroplasticity evoked by inflammation. Auton 
Neurosci Basic Clin 126–127: 264–272, 2006. 

208.   Vera G, Castillo M, Cabezos PA, Chiarlone A, Martín MI, Gori A, Pasquinelli 
G, Barbara G, Stanghellini V, Corinaldesi R, De Giorgio R, Abalo R. Enteric 
neuropathy evoked by repeated cisplatin in the rat. Neurogastroenterol Motil 23: 
370, 2011. 

209.   Verheijden S, De Schepper S, Boeckxstaens GE. Neuron-macrophage 

crosstalk in the intestine: a “microglia” perspective. Front Cell Neurosci 9: 403, 
2015. 

210.   Ward MA, Pierre JF, Leal RF, Huang Y, Shogan B, Dalal SR, Weber CR, 

Leone VA, Musch MW, An GC, Rao MC, Rubin DT, Raffals LE, Antonopoulos 
DA, Sogin ML, Hyman NH, Alverdy JC, Chang EB. Insights into the 
pathogenesis of ulcerative colitis from a murine model of stasis-induced dysbiosis, 
colonic metaplasia, and genetic susceptibility. Am J Physiol - Gastrointest Liver 
Physiol 310: G973–G988, 2016. 

211.   Wardowska A. The epigenetic face of lupus: Focus on antigen-presenting cells. 

Int. Immunopharmacol. 81 Elsevier B.V.: 106262, 2020. 

212.   Wildenberg ME, Vos ACW, Wolfkamp SCS, Duijvestein M, Verhaar AP, Te 

Velde AA, Van Den Brink GR, Hommes DW. Autophagy attenuates the adaptive 
immune response by destabilizing the immunologic synapse. Gastroenterology 
142: 1493-1503.e6, 2012. 

213.   Williams A, Jahreiss L, Sarkar S, Saiki S, Menzies FM, Ravikumar B, 

Rubinsztein DC. Aggregate-Prone Proteins Are Cleared from the Cytosol by 
Autophagy: Therapeutic Implications. Curr. Top. Dev. Biol. 76 Curr Top Dev Biol: 
89–101, 2006. 

214.   Xue L, Farrugia G, Miller SM, Ferris CD, Snyder SH, Szurszewski JH. Carbon 
monoxide and nitric oxide as coneurotransmitters in the enteric nervous system: 
Evidence from genomic deletion of biosynthetic enzymes. Proc Natl Acad Sci U S 
A 97: 1851–1855, 2000. 

215.   Yamauchi T, Moroishi T. Hippo Pathway in Mammalian Adaptive Immune 

System. Cells 8: 398, 2019. 

216.   Yang H, Plevy SE, Taylor K, Tyan D, Fischel-Ghodsian N, McElree C, Targan 
SR, Rotter JI. Linkage of Crohn’s disease to the major histocompatibility complex 
region is detected by multiple non-parametric analyses. Gut 44: 519–526, 1999. 

 
 

56 

217.   Yang L, Liu C, Zhao W, He C, Ding J, Dai R, Xu K, Xiao L, Luo L, Liu S, Li W, 
Meng H. Impaired autophagy in intestinal epithelial cells alters gut microbiota and 
host immune responses. Appl Environ Microbiol 84, 2018. 

218.   Yiu JHC, Dorweiler B, Woo CW. Interaction between gut microbiota and toll-like 
receptor: from immunity to metabolism. J. Mol. Med. 95 Springer Verlag: 13–20, 
2017. 

219.   Yoo BB, Mazmanian SK. The Enteric Network: Interactions between the Immune 

and Nervous Systems of the Gut. Immunity 46: 910–926, 2017. 

220.   Yoshimoto T, Yasuda K, Tanaka H, Nakahira M, Imai Y, Fujimori Y, Nakanishi 

K. Basophils contribute to TH2-IgE responses in vivo via IL-4 production and 
presentation of peptide-MHC class II complexes to CD4+ T cells. Nat Immunol 10: 
706–712, 2009. 

221.   Zhang C, Li L, Feng K, Fan D, Xue W, Lu J. ‘Repair’ Treg Cells in Tissue Injury. 

Cell Physiol Biochem 43: 2155–2169, 2017. 

222.   Zhang M, Sun K, Wu Y, Yang Y, Tso P, Wu Z. Interactions between Intestinal 

microbiota and host immune response in inflammatory bowel disease. Front 
Immunol 8: 1, 2017. 

223.   Zhang N, Weber A, Li B, Lyons R, Contag PR, Purchio AF, West DB. An 

inducible nitric oxide synthase-luciferase reporter system for in vivo testing of anti-
inflammatory compounds in transgenic mice. J Immunol 170: 6307–6319, 2003. 

224.   Zhang W, Sarosi G, Barnhart D, Yule DI, Mulholland MW. Endothelin-activated 
calcium signaling in enteric glia derived from neonatal guinea pig. Am J Physiol - 
Gastrointest Liver Physiol 272, 1997. 

225.   Zhang YZ, Li YY. Inflammatory bowel disease: Pathogenesis. World J 

Gastroenterol 20: 91–99, 2014. 

226.   Zindel J, Kubes P. DAMPs, PAMPs, and LAMPs in Immunity and Sterile 

Inflammation. Annu. Rev. Pathol. Mech. Dis. 15 Annual Reviews Inc.: 493–518, 
2020. 

 

 

 
 

 

57 

Potential roles of enteric glia in bridging neuroimmune communication in the gut 

CHAPTER 2: 

 

 

 

 

 

 

 

 

 

 

 

 

Work presented in this chapter has been published as: 

Chow AK, Gulbransen BD. Potential roles of enteric glia in bridging neuroimmune 

communication in the gut. Am J Physiol Gastrointest Liver Physiol. 2017 Feb 

1;312(2):G145-G152.  

 
 

58 

Abstract 

 

The enteric nervous system (ENS) is a network of neurons and glia that controls 

ongoing gastrointestinal (GI) functions. Damage or injury to the ENS can lead to functional 

GI disorders. Current data support the conclusion that many functional GI disorders are 

caused by an imbalance between gut microbes and the immune system but how the ENS 

is involved in these interactions is less understood. Due to the proximity of the ENS to 

bacteria and other foreign antigens present in the GI tract, it is important to prevent the 

passage of these antigens through the GI epithelium. If any foreign compounds manage 

to  pass  through,  an  immune  response  is  triggered  to  prevent  injury  to  the  ENS  and 

underlying  structures.  However,  careful  modulation  of  the  inflammatory  response  is 

required to allow for adequate elimination of foreign antigens while avoiding inappropriate 

over-activation of the immune system as in autoimmune disorders. Enteric neurons and 

glial  cells  are  capable  of  performing  these  immunomodulatory  functions  to  provide 

adequate  protection  to  the  ENS.  Here,  we  review  recent  studies  examining  the 

interactions between the ENS and the immune system, specifically focusing on enteric 

glial cells and their ability to modulate inflammation in the ENS. 

 

 

 

 

 

 
 

59 

Introduction 

The enteric nervous system (ENS) is the largest division of the autonomic nervous 

system and it is housed entirely within the walls of the gastrointestinal (GI) tract. Neural 

reflexes  mediated  through  the  ENS  are  responsible  for  coordinating  most  ongoing 

activities of the GI tract such as patterns of motility, secretions and local blood flow. Like 

the brain, the ENS is entirely composed of neurons and glial cells that are housed within 

ganglia in two ganglionated plexuses. The neural circuitry in the submucosal plexus is 

responsible  for  coordinating  gut  secretions,  absorption  and  local  blood  flow  while  the 

myenteric plexus is primarily responsible for coordinating contractions and relaxations of 

gut smooth muscle involved in patterns of gut motility. Proper orchestration of GI functions 

requires that both plexuses are intact and maintain healthy interactions with target cells. 

Injuries inflicted on the ENS during local inflammation disrupt the functional connectivity 

of enteric neural networks and create long–lasting changes in ENS function (13). These 

changes  in  the  local  control  of  gut  reflexes  contribute  to  a  broad  range  of  common 

functional GI disorders such as irritable bowel syndrome (IBS) and functional constipation 

and contribute to persistent intestinal dysmotility during remission in inflammatory bowel 

disease (IBD).  

Clearly, cells within the innate and adaptive arms of the immune system are the 

main  drivers  of  gut  inflammation.  Generally  speaking,  inflammation  is  a  protective 

response to invading pathogens that evade the first line of defense posed by the intestinal 

epithelial barrier and defects in the balanced immune response to intestinal bacteria is a 

major contributing factor to gut disease. The acute inflammatory response predominantly 

involves the recruitment and activation of the innate arm of the immune system (30) while 

 
 

60 

chronic inflammation involves both innate and adaptive components. Likewise, an overly 

aggressive  adaptive  immune  response  can  attack  native  antigens  in  the  gut  and 

contribute to the development of inflammation (28). Current animal models used to study 

bowel inflammation are based on these principles and act to either increase epithelial 

permeability,  such  as  the  dextran  sodium  sulfate    colitis  model,  or  to  enhance  T-cell 

stimulation,  such  as  the  trinitrobenzene  sulfonic  acid  (TNBS),  dinitrobenzene  sulfonic 

acid, oxazolone, T-cell adoptive transfer, and interleukin (IL)-10 knockout models (38). 

The understanding of the immune response in colitis is becoming increasingly refined and 

has  led  to  many  important  breakthroughs  in  therapies.  However,  how  inflammation 

produces long–lasting changes in gut function through effects on the ENS is still poorly 

understood. Importantly, new data suggest that bi-directional interactions between cells 

in the ENS and immune cells may actively participate in the modulation of inflammatory 

responses themselves. Here, we provide a short review of recent studies that highlight 

potential roles for the ENS in the regulation of gut inflammation and particularly focus on 

the key roles of enteric glial cells. 

Contribution of enteric neurons to inflammation 

Many immunomodulatory effects of the parasympathetic and sympathetic branches 

of the autonomic nervous system have emerged in recent years and the general picture 

is  that  vagal  parasympathetic  innervation  is  anti-inflammatory  while  α-adrenergic 

sympathetic innervation is pro-inflammatory (6, 10). However, far less is known about 

how  intrinsic  enteric  neurons  influence  immune  responses  in  the  gut.  The  current 

evidence to date strongly suggests that enteric neurons have major immunomodulatory 

capabilities. The relative contributions of direct neuron-immune cell communication and 

 
 

61 

indirect communication via intermediary cells like enteric glia is still largely unknown. It is 

likely  that  many  neuro-immune  interactions  at  the  ganglionic  interface  are  mediated 

through enteric glia because enteric glia delineate the boundaries of enteric ganglia and 

largely  surround  enteric  neuron  cell  bodies.  However,  neuronal  processes  extending 

outside the ganglia have a high potential for direct interactions with various immune cells. 

The capacity of neurons to influence immune responses through both direct and indirect 

pathways  likely  accounts  for  observation  that  enteric  neuron  number  and  density 

influences intestinal inflammation (48). Margolis et al. found that transgenic mice with a 

hyperplastic  ENS  and  higher  density  of  enteric  neurons  developed  greater  colonic 

inflammation when exposed to TNBS as compared to wildtype animals (48). The fact that 

this study used a transgenic animal model where high neuronal density is present prior 

to the administration of TNBS strongly suggests that neuronal hyperplasia is not merely 

a  response  to  induced  colonic  inflammation  but  could,  in  fact,  be  a  risk  factor for  the 

development of colitis. These findings are particularly interesting in light of the discovery 

that enteric glia kill a significant portion of neurons during acute inflammation (14). Given 

the pro-inflammatory effect of enteric neurons, it is tempting to speculate that glial-driven 

neurodegeneration functions to protect against the neuronal potentiation of inflammation 

but this concept is still unproven at this point.  

How higher numbers of enteric neurons translates to worsening inflammation is not 

immediately  clear  but  likely  occurs  through  the  neuronal  release  of  cytokines  and 

neurotransmitters/neuromodulators.  For  example,  enteric  neurons  from  patients  with 

Crohn’s disease demonstrate high expression of pro-inflammatory prostaglandin D2 (44). 

Enteric  neurons  are  also  capable  of  secreting  leukocyte  chemoattractive  factors  that 

 
 

62 

include tumor necrosis factor-α (TNF-α), IL-6, the monocyte chemoattractant protein-1 

and  possibly  IL-8  when  challenged  with  pathogenic  bacteria  (15,  60)  (Fig.2.1). 

Interestingly, enteric neurons do not increase their secretion of these chemoattractive 

factors in response to Gram-positive bacteria despite the ability of both Gram-positive and 

Gram-negative bacteria to activate NF-κβ signal transduction pathways in neurons (15). 

This  may  be  achieved  through  the  effects  of  concurrent  Wnt  signaling  within  enteric 

neurons because the stimulation of the Wnt pathway in the presence of LPS confers an 

anti-inflammatory  phenotype  in  enteric  neurons  in  vitro  (41)  (Fig.2.1).  These  findings 

suggest  that  neurons  are  capable  of  differentiating  between  beneficial  and  harmful 

bacteria and participate in defensive responses to clear potentially pathogenic species. 

However,  the  inherent  ability  of  neurons  to  recruit  leukocytes  could  be  potentially 

detrimental  when  additional  neurons  are  present  and  may  contribute  to  enteric 

ganglionitis. Although untested at this point, aberrant neuronal chemoattractant secretion 

under  these  conditions  could  be  an  important  mechanism  that  contributes  to  a  pro-

inflammatory environment and a predisposition to gastrointestinal disease.  

 

 
 

 

63 

Figure 2.1: Overview of enteric neuron responses to select signals. Pathways 

highlighted in red denote pro-inflammatory pathways while those highlighted in blue 

lead to anti-inflammatory effects. Enteric neuron interactions with other cell types are 

shown with a white background. 

 

 

 
 

 

64 

In addition to their pro-inflammatory potential, enteric neurons may also contribute to 

anti-inflammatory effects via the synthesis and secretion of factors like netrin-1. Netrin-1 

is typically known for its role in neuronal development but its expression is also induced 

during hypoxia-induced tissue damage (59) and the secretion of this molecule can induce 

angiogenesis, the activation of tissue stem cells and bind neutrophil cell surface receptors 

such as A2B adenosine receptors (3, 36). The main effect of netrin-1 on neutrophils is to 

inhibit their migration and accumulation and may induce an egress away from the source 

of netrin-1 (3).  As such, enteric neurons could theoretically synthesize and secrete netrin-

1 as a response to inflammatory damage for neuronal survival and promote regeneration, 

and a loss in netrin-1 secretion could theoretically contribute to inappropriate leukocyte 

recruitment  (4)  (Fig.2.1).  However,  other  major  sources  of  netrin-1  in  the  GI  system 

include  the  colonic  epithelium;  therefore  further  studies  are  required  to  elucidate  the 

extent to which the ENS contributes to netrin-induced anti-inflammatory effects. 

Although activation of the Wnt pathway and secretion of prostaglandin D2 can induce 

inflammation, they can also inhibit the progression of inflammation. As described earlier, 

activation  of  the  Wnt  pathway  with  a  concurrent  LPS  costimulatory  signal  on  enteric 

neurons  cause  anti-inflammatory  effects  via  the  release  of  IL-10  (41).  Neuronal 

prostaglandin  D2  can  be  metabolized  into  15-deoxy-Δ12-14-prostaglandin  J2,  an  anti-

inflammatory compound (44). The dual functioning of the Wnt pathway and prostaglandin 

release suggests that enteric neurons may be important for the selective activation and 

‘fine-tuning’  of  inflammatory  responses  in  the  ENS.  In  addition  to  cytokines  and 

chemokines,  neurotransmitters  present  in  the  ENS  such  as  ATP,  acetylcholine, 

noradrenaline,  vasoactive  intestinal  peptide  (VIP),  neuropeptide  Y,  calcitonin  gene-

 
 

65 

related  peptide,  substance  P,  and  serotonin  can  modulate  the  activities  of  intestinal 

leukocytes (1, 29, 34, 49) (Fig.2.1). These neurotransmitters yield both pro- and anti- 

inflammatory effects and likely have a major influence on inflammatory processes in the 

ENS.  Margolis  and  Gershon  (2016)  recently  reviewed 

the  roles  of  several 

neurotransmitters in the regulation of intestinal inflammation and readers are referred to 

this excellent summary for further details (47). To summarize, the ability for immune cells 

to 

interact  with  neurotransmitters  suggests  a  close  relationship  and 

frequent 

communication between enteric neurons and the immune system.  

In  addition  to  acting  directly  on  immune  cells,  these  neurotransmitters  can  act  on 

intestinal  mucosa  and  alter  epithelial  barrier  permeability.  For  example,  in  vitro 

experiments demonstrate that neuropeptide Y increases the epithelial barrier permeability 

and  exposes  the  ENS  to  potential  pathogens  (17),  whereas  cholinergic  and  VIPergic 

signaling maintains barrier integrity and decreases permeability (18, 39, 55) (Fig.2.1). The 

control of intestinal epithelial barrier function and the exposure of the ENS to potential 

pathogens  can  modulate  the  inflammatory  state  of  the  ENS.  This  may  represent  an 

indirect means by which enteric neurons exert their influence on the immune system, 

further reinforcing the involvement of the ENS in immunomodulation. 

Glia in the healthy intestine 

Enteric glial cells are historically regarded as a network of cells that establish the 

homeostatic environment within enteric ganglia and function to provide both structural 

and  nutritional  support  for  enteric  neurons.  They  are  found  primarily  in  both  the 

submucosal and myenteric plexuses of the ENS, but may also be present outside the 

ENS, such as in the mucosal lamina propria. They are a heterogeneous group of cells 

 
 

66 

that can be subdivided into several types based on morphology and expression profiling 

(8, 56). These glial cell subtypes respond differently to purinergic signaling, suggesting 

different  glial  subtypes  activate  in  varying  situations.  However,  specific  roles  for  the 

distinct  glial  populations  have  yet  to  be  elucidated.  Beyond  structural  and  nutritional 

support, subsets of enteric glial cells also exert influence over other nearby cells of the GI 

tract and have local immunomodulatory effects. These functions largely resemble those 

of astrocytes in the central nervous system (CNS), and in this way, it is thought that certain 

groups of enteric glia fulfill astrocytic-like roles in the ENS.  

The relationship between enteric neurons and enteric glial cells begins early and 

new data show that glial cells are necessary for the proper maturation of neurons in the 

ENS(7). Glial cell guidance of neuronal development occurs through purinergic signaling 

between the two cell types and impaired signaling can lead to decreased enteric neuron 

density  and  complexity  (7).  Purinergic  signaling  between  enteric  glia  and  neurons 

continues into adulthood and plays an important role in neural circuits that coordinate GI 

motility  (33,  50,  51).  These  exciting  discoveries  suggest  that  alterations  to  purinergic 

intercellular signaling between neurons and glia could contribute to gut dysfunction by 

impairing  the  development  and  maturation  of  enteric  neurons  or  by  altering  neuronal 

activity.  In  agreement,  impairing  glial  purinergic  signaling  in  adult  mice  hinders 

coordinated contractions of the GI tract and slows colonic transit time (51).  

Mucosal enteric glial cells also form interactions with non-neuronal cells such as 

enterocytes  and  enteroendocrine  cells  that  are  important  in  the  maintenance  of  GI 

homeostasis. Glial cells influence the development and maturation of enterocytes and 

these roles contribute to the maintenance of an intact GI epithelial barrier (52). Less is 

 
 

67 

known  about  mucosal  glia-enteroendocrine  interactions  but  specialized  connections 

between  the  two  cell  types  suggest  that  glia  may  have  the  potential  to  influence 

gastrointestinal  hormone  release  (9).  The  fundamental  roles  of  barrier  regulation  and 

hormone release in GI physiology suggest that defective interactions between enteric glia 

and enterocytes or enteroendocrine cells could contribute to GI inflammation and possibly 

even metabolic diseases such as diabetes and obesity (9, 65). 

Glial cells are activated by cytokines and immunomodulatory signals 

 

The ability of enteric glial cells to respond to immunomodulatory signals such as 

cytokines,  bacteria  and  neurotransmitters  in  the  extracellular milieu  suggests  that  glia 

mediate a significant amount of the cross talk between the ENS and the immune system. 

For example, glial cells can detect local levels of the pro-inflammatory cytokine IL-1, IL-

4, and TNF-α (12) (Fig.2.2). Binding of these cytokines can elicit glial cell activation similar 

to reactive gliosis in the CNS and contribute to inflammation and GI dysfunction (61). 

Likewise,  the  activation  of  glial  toll-like  receptors  2  and  4  by  LPS  and  other  bacterial 

components  drives  pro-inflammatory  pathways  that  are  important  in  the  protective 

response  against  bacteria  that  successfully  bypass  the  gut  epithelial  barrier  (58,  62). 

Eposito  et  al.  demonstrated  that  LPS-activated  glial  cells  occupy  a  crucial  role  in  the 

creation of a robust inflammatory response in the local ENS environment and that the 

inhibition of the NF-κβ pathway in the glia can effectively ameliorate colonic inflammation 

(24).  However,  LPS-activated  cultured  human  glia  can  also  increase  transcription  of 

growth factors and several anti-inflammatory genes (42). This paradoxical elevation in the 

transcription of growth factors and anti-inflammatory genes with simultaneous expression 

of pro-inflammatory genes hints at a complex regulation of expression that likely involves 

 
 

68 

negative  feedback  pathways.  Interestingly,  Turco  et  al.  showed  that  cultured  human 

enteric glial cells are able to distinguish pathogenic from probiotic bacteria and modulate 

their expression of toll-like receptors accordingly (62). Similarly, Liddo et al. showed that 

exposing mixed  cultures  of  rat  enteric  neurons and  glia  to  a  combination of LPS  and 

Wnt3a induces the secretion of anti-inflammatory agents and inhibits NF-κβ activity in 

vitro (41). These findings suggest that the glial response to bacterial components in the 

ENS  and  the  decision  of  whether  to  undertake  pro-  or  anti-inflammatory  pathways 

depends  on  the  identity  of  the  bacteria  and  which  signaling  molecules  are  being  co-

expressed. 

 

 
 

 

69 

 

Figure 2.2: Overview of enteric glial cell responses to select signals. Pathways 

highlighted with a red background are pro-inflammatory, while those highlighted in blue 

yield anti-inflammatory effects. Glial cell interactions with other cell types are shown with 

a white background. Note that glial response to certain signals such as ATP and LPS 

depend on the context by which the glial cell is stimulated and may yield varying 

responses. 

 

 
 

70 

 

Purines are a major component of the “inflammatory soup” generated during tissue 

injury and purines contribute to the pathophysiology of many inflammatory diseases of 

the intestine. Enteric glia are highly responsive to purines and express receptors for ATP 

and  derivatives  such  as  ADP  and  adenosine.  The  glial  response  to  ADP  through the 

activation  of  P2Y1  receptors  is  the  most  well  characterized  and  is  involved  in  the 

coordination of neural circuits and the promotion of neuronal maturation under normal 

conditions (7, 50, 51). However, glial P2Y1 receptors also play an important role in the 

glial response to neuronal danger cues released during inflammation and the activation 

of glia in this context causes neurodegeneration (14). The cause of the vastly different 

glial responses to P2Y1 receptor activation appears to primarily involve the influence of 

pro-inflammatory mediators, such as nitric oxide, on downstream effectors like connexin-

43 hemichannels (14). Therefore, the response of glia to purinergic agonists depends 

heavily on the context in which they are activated. How the activation of glia by other 

purines  influences  their  response  to  inflammation  is  much  less  clear.  For  example, 

cultured  human  enteric  glia  express  adenosine  receptors  that  primarily  couple  to 

intracellular signal transduction pathways that utilize cAMP (42). Given the potent anti-

inflammatory effect of adenosine in the gut, it is likely that adenosine has similar anti-

inflammatory actions on glia but how the activation of glia by adenosine influences gut 

physiology and pathophysiology is currently unknown.  

 

Finally, enteric glial cells are innervated by both sympathetic and parasympathetic 

neurons of the autonomic nervous system (32, 63) (Fig.2.2). Stimulation of glial cells via 

parasympathetic  vagal  innervation  confers  an  anti-inflammatory  and  neuroprotective 

effect,  similar  to  the  effects  of  parasympathetic  vagal  stimulation  on  intestinal 

 
 

71 

macrophages  (49).  Specifically,  the  activation  of  glial  cholinergic  receptors  in  vitro 

promotes increased epithelial barrier function and inhibits the transcription of the pro-

inflammatory factor NF-κβ (18). These findings support previous data showing that the 

beneficial effects of vagal nerve stimulation on the intestinal epithelium following burn 

injuries  is  mediated  through  effects  on  enteric  glia  (22).  Conversely,  sympathetic-

mediated glia activation occurs through purinergic (32) and noradrenergic (54) signaling. 

As  noted  above,  the  activation  of  glia  by  ATP  may  contribute  to  a  pro-inflammatory 

phenotype but how the activation of glia by norepinephrine influences glia is not known. 

 

In summary, glia respond to a diverse array of immunomodulatory mediators that 

include pro-inflammatory cytokines, bacterial compounds, purines and transmitters from 

the autonomic nervous system. Activated glial cells exhibit some characteristics similar 

to astrocytes undergoing reactive astrogliosis in the CNS such as increased calcium influx 

and signaling and an increase in glial acidic fibrillary protein or S100β expression (12, 18, 

42).  However,  glial  activation  is  context  specific  and  the  outcome  response  of  glia 

depends on integrative processing of all cues available at that particular time.  

Glia express and secrete cytokines and pro-inflammatory signals 

Mounting evidence in recent years suggests that glial cells are not idle bystanders 

during  injury  and  inflammation  in  the  ENS.  What  this  evidence  shows  is  that  when 

activated, glia can contribute to a pro-inflammatory environment by secreting cytokines 

and  other  immunomodulatory  signals.  For  example,  one  response  of  glia  that  are 

activated by inflammatory cues in the gut includes alterations to their morphology and 

expression  and/or  secretion  of  key  proteins  such  as  S100β  and  glial  fibrillary  acidic 

protein, similar to the process of reactive astrogliosis in the CNS (57). Increased secretion 

 
 

72 

of  S100β  by  glia  contributes  to  inflammation  by  activating  receptors  for  advanced 

glycation end-products. This in turn leads to the production of nitric oxide by inducible 

nitric oxide synthase, the formation of reactive oxygen species, increased oxidative stress 

in the local environment and neuronal damage in ex vivo and in vitro studies (20, 23, 42, 

46, 62). It is important to note that extracellular concentrations of S100β in the micromolar 

range  are  needed  for  pro-inflammatory  effects  and  that  basal  S100β  secretion  in  the 

nanomolar range yields neuroprotective effects (23) (Fig.2.2). Interestingly, despite an 

increase  in  local  S100β  during  inflammation  of  the  GI  tract,  Celikbilek  et  al.  show  a 

counterintuitive decrease in serum levels of S100β in patients with ulcerative colitis (16), 

possibly  due  to  damaged  enteric  glial  cells  secreting  reduced  S100β  as  a  result  of 

exposure to prolonged inflammatory stress. Serum S100β levels over time in IBD may 

show fluctuations depending on glial cell viability and may prove useful in determining 

chronicity of inflammation. The stimuli for glial S100β release are incompletely understood 

but do include toll-like receptor agonists (62) and also likely includes purinergic signaling. 

Glial activation by purines during inflammation involves similar pathways as S100β such 

as inducible nitric oxide synthase and the release of ATP from activated glial cells (14). 

Glial S100β and ATP release both have neurotoxic effects on enteric neurons and these 

mediators  are  key drivers  of  ENS  damage during  acute  inflammation  in  vivo  (14,  23) 

(Fig.2.2). 

The  glial  secretion  of  cytokines  and  chemokines  is  of  specific  interest  among  the 

repertoire  of  compounds  secreted  by  enteric  glia  because  of  their  direct  immunologic 

effects. Specifically, recent in vivo and in vitro studies demonstrate that glia secrete a 

number of cytokines and chemokines that include interferon gamma (IFN-γ), chemokine 

 
 

73 

ligand 20, TNF-α and prostaglandin D2 (26, 44, 58) (Fig.2.2). However, conflicting data 

both  support  and  refute  the  concept  that  enteric  glia  secrete  TNF-α.  For  example, 

Coquenlorge et al. showed that glial cell cultures are unable to secrete TNF-α even when 

stimulated  with  LPS  (21). Based on  this  data, they  concluded  that  TNF-α  in  the ENS 

originates from enteric neurons and not from glia. In contrast, Guedia et al. found that 

enteric glial cell lines are capable of secreting TNF-α when stimulated with LPS and that 

the effect is further enhanced when exposed to Tat protein derived from HIV (31). These 

conflicting  results  might  suggest  that  multiple  pathways  can  converge  to  induce 

inflammation  and  that  there  are  various  regulatory  systems  in  place  to  restrict 

inappropriate inflammatory activation. Once activated, the glial secretion of cytokines and 

chemokines likely play an important role in the activation and recruitment of immune cells 

and modulates inflammation. Beyond IFN-γ, chemokine ligand 20, and TNF-α, numerous 

other  pro-inflammatory  cytokines  and  chemokines  undergo  transcriptional  changes  in 

activated  enteric  glia  in  culture.  However,  detectable  protein  secretion  of  these 

compounds has not been measured from activated glia (42, 58).  

Glial cells act as antigen presenting cells 

The many similarities between enteric glial cells and astrocytes hints that enteric glia, 

like astrocytes, may function as antigen presenting cells. New data show that enteric glia 

have phagocytic capabilities because glia transplanted from the ENS into the brain are 

capable of degrading pre-formed Aβ amyloid plaques in a similar manner as resident 

astrocytes (25, 64). In vivo and in vitro studies demonstrate that human enteric glial cells 

from both the myenteric and submucosal plexuses are also capable of expressing major 

histocompatibility complex II molecules, especially after exposure to bacteria or parasites 

 
 

74 

(5, 27, 62). In addition, enteric glia in patients with megacolon due to Chagas disease 

express the T cell co-stimulatory molecules cluster of differentiation (CD)80 and CD86 on 

their cell surface (5). Taken together, these data show that enteric glia have the ability to 

engulf and subsequently, present antigens to innate and adaptive immune cells and hints 

that enteric glial antigen presentation may play an important role at the neuro-immune 

interface. The role of glial antigen presentation in the gut is not currently understood but 

it could theoretically function to prime the adaptive immune system to bacteria in the ENS. 

By the same token, it would be possible for enteric glia to present neuronal debris to the 

adaptive  immune  system,  thereby  triggering  enteric  ganglionitis  and  an  auto-reactive 

immune response against ENS neurons similar to what is seen in multiple sclerosis (45). 

Enteric glia exert immunosuppressive and anti-inflammatory effects  

In  line  with  the  thought  that  glial  cells  play  a  neuroprotective  role  to  maintain 

homeostasis, 

there 

is 

increasing  evidence 

that  glial  cells  are  capable  of 

immunosuppression and anti-inflammatory actions. These anti-inflammatory effects are 

controlled, in part, by the release of soluble glial compounds. Of specific interest are  glia-

related  peptides  and  lipids  that  include  glial  derived  neurotrophic  factor  (GDNF),  glial 

derived  S-nitrosoglutathione  (GSNO)  and  15-deoxy-Δ12,14-prostaglandin  J2  because 

these  compounds  have  beneficial  effects  on 

intestinal  epithelial  permeability, 

inflammation and neuron survival (2, 19, 35, 65) (Fig.2.2). GDNF signals through RET 

and  GFRα  receptors  present  on  innate  immune  cells  and  enterocytes  of  the  gut  to 

decrease expression of pro-inflammatory cytokines and help maintain and strengthen the 

intestinal epithelial barrier, respectively (35, 52, 65). Specifically, Ibiza et al. demonstrated 

via in vivo and in vitro mouse studies that ligands from the GDNF family act on type 3 

 
 

75 

innate  lymphoid  cells,  leading  to  a  release  of  anti-inflammatory  IL-22  and  increased 

expression  of  repair  genes  in  the  gut  epithelium  (35).  In  addition,  RET  and  GFRα 

expression has been demonstrated in B lymphocytes, CD4+ and CD8+ T lymphocytes 

(53) but the direct effects of GDNF on these cells have yet to be described. Recent studies 

with cultured human enteric glial cells derived from the myenteric plexus suggest that 

GDNF contributes to the inhibition of proliferation by previously activated CD8- and CD8+ 

T  lymphocytes.  However,  it  seems  that  direct  cell-to-cell  interactions,  as  opposed  to 

secreted  compounds,  play  a  larger  role  in  preventing  T  lymphocyte  proliferation  (37). 

Furthermore, Kermarrec et al. showed that enteric glia from patients with Crohn’s disease 

exerted a greater immunosuppressant effect than control glia, suggesting that activated 

glial cells play an important role in immunomodulation. Similarly, von Boyen et al. describe 

increased activated mucosal glial cells expressing increased GFAP and GDNF in patients 

with Crohn’s disease. They further show that pro-inflammatory cytokines are an effective 

stimulus for GDNF secretion in vitro (11). Taken together, effects of GDNF contribute to 

an  anti-inflammatory  phenotype  to  reduce  epithelial  damage,  prevent  neuronal  loss, 

suppress activated T lymphocytes and improve gut function, potentially as a negative 

feedback  mechanism  to  limit  the  damage  due  to  inflammation  and  pro-inflammatory 

cytokines (11, 37, 43) (Fig.2.2). Similar to the actions of GDNF, in vitro and in vivo studies 

show that GSNO acts to promote a robust permeability barrier in the intestinal epithelium. 

This is achieved by inducing increased expression of zonula occludens-1 and occludin 

protein to form more tight junctions in the epithelium (19, 40). In addition, GSNO provides 

anti-inflammatory effects via the inhibition of NF-κβ pathways that promote the expression 

of  pro-inflammatory  agents  (40).  Finally,  secretion  of 15-deoxy-Δ12,14-prostaglandin  J2 

 
 

76 

from cultured rat myenteric glia elicits neuroprotective effects through the transcription 

factor NF-E2 related factor 2 pathway leading to neuronal synthesis of glutathione in vitro 

(2).  Therefore,  enteric  glia  can  effectively  modulate  and  control  the  extent  of  local 

inflammation and shift the local ENS environment back towards homeostasis. 

Conclusions 

 

Enteric  glial  cells  have  gained  increasing  attention  because  of  their  diverse 

emerging roles in the modulation of gut health and disease. Increasing evidence supports 

the idea of cross-talk between glial cells and many cells including enterocytes, neurons 

and  immune  cells.  Specifically,  activated  glial  cells  play  a  substantial  role  in  both  the 

progression  and  inhibition  of  inflammation  in  the  ENS.  The  seemingly  contradictory 

actions may be due to the amount of secretion and extracellular concentration of glial 

products, as seen in S100β secretion, or the signaling pathway by which glial cells are 

activated, as  demonstrated by  cholinergic-induced  versus  cytokine-induced  activation. 

Regulation of glial activation has not been well studied but may give insights into the 

specific contexts by which certain activation pathways are chosen to yield either pro- or 

anti-inflammatory effects in the ENS. Furthermore, various pro- and anti- inflammatory 

genes are transcribed in parallel, though many upregulated genes never reach translation 

and expression. This suggests potentially complex expression regulation and that enteric 

glial cells may self-regulate, to an extent, via negative feedback. 

The possible phagocytic ability of enteric glia, along with their capability to induce 

oxidative stress in their local environment and secretion of cytokines likens enteric glial 

cells to that of astrocytes and the innate immune system. Their ability to express major 

 
 

77 

histocompatibility complex II and T-cell costimulatory molecules suggests that glia play 

an important role in antigen presentation to T-cells. Further, the ability of enteric glial cells 

to  modulate  inflammation  and  immune  activation  shows  that  glia  are  an  important 

mediator of neuro-immune interactions. Understanding the specific contribution of glia 

towards  ENS  injury  and  neuronal  viability  will  offer  important  new  insight  into  the 

pathogenesis of inflammatory and functional GI disorders. 

 

 
 

 

78 

REFERENCES

 
 

79 

REFERENCES 

1.   Abad C, Cheung-Lau G, Coûté-Monvoisin A-C, Waschek JA. Vasoactive 
intestinal peptide-deficient mice exhibit reduced pathology in trinitrobenzene 
sulfonic acid-induced colitis. Neuroimmunomodulation 22: 203–12, 2015. 

2.   Abdo H, Mahé MM, Derkinderen P, Bach-Ngohou K, Neunlist M, Lardeux B. 
The omega-6 fatty acid derivative 15-deoxy-Δ12,14-prostaglandin J2 is involved in 
neuroprotection by enteric glial cells against oxidative stress. J Physiol 590: 
2739–50, 2012. 

3.   Aherne CM, Collins CB, Eltzschig HK. Netrin-1 guides inflammatory cell 

migration to control mucosal immune responses during intestinal inflammation. 
Tissue barriers 1: e24957, 2013. 

4.   Aherne CM, Collins CB, Masterson JC, Tizzano M, Boyle T a, Westrich J a, 

Parnes J a, Furuta GT, Rivera-Nieves J, Eltzschig HK. Neuronal guidance 
molecule netrin-1 attenuates inflammatory cell trafficking during acute 
experimental colitis. Gut 61: 695–705, 2012. 

5.   Barcelos Morais Da Silveira A, De Oliveira EC, Neto SG, Luquetti AO, Toshio 

Fujiwara R, Correa Oliveira R, Brehmer A, Barcelos A, Oliveira EC De, Neto 
SG, Luquetti AO, Toshio R, Correa R, Brehmer A. Enteroglial cells act as 
antigen-presenting cells in chagasic megacolon. Hum Pathol 42: 522–532, 2011. 

6.   Bellinger DL, Lorton D. Autonomic regulation of cellular immune function. Auton 

Neurosci 182: 15–41, 2014. 

7.  

Le Berre-Scoul C, Chevalier J, Oleynikova E, Cossais F, Talon S, Neunlist M, 
Boudin H. A novel enteric neuron-glia coculture system reveals the role of glia in 
neuronal development. J Physiol 1542: 33–36, 2016. 

8.   Boesmans W, Lasrado R, Vanden Berghe P, Pachnis V. Heterogeneity and 

phenotypic plasticity of glial cells in the mammalian enteric nervous system. Glia 
63: 229–241, 2015. 

9.   Bohórquez D V., Samsa LA, Roholt A, Medicetty S, Chandra R, Liddle RA. An 

enteroendocrine cell - Enteric glia connection revealed by 3D electron 
microscopy. PLoS One 9, 2014. 

10.   Bonaz, Bruno; Sinniger, Valerie; Pellissier S. Anti-inflammatory properties of 
the vagus nerve: potential therapeuitc implications of vagus nerve stimulation. J 
Physiol 82: 1496–1514, 2011. 

11.   Von Boyen GBT, Steinkamp M, Geerling I, Reinshagen M, Schäfer KH, Adler 
G, Kirsch J. Proinflammatory cytokines induce neurotrophic factor expression in 
enteric glia: A key to the regulation of epithelial apoptosis in Crohn’s disease. 

 
 

80 

Inflamm Bowel Dis 12: 346–354, 2006. 

12.   von Boyen GBT, Steinkamp M, Reinshagen M, Schäfer K-H, Adler G, Kirsch 
J. Proinflammatory cytokines increase glial fibrillary acidic protein expression in 
enteric glia. Gut 53: 222–8, 2004. 

13.   Brierley SM, Linden DR. Neuroplasticity and dysfunction after gastrointestinal 

inflammation. Nat Rev Gastroenterol Hepatol 11: 611–27, 2014. 

14.   Brown IAM, McClain JL, Watson RE, Patel BA, Gulbransen BD. Enteric Glia 

Mediate Neuron Death in Colitis Through Purinergic Pathways That Require 
Connexin-43 and Nitric Oxide. C Cell Mol Gastroenterol Hepatol 2: 77–91, 2016. 

15.   Burgueño JF, Barba A, Eyre E, Romero C, Neunlist M, Fernández E. TLR2 

and TLR9 modulate enteric nervous system inflammatory responses to 
lipopolysaccharide. J Neuroinflammation 13: 187, 2016. 

16.   Celikbilek A, Celikbilek M, Sabah S, Tanık N, Borekci E, Dogan S, Akin Y, 

Baldane S, Deniz K, Yilmaz N, Ozbakir O, Yucesoy M. The Serum S100B Level 
as a Biomarker of Enteroglial Activation in Patients with Ulcerative Colitis. Int J 
Inflam 2014: 986525, 2014. 

17.   Chandrasekharan B, Jeppsson S, Pienkowski S, Belsham DD, Sitaraman S 

V, Merlin D, Kokkotou E, Nusrat A, Tansey MG, Srinivasan S, Author C. 
Tumor necrosis factor (TNF)-neuropeptide Y (NPY) crosstalk regulates 
inflammation, epithelial barrier functions and colonic motility. Inflamm Bowel Dis 
19: 2535–2546, 2013. 

18.   Cheadle G a, Costantini TW, Bansal V, Eliceiri BP, Coimbra R. Cholinergic 

Signaling in the Gut: A Novel Mechanism of Barrier Protection through Activation 
of Enteric Glia Cells. Surg Infect (Larchmt) 15: 387–93, 2014. 

19.   Cheadle GA, Costantini TW, Lopez N, Bansal V, Eliceiri BP, Coimbra R. 

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier 
Breakdown. PLoS One 8: 1–11, 2013. 

20.   Cirillo C, Sarnelli G, Turco F, Mango A, Grosso M, Aprea G, Masone S, 

Cuomo R. Proinflammatory stimuli activates human-derived enteroglial cells and 
induces autocrine nitric oxide production. Neurogastroenterol Motil 23, 2011. 

21.   Coquenlorge S, Duchalais E, Chevalier J, Cossais F, Rolli-Derkinderen M, 

Neunlist M. Modulation of lipopolysaccharide-induced neuronal response by 
activation of the enteric nervous system. J Neuroinflammation 11: 202, 2014. 

22.   Costantini TW, Bansal V, Krzyzaniak M, Putnam JG, Peterson CY, Loomis 
WH, Wolf P, Baird A, Eliceiri BP, Coimbra R. Vagal nerve stimulation protects 
against burn-induced intestinal injury through activation of enteric glia cells. Am J 
Physiol Gastrointest Liver Physiol 299: G1308–G1318, 2010. 

 
 

81 

23.   Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, Brozzi F, Tubaro C, 

Giambanco I. S100B’s double life: Intracellular regulator and extracellular signal. 
Biochim Biophys Acta - Mol Cell Res 1793: 1008–1022, 2009. 

24.   Esposito G, Capoccia E, Turco F, Palumbo I, Lu J, Steardo A, Cuomo R, 
Sarnelli G, Steardo L. Palmitoylethanolamide improves colon inflammation 
through an enteric glia/toll like receptor 4-dependent PPAR-α activation. Gut 
(2013). doi: 10.1136/gutjnl-2013-305005. 

25.   Esposito G, Sarnelli G, Capoccia E, Cirillo C, Pesce M, Lu J, Calì G, Cuomo 
R, Steardo L. Autologous transplantation of intestine-isolated glia cells improves 
neuropathology and restores cognitive deficits in β amyloid-induced 
neurodegeneration. Sci Rep 6: 22605, 2016. 

26.   Fagbemi AO, Torrente F, Puleston J, Lakhoo K, James S, Murch SH. Enteric 

Neural Disruption in Necrotizing Enterocolitis Occurs in Association With 
Myenteric Glial Cell CCL20 Expression. J Pediatr Gastroenterol Nutr 57: 788–
793, 2013. 

27.   Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, Vantrappen G, 

Desmet VJ. Major histocompatibility class II expression on the small intestinal 
nervous system in Crohn’s disease. Gastroenterology 103: 439–447, 1992. 

28.   Geremia A, Biancheri P, Allan P, Corazza GR, Di Sabatino A. Innate and 
adaptive immunity in inflammatory bowel disease. Autoimmun Rev 13: 3–10, 
2014. 

29.   Di Giovangiulio M, Verheijden S, Bosmans G, Stakenborg N, Boeckxstaens 

GE, Matteoli G. The neuromodulation of the intestinal immune system and its 
relevance in inflammatory bowel disease. Front Immunol 6, 2015. 

30.   van der Gracht E, Zahner S, Kronenberg M, van der Gracht E, Zahner S, 

Kronenberg M, Zahner S, Kronenberg M. When Insult Is Added to Injury: Cross 
Talk between ILCs and Intestinal Epithelium in IBD. Mediators Inflamm 2016, 
2016. 

31.   Guedia J, Brun P, Bhave S, Fitting S, Kang M, Dewey WL, Hauser KF, 

Akbarali HI. HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release 
via TLR4 signaling in the enteric nervous system. Sci Rep 6: 31203, 2016. 

32.   Gulbransen BD, Bains JS, Sharkey KA. Enteric Glia Are Targets of the 

Sympathetic Innervation of the Myenteric Plexus in the Guinea Pig Distal Colon. J 
Neurosci 30: 6801–6809, 2010. 

33.   Gulbransen BD, Sharkey KA. Purinergic Neuron-to-Glia Signaling in the Enteric 

Nervous System. Gastroenterology 136: 1349–1358, 2009. 

34.   Hassani H, Lucas G, Rozell B, Ernfors P. Attenuation of acute experimental 

 
 

82 

colitis by preventing NPY Y1 receptor signaling. Am J Physiol Gastrointest Liver 
Physiol 288: G550–G556, 2005. 

35.  

Ibiza S, García-cassani B, Ribeiro H, Carvalho T, Almeida L, Marques R, 
Misic AM, Bartow-McKenney C, Larson DM, Pavan WJ, Eberl G, Grice EA, 
Veiga-Fernandes H, Eberi G, Grice EA, Veiga-Fernandes H, Eberl G, Grice 
EA, Veiga-Fernandes H. Glial-cell-derived neuroregulators control type 3 innate 
lymphoid cells and gut defence. Nature 535: 440–443, 2016. 

36.   Ke T, Wu Y, Li L, Liu Y, Yao X, Zhang J, Kong D, Li C. Netrin-1 Ameliorates 
Myocardial Infarction-Induced Myocardial Injury: Mechanisms of Action in Rats 
and Diabetic Mice. 797: 787–797, 2014. 

37.   Kermarrec L, Durand T, Neunlist M, Naveilhan P, Neveu I. Enteric glial cells 
have specific immunosuppressive properties. J Neuroimmunol 295–296: 79–83, 
2016. 

38.   Kiesler P, Fuss IJ, Strober W. Experimental Models of Inflammatory Bowel 

Diseases. Cell Mol Gastroenterol Hepatol 1: 154–170, 2015. 

39.   Lesko S, Wessler I, Gäbel G, Petto C, Pfannkuche H. Cholinergic modulation of 

epithelial integrity in the proximal colon of pigs. Cells Tissues Organs 197: 411–
420, 2013. 

40.   Li Z, Zhang X, Zhou H, Liu W, Li J. Exogenous S-nitrosoglutathione attenuates 
inflammatory response and intestinal epithelial barrier injury in endotoxemic rats. 
J Trauma Acute Care Surg 80: 977–984, 2016. 

41.   Di Liddo R, Bertalot T, Schuster A, Schrenk S, Tasso A, Zanusso I, Conconi 

MT, Schäfer KH. Anti-inflammatory activity of Wnt signaling in enteric nervous 
system: in vitro preliminary evidences in rat primary cultures. J Neuroinflammation 
12: 1–19, 2015. 

42.   Liñán-Rico A, Turco F, Ochoa-Cortes F, Harzman A, Needleman BJ, 

Arsenescu R, Abdel-Rasoul M, Fadda P, Grants I, Whitaker E, Cuomo R, 
Christofi FL. Molecular Signaling and Dysfunction of the Human Reactive Enteric 
Glial Cell Phenotype. Inflamm Bowel Dis 22: 1, 2016. 

43.   Liu GX, Yang YX, Yan J, Zhang T, Zou YP, Huang XL, Gan HT. Glial-derived 
neurotrophic factor reduces inflammation and improves delayed colonic transit in 
rat models of dextran sulfate sodium-induced colitis. Int Immunopharmacol 19: 
145–152, 2014. 

44.   Le Loupp A-G, Bach-Ngohou K, Bourreille A, Boudin H, Rolli-Derkinderen M, 
Denis MG, Neunlist M, Masson D. Activation of the prostaglandin D2 metabolic 
pathway in Crohn’s disease: involvement of the enteric nervous system. BMC 
Gastroenterol 15: 112, 2015. 

 
 

83 

45.   Lüssi F, Zipp F, Witsch E. Dendritic cells as therapeutic targets in 

neuroinflammation. Cell. Mol. Life Sci. (2016). doi: 10.1007/s00018-016-2170-9. 

46.   MacEachern SJ, Patel BA, Keenan CM, Dicay M, Chapman K, McCafferty D-
M, Savidge TC, Beck PL, MacNaughton WK, Sharkey KA. Inhibiting Inducible 
Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice 
With Colitis. Gastroenterology 149: 445–55.e3, 2015. 

47.   Margolis KG, Gershon MD. Enteric Neuronal Regulation of Intestinal 

Inflammation. Trends Neurosci xx: 1–11, 2016. 

48.   Margolis KG, Stevanovic K, Karamooz N, Li ZS, Ahuja A, D’Autréaux F, 

Saurman V, Chalazonitis A, Gershon MD. Enteric neuronal density contributes 
to the severity of intestinal inflammation. Gastroenterology 141: 588–98, 598.e1–
2, 2011. 

49.   Matteoli G, Gomez-Pinilla PJ, Nemethova A, Di Giovangiulio M, Cailotto C, 

van Bree SH, Michel K, Tracey KJ, Schemann M, Boesmans W, Vanden 
Berghe P, Boeckxstaens GE. A distinct vagal anti-inflammatory pathway 
modulates intestinal muscularis resident macrophages independent of the spleen. 
Gut (2013). doi: 10.1136/gutjnl-2013-304676. 

50.   McClain JL, Fried DE, Gulbransen BD. Agonist-Evoked Ca2+ Signaling in 

Enteric Glia Drives Neural Programs That Regulate Intestinal Motility in Mice. C 
Cell Mol Gastroenterol Hepatol 1: 631–645, 2015. 

51.   McClain JL, Grubišić V, Fried D, Gomez-Suarez RA, Leinninger GM, Sévigny 

J, Parpura V, Gulbransen BD. Ca2+ responses in enteric glia are mediated by 
connexin-43 hemichannels and modulate colonic transit in mice. Gastroenterology 
146: 497-507.e1, 2014. 

52.   Meir M, Flemming S, Burkard N, Bergauer L, Metzger M, Germer C-T, 

Schlegel N. Glial cell line-derived neurotrophic factor promotes barrier maturation 
and wound healing in intestinal epithelial cells in vitro. Am J Physiol - Gastrointest 
Liver Physiol 309: G613–G624, 2015. 

53.   Meir M, Flemming S, Burkard N, Wagner J, Germer C-T, Schlegel N. The glial 
cell-line derived neurotrophic factor: a novel regulator of intestinal barrier function 
in health and disease. Am J Physiol - Gastrointest Liver Physiol 310: G1118–
G1123, 2016. 

54.   Nasser Y, Ho W, Sharkey KA. Distribution of adrenergic receptors in the enteric 
nervous system of the guinea pig, mouse, and rat. J Comp Neurol 495: 529–553, 
2006. 

55.   Neunlist M, Toumi FF, Oreschkova T, Denis M, Leborgne J, Laboisse CL, 
Galmiche J-P, Jarry A. Human ENS regulates the intestinal epithelial barrier 
permeability and a tight junction-associated protein ZO-1 via VIPergic pathways. 

 
 

84 

Am J Physiol - Gastrointest Liver Physiol 285: G1028–G1036, 2003. 

56.   Rao M, Nelms BD, Dong L, Salinas-Rios V, Rutlin M, Gershon MD, Corfas G. 

Enteric glia express proteolipid protein 1 and are a transcriptionally unique 
population of glia in the mammalian nervous system. Glia 63: 2040–2057, 2015. 

57.   Robel S, Sontheimer H. Glia as drivers of abnormal neuronal activity. Nat 

Neurosci 19: 28–33, 2016. 

58.   Rosenbaum C, Schick MA, Wollborn J, Heider A, Scholz C-J, Cecil A, Niesler 

B, Hirrlinger J, Walles H, Metzger M. Activation of Myenteric Glia during Acute 
Inflammation In Vitro and In Vivo. PLoS One 11: e0151335, 2016. 

59.   Rosenberger P, Schwab JM, Mirakaj V, Masekowsky E, Mager A, Morote-

Garcia JC, Unertl K, Eltzschig HK. Hypoxia-inducible factor-dependent 
induction of netrin-1 dampens inflammation caused by hypoxia. Nat Immunol 10: 
195–202, 2009. 

60.   Rumio C, Besusso D, Arnaboldi F, Palazzo M, Selleri S, Gariboldi S, Akira S, 

Uematsu S, Bignami P, Ceriani V, Ménard S, Balsari A. Activation of smooth 
muscle and myenteric plexus cells of jejunum via Toll-like receptor 4. J Cell 
Physiol 208: 47–54, 2006. 

61.   Stoffels B, Hupa KJ, Snoek SA, Van Bree S, Stein K, Schwandt T, Vilz TO, 

Lysson M, Veer CVT, Kummer MP, Hornung V, Kalff JC, De Jonge WJ, 
Wehner S. Postoperative ileus involves interleukin-1 receptor signaling in enteric 
glia. Gastroenterology 146: 176-187.e1, 2014. 

62.   Turco F, Sarnelli G, Cirillo C, Palumbo I, De Giorgi F, D’Alessandro A, 
Cammarota M, Giuliano M, Cuomo R. Enteroglial-derived S100B protein 
integrates bacteria-induced Toll-like receptor signalling in human enteric glial 
cells. Gut 63: 105–15, 2014. 

63.   Uesaka T, Young HM, Pachnis V, Enomoto H. Development of the intrinsic and 

extrinsic innervation of the gut. Dev. Biol. (2016). doi: 
10.1016/j.ydbio.2016.04.016. 

64.   Wyss-Coray T, Loike JD, Brionne TC, Lu E, Anankov R, Yan F, Silverstein 
SC, Husemann J. Adult mouse astrocytes degrade amyloid-beta in vitro and in 
situ. Nat Med 9: 453–457, 2003. 

65.   Xiao W, Wang W, Chen W, Sun L, Li X, Zhang C, Yang H. GDNF is Involved in 

the Barrier-Inducing Effect of Enteric Glial Cells on Intestinal Epithelial Cells 
Under Acute Ischemia Reperfusion Stimulation. Mol Neurobiol 50: 274–289, 
2014. 

  

 
 

85 

CHAPTER 3: 

Enteric glial cells induce Th17 and Treg activation via autophagy-mediated MHC II 

expression 

 

86 

 

 
 

Abstract 

Background and Aims 

 

Enteric glial cells express type II major histocompatibility complex (MHC II) 

molecules in Crohn’s disease and Chagas disease, but it is unclear whether the 

expressed molecules are functional. We examined the capabilities of enteric glia to act 

as an antigen presenting cell in vivo and have immunomodulatory effects.  

Methods 

 

We used a tamoxifen-inducible glial-specific MHC II knockout 

(Sox10CreERT2;IABfl/fl) to isolate effects of enteric glial antigen presentation. We tested 

potential peptide sources to be loaded onto glial MHC II by measuring phagocytic 

activity and autophagy activation. We also determined enteric glial immunomodulatory 

capabilities by measuring T-lymphocyte activation and serum and colonic tissue 

cytokine levels. 

Results 

 

Enteric glia express MHC II molecules in response to low dose IFNγ and LPS 

exposure in the absence of intestinal tissue damage or inflammation. Glial MHC II 

expression increases activation in CD4+ T-lymphocytes, particularly in the Th17 and Treg 

subtypes, but not in Th1 or Th2 subtypes. Enteric glial MHC II does not influence serum 

or regional colonic tissue cytokine levels but may influence local cytokine levels. 

Autophagy activation is necessary, but insufficient to induce enteric glial MHC II 

expression. 

 
 

87 

Conclusions 

 

Enteric glial MHC II expression is induced by pro-inflammatory stimuli IFNγ and 

LPS and is dependent on autophagy activation. The expression of MHC II modulates T-

lymphocyte activation, particularly in the Th17 and Treg subpopulations, but does not 

lead to changes in serum or colonic tissue cytokine levels. 

Keywords: enteric glial cell, autophagy, major histocompatibility complex type II, T-

lymphocyte 

 

 
 

88 

Introduction 

Antigen presentation is a crucial process that maintains immune homeostasis by 

dictating T-lymphocyte activation at the transition between the innate and adaptive arms 

of the immune system. Dysfunction in antigen presentation can lead to the under- or 

over-activation of the immune system, or immune activation against inappropriate 

targets(8, 74). As a consequence, presenting the correct antigens and directing immune 

activity against the proper targets is of prime importance in the gastrointestinal (GI) tract 

where bacteria, viruses, parasites, and other foreign particles are constant challenges. 

Dysfunction in antigen presentation is a major contributing factor to immune 

dysregulation in diseases such as systemic lupus erythematosus, multiple sclerosis, 

and inflammatory bowel disease (IBD) (8, 48, 53, 74, 80).  

Professional antigen presenting cells are the primary cell types responsible for 

the process of antigen presentation and utilize major histocompatibility complex (MHC) 

II molecules to present specific peptides to T-lymphocytes. The identity of presented 

peptides activates specific T-lymphocytes, and therefore modulates immune activation. 

Since this process is sensitive to slight changes in peptides presented on MHC II 

molecules (4, 28), CD74 molecules are utilized to block peptide loading onto newly 

synthesized MHC II until their fusion with phagolysosomes (60). Although phagocytosis 

of extracellular foreign particles is the primary source of peptides for presentation MHC 

II molecules, autophagolysosomes can also contribute intracellular and ‘self’ peptides 

for loading onto MHC II molecules (65, 67). Peptide-loaded MHC II molecules are then 

targeted to the cell surface for presentation to T-lymphocytes. 

 
 

89 

In addition to professional antigen presenting cells expressing MHC II molecules, 

a set of ‘atypical’ MHC II-expressing cells, such as endothelial cells, epithelial cells, 

mast cells, and glial cells demonstrate variable levels of MHC II expression (35). Among 

these, enteric glia have gained considerable interest based on their ability to affect both 

T-lymphocyte activation and neuronal signaling (17, 24, 36). Enteric glial cells express 

MHC II molecules in an inducible manner, and share similar characteristics with antigen 

presenting cells by expressing T-lymphocyte costimulatory molecules CD80 and CD86 

(3, 23, 70). Enteric glia are capable of interacting with T-lymphocytes when expressing 

neoantigens (15), and co-culture studies suggest that enteric glia modulate T-

lymphocyte activation and regulate the activity of innate lymphoid type 3 cells (31, 36). 

Enteric glia also express both pro-inflammatory (20, 46, 62) and anti-inflammatory (1, 

10, 31, 79) molecules in vitro, and may exhibit immune-modulating functions similar to 

those of related glial populations in the central nervous system (13). Although glial-T-

lymphocyte interactions have been alluded to by in vitro studies, whether enteric glia 

affect play a significant role as antigen presenting cells in vivo is not known.  

The goal of this study was to test the hypothesis that glial antigen presentation 

modulates immune homeostasis in the intestine through interactions with CD4+ T-

lymphocytes. We tested our hypothesis using a mild proinflammatory stimulus 

composed of IFNγ and LPS to perturb immune homeostasis without driving overt 

inflammation and tested the role of glial MHC II by generating mice with a conditional 

ablation of MHC II in enteric glia. The data show that enteric glia express MHC II in 

response to IFNγ and LPS and that glial MHC II expression is necessary for adequate 

CD4+ T-lymphocyte activation. Ablating glial antigen presentation impaired the 

 
 

90 

activation of T-lymphocyte subsets expressing IL-17 and FoxP3, suggesting that glial 

antigen presentation influences Th17 and Treg activation. Glial antigen presentation was 

independent of an increase in phagocytic activity and required the activation of 

autophagy. Taken together, these results show that glial autophagy triggered in 

response to proinflammatory stimuli induces glial antigen presentation, which functions 

to modulate the activation of T-lymphocyte subsets involved in tolerance.  

 

Animals 

Materials and Methods 

All protocols involving animals were approved by the Michigan State University 

Institutional Animal Care and Use Committee. Male and female C57BL/6 mice between 

14 to 16-week-old were used unless stated otherwise. Animals were housed in 

ventilated cages with a 12-hour light-dark cycle, and access to food and water ad 

libitum. Mice with a tamoxifen-inducible conditional deletion of major histocompatibility 

complex II (MHC II) in enteric glia (Sox10CreERT2;IABfl/fl) were generated in-house by 

breeding Sox10CreERT2+/- mice ((42), gift from Dr. Vassilis Pachnis, The Francis Crick 

Institute, London, England) with floxed IAB mice (B6.129X1-H2-Ab1tm1Koni/J, 

RRID:IMSR_JAX:013181, The Jackson Laboratory, Bar Harbor, ME). RiboTag mice 

(Rpl22tm1.1Psam/J) expressing hemagglutinin on ribosomal protein L22 (Rpl22; 

011029;RRID:IMSR_JAX:011029; The Jackson Laboratory, Bar Harbor, ME) were bred 

with the glial Sox10CreERT2 driver line to generate Sox10CreERT2+/-;Rpl22tm1.1Psam/J mice for 

experiments assessing glial transcriptional profiles as described previously (16). Enteric 

 
 

91 

glial reporter mice (Sox10CreERT2+/-;tdTomato) were generated by breeding 

Sox10CreERT2+/- mice with Ai14 mice (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; RRID: 

IMSR_JAX:007914; The Jackson Laboratory, Bar Harbor, ME). CreERT2 activity was 

activated by feeding animals a 400mg/kg tamoxifen citrate diet for 2 weeks, and used 

for experiments immediately after the 2-week tamoxifen diet. 

Induction of glial MHC II 

Mice were injected with mouse recombinant interferon-gamma (IFNγ) (1µg/mouse, 

intraperitoneal (IP), cat# BMS326, eBioscience, Grand Island, NY), followed by an IP 

injection of E.coli O111:B4 lipopolysaccharide (LPS) (300µg/kg, cat# L4391, Millipore 

Sigma, St. Louis, MO) 2 hours afterwards to induce MHC II expression in enteric glia. 

Animals were scored for sickness severity and weights were measured at injection, and 

prior to sacrifice. Scoring for sickness severity was adapted from Shrum et al. (66). 

Briefly, animals were scored from 0-4 on each of the following categories: appearance, 

level of consciousness, activity, response to stimulus, eyes appearance, respiration 

rate, and respiration quality. Animals that had a sickness severity score of 21 or greater 

or lost >15% of body weight were euthanized and not included in analysis. Animals 

were euthanized and colons harvested at stated timepoints following LPS injection. 

Phagocytosis assay 

Phagocytic activity was assessed in longitudinal muscle-myenteric plexus (LMMP) 

whole mount preparations in vitro. Colons were isolated from transgenic mice 

expressing tdTomato in glia (Sox10CreERT2+/-;tdTomato) and whole mounts of myenteric 

plexus were prepared by removing the mucosa, submucosa, and circular muscle layers 

 
 

92 

by microdissection. Dissected tissues were incubated in Dulbecco’s Modified Eagle 

Medium (DMEM) containing IFNγ (10ng/mL, cat# BMS326, eBioscience, Grand Island, 

NY), LPS (5μg/mL, cat# L4391, Millipore Sigma, St. Louis, MO), and AlexaFluor488 

conjugated mouse anti-CD45.2 antibody (cat#109816, BioLegend, San Diego, CA) and 

phagocytic substrates. Substrates were green pHrodo conjugated E. coli BioParticles 

(1mg/mL, cat# P35366, Thermo Fisher Scientific, Waltham, MA) AlexaFluor 488 labeled 

LPS (cat#L23351, Thermo Fisher Scientific, Waltham, MA) or FluoSpheres (1.0µm, 

cat#F8803, Thermo Fisher Scientific, Waltham, MA) for 2 hours at 37oC. pHrodo 

conjugated E. coli BioParticles fluoresce only in low pH environments such as within 

phagolysosomes. Fluorescence was visualized by confocal or epifluorescence and 

brightfield microscopy.  

Whole-Mount Immunofluorescence 

Colons were harvested from mice, fecal pellets flushed with phosphate buffered saline 

(PBS), and tissues fixed with Zamboni’s fixative at 4oC overnight. Fixed tissues were 

micro-dissected to remove the mucosa, submucosal plexus, and circular muscle layers. 

The remaining longitudinal muscle-myenteric plexus preparation were processed via 

immunofluorescence as described in prior work (24). Briefly, tissue preparations were 

washed three times at room temperature with Triton X-100 (0.1%, Sigma-Aldrich, St. 

Louis, MO) in PBS for 10 minutes each. Tissue were then incubated in blocking solution 

containing 4% normal goat serum, 0.4% Triton X-100, and 1% bovine serum albumin in 

PBS for 45 minutes at room temperature. Following blocking, samples were incubated 

with primary antibodies for 72 hours at 4oC. Tissues underwent three 10-minute washes 

in PBS prior to a 2-hour incubation with secondary antibodies at room temperature. 

 
 

93 

Following two 10-minute washes in PBS, labeled tissues were washed once in 0.1M 

phosphate buffer for 10 minutes and mounted onto microscope slides. Antibodies and 

dilutions used are listed in Table 3.1. Labeled tissues were visualized by 

epifluorescence microscopy with a 40X objective (0.75 numerical aperture; Plan Fluor, 

Nikon, Melville, NY) on a Nikon Eclipse Ni upright microscope (Melville, NY) using a 

QImaging Retiga 2000R camera (Teledyne Photometrics, Tucson, AZ), or by confocal 

microscopy using an Olympus Fluoview FV1000 microscope (Olympus, Center Valley, 

PA) with a 60x Plan-Apochromat oil immersion objective (1.42 numerical aperture). 

Immunofluorescence was quantified using Fiji software (https://imagej.nih.gov/ij/). Control 

experiments were performed prior to immunofluorescent labeling of samples by omitting 

primary antibodies in labeling protocols in order to control for non-specific binding and 

autofluorescence. 

H&E Staining 

Colon tissues were fixed overnight in Zamboni’s fixative at 4oC and washed with PBS. 

Tissues were embedded in paraffin and tissue sections (4-5µm thickness) were stained 

with hematoxylin and eosin by the MSU Investigative HistoPathology Laboratory.  

Frozen section Immunofluorescence 

Mesenteric lymph nodes and spleen were harvested from animals and fixed in 

Zamboni’s fixative at 4oC overnight. Fixed samples were washed with PBS and 

incubated in a 30% sucrose solution in 0.1M phosphate buffer at 4oC for 72 hours. 

Samples were submitted to the Michigan State University Histology Core for cryo-

sectioning. Microscope slides with 6µm thick tissue sections were first warmed to room 

 
 

94 

temperature, rehydrated in distilled water, and warmed to 60oC. Following a 10-minute 

incubation in PBS at room temperature, tissue sections were labeled in a similar fashion 

as described above. Briefly, slides were incubated in PBS for 10 minutes at room 

temperature followed by two incubations of Triton X-100 (0.1%, Sigma-Aldrich, St. 

Louis, MO) in PBS for 10 minutes at room temperature. Slides were incubated with 

blocking solution containing 4% normal goat serum, 0.4% Triton X-100, and 1% bovine 

serum albumin in PBS for 45 minutes at room temperature. Following blocking, samples 

were incubated with primary antibodies for 72 hours at 4oC in a humidified slide 

chamber. Slides underwent three 10-minute washes in PBS prior to a 2-hour incubation 

with secondary antibodies at room temperature. Following two 10-minute washes in 

PBS, labeled slides were washed once in 0.1M phosphate buffer for 10 minutes, and 

mounted onto microscope slides. Antibodies and dilutions used are listed in Table 3.1. 

Flow cytometry 

Mouse mesenteric lymph nodes were harvested, and cells were isolated by mechanical 

dissociation and kept in FACS buffer consisting of PBS with 1% bovine serum albumin. 

Cells were passed through a 40μm cell strainer (Corning Life Sciences, Tewksbury MA) 

and centrifuged at 350g for 5 minutes at 4oC. Supernatant was removed and the cell 

pellet is washed once and resuspended in 1mL of ice-cold FACS buffer. To label cell 

surface markers, cells were incubated with conjugated antibodies for 30min at 4oC in 

the dark. Labeled cells were washed once more in FACS buffer before analysis with a 

BD LSR II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ). To 

label intracellular targets for flow cytometric analysis, mesenteric lymph node cells are 

first processed for cell surface labelling. Surface-labeled cells were fixed and 

 
 

95 

permeabilized with FoxP3 Fixation / Permeabilization buffer (eBioscience 00-5523-00, 

Grand Island, NY) overnight at 4oC in the dark. Fixed cells were washed with 

permeabilization buffer prior to incubation with intracellular-specific antibodies for 30min 

at 4oC in the dark. Cells were washed once more with permeabilization buffer prior to 

resuspension in ice-cold FACS buffer and analyzed with a BD LSR II flow cytometer 

(Becton, Dickinson and Company, Franklin Lakes, NJ). Flow cytometry data was 

analyzed with FCS Express 7 Research Edition (De Novo Software, Pasadena, CA). 

Antibodies and dilutions used are listed in Table 3.1. We eliminated doublets from flow 

cytometric analysis by plotting cells of interest on a forward scatter area vs height plot 

and gating for singlets. Resultant cells were plotted on a side scatter width vs height plot 

to ensure elimination of doublets. To properly control flow cytometric analysis of 

mesenteric lymph nodes, we incorporated unstained samples consisting of mesenteric 

lymph node cells incubated with FACS buffer without labeled antibodies to determine 

autofluorescence of mesenteric lymph node cells in each channel. Single stained 

controls consisted of UltraComp eBeads (Thermo Fisher Scientific, Waltham, WA) 

incubated with a single labeled antibody of each antigen of interest to calculate 

fluorescent compensation between each fluorophore. To identify the boundary between 

positive and negative fluorescence for each antigen of interest, we incorporated a panel 

of fluorescence minus one controls by labeling mesenteric lymph node cells with all 

fluorophores except the one being measured. 

Gene expression 

Sox10-RiboTag mice were used to quantify gene expression levels in enteric glial cells. 

Mice were treated with a 400mg/kg tamoxifen citrate diet for 2 weeks to induce glial-

 
 

96 

specific ribosomal HA-tag expression. Colons were harvested, fecal pellets flushed with 

PBS, and tissue snap frozen on dry ice and stored at -80oC. The RNA extraction 

protocol was performed as described by Delvalle et al. with minor modifications (16). 

Briefly, homogenization buffer was made by mixing 7.38mL RNAse-free water with 1mL 

Nonident P-40 (cat#: 21-3277, Millipore Sigma, St. Louis, MO), 1mL of 1M potassium 

chloride, 500µL of pH 7.4 Tris 1.0, 120µL of 1M magnesium chloride, and 100mg of 

sodium deoxycholate. Frozen colon tissues were placed in 1mL of ice-cold 

supplemented homogenization buffer containing 949μL homogenization buffer, 40µL 

cycloheximide (Millipore Sigma, St. Louis, MO), 40µL protease inhibitor (cat#: P8340, 

Millipore Sigma, St. Louis, MO), 20µL RNAse inhibitor (cat#: AM2684, Thermo Fisher 

Scientific, Waltham, WA), 2µL dithiothreitol (cat#: 646563, Millipore Sigma, St. Louis, 

MO), and 3mg/mL heparin (cat#: H3149, Millipore Sigma, St. Louis, MO). Frozen 

tissues were homogenized using a GentleMACS Dissociator (Miltenyi Biotech, Auburn, 

CA) in M-tubes running the program RNA_02. Tissue homogenates were centrifuged at 

10,000g at 4oC for 10min, supernatants collected, and incubated with 5μL of 

monoclonal anti-hemagglutinin (HA) antibody (16B12, BioLegend, San Diego, CA) at 

4oC for 4 hours on a tube rotator. During antibody incubation, magnetic protein A/G 

beads (Thermo Fisher Scientific, Waltham, WA) were washed twice in a magnet with 

500μL homogenization buffer. HA-labeled tissue homogenate supernatants were then 

incubated with the washed magnetic beads on a tube rotator at 4oC overnight. Following 

incubation, samples were washed three times in a magnet with 800μL of high-salt 

buffer. 350μL of RLT lysis buffer (Qiagen, Germantown, MD) were added to samples, 

and RNA released from magnetic beads by vortexing for 30 seconds. RNA was 

 
 

97 

collected by placing sample on a magnet and collecting the supernatant. Total RNA was 

purified using a RNeasy Micro kit (Qiagen, Germantown, MD) according to 

manufacturer’s instructions. Purified RNA was flash frozen and submitted to the UCLA 

Center for Systems Biomedicine core facility (UCLA, Los Angeles, CA) for analysis via a 

custom NanoString nCounter multiplex panel (NanoString, Seattle, WA). Gene 

expression levels were quantified by based on counts of fluorescent barcodes specific 

to individual genes. 

Cytokine quantification 

Serum and homogenized colon tissue samples were processed for cytokine 

quantification as described in prior work by Hernandez et al. with minor modifications 

(27). Briefly, serum samples were prepared by first harvesting whole blood from mice by 

cardiac puncture and allowed to clot by incubating at room temperature undisturbed for 

15min in a covered test tube. Tubes were then centrifuged at 1500g at 4oC for 10 

minutes. Serum was collected from the supernatant, diluted 2-fold with PBS to a final 

volume of 80µL, frozen at -80oC, and submitted to Eve Technologies (Calgary, AB, 

Canada) on dry ice for analysis. For tissue cytokine analysis, colon tissues were 

collected, and fecal pellets flushed prior to snap freezing on dry ice and storage at -

80oC. Frozen tissue samples are weighed and placed in 10% w/v of tris-buffered saline 

containing 0.05% tween-20, 25mM Tris-HCl, 130mM sodium chloride, 2.7mM potassium 

chloride, and SIGMAFAST protease inhibitor cocktail (1 tablet, cat#: S8820, Millipore 

Sigma, St. Louis, MO). Tissues are homogenized using a GentleMACS Dissociator 

(Miltenyi Biotech, Auburn, CA) in M tubes running the program Protein_01. Tissue 

homogenates were centrifuged at 4,000g at 4oC for 5min, and supernatants collected. 

 
 

98 

Supernatants were diluted with homogenization buffer to a protein concentration of 

4mg/mL in a final volume of 75μL. Samples were frozen at -80oC prior to submission to 

Eve Technologies (Calgary, AB, Canada) for analysis via the mouse 31-plex multiplex 

cytokine array. 

Autophagy induction and detection 

Live whole mount preparations of myenteric plexus were prepared as described above 

and in prior work (24). Autophagy was induced by incubating samples with DMEM 

containing Rapamycin (50nM,cat#13346, Cayman Chemical, Ann Arbor, MI) for 16 

hours at 37oC. Autophagy was inhibited by incubating samples with DMEM containing 

3-MA (5mM, cat#13242, Cayman Chemical, Ann Arbor, MI) for 16 hours at 37oC.  

Autophagy was detected in enteric neurons and glia by measuring P62 

immunofluorescence and LC3B puncta per cellular area. LC3B puncta was detected 

using FIJI by first thresholding the LC3B channel using the auto setting of the 

Intermodes algorithm in the ‘Threshold’ function of FIJI. LC3B puncta in enteric glia and 

neurons was measured in FIJI using the ‘Analyze Particles’ function, setting particle size 

of interest from 0 to Infinity and circularity from 0 to 1.  

Data Analysis 

Confocal immunofluorescent z-stack images were imported to FIJI software 

(https://imagej.nih.gove/ij/) as a Hyperstack using the Bio-Formats plugin (Open 

Microscopy Environment, University of Dundee, UK). To measure fluorescent intensity 

of a specific antigen within enteric glia or neurons, we first created a region of interest 

outlining GFAP-positive cells in each ganglion to isolate enteric glial expression. The 

 
 

99 

GFAP-positive region of interest was constructed in FIJI using the auto setting of the 

Otsu algorithm in the ‘Threshold’ function of FIJI. The resultant selection was converted 

into a region of interest in FIJI using the ‘Create Selection’ function. Total ganglionic 

expression of a given labeled antigen was measured by creating a ganglionic region of 

interest by utilizing the GFAP channel to outline the total ganglionic area. Glial 

fluorescent intensity or particle number was subtracted from total fluorescent intensity or 

particle number within the ganglion to calculate levels within enteric neurons. To 

calculate Mander’s overlap coefficient between two fluorophores, we utilized the FIJI 

plugin ‘Just Another Colocalisation Plugin’ (6), and adjusted thresholding parameters to 

include GFAP+ glial processes without incorporating background noise, or MHC II and 

CD74 signal without incorporating background noise. Additionally, using Metamorph 

software, we performed line-scans across enteric ganglion confocal z-projection 

images. Confocal z-stack images were processed by first converting z-stacks into 

maximum projection images. Background subtraction was applied in each channel to 

remove noise. Maximum gray values of labeled antigens were measured along the 

length of a line drawn within enteric ganglia to visualize co-presence of fluorophores. 

We used the 3D Viewer plugin in FIJI to create a 3-dimensional reconstruction of GFAP-

positive ganglia and MHC II expression. 3-dimensional reconstruction is displayed as a 

volume with a resampling factor of 2. We analyzed flow cytometric data using FCS 

Express 7 Research Edition (De Novo Software, Pasadena, CA) and determined gating 

cutoffs for CD4, CD69, IL4, IL17, IFNγ, and FoxP3 markers using compensation and 

fluorescence minus one controls as described above. We determined the activated 

fraction of each T-lymphocyte subtype by calculating the proportion of T-lymphocyte 

 
 

100 

subtype marker and CD69 double positive cells within the total number of subtype 

marker positive cells. We used IFNγ as a marker for Th1, IL4 for Th2, IL17 for Th17, and 

FoxP3 for Treg subtypes. Nanostring gene expression data was processed by nSolver 

v4.0 software (NanoString Technologies, Seattle, WA). Data was curated to remove 

entries flagged by nSolver quality control or entries that have fewer reads than negative 

controls. Data is represented as fold change compared to the mean number of reads in 

saline control condition. For cytokine quantification, a four-parameter logistic regression 

was utilized to calculate the concentration of each analyte, and samples below or above 

the logistic regression were designated as 0 or the highest standard curve value, 

respectively.  

Statistics 

 

All data are represented as mean ± SEM and were analyzed by GraphPad Prism 

7 (GraphPad Software, San Diego, CA). Immunofluorescence, flow cytometry, and 

quantified cytokine data were compared between genotype and treatment conditions 

using unpaired two-way ANOVA with Tukey-corrected multiple comparisons. Gene 

expression data retrieved from RNAseq databases are expressed as transcripts per 

million (tpm) and were compared by one-way ANOVA with Tukey-corrected multiple 

comparisons. A level of P < .05 was set as the cutoff for statistical significance. P values 

are denoted as follows: < .05 (*), < .005 (**), < .0005 (***), <.0001 (****). Outliers were 

detected in GraphPad Prism 7 using the ROUT method with Q=1%. 

 

 
 

 

101 

Antigen 
Target 

GFAP 

Host 
Species 

Dilution 

Conjugate 

Manufacturer 

Product no. 

Chicken 

1:1000 

n/a (primary Ab) 

Abcam 

ab4674 

MHC II 

Rat 

CD74 

P62 

LC3B 

Anti-chicken 
IgY 

Rabbit 

Rabbit 

Rabbit 

Goat 

1:200 

1:200 

1:200 

1:200 

1:400 

n/a (primary Ab) 

Novus 

NBP2-21789 

n/a (primary Ab) 

Bioss 

BS-2518R 

n/a (primary Ab) 

Abcam 

ab109012 

n/a (primary Ab) 

Abcam 

ab192890 

Dylight405 

Jackson 

103-475-155 

Anti-rat IgG 

Goat 

1:400 

AlexaFluor 594 

Jackson 

112-585-003 

Anti-rabbit IgG  Goat 

1:400 

AlexaFluor 488 

Invitrogen 

A-11034 

CD45.2 

Mouse 

1:200 

AlexaFluor488 

BioLegend 

109816 

 
Table 3.1: Antibodies used in Chapter 3. 

 

102 

Table 3.1 (cont’d) 

CD4 

CD8 

CD69 

IFNg 

IL-4 

IL-17 

FoxP3 

Rat 

Rat 

Armenian 
Hamster 

Rat 

Rat 

Rat 

Rat 

1:200 

1:200 

1:200 

1:100 

1:100 

1:20 

FITC 

BioLegend 

100509 

APC/Cy7 

BioLegend 

100714 

PE/Cy7 

BioLegend 

104512 

PE 

APC 

BioLegend 

505808 

BioLegend 

504105 

Brilliant Violet 510 

BioLegend 

506933 

1:100 

Pacific Blue 

BioLegend 

126410 

 

 

 

103 

Results 

Conditional ablation of glial MHC II in Sox10CreERT2;IABfl/fl mice and IFNγ + LPS 

challenge 

Enteric glia express MHC II in response to proinflammatory stimuli such as LPS 

and IFNγ (Fig.3.1a, d-f), similar to findings by Turco et al. (70). These stimuli also elicit 

antigen presentation by professional immune cells which can mask the contribution of 

glia. Therefore, we used a minimal pro-inflammatory stimulus (IFNγ 1µg / mouse; LPS 

0.3mg/kg; i.p.) to study glial effects in vivo without an overwhelming inflammatory 

response from other immune cells (Fig.3.1c). This challenge is sufficient to induce glial 

MHC II (Fig.3.1d-f) and causes minor weight loss (Fig.3.2a) but does not increase 

sickness behavior (Fig.3.2b), alter tissue architecture (Fig.3.2c), or cause significant 

histological damage (Fig.3.2d).  

Murine MHC II molecules are composed of gene products from the I-A or I-E loci. 

C57BL/6 mice have a H-2b MHC haplotype due to a deficient I-E loci and rely solely on 

the I-A loci for MHC II expression (29, 30, 39). We isolated the specific role of enteric 

glia by creating a tamoxifen-inducible, glial-specific MHC II knockout (KO) mouse model 

(Sox10CreERT2;IABfl/fl) where the IAB gene, a crucial component in the MHC II I-A loci, is 

deleted in Sox10+ enteric glia (Fig.3.1b,c).  

 

 
 

 

104 

 

Figure 3.1: Enteric glial cells express MHC II when stimulated with IFNγ and LPS.  
(A) Schematic depicting enteric glial expressing MHC II molecules as a response to 

exposure to 1µg of IFNγ and 0.3mg/kg LPS. (B) Schematic depicting gene targeting 

strategy in Sox10CreERT2;IABfl/fl  mice. Tamoxifen-sensitive CreERT2 expression is 

confined to glia by the Sox10 promoter and excises the floxed H2-Ab1 exon 1 loci. (C) 

Schematic depicting exposure protocol. Wildtype and MHC II KO mice are fed  

 
 

105 

Figure 3.1 (cont’d): 

400mg/kg tamoxifen in chow for 14 days to induce Cre recombinase and MHC II 

ablation. Animals are injected via IP with pro-inflammatory stimuli consisting of 1µg of 

IFNγ followed by 0.3mg/kg LPS 2 hours afterwards or 1µg of IFNγ and volume-matched 

saline as controls. Tissues were harvested 16 hours post-injection. (D) Representative 

confocal images of GFAP (grey) and MHC II (red) immunofluorescence labeling in 

enteric ganglia. Enteric glial cells express MHC II when stimulated with IFNγ and LPS. 

Muscularis macrophages (Mac) bordering enteric ganglia show constitutive MHC II 

expression in all treatment conditions (scale bar: 50µm for all images). (E) Orthogonal 

views of confocal z-stacks confirm MHC II (red) labeling in GFAP-expressing (grey) 

enteric glia (scale bar: 50µm). (F) Line-scans through enteric ganglia demonstrate co-

expression of MHC II (red) and GFAP (blue) labeling. (G) Quantified mean fluorescent 

intensity of MHC II labeling on GFAP-expressing enteric glia in the myenteric plexus (n= 

7-11 mice; 2-way analysis of variance with Tukey-corrected multiple comparisons; *P ≤ 

.05, ****P ≤ .0001). (H) Representative confocal images of GFAP (grey), MHC II (red) 

and CD74 (cyan) immunolabeling in wildtype animals treated with IFNγ and LPS show a 

neuronal and glial distribution of CD74 (scale bar: 50µm for all images). (I) Line-scans 

through enteric ganglia show minimal overlap between MHC II (red) and CD74 (green) 

in GFAP-expressing (blue) enteric glia. 

 

 
 

 

106 

 

Figure 3.2: Exposure to IFNγ and LPS causes weight loss but does not cause 

significant tissue damage or observable inflammation. (A) Animals show weight 

loss when treated with IFNγ + LPS in wildtype (P = .0054) and knockout (P < .0001) 

animals (n=5-10 mice; 2-way analysis of variance with Tukey-corrected multiple 

comparisons) (B) Sickness scores were not significantly different between IFNγ + LPS 

challenged animals compared to controls. (C) No tissue architecture changes were  

 

 
 

107 

Figure 3.2 (cont’d): 

observed in any experimental group. (D) No elevation in tissue histology scores was 

present in any condition. 

 

 

 
 

 

108 

Enteric glia express mature MHC II in response to IFNγ + LPS  

Enteric glia increased MHC II expression in animals challenged with IFNγ and 

LPS despite no overt inflammation or tissue architecture changes (Fig.3.1d; Fig.3.2). 

Within enteric ganglia, MHC II expression is limited to glia and no neuronal expression 

was observed (Fig.3.1d). Confocal image analyses confirm that MHC II primarily 

colocalizes with GFAP-labeled enteric glia in enteric ganglia (Mander’s overlap 

coefficient of 90%; Fig.3.1e-f). Treatment with IFNγ alone was not sufficient to induce 

glial MHC II expression (Fig.3.1d). Quantification of MHC II immunolabeling shows that 

enteric glia significantly (P = .0370) upregulate MHC II expression in response to IFNγ 

and LPS in wildtype animals, and that this effect is absent in animals that lack glial MHC 

II (Sox10CreERT2;IABfl/fl) (Fig.3.1g). Muscularis macrophages at the level of the myenteric 

plexus demonstrate constitutive expression of MHC II (54), and exposure to IFNγ and 

LPS stimuli did not alter expression level or pattern in wildtype or glial MHC II KO 

animals (Fig.3.3a,b). These observations are consistent with prior data showing that 

LPS stimulates enteric glial MHC II expression through TLR4 receptor pathways (62, 

70) and that enteric neurons lack expression of TLR4 and MHC II (19). Importantly, 

these results show that the MHC II KO mouse model effectively ablates MHC II 

expression specifically in enteric glia and not within immune cells, thereby isolating any 

changes in immune activation to enteric glia. 

In antigen presenting cells, immature MHC II molecules contain CD74 bound 

within their peptide-binding groove until fusion with phagolysosomes when the CD74 

dissociates to allow loading of processed peptides (58, 60). We tested whether the 

MHC II molecules expressed by enteric glia are mature by labeling for CD74. CD74 

 
 

109 

immunoreactivity was present in both enteric neurons and glia (Fig.3.1h) and this is 

consistent with the pattern of CD74 gene expression in enteric neurons and glia (ADD 

REFS for databases here). However, no co-labeling of enteric glial MHC II and CD74 

was detected (Fig.3.1h). Furthermore, line-scans through enteric ganglia show little 

colocalization between CD74 and MHC II (Mander’s overlap coefficient of 2%; Fig.3.1i). 

Together, these results show that enteric glia are sensitive to pro-inflammatory stimuli 

and respond to low doses of IFNγ and LPS by expressing mature MHC II molecules. 

 

 
 

 

110 

 

Figure 3.3: Pattern and level of MHC II expression in macrophages does not 

change when treated with IFNγ and LPS. (A) Representative confocal images of 

MHC II (red) immunofluorescent labeling in enteric ganglion (scale bar: 50µm for all 

images). (B) Quantified mean fluorescent intensity of MHC II labeling in muscularis 

macrophages bordering enteric ganglion (n = 4-5 mice; 2-way analysis of variance with 

Tukey-corrected multiple comparisons). 

 

 
 

 

111 

Enteric glial MHC II expression contributes to CD4+ T-cell activation in mesenteric 

lymph nodes 

MHC II molecules expressed by professional antigen presenting cells play an 

important role in interactions that activate T-lymphocytes. Therefore, we tested whether 

glial MHC II molecules are functionally relevant in antigen presentation to T-

lymphocytes. We began by using immunolabeling to examine whether enteric glia and 

T-lymphocytes are within sufficient proximity for potential interactions. In support, CD4 + 

T-lymphocytes were observed in close proximity to enteric glia at the level of the 

myenteric plexus (Fig.3.4). Next, we tested whether glial MHC II mediates functional 

interactions with T-lymphocytes that influence their activation state by measuring CD69 

expression in T-lymphocytes in control and glial MHC II KO animals. The majority of 

CD4+ cells are in an inactive state in wildtype control animals and only 38% of CD4+ 

cells expressed the acute activation marker CD69 (Fig.3.5b,c). Exposure to IFNγ and 

LPS expanded the population of activated CD4+ cells by 29% and increased the 

percentage of CD4+ cells expressing CD69 to 67% (P = .0010). Ablating glial MHC II 

significantly (P = .0309) blunted the effect of IFNγ and LPS and decreased the 

proportion of CD4+ cells expressing CD69 to 46%, which is similar to the proportion of 

active CD4+ cells observed in saline treated control animals (Fig.3.5c). Glial MHC II 

ablation had no effect on the percentage of activated CD4+ cells in saline treated 

control animals (38%)(Fig.3.5b,c), and this is expected given that glial MHC II 

expression is only observed in animals challenged with LPS (Fig.3.1) . Enteric glial 

MHC II expression also affected CD8+ T-lymphocyte activation in mesenteric lymph 

nodes (Fig.3.6a). In healthy wildtype control animals, 61% of CD8+ T-lymphocytes 

 
 

112 

expressed CD69 and this proportion significantly increased to 87% in wildtype animals 

challenged with IFNγ and LPS (P = .0132) (Fig.3.6b). However, the IFNγ and LPS 

challenge did not affect CD8+ T-lymphocyte activation in KO animals (Fig.3.6b). 

Together, these results show that enteric glial antigen presentation mediated by MHC II 

plays an important role in regulating immune activity through effects on CD4+ and CD8+ 

T-lymphocytes. 

Since enteric glial MHC II ablation influences the activation state of CD4+ T-

lymphocytes, we examined whether a specific T-lymphocyte subtype is preferentially 

affected by glial MHC II. The baseline activated proportions of T-lymphocyte subtypes 

expressing CD69 in wildtype control animals were 66% of IFNγ+ cells, 48% of IL4+ 

cells, 46% of IL17+ cells, and 43% of FoxP3+ cells (Fig.3.5d, Fig.3.7). Challenge with 

IFNγ and LPS in wildtype animals did not change the activated portion of IFNγ+ or IL4+ 

cells, but significantly elevated the activated portions of IL17+ (P = .0025) and FoxP3+ 

(P = .0121) cells to 83% and 77%, respectively (Fig.3.5d, Fig.3.7). Glial MHC II ablation 

in stimulated animals did not significantly alter the activated portions of IFNγ+ or IL4+ 

cells, but significantly decreased the activated portions of IL17+ (P = .0021) and FoxP3+ 

(P = .0057) cells to levels comparable to healthy wildtype controls. Glial MHC II ablation 

in control animals had no effect on T-lymphocyte subtype activation. Taken together, 

these results show that enteric glial MHC II primarily affects the activation state of Th17 

and Treg subtypes within the CD4+ population of T-lymphocytes in mesenteric lymph 

nodes. 

 

Given the major effect of glial MHC II ablation on T-lymphocyte activation in 

mesenteric lymph nodes, we wondered if local Sox10+ cells might exist within 

 
 

113 

mesenteric lymph nodes that would be susceptible to MHC II ablation and mediate the 

observed effects. We assessed this possibility by using Sox10CreERT2+/-;tdTomato 

reporter mice to screen for potential Sox10+ cells in the mesenteric lymph nodes. We 

did not detect any tdTomato-expressing Sox10+ cells within mesenteric lymph nodes in 

either resting conditions or when stimulated with IFNγ and LPS (data not shown). The 

absence of Sox10+ cells in mesenteric lymph nodes suggests that changes in T-

lymphocyte activation state are due to changes in enteric glial MHC II expression in the 

intestine and not other cell types residing in the mesenteric lymph node. 

 

 
 

 

114 

 

Figure 3.4: CD4 expressing T-lymphocytes are in close proximity with GFAP 

expressing enteric glia in the myenteric plexus. Representative image of GFAP 

(grey) and CD4 (red) immunofluorescent labeling in a colon cross section. Enlarged 

image emphasizes the presence of CD4+ T-lymphocytes within the myenteric plexus. 

The top region of image represents mucosal surface and bottom represents serosal 

surface (scale bar: 50µm) 

 

 
 

 

115 

 

Figure 3.5: Enteric glial MHC II expression affects T-lymphocyte activation. (A) 

Schematic depicting enteric glial cells expressing MHC II when stimulated with IFNγ and 

LPS. MHC II molecules interact with T-lymphocytes and cause changes in activation 

state as measured by CD69 expression. (B) Representative flow cytometric contour 

plots of mesenteric lymph nodes labeled with CD4 and CD69. Proportion of total gated 

cells in each quadrant expressed as percentage. (C) Levels of activated CD4+ cells 

quantified as a percentage of total CD4+ cells that are CD4+CD69+ (n=8-10 mice; 2- 

 
 

116 

Figure 3.5 (cont’d): 

way analysis of variance with Tukey-corrected multiple comparisons; *P ≤ .05, ***P ≤ 

.001). (D) Levels of activated IFNγ+, IL-4+, IL-17+, or FoxP3+ cells quantified as a 

percentage of total subtype-marker expressing cells that are also expressing CD69 

(n=4-7 mice; 2-way analysis of variance with Tukey-corrected multiple comparisons; *P 

≤ .05, **P ≤ .005). 

 

 

 
 

 

117 

 

Figure 3.6: Activation of CD8+ T-lymphocytes decreased when MHC II ablated in 

enteric glia. (A) Representative contour plots of mesenteric lymph nodes labeled with 

CD8 and CD69. Proportion of gated cells in each quadrant expressed as percentage. 

(B) Proportion of total CD8+ T-lymphocytes expressing CD69 quantified in each 

treatment condition (n=3-4 mice; 2-way analysis of variance with Tukey-corrected 

multiple comparisons; * P ≤ .05). 

 

 
 

 

118 

 

Figure 3.7: Enteric glial MHC II expression affect FoxP3+ and IL-17+ cell 

populations to a greater degree than IFNγ+ and IL-4+ cell populations. 

Representative contour plots of mesenteric lymph nodes labeled with FoxP3, IFNγ, IL-4, 

IL-17, and CD69. Proportion of total gated cells in each quadrant expressed as 

percentage. 

 

 
 

 

119 

Enteric glia affect local, but not serum or colonic cytokine levels 

Enteric glia express pro-inflammatory cytokines when stimulated with IFNγ and LPS 

in vitro (45). We tested whether IFNγ and LPS stimulation in wildtype animals in vivo 

causes enteric glial gene expression of select cytokines. Gene expression in majority of 

cytokines examined were not significantly affected by IFNγ and LPS challenge. 

However, a significant (P = .0001) increase in IL-1 gene expression and a trend towards 

increased tumor necrosis factor (P = .0595) was observed (Fig.3.8). Our data suggests 

that enteric glia respond to in vivo IFNγ and LPS challenge by altering gene expression 

to select cytokines and may alter cytokine levels in the local microenvironment. 

Activation of T-lymphocytes during inflammation has profound effects on tissue and 

serum cytokine levels (64, 82). Since enteric glial MHC II expression alters T-

lymphocyte activation, we examined whether enteric glial MHC II expression alters 

cytokine levels as a downstream effect. The majority of molecules examined in a panel 

of 31 cytokines and chemokines were not significantly affected by the IFNγ and LPS 

stimulus (Fig.3.9b,c). However, significant increases were observed in colonic tissue 

levels of leukemia inhibitory factor and CCL5 (Fig.3.10), and serum levels of 

granulocyte colony stimulating factor, macrophage colony stimulating factor, IL-6, 

CXCL-1, and CCL4 (Fig.3.11). Ablating enteric glial MHC II had little effect on colonic 

tissue and serum cytokine levels and expression in MHC II KO animals did not differ 

significantly from wildtype animals (Fig.3.10, 3.11). Therefore, while glial antigen 

presentation does affect the activation profiles of immune cells in mesenteric lymph 

nodes, serum and regional cytokine profiles are mainly driven by other immune cells in 

this model. 

 
 

120 

It is possible that the lack of effects on serum cytokines in Sox10CreERT2;IABfl/fl mice is 

complicated by also ablating MHC II in Sox10+ glia in the spleen (73). In agreement 

with prior work, we observed that Sox10+ spleen glia do express MHC II (Fig.3.12). 

However, Sox10 expressing cells in the spleen were rare and their calculated density is 

only 1.5 cells / mm2 in splenic tissue sections (data not shown). Therefore, it is unlikely 

that MHC II ablation in Sox10+ cells of the spleen has a major impact that would 

confound our cytokine measurements in the serum.  

 

 
 

 

121 

 

Figure 3.8: Enteric glial gene expression. Gene expression of select cytokines /  

 
 

122 

Figure 3.8 (cont’d): 

chemokines in RiboTag animals stimulated with IFNγ + LPS or controls (n = 2-6 mice; 

unpaired student’s t-test; **P ≤ .005, ***P ≤ .0005). 

 

 

 

 
 

 

123 

Figure 3.9: Enteric glial MHC II expression has limited effects on serum and colonic tissue cytokine levels. (A) 

Schematic depicting potential source of cytokines release from T-lymphocytes after activation via binding enteric glial 

MHC II. (B) Heat map depicting fold change compared to saline controls in quantified cytokines and chemokines in  

 
 

   

124 

 

Figure 3.9 (cont’d): 

colonic tissue homogenates of male mice (n=4-10 mice). (C) Heat map depicting fold 

change compared to saline controls in quantified cytokines and chemokines in serum 

samples of male mice (n=4-8 mice). 

 

 
 

125 

Figure 3.10: MHC II ablation in enteric glial cells do not show changes in colonic 

tissue level of select cytokines. Enteric glial MHC II KO animals challenged with IFNγ  

 
 

126 

 

Figure 3.10 (cont’d):  

+ LPS do not display significantly lower levels of regional colonic cytokines compared to 

wildtype counterparts challenged with IFNγ + LPS. 

 

 

 

 
 

127 

Figure 3.11: MHC II ablation in enteric glial cells do not show changes in serum 

level of select cytokines. Enteric glial MHC II KO animals challenged with IFNγ + LPS  

 

 

 
 

128 

Figure 3.11 (cont’d): 

do not display significantly lower levels of serum cytokines compared to wildtype 

counterparts challenged with IFNγ + LPS. 

 

 

 

 
 

129 

Figure 3.12: Sox10 expressing cells are present in sparse numbers in spleen 

tissue and express MHC II. (A) Representative confocal images of tdTomato (white)  

 

 
 

130 

Figure 3.12 (cont’d): 

and MHC II (red) immunofluorescent labeling in spleen sections in IFNγ + LPS treated 

and control animals (scale bar: 50µm for all images). (B) Quantified MHC II mean 

fluorescent intensity in Sox10tdTomato expressing cells (C-D) Line-scan of MHC II (red) 

and Sox10tdTomato (blue) labeled spleen tissue from (C) control or (D) IFNγ + LPS 

treated mice. 

 

 
 

 

131 

Pro-inflammatory stimuli IFNγ and LPS do not trigger phagocytosis in enteric glia 

The effects of glial MHC II on T-lymphocyte activation state suggest that glia 

MHC II molecules are not empty and are loaded with peptides(63, 75). In professional 

antigen presenting cells, peptides loaded onto MHC II are typically derived from the 

phagocytosis and processing of foreign antigens (60). We tested whether enteric glia 

present phagocytosed antigens onto MHC II molecules by first examining the 

phagocytic potential of enteric glia. Exposure to pHrodo BioParticles elicited 

phagocytosis in CD45+ immune cells neighboring enteric ganglia (Fig.3.13c), but 

phagocytosis or internalization of labeled E.coli particles was never observed in 

tdTomato+ enteric glia in 18 separate experiments (Fig.3.13b,c). We reasoned that this 

lack of effect might be due to using pHrodo labeled E.coli particles as the phagocytic 

substrate and that glia might engulf material through other mechanisms such as 

endocytosis. Therefore, we used labeled LPS molecules and 1.0µm fluorescent 

polystyrene microspheres to test whether TLR4-mediated endocytosis or size are 

contributing factors to glial phagocytosis. Incubation with fluorescently labeled LPS or 

fluorescent polystyrene microspheres did not elicit any uptake within enteric glial cells 

(data not shown), suggesting that enteric glia are either incapable of phagocytosis or 

that there is insufficient stimulation to trigger phagocytic behavior in enteric glia. Taken 

together, our results demonstrate that expression of MHC II molecules on enteric glia 

when stimulated with low dose IFNγ and LPS is not due to the phagocytosis of foreign 

antigens but is derived from other pathways. 

 

 
 

132 

 
Figure 3.13: Enteric glia do not exhibit phagocytic activity when stimulated with IFNγ and LPS. (A) Schematic 

depicting that enteric glial cells do not exhibit phagocytic activity when stimulated with IFNγ and LPS. (B) Representative 

brightfield and epifluorescent image of tdTomato-expressing Sox10+ cells (blue) and pHrodo labeled E.coli BioParticles 

(green). (C) Representative confocal image of tdTomato-expressing Sox10+ cells (blue), pHrodo labeled E.coli 

BioParticles (green), and CD45 immunolabeling (magenta) (scale bar: 50µm). 

 

 

   

133 
 

Glial autophagy regulates MHC II expression  

Our data show that glial MHC II expression occurs independently of 

phagocytosis. Therefore, we examined alternative pathways for MHC II induction. In 

antigen presenting cells such as B cells, a deficiency in autophagic protein Atg5 

decreases MHC II expression levels, suggesting that autophagy regulates MHC II 

expression levels in antigen presenting cells (78). We tested whether a similar 

autophagy mechanism controls enteric glial MHC II expression by measuring levels of 

the autophagy proteins p62 and LC3 over a 16-hour time-course. As described above, 

MHC II expression was detected in enteric glia by 4 hours post injection (Fig.3.14b). 

Expression of autophagic proteins LC3 and p62 was not limited to enteric glia and is 

distributed in both enteric neurons and glia. The punctate nature of LC3 labeling shows 

a greater density in enteric glia, while p62 labeling is more prominent in neurons 

compared to glia, consistent with findings in single-cell RNA seq databases (19) 

(Fig.3.14b,c). Glial MHC II expression significantly (P = .0021) increased by 8 hours 

post-injection with IFNγ and LPS and further increased by 16 hours (P < .0001) (Fig. 

3.14b,c,d). LC3+ puncta density in both enteric glia (Fig.3.14f) and enteric neurons 

(Fig.3.14i) trended towards increased levels over 16 hours, while p62 expression was 

significantly elevated in enteric glia (P = 0.0060) and neurons (P = .0270) by 4 hours 

post-injection and subsequently decreased to pre-injection levels by 16 hours 

(Fig.3.14e,h). The spike and subsequent decrease in p62 labeling, with the concurrent 

trend in increasing LC3 puncta over 16 hours suggests that autophagy is active in both 

enteric glia and neurons (55). The timing of MHC II upregulation in enteric glia coincides 

with autophagy-mediated consumption of p62 molecules and accumulation of LC3 

 

134 

 

deposits, showing that enteric glial MHC II expression occurs concurrently with 

autophagy pathways.  

Since autophagy can lead to the presentation of intracellular antigens on MHC II 

molecules (18), we tested whether our findings are indicative of autophagy-induced glial 

MHC II expression. We used a combination of IFNγ and LPS, the autophagy stimulator 

rapamycin, and the autophagy inhibitor 3-methyladenine (3-MA) to test the regulation of 

glial MHC II expression by autophagy. A 16-hour incubation with IFNγ and LPS 

significantly (P = 0.0015) increased MHC II levels in enteric glia, similar to our in vivo 

findings (Fig.3.14g). Combining IFNγ and LPS with rapamycin further increased glial 

MHC II expression level compared to control (P < .0001), but interestingly, incubation 

with rapamycin alone did not increase MHC II levels compared to control (P = .8354) 

(Fig.3.14g). However, tissues incubated with a combination of IFNγ, LPS, and 3-MA, or 

IFNγ, LPS, rapamycin, and 3-MA had significantly decreased levels of glial MHC II 

expression compared to non-3-MA treated counterparts (P = .0047 and P < .0001, 

respectively) and reduced glial MHC II expression to levels comparable to untreated 

controls (Fig.3.14g). Taken together, our data show that inhibition of glial autophagy 

effectively eliminates MHC II labeling, suggesting that glial MHC II expression requires 

autophagy activation. However, the inability for rapamycin alone to induce glial MHC II 

suggests that the activation of autophagy alone is insufficient to trigger glial MHC II 

 

expression. 

 

 

135 

 

Figure 3.14: Enteric glia express MHC II via autophagy pathways. (A) Schematic depicting enteric glial MHC II loading 

136 
 

Figure 3.14 (cont’d): 

and expression of autophagy-derived peptides when stimulated with IFNγ and LPS. (B) 

Representative confocal images of GFAP (grey), MHC II (red), and LC3 (cyan) 

immunofluorescent labeling in enteric ganglia in wildtype animals injected with IFNγ and 

LPS over a 16-hour time-course. LC3 show punctate labeling and is distributed in a 

neuronal and glial pattern (scale bar: 50µm for all images). (C) Representative confocal 

images of GFAP (grey), MHC II (red), and P62 (cyan) immunofluorescent labeling in 

enteric ganglia in wildtype animals injected with IFNγ and LPS over a 16-hour time-

course. P62 labeling is prominent in enteric neurons but is distributed in both neurons 

and glia (scale bar: 50µm for all images). (D,E) Quantified mean fluorescent intensity of 

MHC II and p62 labeling on GFAP-expressing enteric glia in the myenteric plexus. 

Wildtype animals were injected with IFNγ and LPS and colons harvested over a 16-hour 

time-course ((D) n=5-8 mice and (E) n=3-4 mice; 1-way analysis of variance with Sidak-

corrected multiple comparisons; *P ≤ .05, **P ≤ .005, ****P ≤ .0001). (F) Quantified 

density of LC3 puncta labeling over the area of GFAP-expressing enteric glia in the 

myenteric plexus. Wildtype animals were injected with IFNγ and LPS and colons 

harvested over a 16-hour time-course (n=2-3 mice; 1-way analysis of variance with 

Sidak-corrected multiple comparisons). (G) Quantified mean fluorescent intensity of 

MHC II labeling on GFAP-expressing enteric glia in the myenteric plexus. LMMP tissue 

preparations from wildtype animals were incubated with a combination of IFNγ and LPS, 

Rapamycin, and 3-MA for 16 hours prior to fixation and immunolabeling. (n=4-5 mice; 1-

way analysis of variance with Sidak-corrected multiple comparisons; **P ≤ .005, ****P ≤ 

.0001). (H) Quantified mean fluorescent intensity of p62 labeling within enteric ganglion  

 

137 

 

Figure 3.14 (cont’d): 

outside of GFAP-expressing enteric glia in the myenteric plexus. Wildtype animals were 

injected with IFNγ and LPS and colons harvested over a 16-hour time-course (n=3-4 

mice; 1-way analysis of variance with Sidak-corrected multiple comparisons). (I) 

Quantified density of LC3 puncta labeling within enteric ganglion outside of GFAP-

expressing enteric glia in the myenteric plexus. Wildtype animals were injected with 

IFNγ and LPS and colons harvested over a 16-hour time-course (n=2-3 mice; 1-way 

analysis of variance with Sidak-corrected multiple comparisons).

 

138 

 

Discussion 

The notion that enteric glia function as antigen presenting cells stems from 

observations in Crohn’s and Chagas disease where glia increase MHC II expression 

(REFS). The functional significance of these observations has remained in question 

ever since. Here, we show that antigen presentation by enteric glia functions to 

modulate immune responses through interactions with T-lymphocytes. Enteric glia are 

sensitive to inflammatory stimuli and respond to low dose IFNγ and LPS by expressing 

MHC II molecules. Enteric glial MHC II molecules are functional and contribute to CD4+ 

T-lymphocyte activation, particularly in the Th17 and Treg subpopulations. These effects 

do not involve large shifts in proinflammatory mediators. Glial MHC II expression is not 

phagocytosis-mediated and is instead, dependent on autophagy activation. However, 

autophagy activation alone is not sufficient to elicit glial MHC II expression. 

 

MHC II is primarily expressed by professional antigen presenting cells and plays 

an important role in their interactions with T-lymphocytes. We show that enteric glia 

express MHC II in an inducible manner when exposed to proinflammatory stimuli, 

consistent with prior in vitro and in vivo human studies which show that glial MHC II 

expression is induced by enteroinvasive E.coli or inflammation (3, 23, 70). Furthermore, 

the punctate distribution of MHC II along glial processes is consistent with findings in 

Chagas disease (3). In our study, enteric glia expressed MHC II in the absence of 

inflammation or observable tissue damage. This suggests that glial antigen presentation 

may act as an early responder to inflammatory stress. This conclusion is consistent with 

observations in Crohn’s disease showing that enteric glia express MHC II in diseased 

areas and macroscopically uninvolved areas (23). Interestingly, occasional glial MHC II 

 

139 

labeling is also observed in samples from healthy individuals (3, 23). We did not 

observe glial MHC II expression in healthy animals and it is not observed in cultured 

human enteric glia in vitro (70). This discrepancy in baseline glial MHC II expression 

may be due to the lack of a microbiome in vitro, and potentially due to murine tolerance 

to LPS and bacterial components (14). However, baseline glial expression of MHC II in 

humans suggests that enteric glia constantly monitor the ENS and modulate immune 

activation in response to the constant presence of microbiome-derived antigens. The 

baseline MHC II expression also suggests that enteric glial MHC II may have anti-

inflammatory effects. Anti-inflammatory actions of MHC II may contribute to 

homeostasis by preventing repeated immune activation and inflammation against the 

normal microbiome. Similar anti-inflammatory effects of MHC II expression by intestinal 

epithelial cells limit Th1 cell infiltration and modulate T-lymphocyte subtype distribution in 

the intestine (69). 

The presence of enteric glial MHC II expression in vivo and in vitro (3, 23, 70) 

suggests potential interactions with T-lymphocytes. Enteric glial interactions with T-

lymphocytes in vivo when expressing neoantigens (15) suggest that T-lymphocytes can 

recognize enteric glia. Moreover, enteric glia inhibit T-lymphocyte activation in co-

cultures (36). However, the functional role of antigen presentation by enteric glia and 

their potential to influence T-lymphocytes in vivo is not understood. Here, we show that 

enteric glial MHC II expression plays an important role in the activation of CD4+ T-

lymphocytes in mesenteric lymph nodes. We also noted a surprisingly large population 

of CD4- cells expressing CD69 in stimulated animals. A portion of those cells 

responding to glial MHC II represent CD69-expressing CD8+ T-lymphocytes (51, 57, 

 

140 

59). The decreased CD8+ T-lymphocyte activation may contribute to the lack of 

histological tissue damage. Additional cell types may include natural killer cells (11, 33) 

and dendritic cells (2, 40), but further study is warranted to further elucidate this cell 

population. Our data show that glial MHC II expression has a propensity towards 

affecting Th17 and Treg subpopulations moreso than Th1 and Th2 subpopulations. This 

apparent conflict in immune regulation by activating both pro- and anti-inflammatory T-

lymphocyte subpopulations suggest a feedback inhibition to limit Th17 activity (9). 

Alternatively, the Th17 subpopulation may exhibit anti-inflammatory actions by secreting 

anti-inflammatory cytokines (22, 52, 56, 84). Furthermore, enteric glia may contribute to 

an anti-inflammatory phenotype by destabilizing interactions with certain T-lymphocyte 

subtypes to inhibit proliferation, similar to effects seen in dendritic cells (76). Despite 

enteric glial MHC II expression having no apparent effect on regional tissue cytokine 

levels, gene expression changes suggest that enteric glia may have a mixed role in the 

local ENS microenvironment by expressing IL-1 and tumor necrosis factor, while 

decreasing IL-12b. The apparent expression of certain pro-inflammatory cytokines 

despite decreasing others may be due to the concurrent activation of separate 

pathways. While IL-1 and tumor necrosis factor gene expression may be driven by 

direct binding of LPS molecules onto TLR4 receptors on enteric glia, a decrease in IL-

12b may be due to the activation of other separate signaling pathways. In support, 

enteric glia show strong immunosuppressive effects that inhibit T-lymphocyte 

proliferation in co-culture models and this effect is strengthened in enteric glia derived 

from Crohn’s patients (36). In contrast, enteric glia can contribute to neuronal cell death 

(7), and cultured human enteric glia challenged with IFNγ and LPS increase gene 

 

141 

expression of various pro-inflammatory cytokines (45). This suggests that the dynamic 

immunomodulatory capabilities of enteric glia depend on the surrounding environment.  

Our current data focuses on the glial MHC II KO (Sox10CreERT2;IABfl/fl) model and 

its effects on T-lymphocytes of the mesenteric lymph nodes. Since we did not detect 

any Sox10+ cells that would be susceptible to the MHC II KO in mesenteric lymph 

nodes, how enteric glia in the ENS affect T-lymphocyte activation while in separate 

anatomical locations remains to be understood. Given the proximity of local CD4+ T-

lymphocytes to enteric glia, it is possible that enteric glia influence the activation of local 

T-lymphocytes via cell interactions or secreted compounds, and the migration of 

activated lymphocytes could subsequently have a wider reaching effect in regional 

lymph nodes (36). Similarly, enteric glia may potentially influence nearby dendritic cells 

to bring about lymph node effects. Enteric glial expression of 15-deoxy-Δ12,14-

prostaglandin J2 (1) can conceivably influence dendritic cells to stimulate activation of 

type 1 regulatory T-lymphocytes (72), but this interaction has yet to be described. 

Phagocytosis is an important pathway for professional antigen presenting cells to 

uptake foreign antigens for processing and presentation. In our study, we did not find 

evidence that enteric glia uptake labeled E.coli, labeled LPS, or fluorescent latex beads. 

The lack of phagocytosis in our study, however, may be due to the strength of the 

proinflammatory stimuli used, since others have successfully demonstrated phagocytic 

potential in glia in other organ systems (49) and in other related glial populations in the 

CNS (21, 34, 38, 71). Therefore, stronger stimuli or the dysfunction and death of nearby 

cells may be necessary for enteric glia to demonstrate phagocytic activity. Given their 

shared characteristics with astrocytes (12), their proximity to enteric neurons (5) and 

 

142 

their ability to contribute to neuronal demise (7), it is conceivable that enteric glia also 

uptake neuronal fragments during neuronal injury or death. The presentation of 

neuronal fragments to the adaptive immune system could lead to the generation of 

autoreactive antibodies and may be a mechanism contributing to the production of anti-

HuC/D antibodies in patients with irritable bowel syndrome (77). The expression of 

enteric glial MHC II in situations of inflammation and neuron loss, such as in Chagasic 

megacolon or Crohn’s disease (3, 23), suggest that enteric glia play a role in these 

disease states. Whether enteric glia contribute to neuronal death and inflammation 

through MHC II expression or promote anti-inflammatory pathways to maintain 

homeostasis remains to be studied. 

The ability of enteric glia to express MHC II molecules in the absence of 

phagocytosis suggests that glia, like B-lymphoma cell lines, use alternative pathways 

such as autophagy promote the loading of intracellular and self-antigens onto MHC II for 

presentation (18, 67). Enteric glial MHC II expression is dependent on autophagy 

activation and the timing of MHC II expression coincides with autophagy activation, 

which raises the possibility that enteric glia present autophagy-derived peptides on their 

MHC II molecules. Although the presentation of autophagy-derived self-antigens on 

MHC II molecules suggest the potential development of autoimmune reactions, our 

findings suggest otherwise, as no pro-inflammatory cytokine levels were changed when 

glial MHC II expression status changed and no inflammatory cell recruitment was 

observed in colonic tissue. These data are consistent with findings by other groups 

demonstrating anti-inflammatory effects from autophagy and autophagy-induced MHC II 

 

143 

expression (76, 81). However, mass spectrometry analysis of glial MHC II molecules is 

necessary to conclusively determine the identity of presented peptides.  

The autophagic response to IFNγ and LPS differed between enteric neurons and 

glia in their expression of LC3 and p62, with more prominent labeling of p62 in enteric 

neurons and greater LC3 puncta in enteric glia. Although unclear, the more prominent 

p62 labeling may indicate that enteric neurons exhibit a higher sensitivity to 

inflammatory stimuli than enteric glia. The observed elevation in density of glial LC3 

puncta may in fact be related to glial phagocytosis and antigen presentation in the form 

of LC3-associated phagocytosis via TLR4 activation (25, 26, 61). However, as we did 

not detect any phagocytic activity, a more detailed examination specifically into LC3-

associated phagocytosis is needed to test this hypothesis (50). Taken in the context of 

IBD, which is considered having dysfunctional autophagy pathways (32, 41), the 

activation of autophagy in enteric glia and neurons may in fact be a compensatory 

mechanism to limit potential inflammatory damage to the ENS. This potential 

mechanism is indeed supported by findings from numerous groups demonstrating the 

activation of autophagy pathways can inhibit intestinal inflammation and reduce colitis 

(37, 47, 81). 

Our data show that autophagy activation is required for enteric glial MHC II 

expression, agreeing with prior studies demonstrating that dendritic cells require 

autophagy machinery to express peptides on MHC II molecules (43). Furthermore, our 

data show that autophagy activation by rapamycin alone is insufficient to induce glial 

MHC II expression. This may be due to the nature of rapamycin, as other groups have 

shown in vitro that rapamycin has varying effectiveness in eliciting autophagy in 

 

144 

different glial cell lines (68). Alternatively, enteric glia may simply require both a stimulus 

with a concomitant activation in autophagy to express MHC II molecules. However, LPS 

as a stimulus may in fact contribute to autophagy inhibition (44, 83). Taken together with 

the upregulation of pro-inflammatory cytokine gene expression, our data suggest that 

two separate pathways may be concurrently activated by IFNγ and LPS challenge. 

Direct LPS binding onto enteric glial TLR4 receptors may contribute to a pro-

inflammatory phenotype in enteric glia, while the activation of a separate pathway 

induces autophagy and an anti-inflammatory phenotype.  

In conclusion, our results show that enteric glia respond to inflammatory stimuli 

by expressing functional MHC II molecules to modulate T-lymphocyte activation 

(Fig.3.15). Furthermore, we show that autophagy activation is necessary, but 

insufficient, to induce enteric glial MHC II expression. Together, these observations 

provide evidence for enteric glia being an important immune regulator during 

inflammatory stress and suggest that they may play an anti-inflammatory role during 

intestinal inflammation and IBD in an attempt to maintain homeostasis. 

 

 

 

145 

 

Figure 3.15: Schematic mechanism of enteric glial MHC II expression. Enteric glia 

exposed to pro-inflammatory stimuli IFNγ and LPS upregulate expression of MHC II 

molecules on their cell surface. Enteric glial MHC II is not derived from phagocytosed 

foreign antigens, but rather derived from products of autophagy. Enteric glial MHC II are 

functional molecules that can interact with CD4+ T-lymphocytes and modulate CD69 

expression levels primarily in IL-17 and FoxP3 expressing CD4+ T-lymphocytes. 

 

 

 

146 

REFERENCES 

 

147 

REFERENCES 

1.   Abdo H, Mahé MM, Derkinderen P, Bach-Ngohou K, Neunlist M, Lardeux B. 
The omega-6 fatty acid derivative 15-deoxy-Δ12,14-prostaglandin J2 is involved in 
neuroprotection by enteric glial cells against oxidative stress. J Physiol 590: 
2739–50, 2012. 

2.   Alari-Pahissa E, Notario L, Lorente E, Vega-Ramos J, Justel A, López D, 

Villadangos JA, Lauzurica P. CD69 Does Not Affect the Extent of T Cell 
Priming. PLoS One 7, 2012. 

3.   Barcelos Morais Da Silveira A, De Oliveira EC, Neto SG, Luquetti AO, Toshio 

Fujiwara R, Correa Oliveira R, Brehmer A, Barcelos A, Oliveira EC De, Neto 
SG, Luquetti AO, Toshio R, Correa R, Brehmer A. Enteroglial cells act as 
antigen-presenting cells in chagasic megacolon. Hum Pathol 42: 522–532, 2011. 

4.   Birnboim HC, Lemay A-M, Lam DKY, Goldstein R, Webb JR. Cutting edge: 

MHC class II-restricted peptides containing the inflammation-associated marker 3-
nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune 
response. J Immunol 171: 528–32, 2003. 

5.   Boesmans W, Martens MA, Weltens N, Hao MM, Tack J, Cirillo C, Vanden 

Berghe P. Imaging neuron-glia interactions in the enteric nervous system. Front 
Cell Neurosci 7: 183, 2013. 

6.   Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in 

light microscopy. J. Microsc. 224 Blackwell Publishing Ltd: 213–232, 2006. 

7.   Brown IAM, McClain JL, Watson RE, Patel BA, Gulbransen BD. Enteric Glia 

Mediate Neuron Death in Colitis Through Purinergic Pathways That Require 
Connexin-43 and Nitric Oxide. C Cell Mol Gastroenterol Hepatol 2: 77–91, 2016. 

8.   Chastain EML, Duncan DS, Rodgers JM, Miller SD. The role of antigen 

presenting cells in multiple sclerosis. Biochim. Biophys. Acta - Mol. Basis Dis. 
1812 NIH Public Access: 265–274, 2011. 

9.   Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich J, Jack 

RS, Wunderlich FT, Brüning JC, Müller W, Rudensky AY. Interleukin-10 
signaling in regulatory T cells is required for suppression of Th17 cell-mediated 
inflammation. Immunity 34: 566–78, 2011. 

10.   Cheadle GA, Costantini TW, Lopez N, Bansal V, Eliceiri BP, Coimbra R. 

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier 
Breakdown. PLoS One 8: 1–11, 2013. 

11.   Cibrián D, Sánchez-Madrid F. CD69: from activation marker to metabolic 

gatekeeper. Eur. J. Immunol. 47 Wiley-VCH Verlag: 946–953, 2017. 

 

148 

12.   Coelho-Aguiar J de M, Bon-Frauches AC, Gomes ALT, Verissimo CP, Aguiar 

DP, Matias D, Thomasi BB de M, Gomes AS, Brito GA de C, Moura-Neto V. 
The enteric glia: Identity and functions. Glia 63: 921–935, 2015. 

13.   Colombo E, Farina C. Astrocytes: Key Regulators of Neuroinflammation. Trends 

Immunol 37: 608–620, 2016. 

14.   Copeland S, Warren HS, Lowry SF, Calvano SE, Remick D. Acute 

inflammatory response to endotoxin in mice and humans. Clin Diagn Lab Immunol 
12: 60–67, 2005. 

15.   Cornet A, Savidge TC, Cabarrocas J, Deng W-L, Colombel J-F, Lassmann H, 

Desreumaux P, Liblau RS. Enterocolitis induced by autoimmune targeting of 
enteric glial cells: A possible mechanism in Crohn’s disease? Proc Natl Acad Sci 
98: 13306–13311, 2001. 

16.   Delvalle NM, Dharshika C, Morales-Soto W, Fried DE, Gaudette L, 
Gulbransen BD. Communication Between Enteric Neurons, Glia, and 
Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation. CMGH 6: 
321–344, 2018. 

17.   Delvalle NM, Fried DE, Rivera-Lopez G, Gaudette L, Gulbransen BD. 

Cholinergic activation of enteric glia is a physiological mechanism that contributes 
to the regulation of gastrointestinal motility. Am J Physiol - Gastrointest Liver 
Physiol 315: G473–G483, 2018. 

18.   Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Müller M, Kreymborg K, 

Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen C, 
Rammensee H, Stevanovic S. Autophagy promotes MHC class II presentation of 
peptides from intracellular source proteins. Proc Natl Acad Sci U S A 102: 7922–
7, 2005. 

19.   Drokhlyansky E, Smillie CS, Van Wittenberghe N, Ericsson M, Griffin GK, 

Eraslan G, Dionne D, Cuoco MS, Goder-Reiser MN, Sharova T, Kuksenko O, 
Aguirre AJ, Boland GM, Graham D, Rozenblatt-Rosen O, Xavier RJ, Regev 
A. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell 
182: 1606-1622.e23, 2020. 

20.   Fagbemi AO, Torrente F, Puleston J, Lakhoo K, James S, Murch SH. Enteric 

Neural Disruption in Necrotizing Enterocolitis Occurs in Association With 
Myenteric Glial Cell CCL20 Expression. J Pediatr Gastroenterol Nutr 57: 788–
793, 2013. 

21.   Fricker M, Oliva-Martín MJ, Brown GC. Primary phagocytosis of viable neurons 
by microglia activated with LPS or Aβ is dependent on calreticulin/LRP phagocytic 
signalling. J Neuroinflammation 9: 1–12, 2012. 

22.   Gagliani N, Amezcua Vesely MC, Iseppon A, Brockmann L, Xu H, Palm NW, 

 

149 

de Zoete MR, Licona-Limón P, Paiva RS, Ching T, Weaver C, Zi X, Pan X, Fan 
R, Garmire LX, Cotton MJ, Drier Y, Bernstein B, Geginat J, Stockinger B, 
Esplugues E, Huber S, Flavell RA. Th17 cells transdifferentiate into regulatory T 
cells during resolution of inflammation. Nature 523: 221–5, 2015. 

23.   Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, Vantrappen G, 

Desmet VJ. Major histocompatibility class II expression on the small intestinal 
nervous system in Crohn’s disease. Gastroenterology 103: 439–447, 1992. 

24.   Gulbransen BD, Sharkey KA. Purinergic Neuron-to-Glia Signaling in the Enteric 

Nervous System. Gastroenterology 136: 1349–1358, 2009. 

25.   Heckmann BL, Boada-Romero E, Cunha LD, Magne J, Green DR. LC3-

Associated Phagocytosis and Inflammation. J. Mol. Biol. 429 Academic Press: 
3561–3576, 2017. 

26.   Heckmann BL, Green DR. LC3-associated phagocytosis at a glance. J. Cell Sci. 

132 NLM (Medline): 2019. 

27.   Hernandez S, Fried DE, Grubišić V, McClain JL, Gulbransen BD. 

Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness. 
FASEB J 33: 6168–6184, 2019. 

28.   Herzog J, Maekawa Y, Cirrito TP, Illian BS, Unanue ER. Activated antigen-

presenting cells select and present chemically modified peptides recognized by 
unique CD4 T cells. Proc Natl Acad Sci U S A 102: 7928–7933, 2005. 

29.   Hood L, Steinmetz M, Malissen B. Genes of the major histocompatibility 

complex of the mouse. Annu Rev Immunol 1: 529–68, 1983. 

30.   Hyldig-Nielsen JJ, Schenning L, Hammerling U, Widmark E, Heldin E, Lind 

P, Servenius B, Lund T, Flavell R, Lee JS, Trowsdale J, Schreier PH, 
Zablitzky F, Larhammar D, Peterson PA, Rask L. The complete nucleotide 
sequence of the I-E alpha d immune response gene. Nucleic Acids Res 11: 5055–
71, 1983. 

31.  

Ibiza S, García-cassani B, Ribeiro H, Carvalho T, Almeida L, Marques R, 
Misic AM, Bartow-McKenney C, Larson DM, Pavan WJ, Eberl G, Grice EA, 
Veiga-Fernandes H, Eberi G, Grice EA, Veiga-Fernandes H, Eberl G, Grice 
EA, Veiga-Fernandes H. Glial-cell-derived neuroregulators control type 3 innate 
lymphoid cells and gut defence. Nature 535: 440–443, 2016. 

32.  

Iida T, Onodera K, Nakase H. Role of autophagy in the pathogenesis of 
inflammatory bowel disease. World J Gastroenterol 23: 1944–1953, 2017. 

33.   Jiang YZ, Couriel D, Mavroudis DA, Lewalle P, Malkovska V, Hensel NF, 

Dermime S, Molldrem J, Barrett AJ. Interaction of natural killer cells with MHC 
class II: Reversal of HLA-DR1-mediated protection of K562 transfectant from 

 

150 

natural killer cell-mediated cytolysis by brefeldin-A. Immunology 87: 481–486, 
1996. 

34.   Jones RS, Minogue AM, Connor TJ, Lynch MA. Amyloid-β-induced astrocytic 

phagocytosis is mediated by CD36, CD47 and RAGE. J Neuroimmune Pharmacol 
8: 301–11, 2013. 

35.   Kambayashi T, Laufer TM. Atypical MHC class II-expressing antigen-presenting 
cells: can anything replace a dendritic cell? Nat Rev Immunol 14: 719–30, 2014. 

36.   Kermarrec L, Durand T, Neunlist M, Naveilhan P, Neveu I. Enteric glial cells 
have specific immunosuppressive properties. J Neuroimmunol 295–296: 79–83, 
2016. 

37.   Kökten T, Gibot S, Lepage P, D’Alessio S, Hablot J, Ndiaye NC, Busby-
Venner H, Monot C, Garnier B, Moulin D, Jouzeau JY, Hansmannel F, 
Danese S, Guéant JL, Muller S, Peyrin-Birouleta L. TREM-1 inhibition restores 
impaired autophagy activity and reduces colitis in mice. J Crohn’s Colitis 12: 230–
244, 2018. 

38.   Konishi H, Okamoto T, Hara Y, Komine O, Tamada H, Maeda M, Osako F, 

Kobayashi M, Nishiyama A, Kataoka Y, Takai T, Udagawa N, Jung S, Ozato 
K, Tamura T, Tsuda M, Yamanaka K, Ogi T, Sato K, Kiyama H. Astrocytic 
phagocytosis is a compensatory mechanism for microglial dysfunction. EMBO J. ( 
September 22, 2020). doi: 10.15252/embj.2020104464. 

39.   Lafuse WP, Singh S, Savarirayan S, David CS. DNA restriction fragment 

analysis of E alpha genes in E-negative H-2 recombinant mouse strains. 
Immunogenetics 24: 68–70, 1986. 

40.   Lamana A, Martin P, De La Fuente H, Martinez-Mũoz L, Cruz-Adalia A, 

Ramirez-Huesca M, Escribano C, Gollmer K, Mellado M, Stein J V., 
Rodriguez-Fernandez JL, Sanchez-Madrid F, Del Hoyo GM. CD69 modulates 
sphingosine-1-phosphate-induced migration of skin dendritic cells. J Invest 
Dermatol 131: 1503–1512, 2011. 

41.   Larabi A, Barnich N, Nguyen HTT. New insights into the interplay between 

autophagy, gut microbiota and inflammatory responses in IBD. Autophagy 16: 38–
51, 2020. 

42.   Laranjeira C, Sandgren K, Kessaris N, Richardson W, Potocnik A, Vanden 

Berghe P, Pachnis V. Glial cells in the mouse enteric nervous system can 
undergo neurogenesis in response to injury. J Clin Invest 121: 3412–24, 2011. 

43.   Lee HK, Mattei LM, Steinberg BE, Alberts P, Lee YH, Chervonsky A, 

Mizushima N, Grinstein S, Iwasaki A. In vivo requirement for Atg5 in antigen 
presentation by dendritic cells. Immunity 32: 227–39, 2010. 

 

151 

44.   Lee JW, Nam H, Kim LE, Jeon Y, Min H, Ha S, Lee Y, Kim SY, Lee SJ, Kim 

EK, Yu SW. TLR4 (toll-like receptor 4) activation suppresses autophagy through 
inhibition of FOXO3 and impairs phagocytic capacity of microglia. Autophagy 15: 
753–770, 2019. 

45.   Liñán-Rico A, Turco F, Ochoa-Cortes F, Harzman A, Needleman BJ, 

Arsenescu R, Abdel-Rasoul M, Fadda P, Grants I, Whitaker E, Cuomo R, 
Christofi FL. Molecular Signaling and Dysfunction of the Human Reactive Enteric 
Glial Cell Phenotype: Implications for GI Infection, IBD, POI, Neurological, Motility, 
and GI Disorders. Inflamm Bowel Dis 22: 1812–1834, 2016. 

46.   Le Loupp A-G, Bach-Ngohou K, Bourreille A, Boudin H, Rolli-Derkinderen M, 
Denis MG, Neunlist M, Masson D. Activation of the prostaglandin D2 metabolic 
pathway in Crohn’s disease: involvement of the enteric nervous system. BMC 
Gastroenterol 15: 112, 2015. 

47.   Macias-Ceja DC, Cosín-Roger J, Ortiz-Masiá D, Salvador P, Hernández C, 

Esplugues J V, Calatayud S, Barrachina MD. Stimulation of autophagy 
prevents intestinal mucosal inflammation and ameliorates murine colitis. Br J 
Pharmacol 174: 2501–2511, 2017. 

48.   Mahdi BM. Role of HLA typing on Crohn’s disease pathogenesis. Ann Med Surg 

4: 248–253, 2015. 

1047–1055, 1990. 

49.   Mano T, Puro DG. Phagocytosis by Human Retinal Glial Cells in Culture. 31: 

50.   Martinez J. Detection of LC3‐Associated Phagocytosis (LAP). Curr Protoc Cell 

Biol 87, 2020. 

51.   Matechak EO, Killeen N, Hedrick SM, Fowlkes BJ. MHC class II-specific T cells 
can develop in the CD8 lineage when CD4 is absent. Immunity 4: 337–347, 1996. 

52.   McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, 

McClanahan T, Cua DJ. TGF-β and IL-6 drive the production of IL-17 and IL-10 
by T cells and restrain TH-17 cell-mediated pathology. Nat Immunol 8: 1390–
1397, 2007. 

53.   Middel P, Raddatz D, Gunawan B, Haller F, Radzun HJ. Increased number of 

mature dendritic cells in Crohn’s disease: Evidence for a chemokine mediated 
retention mechanism. Gut 55: 220–227, 2006. 

54.   Mikkelsen HB. Macrophages in the external muscle layers of mammalian 

intestines. Histol Histopathol 10: 719–736, 1995. 

55.   Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy 

research. Cell 140: 313–26, 2010. 

 

152 

56.   Musuraca G, De Matteis S, Napolitano R, Papayannidis C, Guadagnuolo V, 

Fabbri F, Cangini D, Ceccolini M, Giannini MB, Lucchesi A, Ronconi S, 
Mariotti P, Savini P, Tani M, Fattori PP, Guidoboni M, Martinelli G, Zoli W, 
Amadori D, Carloni S. IL-17/IL-10 double-producing T cells: New link between 
infections, immunosuppression and acute myeloid leukemia. J Transl Med 13: 1–
10, 2015. 

57.   Ranasinghe S, Lamothe PA, Soghoian DZ, Kazer SW, Cole MB, Shalek AK, 
Yosef N, Jones RB, Donaghey F, Nwonu C, Jani P, Clayton GM, Crawford F, 
White J, Montoya A, Power K, Allen TM, Streeck H, Kaufmann DE, Picker LJ, 
Kappler JW, Walker BD. Antiviral CD8+ T Cells Restricted by Human Leukocyte 
Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion. 
Immunity 45: 917–930, 2016. 

58.   Riberdy JM, Avva RR, Geuze HJ, Cresswell P. Transport and intracellular 

distribution of MHC class II molecules and associated invariant chain in normal 
and antigen-processing mutant cell lines. J Cell Biol 125: 1225–1237, 1994. 

59.   Rist M, Smith C, Bell MJ, Burrows SR, Khanna R. Cross-recognition of 

HLADR4 alloantigen by virus-specific CD8+ T cells: A new paradigm for self-
/nonself-recognition. Blood 114: 2244–2253, 2009. 

60.   Roche PA, Furuta K. The ins and outs of MHC class II-mediated antigen 

processing and presentation. Nat Rev Immunol 15: 203–16, 2015. 

61.   Romao S, Münz C. LC3-associated phagocytosis. Autophagy 10: 526–528, 2014. 

62.   Rosenbaum C, Schick MA, Wollborn J, Heider A, Scholz C-J, Cecil A, Niesler 

B, Hirrlinger J, Walles H, Metzger M. Activation of Myenteric Glia during Acute 
Inflammation In Vitro and In Vivo. PLoS One 11: e0151335, 2016. 

63.   Rupp B, Günther S, Makhmoor T, Schlundt A, Dickhaut K, Gupta S, 

Choudhary I, Wiesmüller KH, Jung G, Freund C, Falk K, Rötzschke O, Kühne 
R. Characterization of structural features controlling the receptiveness of empty 
class II MHC molecules. PLoS One 6, 2011. 

64.   Santana MA, Esquivel-Guadarrama F. Cell biology of T cell activation and 

differentiation. Int Rev Cytol 250: 217–74, 2006. 

65.   Schmid D, Pypaert M, Münz C. Antigen-loading compartments for major 

histocompatibility complex class II molecules continuously receive input from 
autophagosomes. Immunity 26: 79–92, 2007. 

66.   Shrum B, Anantha R V, Xu SX, Donnelly M, Haeryfar S, McCormick JK, Mele 
T, Bone R, Balk R, Cerra F, Dellinger R, Fein A, Knaus W, Schein R, Sibbald 
W, Martin G, Mannino D, Eaton S, Moss M, Vincent J, Sakr Y, Sprung C, 
Ranieri V, Reinhart K, Gerlach H, Moreno R, Carlet J, Gall J Le, Payen D, 
Levy M, Fink M, Marshall J, Abraham E, Angus D, Cook D, Cohen J, Opal S, 

 

153 

Vincent J, Ramsay G, Buras J, Holzmann B, Sitkovsky M, Marshall J, Esmon 
C, Nemzek J, Hugunin K, Opp M, Vincent J, Rello J, Marshall J, Silva E, 
Anzueto A, Martin C, Moreno R, Lipman J, Gomersall C, Sakr Y, Reinhart K, 
Hubbard W, Choudhry M, Schwacha M, Kerby J, Rue L, Bland K, Chaudry I, 
Wichterman K, Baue A, Chaudry I, Maier S, Traeger T, Entleutner M, 
Westerholt A, Kleist B, Huser N, Holzmann B, Stier A, Pfeffer K, Heidecke C, 
Dejager L, Pinheiro I, Dejonckheere E, Libert C, Knaus W, Draper E, Wagner 
D, Zimmerman J, Huet O, Ramsey D, Miljavec S, Jenney A, Aubron C, Aprico 
A, Stefanovic N, Balkau B, Head G, Haan J de, Chin-Dusting J, Langford D, 
Bailey A, Chanda M, Clarke S, Drummond T, Echols S, Glick S, Ingrao J, 
Klassen-Ross T, Lacroix-Fralish M, Matsumiya L, Sorge R, Sotocinal S, 
Tabaka J, Wong D, Maagdenberg A van den, Ferrari M, Craig K, Mogil J, 
Hyde S, Stith R, McCallum R, Gavrieli Y, Sherman Y, Ben-Sasson S, Hanley 
J, McNeil B, Youden W, Marshall J, Reinhart K, Lee S, Feingold D, Carter J, 
Zhai C, Stapleton G, Gleason N, Whelan R, Otto G, Busch M, Sossdorf M, 
Claus R, Hotchkiss R, Karl I, Hotchkiss R, Opal S, Linch S, Danielson E, 
Kelly A, Tamakawa R, Lee J, Gold J, Castillo E, Schluns K, Jinquan T, Jing 
C, Jacobi H, Reimert C, Millner A, Quan S, Hansen J, Dissing S, Malling H, 
Skov P, Poulsen L, Driscoll K, Fisher B, Kraskauskas D, Martin E, Farkas D, 
Puri P, Massey H, Idowu M, Brophy D, Voelkel N, Fowler A, Natarajan R, Doi 
K, Leelahavanichkul A, Hu X, Sidransky K, Zhou H, Qin Y, Eisner C, 
Schnermann J, Yuen P, Star R, Miyaji T, Hu X, Yuen P, Muramatsu Y, Iyer S, 
Hewitt S, Star R, Yasuda H, Yuen P, Hu X, Zhou H, Star R, Zhang Y, Li M, 
Meng M, Qin C, Hotchkiss R, Swanson P, Freeman B, Tinsley K, Cobb J, 
Matuschak G, Buchman T, Karl I, Hotchkiss R, Tinsley K, Swanson P, 
Schmieg R, Hui J, Chang K, Osborne D, Freeman B, Cobb J, Buchman T, 
Karl I, Zingarelli B, Piraino G, Hake P, O’Connor M, Denenberg A, Fan H, 
Cook J. A robust scoring system to evaluate sepsis severity in an animal model. 
BMC Res Notes 7: 233, 2014. 

67.   Strawbridge AB, Blum JS. Autophagy in MHC class II antigen processing. Curr 

Opin Immunol 19: 87–92, 2007. 

68.   Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB, Kondo S. 
Synergistic augmentation of rapamycin-induced autophagy in malignant glioma 
cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res 65: 
3336–3346, 2005. 

69.   Thelemann C, Eren RO, Coutaz M, Brasseit J, Bouzourene H, Rosa M, Duval 

A, Lavanchy C, Mack V, Mueller C, Reith W, Acha-Orbea H. Interferon-γ 
induces expression of MHC class II on intestinal epithelial cells and protects mice 
from colitis. PLoS One 9: 1–10, 2014. 

70.   Turco F, Sarnelli G, Cirillo C, Palumbo I, De Giorgi F, D’Alessandro A, 
Cammarota M, Giuliano M, Cuomo R. Enteroglial-derived S100B protein 
integrates bacteria-induced Toll-like receptor signalling in human enteric glial 
cells. Gut 63: 105–15, 2014. 

 

154 

71.   Vilalta A, Brown GC. Neurophagy, the phagocytosis of live neurons and 

synapses by glia, contributes to brain development and disease. FEBS J. (2017). 
doi: 10.1111/febs.14323. 

72.   Volchenkov R, Karlsen M, Jonsson R, Appel S. Type 1 regulatory T cells and 

regulatory B cells induced by tolerogenic dendritic cells. Scand J Immunol 77: 
246–254, 2013. 

73.   Wang D, Wu F, Yuan H, Wang A, Kang GJ, Truong T, Chen L, McCallion AS, 

Gong X, Li S. Sox10+ Cells Contribute to Vascular Development in Multiple 
Organs-Brief Report. Arterioscler Thromb Vasc Biol 37: 1727–1731, 2017. 

74.   Wardowska A. The epigenetic face of lupus: Focus on antigen-presenting cells. 

Int. Immunopharmacol. 81 Elsevier B.V.: 106262, 2020. 

75.   Wieczorek M, Abualrous ET, Sticht J, Álvaro-Benito M, Stolzenberg S, Noé 
F, Freund C. Major histocompatibility complex (MHC) class I and MHC class II 
proteins: Conformational plasticity in antigen presentation. Front Immunol 8: 1–16, 
2017. 

76.   Wildenberg ME, Vos ACW, Wolfkamp SCS, Duijvestein M, Verhaar AP, Te 

Velde AA, Van Den Brink GR, Hommes DW. Autophagy attenuates the adaptive 
immune response by destabilizing the immunologic synapse. Gastroenterology 
142: 1493-1503.e6, 2012. 

77.   Wood JD, Liu S, Drossman DA, Ringel Y, Whitehead WE. Anti-enteric 

neuronal antibodies and the irritable bowel syndrome. J Neurogastroenterol Motil 
18: 78–85, 2012. 

78.   Xia F, Deng C, Jiang Y, Qu Y, Deng J, Cai Z, Ding Y, Guo Z, Wang J. IL4 
(interleukin 4) induces autophagy in B cells leading to exacerbated asthma. 
Autophagy 14: 450–464, 2018. 

79.   Xiao W, Wang W, Chen W, Sun L, Li X, Zhang C, Yang H. GDNF is Involved in 

the Barrier-Inducing Effect of Enteric Glial Cells on Intestinal Epithelial Cells 
Under Acute Ischemia Reperfusion Stimulation. Mol Neurobiol 50: 274–289, 
2014. 

80.   Yang H, Plevy SE, Taylor K, Tyan D, Fischel-Ghodsian N, McElree C, Targan 
SR, Rotter JI. Linkage of Crohn’s disease to the major histocompatibility complex 
region is detected by multiple non-parametric analyses. Gut 44: 519–526, 1999. 

81.   Zhao J, Wang H, Yang H, Zhou Y, Tang L. Autophagy induction by rapamycin 
ameliorates experimental colitis and improves intestinal epithelial barrier function 
in IL-10 knockout mice. Int Immunopharmacol 81: 105977, 2020. 

82.   Zhou H, Li B, Li J, Wu T, Jin X, Yuan R, Shi P, Zhou Y, Li L, Yu F. 

Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in 

 

155 

Systemic Lupus Erythematosus. Mediators Inflamm 2019: 8450947, 2019. 

83.   Zhou M, Xu W, Wang J, Yan J, Shi Y, Zhang C, Ge W, Wu J, Du P, Chen Y. 

Boosting mTOR-dependent autophagy via upstream TLR4-MyD88-MAPK 
signalling and downstream NF-κB pathway quenches intestinal inflammation and 
oxidative stress injury. EBioMedicine 35: 345–360, 2018. 

84.   Zielinski CE, Mele F, Aschenbrenner D, Jarrossay D, Ronchi F, Gattorno M, 

Monticelli S, Lanzavecchia A, Sallusto F. Pathogen-induced human T H17 cells 
produce IFN-γ or IL-10 and are regulated by IL-1β. Nature 484: 514–518, 2012. 

 

 

 

 

156 

CHAPTER 4: 

Summary, Key Findings and Future Directions 

 

157 

 

 

Summary 

Enteric glia, as part of the ENS, play important roles in the maintenance of ENS 

health and modulation of enteric neuronal activity. Enteric glial cells express and 

release both pro- and anti-inflammatory mediators (1, 7, 12, 17, 18, 28), and behave 

similarly to atypical antigen presenting cells by expressing MHC II molecules in select 

conditions (2, 9). However, it has not been shown whether the expressed MHC II 

molecules are functional or whether enteric glial cells can carry out other antigen 

presenting cell functions in an in vivo system. Our study here examines enteric glial 

behavior in the context of an in vivo murine system with low inflammatory stress. We 

isolate glial contributions to overall immune and inflammatory responses by using a 

glial-specific inducible MHC II KO (Sox10CreERT2;IABfl/fl). Overall, our findings suggest that 

the enteric glia may be responding to a low dose or initial inflammatory challenge by 

inducing anti-inflammatory effects via autophagy-mediated MHC II expression to 

maintain homeostasis. 

Key Findings 

Enteric glial cells express MHC II in response to low dose IFNγ and LPS 

 

Animals challenged with a low dose of IFNγ and LPS was sufficient in eliciting an 

increase in MHC II expression in enteric glia. We found that the glial MHC II expression 

was similar in morphology to glial MHC II expressed in human Chagas disease patients 

(2), and this MHC II expression was in the absence of macroscopic or microscopic 

intestinal inflammation or tissue damage. Our results suggest that enteric glia are 

sensitive to the local microenvironment and may represent an ‘early responder’ to 

 

158 

inflammatory stress. Together with the findings that normal non-inflamed patients 

occasionally show enteric glial MHC II expression, these results suggest that the early 

expression of MHC II as a response to inflammatory challenge may have anti-

inflammatory effects in order to maintain homeostasis. Compared to previous studies 

that examined enteric glial cells in the context of Chagas disease and Crohn’s disease 

patients, our mouse model did not experience overt activation of the inflammatory 

pathway and phenotypes dominated by the activation of other immune cells (2, 9). 

Therefore, we are able to isolate glial responses inflammatory stress and not their 

response to cytokine signaling from activated immune cells. 

Enteric glial MHC II alter T-lymphocyte activation 

 

Previously, the expression of enteric glial MHC II has not been demonstrated to 

be a functional molecule in modulating T-lymphocyte activation in vivo. Kermarrec et al. 

have shown that enteric glial MHC II have an inhibitory effect on T-lymphocyte 

proliferation, but the study was performed in vitro with primary cultured human enteric 

glial cells co-cultured with activated T-lymphocytes (16). Our results in a mouse in vivo 

system demonstrate that glial MHC II expression are functional, and influence T-

lymphocyte levels as measured in regional mesenteric lymph nodes. In addition, we 

show that the Th17 and Treg subpopulations are the primary subtypes affected by enteric 

glial MHC II expression. Our data suggests that enteric glial MHC II may be involved in 

the pro-inflammatory and subsequent Treg-mediated inhibition of T-lymphocyte 

activation. Alternatively, MHC II expression on enteric glia may induce an anti-

inflammatory phenotype in Th17 T-lymphocytes to contribute alongside Treg activities 

and maintain homeostasis (21, 24). Despite influencing the activation state of T-

 

159 

lymphocytes, our data show that cytokine levels at the tissue or serum level remain 

unchanged, suggesting that enteric glial have more of an impact on the local 

microenvironment around the ENS than at the regional or whole-body level. 

Enteric glial MHC II expression is derived from autophagy pathways 

 

MHC II molecules are responsible for loading and presenting peptide antigens 

that were generally derived from the phagocytosis of extracellular molecules (26). Our 

results show that, despite enteric glial cells expression of MHC II molecules, they do not 

phagocytose extracellular antigens when challenged with IFNγ and LPS. Furthermore, 

our data shows that the activation of autophagy is necessary, but not sufficient to induce 

MHC II expression in enteric glia. Our findings suggest that enteric glia present 

intracellular antigens, and potentially self-antigens, for presentation onto MHC II 

molecules. Given that cells maintain a basal level of autophagic presentation of self-

peptides onto MHC II while maintaining immune tolerance and the stimulation of 

autophagy ameliorates inflammation (19, 30), our data suggest that autophagy 

activation in enteric glia may have an anti-inflammatory effect when challenged with 

IFNγ and LPS. Together with our other findings, this paints a picture that enteric glia are 

actively attempting to maintain homeostasis in the local ENS microenvironment when 

faced with initial pro-inflammatory stimuli. This may help maintain proper ENS 

functioning prior to overt activation of pro-inflammatory immune responses that can lead 

to ENS damage and GI dysfunction. 

 

160 

Study Limitations 

Pharmacological modulators 

 

We used Rapamycin and 3-methyladenine as chemicals to stimulate or inhibit 

the autophagy pathway. Our findings and conclusions from experiments assume that 

these molecules are specific in their target activity and do not have other side effects 

that may confound findings. 

 

Sex of experimental animals 

 

Cytokine quantification experiments were performed in only male mice. As sex is 

an important variable in cytokine levels, the changes in cytokine levels observed in our 

studies may not be applicable to a wider population. 

MHC II knockout animals 

 

Our MHC II KO mouse strain is driven by the Sox10 promoter to target enteric 

glia in our experiments. Any cells actively utilizing the Sox10 promoter would be ablated 

in MHC II. We examined mesenteric lymph nodes and spleens and did not see 

evidence of Sox10+ cells. However, whether other Sox10+ cells in the periphery 

influence our findings is not known. 

Future Directions 

Determination of antigens presented on enteric glial MHC II 

 

Our findings showed that the expression of enteric glial MHC II is functional in 

modulating T-lymphocyte activation and is mediated by the autophagy pathway. This 

suggests the possibility that the peptides presented on glial MHC II are intracellular ‘self’ 

 

161 

peptides. Since the presentation of peptides on MHC II molecules can dictate the target 

of immune responses, it would be interesting to see what peptides enteric glial cells are 

presenting on their MHC II molecules. In order to determine the identity of peptides 

expressed to T-lymphocytes, mass spectrometry on peptides eluted from enteric glial 

MHC II would be beneficial.  

Enteric glial autophagy effects in modulating T-lymphocyte activation 

 

As discussed earlier, our results show that enteric glial MHC II expression is 

dependent on the activation of the autophagy pathway. Given that our data also show 

that enteric glial MHC II expression can influence T-lymphocyte activation, the next step 

would be to study the direct effects of autophagy modulation on T-lymphocyte 

activation, and whether a similar level of T-lymphocyte activation is seen in animals with 

inhibited autophagy compared to MHC II KO animals. 

Glial MHC II contributions in overt inflammation 

 

Our studies here were based on using a low dose of IFNγ and LPS as an 

inflammatory stimulus. While this inflammatory challenge is beneficial in isolating 

contributions from enteric glia and not from other immune cells, it would be interesting to 

study whether enteric glial MHC II expression have noticeable effects on the 

inflammatory pathway during stronger inflammatory challenge and overt inflammation. 

Utilizing a stronger inflammatory challenge would be beneficial in studying whether 

enteric glial behavior changes based on inflammation intensity. Furthermore, examining 

the impact of glial MHC II in stronger inflammatory stress and established inflammation 

 

162 

would give relevant findings to the pathogenesis of inflammatory bowel disease Crohn’s 

and ulcerative colitis.  

Sympathetic effects on enteric glial-mediated T-lymphocyte activation 

 

The sympathetic and parasympathetic nervous systems innervate and modulate 

immune activation and ENS activity (23, 25). Furthermore, the autonomic nervous 

system innervates enteric glial cells in the ENS (6, 10). Therefore, a future set of 

experiments could examine the effects autonomic activity has on enteric glial autophagy 

activation, expression of MHC II, and their ability to modulate T-lymphocyte activation. 

We performed pilot studies examining MHC II expression in the wildtype control animals 

after surgical sympathectomy (removal of the celiac and inferior mesenteric ganglia), 

chemical sympathectomy (single subcutaneous injection of 100mg/kg 6-

hydroxydopamine (6-OHDA)), or norepinephrine depletion (single subcutaneous 

injection of 0.5mg/kg reserpine). Tissues harvested 7 days after treatments demonstrate 

a trend in increasing enteric glial MHC II expression in surgical sympathectomy and 

reserpine-treated conditions, but not in 6-OHDA-mediated chemically 

sympathectomized animals (Fig.4.1a,b). Since these results were observed in wildtype 

tissues without inflammatory challenge, changes in autonomic innervation to enteric glia 

in tissues and animals challenged with inflammatory stimuli may show more significant 

results. As mentioned above, it would be beneficial to examine how autonomic activity 

affects autophagy activation in enteric glia, and, by performing sympathectomy 

treatments to MHC II KO animals, we can elucidate the effects of autonomic activity on 

enteric glial activation of T-lymphocytes. 

 

 

 

163 

 
Figure 4.1: Autonomic innervation onto enteric glia may modulate MHC II expression. (A) Representative images of 

GFAP (grey) and MHC II (red) immunofluorescent labeling in enteric glia (scale bar: 50µm for all images) (B) Quantified 

mean fluorescent intensity of MHC II labeling on GFAP-expressing enteric glia in the myenteric plexus (n=3-5 mice; 

student’s unpaired t-test). 

 

164 

Local renin angiotensin effects 

 

Finally, the local renin angiotensin system within the GI tract may contribute to 

intestinal inflammation. Studies within the GI tract and CNS demonstrate that the 

binding of the prorenin receptor by prorenin or renin, along with the binding of 

angiotensin receptor 1 by angiotensin II can elicit pro-inflammatory effects, oxidative 

stress, and neuronal damage (3, 8, 31, 33) (Fig.4.2). In contrast, the binding of 

angiotensin receptor 2 by angiotensin II and the mas receptor by angiotensin (1-7) leads 

to anti-inflammatory effects, decreased oxidative stress, and increased neuronal 

survival (4, 11, 15, 20, 27, 29) (Fig.4.2). We performed pilot studies to examine the 

myenteric plexus and the expression of compounds associated with the renin-

angiotensin pathway. In this collaborative pilot study with Dr. Yumei Feng Earley 

(University of Nevada), we examined the myenteric plexus of mice fed with a high fat 

diet compared to controls. Since a high fat diet in mice can lead to intestinal 

inflammation (5), we examined the expression of angiotensin converting enzyme-2 

within the myenteric plexus as a measure of potential anti-inflammatory behavior in 

enteric glia. Our preliminary results show that the expression of angiotensin converting 

enzyme-2 is in a punctate morphology in enteric glia, and expression is increased in the 

high fat diet condition compared to control animals (P = .0417) (Fig.4.3a,b). These 

preliminary results suggest that enteric glia play a role in shifting the local renin-

angiotensin pathway towards a more anti-inflammatory phenotype in the context of a 

high fat diet to potentially negate inflammatory effects due to elevated dietary fat. 

Furthermore, we will examine the role enteric neurons play in renin-induced 

inflammation with the use of transgenic animals with a neuron-specific KO in the 

 

165 

prorenin receptor, developed by Dr. Yumei Feng Earley (University of Nevada). These 

studies will help further elucidate the contributions of the local renin-angiotensin system 

in inflammatory stress in the GI tract. Although our preliminary study is lacking in power, 

our findings, like others (13, 14, 22, 32), suggest that the local renin-angiotensin system 

is at play in the GI tract and is involved in regulating intestinal inflammation. However, 

the role the ENS plays in this pathway is largely unknown and warrants further study. 

 

Taken together, the data presented in this dissertation unveils a novel role that 

enteric glia play as an immune modulator by interacting and controlling T-lymphocyte 

activation. Together with proposed future experiments, the role enteric glia play as an 

antigen presenting cell can be further elucidated in the context of intestinal 

inflammation, leading to potentially new therapeutic targets for inflammatory bowel 

diseases. 

 

 

 

166 

 

Figure 4.2: The renin-angiotensin pathway neuronal damage and inflammation. 

The renin-angiotensin pathway works through several receptors (prorenin receptor, 

angiotensin receptor 1, angiotensin receptor 2, and mas receptor) to bring about pro- 

and anti-inflammatory effects. Angiotensin II can bind both angiotensin receptor 1 and 

angiotensin receptor 2 to bring about opposing effects, but the angiotensin II molecule is 

generally regarded as pro-inflammatory. Angiotensin converting enzyme 2 shifts the 

renin-angiotensin pathway away from a pro-inflammatory phenotype to a more anti-

inflammatory phenotype by increasing production of angiotensin (1-7) and binding of 

mas receptor. 

 

 

167 

 

Figure 4.3: Enteric glia increase expression of ACE2 during high fat diet 

conditions. (A) Representative Images of GFAP (grey) and ACE2 (red) 

immunofluorescent labeling in enteric glia. (B) Quantified mean fluorescent intensity of 

ACE2 labeling on GFAP-expressing enteric glia in the myenteric plexus (n=3 mice; 

student’s unpaired t-test; *P < .05) 

 

 

 

168 

REFERENCES

 

169 

REFERENCES 

1.   Abdo H, Mahé MM, Derkinderen P, Bach-Ngohou K, Neunlist M, Lardeux B. 
The omega-6 fatty acid derivative 15-deoxy-Δ12,14-prostaglandin J2 is involved in 
neuroprotection by enteric glial cells against oxidative stress. J Physiol 590: 
2739–50, 2012. 

2.   Barcelos Morais Da Silveira A, De Oliveira EC, Neto SG, Luquetti AO, Toshio 

Fujiwara R, Correa Oliveira R, Brehmer A, Barcelos A, Oliveira EC De, Neto 
SG, Luquetti AO, Toshio R, Correa R, Brehmer A. Enteroglial cells act as 
antigen-presenting cells in chagasic megacolon. Hum Pathol 42: 522–532, 2011. 

3.   Benicky J, Sánchez-Lemus E, Pavel J, Saavedra JM. Anti-inflammatory effects 
of angiotensin receptor blockers in the brain and the periphery. Cell Mol Neurobiol 
29: 781–792, 2009. 

4.   Bennion DM, Steckelings UM, Sumners C. Neuroprotection via AT2 receptor 
agonists in ischemic stroke. Clin. Sci. 132 Portland Press Ltd: 1055–1067, 2018. 

5.   Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, Burcelin 

R. Changes in gut microbiota control metabolic endotoxemia-induced 
inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 57: 
1470–1481, 2008. 

6.   Costantini TW, Bansal V, Krzyzaniak M, Putnam JG, Peterson CY, Loomis 
WH, Wolf P, Baird A, Eliceiri BP, Coimbra R. Vagal nerve stimulation protects 
against burn-induced intestinal injury through activation of enteric glia cells. Am J 
Physiol Gastrointest Liver Physiol 299: G1308–G1318, 2010. 

7.  

Fagbemi AO, Torrente F, Puleston J, Lakhoo K, James S, Murch SH. Enteric 
Neural Disruption in Necrotizing Enterocolitis Occurs in Association With 
Myenteric Glial Cell CCL20 Expression. J Pediatr Gastroenterol Nutr 57: 788–793, 
2013. 

8.   Garg M, Royce SG, Tikellis C, Shallue C, Batu D, Velkoska E, Burrell LM, 
Patel SK, Beswick L, Jackson A, Britto K, Lukies M, Sluka P, Wardan H, 
Hirokawa Y, Tan CW, Faux M, Burgess AW, Hosking P, Monagle S, Thomas 
M, Gibson PR, Lubel J. Imbalance of the renin-angiotensin system may 
contribute to inflammation and fibrosis in IBD: A novel therapeutic target? Gut 69: 
841–854, 2020. 

9.   Geboes K, Rutgeerts P, Ectors N, Mebis J, Penninckx F, Vantrappen G, 

Desmet VJ. Major histocompatibility class II expression on the small intestinal 
nervous system in Crohn’s disease. Gastroenterology 103: 439–447, 1992. 

10.   Gulbransen BD, Bains JS, Sharkey KA. Enteric Glia Are Targets of the 

Sympathetic Innervation of the Myenteric Plexus in the Guinea Pig Distal Colon. J 

 

170 

Protective in Lipopolysaccharide‐Induced Inflammation and Renal Injury. FASEB 

11.   Hussain T, Dhande I, Patel SN, Ali Q. Angiotensin AT2 Receptor Stimulation is 

Neurosci 30: 6801–6809, 2010. 

J 32: 849.8-849.8, [date unknown]. 

12.  

Ibiza S, García-cassani B, Ribeiro H, Carvalho T, Almeida L, Marques R, 
Misic AM, Bartow-McKenney C, Larson DM, Pavan WJ, Eberl G, Grice EA, 
Veiga-Fernandes H, Eberi G, Grice EA, Veiga-Fernandes H, Eberl G, Grice 
EA, Veiga-Fernandes H. Glial-cell-derived neuroregulators control type 3 innate 
lymphoid cells and gut defence. Nature 535: 440–443, 2016. 

13.  

Inokuchi Y, Morohashi T, Kawana I, Nagashima Y, Kihara M, Umemura S. 
Amelioration of 2,4,6-trinitrobenzene sulphonic acid induced colitis in 
angiotensinogen knockout mice. Gut 54: 349–356, 2005. 

14.   Jahovic N, Ercan F, Gedik N, Yüksel M, Şener G, Alican I. The effect of 

angiotensin-converting enzyme inhibitors on experimental colitis in rats. Regul 
Pept 130: 67–74, 2005. 

15.   Kamel AS, Abdelkader NF, Abd El-Rahman SS, Emara M, Zaki HF, Khattab 

MM. Stimulation of ACE2/ANG(1–7)/Mas Axis by Diminazene Ameliorates 
Alzheimer’s Disease in the D-Galactose-Ovariectomized Rat Model: Role of 
PI3K/Akt Pathway. Mol Neurobiol 55: 8188–8202, 2018. 

16.   Kermarrec L, Durand T, Neunlist M, Naveilhan P, Neveu I. Enteric glial cells 
have specific immunosuppressive properties. J Neuroimmunol 295–296: 79–83, 
2016. 

17.   Liñán-Rico A, Turco F, Ochoa-Cortes F, Harzman A, Needleman BJ, 

Arsenescu R, Abdel-Rasoul M, Fadda P, Grants I, Whitaker E, Cuomo R, 
Christofi FL. Molecular Signaling and Dysfunction of the Human Reactive Enteric 
Glial Cell Phenotype: Implications for GI Infection, IBD, POI, Neurological, Motility, 
and GI Disorders. Inflamm Bowel Dis 22: 1812–34, 2016. 

18.   Le Loupp A-G, Bach-Ngohou K, Bourreille A, Boudin H, Rolli-Derkinderen M, 
Denis MG, Neunlist M, Masson D. Activation of the prostaglandin D2 metabolic 
pathway in Crohn’s disease: involvement of the enteric nervous system. BMC 
Gastroenterol 15: 112, 2015. 

19.   Macias-Ceja DC, Cosín-Roger J, Ortiz-Masiá D, Salvador P, Hernández C, 

Esplugues J V, Calatayud S, Barrachina MD. Stimulation of autophagy 
prevents intestinal mucosal inflammation and ameliorates murine colitis. Br J 
Pharmacol 174: 2501–2511, 2017. 

20.   Matavelli LC, Siragy HM. AT2 receptor activities and pathophysiological 

implications. J Cardiovasc Pharmacol 65: 226–232, 2015. 

 

171 

21.   McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, 

McClanahan T, Cua DJ. TGF-β and IL-6 drive the production of IL-17 and IL-10 
by T cells and restrain TH-17 cell-mediated pathology. Nat Immunol 8: 1390–
1397, 2007. 

22.   Mizushima T, Sasaki M, Ando T, Wada T, Tanaka M, Okamoto Y, Ebi M, 
Hirata Y, Murakami K, Mizoshita T, Shimura T, Kubota E, Ogasawara N, 
Tanida S, Kataoka H, Kamiya T, Alexander JS, Joh T. Blockage of angiotensin 
II type 1 receptor regulates TNF-α-induced MAdCAM-1 expression via inhibition of 
NF-κB translocation to the nucleus and ameliorates colitis. Am J Physiol - 
Gastrointest Liver Physiol 298, 2010. 

23.   Mourad FH, Saadé NE. Neural regulation of intestinal nutrient absorption. Prog 

Neurobiol 95: 149–162, 2011. 

24.   Musuraca G, De Matteis S, Napolitano R, Papayannidis C, Guadagnuolo V, 

Fabbri F, Cangini D, Ceccolini M, Giannini MB, Lucchesi A, Ronconi S, 
Mariotti P, Savini P, Tani M, Fattori PP, Guidoboni M, Martinelli G, Zoli W, 
Amadori D, Carloni S. IL-17/IL-10 double-producing T cells: New link between 
infections, immunosuppression and acute myeloid leukemia. J Transl Med 13: 1–
10, 2015. 

25.   De Ponti F, Giaroni C, Cosentino M, Lecchini S, Frigo G. Adrenergic 

mechanisms in the control of gastrointestinal motility: from basic science to clinical 
applications. Pharmacol Ther 69: 59–78, 1996. 

26.   Roche PA, Furuta K. The ins and outs of MHC class II-mediated antigen 

processing and presentation. Nat Rev Immunol 15: 203–16, 2015. 

27.   Rodrigues Prestes TR, Rocha NP, Miranda AS, Teixeira AL, Simoes-e-Silva 

AC. The Anti-Inflammatory Potential of ACE2/Angiotensin-(1-7)/Mas Receptor 
Axis: Evidence from Basic and Clinical Research. Curr Drug Targets 18, 2017. 

28.   Rosenbaum C, Schick MA, Wollborn J, Heider A, Scholz C-J, Cecil A, Niesler 

B, Hirrlinger J, Walles H, Metzger M. Activation of Myenteric Glia during Acute 
Inflammation In Vitro and In Vivo. PLoS One 11: e0151335, 2016. 

29.   Sabuhi R, Ali Q, Asghar M, Al-Zamily NRH, Hussain T. Role of the angiotensin 
II AT 2 receptor in inflammation and oxidative stress: opposing effects in lean and 
obese Zucker rats. Am J Physiol Physiol 300: F700–F706, 2011. 

30.   Schmid D, Pypaert M, Münz C. Antigen-loading compartments for major 

histocompatibility complex class II molecules continuously receive input from 
autophagosomes. Immunity 26: 79–92, 2007. 

31.   Shi P, Grobe JL, Desland FA, Zhou G, Shen XZ, Shan Z, Liu M, Raizada MK, 
Sumners C. Direct pro-inflammatory effects of prorenin on microglia. PLoS One 
9: 92937, 2014. 

 

172 

32.   Spencer AU, Yang H, Haxhija EQ, Wildhaber BE, Greenson JK, Teitelbaum 

DH. Reduced severity of a mouse colitis model with angiotensin converting 
enzyme inhibition. Dig Dis Sci 52: 1060–1070, 2007. 

33.   Worker CJ, Li W, Feng CY, Souza LAC, Gayban AJB, Cooper SG, Afrin S, 
Romanick S, Ferguson BS, Earley YF. The neuronal (pro)renin receptor and 
astrocyte inflammation in the central regulation of blood pressure and blood 
glucose in mice fed a high-fat diet. Am J Physiol - Endocrinol Metab 318: E765–
E778, 2020. 

 

 

 

 

 

 

 

173