ENTERIC GLIAL CELL REGULATION OF OXIDATIVE STRESS AND
IMMUNE HOMEOSTASIS DURING GASTROINTESTINAL
INFLAMMATION
By
Isola Angella Maria Brown

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Pharmacology and Toxicology-Environmental Toxicology-Doctor of Philosophy
2017

ABSTRACT
ENTERIC GLIAL CELL REGULATION OF OXIDATIVE STRESS AND IMMUNE
HOMEOSTASIS DURING GASTROINTESTINAL INFLAMMATION
By
Isola Angella Maria Brown
Gastrointestinal (GI) motility dysfunction is a debilitating feature that presents as
a symptom in a number of conditions. These include primary GI disorders like
inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) but also
secondary conditions like diabetes mellitus, Parkinson’s Disease and simply as a
consequence of physiological aging. Although as many as 1 in 4 persons worldwide are
affected by GI motility dysfunction, there is a significant lack of safe and effective drugs,
due to a gap in the knowledge regarding the cellular mechanisms that contribute to GI
motility dysfunction.
Normal gut function is controlled by the enteric nervous system (ENS), an
intrinsic neuronal network comprised of enteric neurons and supporting glial cells, which
is embedded within the walls of the GI tract. Death of enteric neurons, and the resulting
disruption of this neuronal network, contributes to motility dysfunction during
inflammation. In this dissertation, we investigate how increased oxidative stress and
imbalanced immune homeostasis, key factors associated with GI inflammation,
contribute to enteric neuropathy. We hypothesize a role for enteric glial cells, which are
capable of modulating ENS oxidative stress, and also have an important
immunomodulatory role in the ENS.
The work in this dissertation used a combination of transgenic animal strains,
immunohistochemistry (IHC), pharmacological modulators, Ca2+ imaging and in situ and

in vivo models of colitis to investigate our hypothesis. We show a key role for enteric
glial regulation of oxidative stress in mediating neuronal loss. During inflammation,
purinergic activation of enteric glia drives enteric neuron death through a pathway that
requires glial nitric oxide (NO) production and glial Cx43 hemichannels and
demonstrates a novel pathogenic role for enteric glia. Further, glial production of the
antioxidant reduced glutathione (GSH) is necessary for neuronal survival in situ,
although whole body GSH depletion has novel protective roles against key features of
murine colitis. Mice with disrupted in T cell signaling of the anti-inflammatory cytokine
TGFβ had altered GI function, immune homeostasis and glial activation at the level of
the myenteric plexus. Lastly, we show that key pro-inflammatory mediators differentially
alter glial Cx43 channel opening in quiescent versus activated enteric glia.
Our data provide new evidence for an active role for enteric glial cells in GI
(patho)physiology. Specifically, we demonstrate that enteric glia are involved in
mediating the effects of oxidative stress and immune imbalance during GI inflammation
and propose novel targets for the development of improved therapeutics to treat GI
motility dysfunction.

For my mom

iv

ACKNOWLEDGEMENTS

This dissertation is the end result of five years of hard work, long days and even
longer nights. The journey was made that much easier because of the support of a
number of people who I would like to thank here.
I’d first like to thank my PhD mentor Dr. Brian Gulbransen. I had the honor of
being Brian’s first trainee and I couldn’t have asked for a better mentor to guide me
through my PhD program. Brian’s confidence and belief in me was often greater than
what I had in myself and with his help, I’ve achieved so much more than I thought I
could and realized new limits for myself. Brian was always supportive of my ideas and
gave me the freedom to grow as a scientist and try new things. Thanks to him, I now
approach new challenges with an “eh – just go for it, we’ll see what happens” attitude.
He was always supportive of my activities outside of the lab and helped me to realize
their importance for my development as a well-rounded scientist and person. I thank
him for his patience on this journey and for putting up with my procrastination and last
minute recommendation letter requests .
I’d also like to thank the other members of my guidance committee – Dr. James
(Jim) Galligan, Dr. Colleen Hegg and Dr. Cheryl Rockwell. Thank you all for challenging
me and pushing me to think critically about my work. After each committee meeting with
you, I returned to lab with new ideas and new energy towards my work. This
dissertation would not have been possible without your scholarship and expertise, but
more importantly – your support.

v

I feel an immense amount of gratitude to past and present members of the
Gulbransen Lab: Jonathon, David, Vlado, Siomara, Ninotchska, Aaron, Christine, RJ,
Becca, Anne and Erika. I couldn’t have asked for a better group of people to work with.
Thank you for your help and support – for listening to me when I talked through
calculations and experimental design, for switching buffers when I was busy and had to
leave early, and for helping me figure out some new protocol or equipment. I’ll
especially miss our time together outside the lab. Our annual Lugnuts game, Secret
Santa gift exchanges and lab potlucks helped make this journey so much easier and I’ll
always treasure those memories. To Anne and Erika, the two undergrads I had the
honor of mentoring, thanks for trusting me to guide you through this crazy science
world.
I owe additional thanks to the Michigan State University (MSU) chapter of
Graduate Women in Science (GWIS), the MSU Alliance for Graduate Education and the
Professoriate (AGEP) and the Pharm/Tox Graduate Student Association (GSO). Thank
you for serving as a much-needed distraction from the lab and for helping me grow as a
well rounded scientist and person. My time in leadership in these groups, and the
relationships I formed, will stick with me forever.
I’d like to thank the Department of Pharmacology and Toxicology, Department of
Physiology, the Institute of Integrative Toxicology and their staffs. Their behind the
scene support and help with paper work and the nitty gritty aspects of grad school
allowed me to be a more productive graduate student. I owe a special thank you to Dr.
Anne Dorrance, the Pharm/Tox graduate program director, for always having an open
door, open arms and a full box of Kleenexes. Special thanks for Drs. Melinda Frame

vi

and Sandra O’Reilly for their technical guidance and expertise in many experiments in
this dissertation.
To my friends, both near and far – thank you! I won’t name you all individually for
fear of forgetting someone but know you are all appreciated. Each of you has helped
get me through this journey. Thanks for listening to me complain, encouraging me when
the work got tough, celebrating with me when things were going well and just being
there these last five year. You were all a much needed break from the craziness and
know that you are appreciated.
Lastly, I’d like to thank my family. To my extended family, your love and support
is felt from where you are - I know you all are proud. To Collin, Georgetta and Enid –
thank you for being the best role models a little sister could ask for. No matter how old I
am, know that I will always look up to your three as motivation to go a little bit further
and try a little bit harder. Collin, you’re the best big brother a girl could ask for. Thank
you for your late night homework help in high school and for reading all those grad
school admissions essays and for always reminding me: “Another One”. Enid, thank you
for being my toughest critic and biggest supporter. As much as you fight with me, I know
you fight twice as hard for me. Georgie, thanks for being the catalyst for this grad school
journey and for pushing me to apply for that first summer research program. Because of
you, my passion for research was ignited. You go above and beyond for me, and it’s
appreciated. To mommy, this is dedicated to you because none of it would be possible
without you. Thank you for the years of sacrifice and support that got me here – we did
it! This PhD was a group project and wouldn’t have been possible without those named
here and so many others. Thank you for your love and light on this journey.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................... xiii
LIST OF FIGURES ............................................................................................. xiv
KEY TO ABBREVIATIONS ............................................................................... xvii
CHAPTER 1 Introduction ......................................................................................1
Overview ....................................................................................................2
The Enteric Nervous System (ENS) ...........................................................3
The Ganglionated Plexuses of the ENS .....................................................4
Enteric Neurons ..........................................................................................6
Morphological Classification of Enteric Neurons ..............................6
Electrophysical Classification of Enteric Neurons ............................8
Immunohistochemical Classification of Enteric Neurons..................8
Enteric Neural Circuitry .............................................................................11
Enteric Neuropathies ................................................................................14
Congenital Neuropathies ...............................................................14
Inflammatory Neuropathies ............................................................14
Secondary Enteric Neuropathies ...................................................15
Oxidative Stress in Enteric Neuropathies .................................................15
Inflammatory Mediators and Cytokines in Enteric Neuropathies ..............17
Enteric Glia ...............................................................................................18
Enteric glial receptors ....................................................................20
Enteric glial regulation of neurotransmitters ...................................21
Enteric glial channels .....................................................................21
Enteric glial regulation of oxidative stress .................................................22
Enteric glial cells as immunomodulators ...................................................23
Summary and Aims of Dissertation ..........................................................24
REFERENCES .........................................................................................26
CHAPTER 2 Enteric glia mediate neuron death in colitis through purinergic pathways
that require connexin-43 and nitric oxide.............................................................42
Abstract ....................................................................................................43
Introduction ...............................................................................................44
Materials and Methods .............................................................................45
Animals ..........................................................................................45
Human tissue .................................................................................46
Whole-mount Immunohistochemistry .............................................46
Quantification of neuronal thiol oxidation .......................................47
Dihydroethidium (DHE) staining .....................................................48
In situ model of neuroinflammation ................................................48
Ca2+ and NO imaging.....................................................................48

viii

Determination of Cell Viability ........................................................49
Detection of ATP release ...............................................................49
Induction of colitis ..........................................................................50
Chemicals/ Drugs ..........................................................................50
Solutions ........................................................................................51
Data and Statistical Analysis ..........................................................51
Results .....................................................................................................52
Purinergic activation of enteric glial cells drives glial Ca2+ responses
and enteric neuron death ...............................................................52
In situ glial-driven neuron death requires Cx43 hemichannels.......55
Glial Cx43 hemichannels are required for in vivo neuron death
during inflammation .......................................................................56
Stimulation of enteric glial P2Y1Rs elicits Cx43-dependent ATP
release ...........................................................................................58
Oxidative stress coincides with neuron death during in vivo
inflammation and is required for P2X7R-driven neuron death
in situ .............................................................................................60
Enteric glia contribute to oxidative stress via local nitric oxide (NO)
production during inflammation ......................................................63
NO potentiates glial ATP release and promotes enteric neuron
death through a Cx43-dependent mechanism ...............................66
Mechanisms of neuron death downstream of glial P2Y1R activation
involve glial iNOS and neuronal P2X7Rs .......................................67
NO positively modulates glial Cx43 hemichannels and negatively
modulates neuronal panx1 hemichannels......................................67
Discussion ................................................................................................74
APPENDIX ...............................................................................................80
REFERENCES .........................................................................................83
CHAPTER 3 The antioxidant glutathione protects against enteric neuron death
in situ, but its depletion is protective during colitis ..............................................89
Abstract ....................................................................................................90
Introduction ...............................................................................................91
Materials and Methods .............................................................................92
Animals ..........................................................................................92
In Vivo Colitis .................................................................................92
In Vivo Glutathione Depletion ........................................................93
In Situ Model of Neuroinflammation ...............................................93
Whole-mount Immunohistochemistry (IHC) ...................................94
Dual-Labeling with Primary Antibodies Raised in the Same Host ..94
Quantification of Neuronal Thiol Oxidation.....................................95
Chemicals/Drugs ...........................................................................95
Solutions and Media ......................................................................96
Data and Statistical Analysis ..........................................................96
Results .....................................................................................................97
Glutathione synthesis enzymes are expressed in myenteric

ix

ganglia ...........................................................................................97
In situ inhibition of GSH synthesis with L-Buthionine Sulfoximine
decreases myenteric neuronal density...........................................98
In vivo inhibition of glutathione synthesis does not cause overt
inflammation but is protective against macroscopic measures of
inflammation ................................................................................104
Glutathione depletion decreases neutrophil infiltration during
DNBS-colitis but does not alter reactivity of MHC-II expressing
macrophages or neuronal thiol oxidation levels ...........................108
In vivo inhibition of glutathione synthesis decreases neuronal
density and alters myenteric plexus neurochemical coding .........111
Inflammation alters the expression of the glutathione synthesis
enzymes, glutamate-cysteine ligase and glutathione synthetase,
in the ENS....................................................................................112
Discussion ..............................................................................................117
APPENDIX .............................................................................................123
REFERENCES .......................................................................................125
CHAPTER 4 The effect of disrupted T cell TGFβ signaling on myenteric plexus
structure and function ........................................................................................131
Abstract ..................................................................................................132
Introduction .............................................................................................133
Materials and Methods ...........................................................................134
Animals ........................................................................................134
Distal Colon Motility (Colon Bead) Assay.....................................135
Whole-mount Immunohistochemistry (IHC) .................................135
Scoring of Colonic Macroscopic Damage ....................................136
Data and Statistical Analysis........................................................136
Results ...................................................................................................136
Mice with disrupted T cell TGFβRII signaling have increased distal
colon transit but no overt markers of colonic inflammation ..........136
Expression of a dominant negative TGFβRII on CD4+ and CD8+
T cells does not alter myenteric plexus neuronal density or
neurochemical coding ..................................................................140
Disrupted TGFβRII signaling induces reactive gliosis in the
myenteric plexus enteric glia ........................................................141
dnTGFβRII mice have increased activation of MHC-II positive
muscularis macrophages but no change in ganglionic IL-1β
expression ...................................................................................144
Mice with disrupted TGFβRII have dysregulated innate and
adaptive immune responses at the level of the
myenteric plexus ..........................................................................145
The transforming growth factor beta receptor type II (TGFβRII) is
expressed in enteric neurons, but not enteric glial cells,
in mouse distal colon myenteric plexus........................................149
Discussion ..............................................................................................151

x

APPENDIX .............................................................................................156
REFERENCES .......................................................................................159
CHAPTER 5 The effects of pro-inflammatory mediators on glial
hemichannel activity ..........................................................................................164
Abstract ..................................................................................................165
Introduction .............................................................................................166
Materials and Methods ...........................................................................167
Animals........................................................................................167
Murine Model of Colitis ................................................................167
Whole-mount Immunohistochemistry (IHC) for
Interleukin 1 beta (IL-1β) .............................................................168
Multiplex Protein Analysis ............................................................168
Ethidium Bromide Dye Uptake Assay ..........................................169
Chemicals, Reagents and Solutions ............................................170
Data and Statistical Analysis ........................................................170
Results ...................................................................................................171
Activation of glial P2Y1 receptors leads to connexin-43 (Cx43)
hemichannel opening ...................................................................171
Pro-inflammatory cytokine levels are altered in the distal colon
during GI inflammation .................................................................173
Pro-inflammatory mediators increase glial connexin-43 (Cx43)
hemichannel opening in the absence of
purinergic glial stimulation............................................................175
Stimulation of glial P2Y1 receptors increase glial EtBr uptake.....176
Nitric Oxide (NO) decreases glial EtBr uptake through a Cx43independent pathway ...................................................................179
IL-1β potentiation of glial EtBr uptake is sensitive to connexin-43
blockade with 43Gap26 ...............................................................179
Interleukin 17 (IL-17) does not alter ADP-stimulated dye uptake
and in the presence or absence of the Cx43 mimetic peptide
43Gap26 ......................................................................................180
Interferon-γ (IFN-γ) does not potentiate ADP-stimulated dye
uptake and is insensitive to blockade with the connexin-43
mimetic peptide ............................................................................180
Discussion ..............................................................................................185
APPENDIX .............................................................................................189
REFERENCES .......................................................................................191
CHAPTER 6 Summary and Future Directions ...................................................196
Summary ................................................................................................197
Novel Findings ........................................................................................199
Enteric neurons express the cellular machinery necessary for
glutathione synthesis ...................................................................199
Protective role of in vivo depletion of glutathione during
inflammation ...............................................................................199

xi

The TGFbeta type II receptor is expressed in enteric neurons ....200
Study Limitations ....................................................................................200
DNBS Model of Colitis .................................................................200
Use of Pharmacological Modulators ............................................201
Sex of Experimental Animals .......................................................201
Future Directions ....................................................................................201
Cell-specific Inhibition of Glial NO Production..............................202
Cell-specific Inhibition of Glial GSH Production ...........................204
The Role of Neuronal TGF-beta Signaling in Colonic
Inflammation ................................................................................204
Modulation of Cx43 Channel Opening by Pro-Inflammatory
Cytokines and Mediators .............................................................205
Mechanisms of Glial GSH Release and Regulation .....................205
REFERENCES .......................................................................................207

xii

LIST OF TABLES

Table 1.1: Types of neurons in the myenteric plexus ............................................9
Table 2.1: Primary Antibodies used in Chapter 2 ................................................81
Table 2.2: Secondary Antibodies used in Chapter 2 ...........................................82
Table 3.1: Primary Antibodies used in Chapter 3 ..............................................123
Table 3.2: Secondary Antibodies used in Chapter 3 .........................................124
Table 4.1: Primary Antibodies used in Chapter 4 ..............................................157
Table 4.2: Secondary Antibodies used in Chapter 4 .........................................158
Table 5.1: Cytokines and Chemicals used in the Ethidium Bromide Dye
Uptake Assay ....................................................................................................190

xiii

LIST OF FIGURES

Figure 1.1: Organization of the enteric nervous system (ENS) in humans and
higher order mammals .........................................................................................5
Figure 1.2: Morphological Classification of Neurons in the ENS ...........................7
Figure 1.3: The enteric neural circuitry controlling peristalsis in the GI tract .......13
Figure 1.4: Enteric glial cells are morphologically similar to astrocytes ...............19
Figure 2.1: Activation of enteric glial P2Y1Rs drives neuron death through a
Cx43-dependent mechanism ..............................................................................53
Figure 2.2: Genetic ablation of glial Cx43 limits neuropathy without modifying
overt inflammation ...............................................................................................57
Figure 2.3: Stimulation of enteric glial P2Y1Rs elicits Cx43-dependent ATP
release ................................................................................................................59
Figure 2.4: Oxidative stress coincides with neuron death during in vivo
inflammation and is required for P2X7R-driven neuron death in situ ..................61
Figure 2.5: Enteric glia contribute to oxidative stress by producing nitric oxide
(NO) during inflammation ....................................................................................64
Figure 2.6: Nitric oxide (NO) potentiates ATP release from glia and
promotes neuron death in situ through a mechanism that involves glial Cx43
hemichannels ......................................................................................................68
Figure 2.7: Nitric Oxide positively modulates glial Cx43 hemichannels and
negatively modulates neuronal panx1 hemichannels ..........................................72
Figure 2.8: Proposed model of the mechanisms involved in glial cell-driven
enteric neuron death during acute inflammation .................................................73
Figure 3.1: Expression of the glutathione synthesis enzymes,
glutamate-cysteine ligase and glutathione synthetase, in the enteric
nervous system ..................................................................................................99
Figure 3.2: Glutathione (GSH) synthesis is required for enteric neuron
survival .............................................................................................................102
Figure 3.3: Inhibition of glutathione synthesis alone is not sufficient to cause

xiv

overt inflammation but does alter susceptibility to inflammatory insults ............106
Figure 3.4: Effects of glutathione depletion on neutrophil infiltration into the
myenteric plexus, MHC II-positive muscularis macrophage infiltration and
neuronal thiol oxidation levels during DNBS colitis in mice ...............................109
Figure 3.5: Inhibition of glutathione synthesis in vivo decreases enteric
neuron survival and alters myenteric plexus neurochemical coding ..................113
Figure 3.6: Inflammation alters the expression of the glutathione synthesis
enzymes, glutamate-cysteine ligase and glutathione synthetase, in the
enteric nervous system .....................................................................................116
Figure 4.1: dnTGFβRII mice have disrupted TGFβ signaling in CD4 and CD8
positive T cells compared to wild-type (WT) mice .............................................138
Figure 4.2: dnTGFβRII mice have increased distal colon transport time but no
significant changes in macroscopic measures of inflammatory damage ..........139
Figure 4.3: Disrupted TGFβRII signaling does not alter myenteric neuronal
density or neurochemical coding .......................................................................142
Figure 4.4: Disrupted TGFβRII signaling induces reactive gliosis in the
myenteric plexus ...............................................................................................143
Figure 4.5: dnTGFβRII mice have increased activation of MHC-II positive
muscularis macrophages but no changes in IL-1β immunoreactivity ................146
Figure 4.6: Mice with disrupted TGFβRII have dysregulated innate and
adaptive immune responses at the level of the myenteric plexus .....................148
Figure 4.7: TGFβRII is expressed in murine myenteric neurons .......................150
Figure 5.1: Purinergic glial activation leads to connexin-43 (Cx43)
hemichannel opening ........................................................................................172
Figure 5.2: Pro-inflammatory cytokine levels are altered in the distal colon
during GI inflammation ......................................................................................174
Figure 5.3: Effects of the pro-inflammatory mediators NO, IL-1β, IL-17 and
IFN-γ on Cx43 hemichannel opening in unstimulated enteric glia .....................177
Figure 5.4: Effects of Nitric Oxide (NO) on EtBr dye uptake in ADP-stimulated
enteric glia .........................................................................................................178
Figure 5.5: Effects of Interleukin 1-β (IL-1β) on dye uptake in ADP-stimulated

xv

enteric glia .........................................................................................................181
Figure 5.6: Effect of Interleukin 17 (IL-17) on ADP-stimulated dye uptake in
enteric glia .........................................................................................................182
Figure 5.7:Effects of the pro-inflammatory cytokine Interferon-γ (IFN-γ) of
Cx43-mediated dye uptake in stimulated enteric glia ........................................184
Figure 6.1: Myenteric ganglia in Sox10-CreERT2 mice and Cre- controls............204

xvi

KEY TO ABBREVIATIONS

γ-GC – gamma glutamyl cysteine
15d-PGJ2 – 15-deoxy-Δ12,14-prostaglandin J2
AAV – adeno-associated virus
ACh – acetylcholine
ADP – adenosine diphosphate
AFU – arbitrary fluorescence units
AH – afterhyperpolarization
APC – antigen-presenting cell
ATP – adenosine triphosphate
BSO – L-buthionine sulfoximine
BN – bombesin
BzATP - 2’(3’)-O-(4-benzoylbenzoyl)adenosine-5’-triphosphate triethylammonium salt
Ca2+ - Calcium
ChAT – choline acetyltransferase
CIPO – chronic idiopathic pseudo-obstruction
CNS – Central Nervous System
Cx43 – Connexin 43
DHE - dihydroethidium
DMEM – Dulbecco’s Modified Eagle Medium
DNBS – 2,4-dinitrobenzene sulfonic acid
ENK – encephalin

xvii

ENS – enteric nervous system
EtBr – Ethidium Bromide
FLEX – flip excision
GABA – gamma amino butyric acid
GCL – glutamate-cytsteine ligase
GDNF – glial-derived neurotrophic factor
GFAP – glial fibrillary acidic protein
GI – gastrointestinal
GSH – reduced glutathione
GPX – glutathione peroxidase
GS – glutathione synthethase
GSNO – S-nitrosoglutathione
IBD – inflammatory bowel disease
ICC – interstitial cells of Cajal
IFN-γ – interferon gamma
IHC – immunohistochemistry
IL – interleukin
iNOS – inducible nitric oxide synthase
IPANs – intrinsic primary afferent neurons
LMMP – longitudinal muscle myenteric plexus
MHC – major histocompatibility complex
miRNA – microRNA
MPO – myeloperoxidase

xviii

NAC – N-acetyl cysteine
NDS – normal donkey serum
NEM – N-ethyl maleimide
NGS – normal goat serum
NK3 – neurokinin 3
nNOS – neuronal nitric oxide synthase
NO – nitric oxide
NPY – neuropeptide Y
Nrf2 – nuclear factor-erythroid 2- relatated factor 2
nTYR – nitrotyrosine
panx1 – pannexin-1
PACAP – pituitary adenylyl cyclase activating peptide
PAPA NONOate – Propylamine Propylamine NONOate
PAR1 – protease –activated receptor 1
SMP – submucosal plexus
SP – substance P
TK - tachykinin
TLR4 – Toll-like receptor 4
TNFα – tumor necrosis factor alpha
UC – ulcerative colitis
UTP – uridine triphosphate
VIP – vasoactive intestinal peptide

xix

CHAPTER 1
Introduction

1

Overview
The gastrointestinal (GI) tract is the organ system responsible for controlling
digestion and the movement of food through the body. Disorders and diseases of the GI
tract may impact anywhere along the alimentary canal and are often debilitating. GI
diseases are estimated to affect as many as 1 in 4 persons worldwide 1-3, with
prevalence increasing annually. Despite this, current drug treatments for GI diseases
are few, are often ineffective in large patient populations, and pose serious side effects 4.
This is in part due to the lack of understanding of the underlying cellular mechanisms
that contribute to GI motility dysfunction.
The pathology of many GI diseases involves alterations within the enteric
nervous system (ENS)5; the heterogeneous neuronal network embedded within the gut
wall that controls intrinsic GI function 6-8. Disruption of this neuronal network, by
irreversible neuron death, is an underlying cause of motility dysfunction in a number of
GI disorders including Inflammatory Bowel Disease (IBD)9,10, infectious diseases11,12,
irritable bowel syndrome (IBS)13, the congenital condition Hirschsprung’s Disease14 and
chronic idiopathic pseudo-obstruction (CIPO)15. GI dysfunction and enteric neuropathy
are also secondary symptoms in other conditions such as diabetes mellitus16,17, central
nervous system diseases18 including Parkinson’s Disease19,20 and Alzheimer’s
Disease21, and simply as a consequence of physiological aging22,23.
Research has begun to identify some mechanisms that contribute to enteric
neuropathy24. However, a number of other factors are associated with neuron death and
GI dysfunction, and their mechanisms of action are less clearly understood. For
example, increased oxidative stress in the ENS is a key factor associated with enteric

2

neuron death in IBD25-27, aging28,29 and diabetes mellitus17. In addition, inflammatory
mediators and disruption of the immune balance in the gut are also associated with
enteric neuropathies30-32. Enteric glial cells, specialized cells that surround and support
enteric neurons and maintain the neuronal extracellular environment33-35, can regulate
ENS oxidative stress by regulating antioxidant36,37 and oxidant production38-40 and also
interact with immune signaling molecules and components41-44. This dissertation
investigates how ENS oxidative stress and immune dysfunction contribute to enteric
neuropathy during inflammation, and the role of enteric glial cells in mediating those
effects.

The Enteric Nervous System (ENS)
The enteric nervous system (ENS) or “brain of the gut” is one of three
components of the autonomic nervous system45 and is responsible for local and
moment-to-moment control of gastrointestinal (GI) functions such as motility, blood flow
and luminal absorptions and secretions. Early work by Auerbach (1862)46 identified the
existence of nerve cells within the wall of the GI tract. Bayliss and Sterling47 showed that
these intrinsic nerves are capable of controlling the reflexive behaviors of the intestine,
without innervation from the CNS, writing: “Local stimulation of the gut produces
excitation above and inhibition below the excited spot. These effects are dependent on
the activity of the local nervous mechanism”. Ultimately, it was British physiologist John
Newport Langley who coined the term ‘enteric nervous system’ to describe this intrinsic
nerve network45.

3

The progenitor cells of the ENS are derived from the vagal and sacral neural
crest48 and colonize the gut in a rostral to caudal manner, moving from the foregut to the
hindgut49 in a process completed by embryonic day 1550. Following this initial
colonization, progenitor cells actively proliferate and differentiate into the neural
components of the ENS. Improper colonization results in a lack of enteric neural cells
(aganglionosis) in the hindgut, as seen in Hirschsprung’s disease patients51.

The Ganglionated Plexuses of the ENS
The neural cells of the ENS are organized into enteric ganglia, or collections of
neural bodies, that are connected by interganglionic fiber tracts to form ganglionated
networks or plexuses. The ENS is subdivided into two plexuses: the submucosal plexus
(SMP) and the myenteric plexus (Figure 1.1). The SMP, as its name suggests, is
located in the submucosal space between the mucosal and circular muscle layers of the
gut (Figure 1.1) and is primarily responsible for the control of local blood flow and
luminal absorptions and secretions. SMP innervation is only present within the small
and large intestines and does not extend to the esophagus or stomach. In higher order
animals with a larger ENS52, the SMP is organized into two distinct layers: an inner
(Meissner’s) and an outer (Schabadasch or Henle’s) SMP 53-56.
The myenteric (or Auerbach’s) plexus is the larger of the two ganglionated
plexuses and contains two-thirds of the neurons in the ENS. Unlike the SMP, the
myenteric plexus innervates the entire length of the GI tract from esophagus to anus
6,57.

It is located between the circular and longitudinal smooth muscle layers within gut

(Figure 1.1) and controls smooth muscle contractions and relaxations, and thus exerts

4

Figure 1.1: Organization of the enteric nervous system (ENS) in humans and higher order mammals. The ENS is
comprised of two ganglionated plexuses the SMP, which regulates blood flow and luminal absorptions, and the myenteric
plexus, which controls motility. In larger mammals, the SMP is divided into an inner and outer SMP. Figure from Furness
(2012)6.

5

control over GI motility6,57,58. Accordingly, changes or alterations to the myenteric plexus
are associated with changes in GI motility59,60. Ganglia in both plexuses are comprised
of two primary cell types: enteric neurons and enteric glial cells.

Enteric Neurons
The ENS is estimated to contain up to 100 million enteric neurons – as many as
found in the spinal cord52. Enteric neurons are the primary building block of the neural
circuits of the ENS and are a heterogeneous cellular population with an estimation of up
20 different types of neurons in the ENS61-63. Enteric neurons have been traditionally
classified according to their morphology and/or electrophysical properties, and in more
recent years, by neurochemical coding.
Morphological Classification of Enteric Neurons
Before the methodological advance of immunohistochemistry, enteric neurons were
primarily classified according to cellular morphology as described by Dogiel in 1899 64,65
(Figure 1.2). Dogiel Type I neurons have multiple short dendrites and a single long
axon which projects to neighboring ganglia or to the musculature. Type I neurons can
be further classified by the direction of their axonal projection as either orally projection
or aborally projecting. This feature was important in identifying at least some Type I
neurons as motor neurons, responsible for innervating and controlling smooth muscle
contraction and relaxation. Dogiel Type II neurons are identified by multiple short and
long processes, which project circumferentially from the entire cell body. Dogiel type II
neurons feature a large cell body and at least one dendrite with ends residing in the
submucosa or mucosa, lending to their classification as sensory neurons capable of

6

Dogiel Type I

Dogiel Type II

Figure 1.2: Morphological Classification of Neurons in the ENS. Dogiel Type I neurons are primarily identified by their
uniaxonal morphology while Dogiel Type II neurons have a large cell body and multipolar morphology with circumferential
projection of processes. Figure modified from Enteric Nervous System (Wood, 2011)66.

7

detecting mechanical or chemical stimuli in the mucosa67.
Electrophysical Classification of Enteric Neurons
Enteric neurons can also be classified according to electrophysical properties
into two broad classes: AH-type neurons and S-type neurons68,69. After the firing of an
action potential, AH neurons produce an extended afterhyperpolarisation lasting on the
magnitude of seconds70. This characteristic electrophysiological feature is only evident
in neurons with Dogiel Type II morphology69,71,72. The extended hyperpolarization limits
the firing rate of these AH-type neurons and is further evidence for the designation of
Type II/AH neurons as sensory neurons73. S-type neurons are distinguished from AH
neurons by the lack of the prominent afterhyperpolarisation. Instead, they are
characterized by a fast excitatory synaptic input, which results in an excitatory post
synaptic potential68. S-type neurons have Dogiel Type I morphology and S-type
electrophysiology is seen in multiple classes of enteric neurons including motor neurons
and interneurons (described further below).
Immunohistochemical Classification of Enteric Neurons
Currently, enteric neurons are primarily classified by immunohistochemistry/
neurochemical coding 74,75 (Table 1.1). Three broad classes of neurons involved in the
control of GI motility can be described based on neurochemical coding: motor neurons
(excitatory and inhibitory), interneurons and sensory neurons. Excitatory motor neurons
project to orally or locally to circular and longitudinal smooth muscle where they cause
muscle contraction via release of excitatory neurotransmitters acetylcholine (ACh) and
substance P (SP)76. These neurons are immunoreactive for choline acetyltransferase
(ChAT) and the calcium binding protein calretinin. The primary neurotransmitters in

8

Proportion

Neurochemical Coding

Shape

12%

ChAT/TK/ENK/GABA

Dogiel type I

16%

NOS/VIP/PACAP/ENK/NPY/GABA Dogiel type I

25%

ChAT/calretinin/TK

Dogiel type I

~2%

NOS/VIP/GABA

Dogiel type I

Ascending interneurons

5%

ChAT/calretinin/TK/ENK

Dogiel type I

Descending interneurons

5%

ChaT/NOS/VIP/BN/NPY

Dogiel type I

Myenteric IPANs

26%

ChAT/TK/NK3 receptor/calbindin

Dogiel type II

Excitatory circular muscle
motor neurons
Inhibitory circular muscle
motor neurons
Excitatory longitudinal
muscle motor neurons
Inhibitory longitudinal
muscle motor neurons

Table 1.1: Types of neurons in the myenteric plexus. This table lists a subset of neurons found in the myenteric plexus
of guinea-pig small intestine. The neurons types highlighted are involved in local control of the peristaltic reflex. Table
modified from The Enteric Nervous System (Furness, 2006)77.

9

Table 1.1 (cont’d)
ChAT (choline acetyltransferase), BN (bombesin/gastrin-releasing hormone/GRP), ENK (encephalin), GABA (gamma
amino butyric acid), NK (neurokinin), NOS (nitric oxide synthase), NPY (neuropeptide Y), PACAP (pituitary adenylyl
cyclase activating peptide), TK (tachykinin), VIP (vasoactive intestinal peptide)

10

inhibitory motor neurons are nitric oxide (NO), vasoactive intestinal peptide (VIP) and
ATP. Thus, these neurons are identified by immunoreactivity for neuronal nitric oxide
synthase (nNOS) and VIP. Ascending and descending interneurons, which form
neuronal chains along the length of the gut, signal via nicotinic ACh receptors and these
neurons are also immunoreactive for ChAT. However, co-staining for other proteins and
neurotransmitters such as somatostatin, serotonin (5-HT), gastrin-releasing peptide
(GRP or bombesin/BN) and GABA allow for the distinction between different classes of
interneurons that are involved in different reflex pathways. The final class of neurons,
sensory or intrinsic primary afferent neurons (IPANS), are immunoreactive for ChAT,
tachykininins and the neurokinin 3 (NK3) receptor (Table 1.1).
The ENS also contains other classes of neurons that do not regulate GI motility
but contribute to neural control of other GI functions. These include 1) viscerofugal
neurons, which have cell bodies within the ENS but project out of the gut to sympathetic
ganglia and do not synapse with any enteric neurons78,79, 2) vasomotor and
secretomotor neurons which control blood vessel vasodilation and epithelial cell
secretions respectively80-82 and 3) motor neurons that innervate enteroendocrine cells 77.

Enteric Neural Circuitry
The various classes of enteric neurons are arranged in an intricate circuit that
controls the internal reflexes of the GI tract (Figure 1.3)83. The primary enteric reflex,
peristalsis, is activated when a bolus of food increases intraluminal volume and
activates intrinsic primary afferent neurons (IPANs) in the gut wall

73.

IPANs have cell

bodies in the SMP and myenteric plexus with projections to the mucosal layer of the gut.

11

Individual subtypes of IPANs have axons that synapse onto other IPANs, motor neurons
or onto ascending and descending interneurons57; the latter activates ascending
excitatory and descending inhibitory pathways respectively. Neuron-to-neuron synapses
between interneurons form ascending and descending neural chains oral and aboral to
the site of distention, respectively. The ascending/excitatory tract culminates with
cholinergic excitatory neurons that synapse onto longitudinal and circular smooth
muscle and causes smooth muscle contraction oral to the food bolus. Simultaneously,
the descending inhibitory pathway terminates with inhibitory nitregic motor neurons that
cause smooth muscle relaxation aboral to the food bolus. Synchronized smooth muscle
contraction and relaxation propels the food bolus in an aboral direction, as described by
Bayliss and Sterling (1899) in their law of the intestine 47,84: “Excitation at any point of
the gut excites contraction above, inhibition below”. The ENS is also responsible for
controlling other motor patterns and behaviors of the gut including accommodation to
increased fluid content 85 and the non-propulsive colonic migrating motor complexes,
which are proposed to be important for mixing and promoting absorption 86-88.
In addition to enteric neurons, other non-neuronal components are important for
regulation of GI motility. These include the interstitial cells of Cajal (ICCs) which function
as pacemaker cells to regulate control GI smooth muscle contraction

89-91

and are

hypothesized to at least partially mediate both excitatory and inhibitory smooth muscle
neurotransmission 92-94. The second non-neuronal cell type of interest are enteric glial
cells, a unique class of peripheral glia that will be discussed in further detail later in this
chapter 33-35.

12

Figure 1.3: The enteric neural circuitry controlling peristalsis in the GI tract. Distention caused by a bolus of food in
the GI tract activates IPANs, which have their projections in the mucosa. IPANs activated ascending and descending
motor pathways by synapsing on ascending and descending interneurons, respectively or directly to excitatory or
inhibitory motor neurons. Finally, motor neurons regulate smooth muscle contraction (oral to the food bolus) and
relaxation (aboral to the bolus) to cause propulsion. Figure from The Enteric Nervous System (Furness, 2006) 77.

13

Enteric Neuropathies
Disruption of the enteric reflex circuit is a primary cause of gut dysfunction in GI
motility disorders31,95. This disruption may be due to either a loss of the functional units
of the circuit or functional impairment without cell death. Here, we focus on enteric
neuropathies where an irreversible loss of enteric neurons contributes to GI motility
dysfunction. Of note, enteric neuron death may be genetic/congenital or acquired, or
present as a secondary symptom of another condition, and cause dysfunction of varied
severity and pathologies96.
Congenital Neuropathies
The most well understood congenital neuropathy, Hirschsprung’s disease, is
characterized by aganglionosis of the myenteric and submucosal plexuses in the distal
colon and rectum. Hirschsprung’s is estimated to affect about 1 in 5000 children 14 and
about 20% of cases are defined by genetic abnormalities that prevent the proper
migration of neural-crest derived enteric progenitor cells into the hindgut97-99.
Inflammatory Neuropathies
A large percentage of enteric neuron death is associated with gut inflammation
and the infiltration of immune cells into the GI tract. These aptly named inflammatory
neuropathies include conditions such as the Inflammatory Bowel Diseases (IBD)
Crohn’s and Ulcerative Colitis9,100, Chagas’ disease101 and Irritable Bowel Syndrome
(IBS)24,102,103. Significant neuron death is reported in both animal models of IBD and in
biopsies from human patients13,104 and is widely shown to directly contribute to the
motility dysfunction seen in these conditions. NOS-immunoreactive inhibitory motor
neurons are selectively targeted in animal models of colitis, allowing us to speculate to

14

the mechanisms that cause motility dysfunction during colitis, as will be addressed in
this dissertation.
Secondary Enteric Neuropathies
This terminology refers to diseases or syndromes where GI motility dysfunction is
present but the gut is not a primary target of disease pathology. A common example is
in diabetes mellitus patients where dysfunction throughout the length of the GI tract is
present105. In an animal model of diabetes mellitus, altered colonic transit time is
associated with a preferential loss of inhibitory nitregic myenteric neurons104.
Constipation is frequently seen in Parkinson’s disease patients106 where enteric neuron
death, and impairment of intrinsic and extrinsic enteric nerve innervation, are thought to
contribute to GI motility dysfunction. Lastly, pronounced gut dysfunction, particularly
constipation, is a symptom of general physiological aging and is associated with enteric
neuropathy107. In opposition to neuron loss in diabetes mellitus and IBD, cholinergic
neurons are particularly vulnerable in age-related neuron loss24.
Recent work has begun to identify the cellular mechanisms that can contribute to
enteric neuron loss, particularly during inflammation24. What is still unclear is how
factors such as increased oxidative stress108,109 and inflammatory mediators110, which
are associated with enteric neuropathy: 1) contribute to neuron loss and 2) are
regulated by the cellular components of the ENS during inflammation.

Oxidative Stress in Enteric Neuropathies
Oxidative stress is a prominent feature associated with motility dysfunction in
both primary and secondary GI disorders17,25,27,28,111-116. Evidence in support of this

15

includes the increased production of oxidants117, and the concurrent depletion of
antioxidants118, in animal models of IBD. Mice with genetic disruptions in the antioxidant
glutathione peroxidase genes (GPX-1 and GPX-2)119,120 or the redox-sensitive
transcription factor nuclear factor-erythroid 2–related factor 2 (Nrf2)121 develop
spontaneous ileocolitis and have an increased susceptibility to chemical models of
colitis, respectively. Treatment with antioxidant L-glutathione was protective against
enteric neuropathy in the jejunem of diabetic rats122, further supporting a role for
oxidative stress in enteric neuropathy in animal models.
Evidence also supports a role for oxidative stress in human IBD patients 27. IBD
patients have decreased mucosal content of antioxidants123 and increased biomarkers
of oxidative stress124. Genetic polymorphisms associated with decreased expression/
activity of antioxidant genes are also related to IBD incidence and considered an
important genetic predictor for disease development125,126. Consistent with our animal
model, genetic polymorphisms in Nrf2 gene were associated with ulcerative colitis in a
human study127. Human trials investigating the efficacy of antioxidant treatment as a
therapy for human IBD have yielded more inconclusive results than animal studies. In a
subset of trials, while antioxidant treatment improved patient oxidative status and
decreased overall oxidative stress, disease index and patient health were not
significantly improved128-130. These findings demonstrate the need for further study to
elucidate the potential causative relationship between oxidative stress and IBD, identify
enteric mechanisms that contribute to increased oxidative stress, and determine the
downstream pathways by which oxidative stress may lead to neuron death.

16

Inflammatory Mediators and Cytokines in Enteric Neuropathies
The infiltration of inflammatory mediators into the gut, and the disruption of
immune balance within the gut, are also associated with enteric neuron loss and motility
dysfunction of the GI tract30-32. Recent studies have begun to identify a role for the
immune system in GI disorders such as IBS131,132 and Chagas’ disease133, where
inflammation was not thought to play a significant role. Conversely, a role for
inflammatory cytokines and the immune system in the pathology of IBDs Crohn’s
disease and ulcerative colitis (UC) has been long established and much better studied.
Both Crohn’s and UC are characterized by persistanr GI inflammation but have key
differences in their pathology and presentation134. For example, Crohn’s disease
presents with transmural inflammation anywhere along the GI tract, while in UC,
inflammation is restricted to the colon and primarily mucosal.
The pro-inflammatory cytokines associated with Crohn’s and UC also differ
considerably30,32,135. UC is characterized as a Th2-mediated disease associated with
increased interleukins 3, 4, 5 and 13136. Conversely, Crohn’s Disease is traditionally
classified as a Th1-mediated disease with increased IL-12, Interferon-γ, IL-1β and tumor
necrosis factor α (TNF-α). However, recent studies propose a mixed Th1/Th17
profile137. Although enteric neurons have the ability to produce inflammatory cytokines
and mediators138,139, there has been little study of their ability to respond to directly
respond to extracellular cytokines and the expression of such receptors. This suggests
that an intermediate cell type mediates the immunomodulatory effects of proinflammatory cytokines on enteric neurons in IBD.

17

Enteric Glia
Enteric glial cells are a specialized type of peripheral glia found within enteric
ganglia33,34,140 and have the unique ability to both regulate ENS oxidative stress and
mediate the immunomodulatory effects associated with enteric neuropathy. Like their
CNS counterpart, enteric glia were historically considered to simply be passive support
cells and the “glue” of the ENS. Increasing evidence supports a more dynamic role for
enteric glia in not only regulating neuronal health, but participating in neuro- and
gliotransmission and regulating GI motility35,43.
The first detailed anatomical analysis of enteric glial cells was presented by
Giorgio Gabella in 1972141. Here, Gabella noted important characteristics of enteric glia
including cell and nucleus size and the ratio of enteric glia to neuronal cell bodies.
Gabella also demonstrated that enteric glia were non-myelinating, differentiating them
from other peripheral glia and Schwann cells141. These findings were confirmed by other
investigators142,143 and Gabella himself144, who later coined the term “enteric glial cell”.
Enteric glial cells are morphologically and molecularly similar to astrocytes in
central nervous system , further emphasizing the parallel of the ENS as the “brain of the
gut”. Intraganglionic or Type I enteric glia are identified by their characteristic starshaped morphology with prominent glial processes extending circumferentially from the
glial cell body35,145 (Figure 1.4). Type I enteric glia differ considerably in shape, and
possible function, from interganglionic (Type II), mucosal (Type III) and intramuscular
(Type IV) enteric glia, the detailed study of which is well reviewed by Gulbransen and
Sharkey (2012)140. Enteric glial cells also have profound molecular similarities to CNS
astrocytes. Immunohistochemical identification of enteric glia is achieved using

18

Figure 1.4: Enteric glial cells are morphologically similar to astrocytes. (A) A
classic drawing of astrocyte surrounding a neuronal cell body. (B,C) Myenteric plexus
glial cells stained with the prototypical astrocytic marker GFAP (black in B, green in C).
Figure from Enteric Glia (Gulbransen, 2014)146.

19

prototypical astrocyte markers such as the filamentous proteins glial fibrillary acidic
protein (GFAP)147, vimentin148 and the astrocyte marker S-100beta149 (Figure 1.4).
Enteric glia are also immunoreactive for the transcription factor Sox10 which is
necessary for proper ENS development from neural crest cells150.
The number of enteric glia, and hence the neuron to glia ratio, varies
considerably in different species of animals and in the different plexuses52,150. In mice,
the laboratory animal used in this dissertation, enteric neurons and glia present in equal
numbers in the myenteric plexus52. Guinea pigs, which are a more widely studied rodent
in gastroenterology, have a glia:neuron ratio closer to 2:1 in myenteric ganglia. This
ratio continually increases with animal size, with humans having up to seven times more
myenteric glia than neurons150. The heterogeneity of glia:neuron ratio may highlight
increasingly complex roles of enteric glia in higher order mammals, and is worth
considering when translating findings from animal models to human disease.
Recent work has begun to identify a bevy of functional roles for enteric glia based
on 1) the histological expression of receptors, channels, enzymes and signaling
molecules on enteric glia and 2) demonstration of the functional relevance of said
receptor/channel/molecule by a secondary method. Below, I highlight key findings that
verify the role of enteric glial cells as activate participants in the enteric neural circuitry.
Enteric glial receptors
Enteric glia express a number of neurotransmitter receptors allowing them to
“listen to” neuron to neuron communication and dynamically respond to changes in
extracellular neurotransmitter concentrations. Further, these receptors may mediate
glia’s ability to communicate with other glia via gliotransmission151. The class of

20

receptors most widely expressed on enteric glia are purinergic receptors including the
P2Y1 receptors for ADP24,39,151,152, P2Y4 receptors for ATP and UTP102,153 and A2B
receptors for adenosine154,155. Enteric glia also express adrenergic receptors156 and
receptors for glutamate157, although their functional relevance is still unclear. Enteric glia
also express receptors for other bioactive compounds besides neurotransmitters
including protease-activate receptors (PAR1 and PAR2)158, bradykinin receptors159,160
and endothelin receptors161.
Enteric glial regulation of neurotransmitters
Enteric glial cell can contribute to the concentrations of neurotransmitters in the
ENS through two primary mechanism: 1) synthesis and production of neurotransmitters
and necessary precursors and 2) degradation or sequestration of neurotransmitters.
Enteric glia are immunoreactive for L-arginine162, suggesting that enteric glia are
capable of contributing to nitrergic signaling within the gut. Enteric glia also play a key
role in the degradation and sequestration of neuroactive compounds, and in doing so
contribute to the maintenance of neuronal health by preventing over-excitation of enteric
neurons. Neurotransmitter sequestration occurs through the uptake of neurotransmitters
via the PEPT2 transporter163 and GABA transporter164. In coordination with their
prominent expression of purinergic receptors, enteric glia express ectonucleotidases,
which play an important role in the degradation on ATP to ADP and promotes neuronalglial purinergic signaling in the ENS24,39,165,166.
Enteric glial channels
Analogous to their CNS counterparts, enteric glia express Cx43 hemichannels,
which play a key role in mediating glial purinergic signaling and neuron to glia

21

communication39,151. In addition, glia express both voltage-gated sodium and potassium
channels, although the functional roles of these in glial signaling are still being
elucidated167,168.

Enteric glial regulation of oxidative stress
Enteric glial cells are ideally positioned to regulate the oxidant/antioxidant
balance in the ENS through the production of both oxidants and antioxidants. Enteric
glial produce and secrete antioxidant compounds such as reduced glutathione
(GSH)36,169 and the prostaglandin derivative 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2)37,170, which aid in neuronal defense against oxidative stress and protect against
neuron death. A loss of these antioxidants, and glial derived neurotrophic factors171,172,
may contribute to the neuron death associated with ablation of enteric glial cells 173. In
addition, enteric glia produce oxidants such as nitric oxide (NO) via inducible nitric oxide
synthase (iNOS)40,174,175. During inflammation, glial cells (including enteric glia) convert
to a reactive phenotype176 which can increase iNOS activity177-179. Although increased
iNOS activity and NO production are associated with IBD180,181, studies inhibiting NO
production in animal models of inflammation have produced conflicting results where
depletion of NO is pathogenic while treatment with exogenous NO was both protective
and pathogenic against different aspects of GI inflammatory damage182. These findings
highlight the need for an improved understanding of the role and mechanism of action of
NO during GI inflammation.

22

Enteric glial cells as immunomodulators
Enteric glia are also uniquely able to respond to, and directly modulate, the
extracellular balance of inflammatory mediators such as pro and anti-inflammatory
cytokines. Immunohistochemical staining shows the expression of receptors for the proinflammatory cytokines IL-1β and TNF-α on cultured rat enteric glia183. Further, these
receptors are functional as treatment with IL-1β, IL-4 and TNF-α increased the
expression of GFAP in cultured enteric glia, denoting their conversion to a “reactive” or
“activated” state184. Activation of glia with IL-1β stimulates the production and release of
additional cytokines or neuroprotective factors, further cementing an immunomodulatory
role for enteric glial cells185,186. Enteric glia also express Toll-like receptors 2 and 4,
which may be activated by lipopolysaccharide (LPS) or other bacterial components
during inflammation42,187,188. Glial activation via the TLR4 receptor also induces cytokine
synthesis and release187.
Enteric glia can also transform into antigen-presenting cells in response to
inflammatory mediators during inflammation. As their name suggests, antigenpresenting cells (APCs) express of portion of a foreign antigen on their cell surface,
coupled to one of two classes of major histocompatibility complex (MHC) proteins. The
presentation of an antigen to a T-cell, along with the coupling of co-stimulatory
molecules such as CD80 and CD86, causes T-cell activation and differentiation, and
initiates an inflammatory response. In patients with chagastic megacolon, enteric glial
cell upregulate expression of the MHC class II peptide and the costimulatory proteins
CD80 and CD86189.Increased glial MHC II expression in also seen in human Crohn’s
disease patients 190,191.

23

The regulation of anti-inflammatory cytokines is a key factor in the pathogenesis
of GI inflammation and motility dysfunction. Animals with disrupted signaling of antiinflammatory cytokines such as IL-10 192and TGFβ193 develop spontaneous colitis. The
specific cellular mechanisms contributing to colitis in these animal models on unclear,
but it is possible that they are mediated through the immunomodulatory signaling
pathways of enteric glia.

Summary and Aims of Dissertation
Normal function of the GI tract is controlled by the enteric nervous system (ENS),
which is comprised of enteric neurons and glial cells organized into enteric ganglia 6.
Disruption of this neural network, due to loss of enteric neurons, contributes to motility
dysfunction in a number of GI diseases. While some of the mechanisms that contribute
to neuron loss during inflammation have been elucidated24, our lack of knowledge of
other key mechanisms involved is still incomplete and has led to a lack of therapeutic
targets for disease relief.
Increased oxidative stress and immune dysregulation are two factors associated
with enteric neuropathy and motility dysfunction in GI disorders. Enteric glial cells are
ideally positioned to regulate ENS oxidative balance and mediate immunological
signals. The objective of this dissertation is to investigate how ENS oxidative stress and
immune dysfunction contribute to enteric neuropathy during inflammation, and the role
of enteric glial cells in mediating those effects. Chapters 2 and 3 investigate how enteric
glial cells contribute to oxidative stress regulation during inflammation. Chapter 2
studies glial production of the oxidant nitric oxide (NO) and its downstream signaling

24

pathways. Chapter 3 studies the other arm of oxidative balance, focusing on glial
antioxidant production and how its (dys)regulation mediates enteric neuropathy and GI
inflammation. Chapter 4 studies the effect of disrupted immune homeostasis on
neuronal and glial structure and function. The final data chapter, Chapter 5, directly
measures the effect of pro-inflammatory cytokines and oxidative mediators on a key
glial cell function. Together, these chapters aim to improve the understanding of key
glial mechanisms that contribute to enteric neuropathy and allow the development of
novel therapeutics. Further, this dissertation serves to improve broader understanding
of neuron-glia interactions in the ENS in health and disease.

25

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CHAPTER 2
Enteric glia mediate neuron death in colitis through purinergic
pathways that require connexin-43 and nitric oxide

Work presented in this chapter has been published as 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. Cellular and Molecular
Gastroenterology and Hepatology. 2016;2(1):77-91

42

Abstract
The concept of enteric glia as regulators of intestinal homeostasis is slowly
gaining acceptance as a central concept in neurogastroenterology. Yet how glia
contribute to intestinal disease is still poorly understood. Purines generated during
inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors
(P2X7R), triggering ATP release via neuronal pannexin-1 channels that subsequently
recruits intracellular Ca2+ ([Ca2+]i) in surrounding enteric glia. We tested the hypothesis
that the activation of enteric glia contributes to neuron death during inflammation. We
studied neuroinflammation in vivo using the 2,4-dinitrobenzenesulfonic acid model of
colitis and in situ using whole-mount preparations of human and mouse intestine.
Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP::CreERT2+//Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in
inducible nitric oxide (NO) synthase (iNOS-/-) were used to study NO production. Protein
expression and oxidative stress were measured using immunohistochemistry and in situ
Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i. Purinergic activation
of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial
iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43dependent ATP release was potentiated by NO. Our results show that the activation of
glial cells in the context of neuroinflammation kills enteric neurons. Mediators of
inflammation that include ATP and NO activate neurotoxic pathways that converge on
glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute
to the development of motility disorders.

43

Introduction
Reflex behaviors of the intestine, such as peristalsis, are orchestrated by the
enteric nervous system (ENS); a complex network of neurons and glia embedded in the
gut wall. The basic neural circuitry of the ENS is now well defined and it is generally
accepted that the breakdown of ENS control is a major contributing factor in the
development of functional bowel disorders1. However, it is only recently that we are
beginning to appreciate the potential roles of enteric glial cells in the physiology and
pathophysiology of the ENS2. Despite intense interest in enteric glia as regulators of
enteric neurons, the precise functions of enteric glia remain poorly defined.
Enteric glia are a unique population of peripheral astroglial cells that surround
enteric neurons and are thought to sustain neural signaling and survival. In support,
enteric glia secrete neuroprotective factors3 and the selective ablation of glial signaling
alters the neural control of motility4. Likewise, in vivo models of glial ablation cause
enteric neuron death5,6. Thus, the loss of glial supportive functions is postulated as a
potential mechanism contributing to enteric neuropathy2. However, new data show that
chronic astroglial activation, rather than glial cell loss, is responsible for driving
neurodegeneration during neuroinflammation in the brain 7. Indeed, the conversion of
astroglia to reactive astrocytes can promote the secretion of factors that promote
neuron death8.
We recently discovered that enteric glia are activated by purines released from
enteric neurons prior to neuronal death during colitis9. Specifically, the activation of
neuronal P2X7 purine receptors (P2X7Rs) triggers the release of ATP from neurons
through pannexin-1 (panx1) channels as a signal to enteric glia. In the brain, neuronal

44

ATP release through panx1 is considered a danger signal that glial cells interpret as a
“search and destroy” message; causing glia to execute otherwise healthy neurons.
Given that stimulation of P2Y1Rs is a potent stimulus for reactive astrogliosis in the
central nervous system10, we hypothesized that the activation of glial purine receptors
contributes to neuropathy in the ENS. We tested our hypothesis using a combination of
in vivo models of colitis with inducible and conditional transgenic mice and ex vivo
intestinal preparations to address specific mechanisms. Our data show that glial
activation is sufficient to cause enteric neuron death via a mechanism that depends on
the activation of connexin-43 (Cx43) hemichannels and subsequent ATP release.
Surprisingly, our data show that glial-driven neuron death requires the gating of glial
Cx43 hemichannels by nitric oxide (NO). In all, our results suggest that the activation of
enteric glial cells is a central mechanism in the development of enteric neuropathy.

Materials and Methods
Animals
All work involving animals was approved by the Institutional Animal Care and Use
Committee (IACUC) of Michigan State University. Male mice (8-10 weeks of age) were
maintained on a 12-hour light/dark cycle with access to food and water ad libitum.
C57Bl/6 mice were purchased from Charles River and inducible nitric oxide synthase
(iNOS) null mice (B6.129S2-Nos2tm1MrlN12; Taconic Labs; RRID: MGI_4837857;
hereafter referred to as iNOS-/-) from Taconic11. Transgenic mice with an inducible and
conditional deletion of Cx43 in GFAP-expressing glia (GFAP::CreERT2+/-/Cx43f/f;
hereafter referred to as Cx43i-cKO) and their Cre negative littermate controls

45

(GFAP::CreERT2+/+/Cx43f/f) were generated in-house as previously described4 by
crossing GFAP::CreERT2+/- mice (GFAP-cre/ERT2)505Fmv/J; Jackson Labs; RRID:
IMSR_JAX:012849) with Cx43f/f mice (B6.129S7-Gja1tm1Dlg/J; Jackson Labs; RRID:
IMSR_JAX:008039). Cre recombinase activity was induced by feeding animals
tamoxifen citrate in chow (400 mg/kg) for two weeks. Animals were returned to normal
chow for 1 week to clear tamoxifen prior to beginning experiments.

Human tissue
Work involving human tissue was approved by the Institutional Review Board of
Michigan State University (IRB# 13-945M). Samples of live, full-thickness human
jejunum were collected from a 57-year-old female with hypertension and type 2 diabetes
who underwent elective laparoscopic bariatric surgery for weight loss, and placed in
chilled DMEM/F12 media during transfer to the laboratory. Live longitudinal muscle
myenteric plexus whole-mount preparations were prepared by microdissection for Ca2+
imaging.

Whole-mount Immunohistochemistry
Whole-mount preparations of mouse colonic longitudinal muscle and myenteric plexus
(LMMP) were prepared by microdissection from tissue preserved in Zamboni’s fixative.
Processing of LMMPs via immunohistochemistry was conducted as described
previously4 with the primary and secondary antibodies listed in Tables 2.1 and 2.2,
respectively. Briefly, dissected LMMP preparations underwent three 10 min washes in
0.1% Triton X-100 in phosphate-buffered saline (PBST) followed by a 45 min block in

46

blocking solution containing 4% normal goat serum, 0.4% Triton X-100 and 1% bovine
serum albumin (4% NGS). Preparations were incubated in primary antibodies (listed in
Table 2.1, appendix) for 48h at 4oC and secondary antibodies (listed in Table 2.2,
appendix) for 2h at room temperature before mounting. Antibody specificity was
confirmed by preadsorption with the corresponding control peptides or in knockout mice
as previously described9. Fluorescent labeling was visualized using the 40X (PlanFluor,
0.75 numerical aperture) objective of an upright epifluorescence microscope (Nikon
Eclipse Ni, Melville, NY) with a Retiga 2000R camera (QImaging, Surrey, BC, Canada)
controlled by QCapture Pro 7.0 (QImaging) software or by confocal imaging through the
Plan-Apochromat 60x oil immersion objective (1.42 numerical aperture) of an inverted
Olympus Fluoview FV1000 microscope (Olympus, Center Valley, PA).

Quantification of neuronal thiol oxidation
We quantified neuronal thiol oxidation as a measure of oxidative stress as described
previously12. Reduced (-SH) and oxidized (-SS) thiols were labeled in live LMMP
preparations with 1μM Alexa Fluor 680 C2 maleimide and 1μM Alexa Fluor 546 C5
maleimide, respectively. Fluorescent dyes were dissolved in 4% paraformaldehyde,
0.02% Triton X-100 and 1mM N-ethylmaleimide (NEM) in PBS. Oxidized thiols were
converted to reduced thiols for labeling by washing tissue in 5mM tris(2carboxyethyl)phosphine hydrochloride in PBS for 20 minutes. Images were obtained by
epifluorescence microscopy as described above and the ratio of 546-maleimide/680maleimide (SS/SH) calculated with ImageJ software (NIH).

47

Dihydroethidium (DHE) staining
Superoxide levels were measured by quantifying fluorescence of the superoxide marker
DHE14 in live LMMP ganglia after a 1h incubation with 2 μM DHE (Life Technologies)
dissolved in DMEM at 37oC.

In situ model of neuroinflammation
Enteric neuron death was driven, as previously described9, by incubating live LMMP
preparations with the P2X7R agonist BzATP (300 μM) for 2h in 95% air: 5%CO2 at
37oC. Following this incubation, LMMP preparations were rinsed with fresh buffer,
incubated for an additional 2h in Kreb’s buffer only, and fixed in Zamboni’s fixative
overnight.

Ca2+ and NO imaging
Live LMMPs were loaded with 4 μM Fluo-4-AM or DAF-FM (Life Technologies) for 45
min at 37oC as described previously4 to measure intracellular Ca2+ and NO,
respectively. Loaded tissue was continuously perfused with pre-warmed buffer (34°C; 3
mL/min) and drugs were bath applied. Images were acquired every 1-5s with a Neo
sCMOS digital camera (Andor, South Windsor, CT) through the 40X water-immersion
objective of an upright Olympus BX51W1 fixed-stage microscope (Olympus, Center
Valley, PA) controlled by Andor IQ3 software.

48

Determination of Cell Viability
Cell viability was determined using Calcein-AM; a hydrophobic compound that is
converted to hydrophilic, fluorescent Calcein in live, membrane-intact cells13. The
Calcein-AM cell viability assay is a simple, rapid and accurate method to measure
cytotoxicity that is not dependent upon the mode of cell death and is a true end-point
assay for cell viability. Calcein-AM is a non-fluorescent, hydrophobic compound that
easily permeates live, intact cells. The hydrolysis of the Calcein-AM by intracellular
esterases produces Calcein, a hydrophilic, strongly fluorescent compound that is wellretained in the cell cytoplasm. A loss of membrane integrity in dying cells permits the
dye to escape from the cell and a loss of cellular fluorescence is indicative of neuron
death. Live LMMPs were loaded with 4 μM Calcien-AM for 30 min at 37°C following in
situ induction of neuroinflammation with BzATP (300 μM) or ADP (100 μM) or
incubation with buffer control. Static images of 10 ganglia per whole mount were
acquired as described for Ca2+ imaging and neuronal density calculated based on the
number of live (fluorescent) cells per ganglionic area.

Detection of ATP release
ATP release was measured using an in-house-fabricated, selective ATP sensor14,15.
LMMPs were continuously perfused with buffer (34°C; 6 mL/min) and ATP release was
stimulated by bath application of either BzATP (300 μM) or ADPβS (100 µM) in the
presence or absence of various pharmacological inhibitors. The ATP-selective sensor
was placed directly on a ganglion and a superfusion pipette was located within 100 µm

49

of the sensor. Traces were analyzed by measuring the area under the curve and
converting this to the concentration of ATP using pre-obtained calibration curves14.

Induction of colitis
Colitis was induced in mice via an enema of dinitrobenzene sulfonic acid (DNBS, 5.5
mg/mouse in 0.1mL ethanol/saline administered via a gavage needle inserted 3 cm into
the colon) as previously described9. Animal weight was recorded daily and tissue
harvested 6h and 48h following DNBS treatment. Upon tissue collection, macroscopic
damage was assessed to quantify acute inflammation16. Inflammation is classified as
mild if damage scores were <1, moderate if scores were >1 but <5 and severe if > 5.

Chemicals/ Drugs
2’(3’)-O-(4-Benzoylbenzoyl)adenosine 5’-triphosphate triethylammonium salt (BzATP),
Adenosine 5’-diphosphate monopotassium salt dihydrate (ADP), Adenosine 5’-[βthio]diphosphate trilithium salt] (ADPβS), N-ethylmaleimide (NEM) and tris(2carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St.
Louis, MO) and MRS2365 from Tocris Bioscience (Bristol, UK). 1400W, PTIO, L-NAME
and Propylamine Propylamine NONOate (PAPA NONOate) were purchased from
Cayman Chemical (Ann Arbor, MI). The Cx43 hemichannel mimetic peptide 43Gap26
and panx1 mimetic peptide 10panx were purchased from Anaspec (Fremont, CA).

50

Solutions
Live tissue was maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12
(DMEM/F12; Life Technologies) containing 3 μM nicardipine and 1 μM scopolamine
during microdissection and incubations. LMMPs were perfused with modified Krebs
buffer containing (in mM): 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 10
HEPES, 21.2 NaHCO3, 1 pyruvic acid and 8 glucose (pH adjusted to 7.4 with NaOH)
with 3 μM nicardipine and 1 μM scopolamine.

Data and Statistical Analysis
Average fluorescence intensity was quantified by measuring average fluorescence
using the in-program measure function in ImageJ software, version 1.49 (NIH). Neuron
packing density was determined by counting the number of HuC/D-immunoreactive
neurons per ganglionic area in ten ganglia per LMMP preparation using the cell counter
plug-in tool in ImageJ. All results are presented as mean ± standard error of the mean
(SEM) and statistical differences determined using an ANOVA or t-test as appropriate
with a P value of <0.05 considered significant (GraphPad Prism). For Ca 2+ and NO
imaging, traces represent the average change in fluorescence (ΔF/F) over time for all
glial cells within a single ganglion.

51

Results

Purinergic activation of enteric glial cells drives glial Ca2+ responses and enteric neuron
death
Enteric neuron death during intestinal inflammation requires the activation of neuronal
P2X7Rs and neuronal ATP release through panx1 channels9. Neuronal ATP released
via panx1 is rapidly hydrolysed to ADP by eNTPDase2 and ADP recruits Ca2+
responses in the surrounding enteric glial cells following stimulation of P2Y1Rs (Figure
2.1A)9. In agreement with our previous work in mice, we find that activation of P2Y1Rs
with the agonist ADP stimulates Ca2+ responses in enteric glial cells within the
myenteric plexus of the human jejunum (Figure 2.1B). Likewise, activation of neuronal
P2X7Rs with the agonist BzATP in the human myenteric plexus recruits Ca 2+ responses
in the surrounding enteric glial cells (Figure 2.1B). Previous reports suggest that
P2Y1Rs are expressed by enteric neurons in the guinea pig ENS17 and by enteric glia in
the mouse ENS9,18. Our data support the conclusion that P2Y1Rs are primarily localized
to enteric glia in the mouse myenteric plexus because P2Y1R agonists including ADP
(100 μM), the non-hydrolysable ADP analog ADPβS (100 μM) or MRS2365 (1 μM)
primarily drive cellular activity in enteric glial cells and not neurons (Figure 2.1C-D, data
not shown for ADP and MRS2365). Likewise, immunohistochemistry with specific
antibodies against an extracellular loop of the P2Y1R shows that P2Y1Rs are primarily
localized to enteric glial cell processes in the myenteric plexus (Figure 2.1E).
We tested how direct activation of glial cells with P2Y1R agonists affects neuron
survival in isolated preparations of the mouse ENS. Activation of glial P2Y1Rs with

52

Figure 2.1: Activation of enteric glial P2Y1Rs drives neuron death through a Cx43dependent mechanism. (A) Schematic depicting how activation of neuronal P2X7Rs
elicits Ca2+ responses in enteric glia. (B) Representative traces of in situ Ca2+ imaging
of human myenteric glia. As in mice, human glia respond to the P2Y1R agonist, ADP
and to the neuronal release of ATP stimulated by the P2X7R agonist, BzATP. Gray
traces show responses of individual glial cells within a ganglion and the averaged
response of all glia within the ganglion is overlaid in green. Traces are representative of

53

Figure 2.1 (cont’d)
recordings from at least 4 myenteric ganglia from the human jejunum. (C-D) In situ Ca2+
imaging of the mouse myenteric plexus demonstrates that P2Y1R agonists primarily
elicit Ca2+ responses in enteric glia. (C) Representative images of Fluo-4 fluorescence
in a myenteric ganglion from the mouse colon at rest (baseline) and at peak stimulation
(at time = 60s) with the non-hydrolysable P2Y1R agonist, ADPβS. Arrows point to
representative enteric glia. (D) Averaged Ca2+ responses of glia (green line) and
neurons (magenta dashed line) within a myenteric ganglion from the mouse colon in
response to ADPβS. Traces are representative of responses in over 5 myenteric
ganglia. (E) Representative mouse myenteric ganglion showing immunoreactivity for
P2Y1Rs (magenta). Enteric glia are labeled with the glial cell marker glial fibrillary acidic
protein (GFAP; green) and arrowheads highlight areas of co-localization (scale bar, 30
μM). The boxed region in the overlay image at top right is expanded in the bottom right
panel to highlight a glial cell with dense P2Y1R expression. (F) Mean packing density of
HuC/D-immunoreactive neurons in myenteric ganglia after in situ activation of P2Y1Rs
with the agonists ADP, ADPβS and MRS2365 or activation of P2X7Rs with BzATP in
the presence or absence of the Cx43 inhibitor 43Gap26 (n = 3-5 animals, **P≤0.01,
***P≤0.005 ANOVA). (G) Mean packing density of live enteric neurons in myenteric
ganglia following in situ neuroinflammation with BzATP, ADP or buffer control,
quantified by Calcein-AM fluorescence. Representative images show viable cells,
labeled with fluorescent Calcien-AM (green), in mouse myenteric ganglia from control
and BzATP-treated tissues. Dead (non-fluorescent) neurons are denoted by asterisks
(scale bar, 30 μM), (n = 3-4 animals).

54

ADP, ADPβS or MRS2365 decreased the ganglionic density of HuC/D-immunoreactive
neurons by 24 ± 4%, 23 ± 4% and 19 ± 4%, respectively (Figure 2.1F). Importantly,
P2Y1R activation drove enteric neuron death to an equal extent as activation of
neuronal P2X7Rs with BzATP (300 μM) (23 ± 2%, Figure 2.1F). To confirm that the
loss of HuC/D-immunoreactivity truly reflects a loss of neurons and not merely a loss of
HuC/D-immunoreactivity in neurons, we assessed neuronal viability using the
fluorescent dye Calcien-AM which labels live, membrane-intact cells13. Treatment with
BzATP (300 μM) and ADP (100 μM) decreased neuronal density in myenteric ganglia
by ~50% compared to buffer control (53 ± 4% and 52 ± 3% respectively, Figure 2.1G),
consistent with results determined by HuC/D-immunoreactivity. These results show that
the activation of glial P2Y1Rs is sufficient to induce enteric neuron death driven through
P2X7R activation.

In situ glial-driven neuron death requires Cx43 hemichannels
The activation of glial P2Y1Rs drives Ca2+ responses and triggers mechanisms that
lead to Cx43 hemichannel opening in mice4. Astroglial Cx43 hemichannel opening in
the context of neuroinflammation releases mediators that contribute to the development
of neuropathic pain and neurodegeneration in the central nervous system (CNS) 19,20.
Thus, we tested if glial-driven enteric neuron death requires Cx43 hemichannel opening
by activating glial P2Y1Rs in the presence of the specific Cx43 hemichannel mimetic
peptide, 43Gap26 (100 μM) which inhibits Cx43 hemichannel opening21. The neurotoxic
effects of both P2Y1R agonists (ADP and ADPβS) and P2X7R agonist (BzATP) were
lost in the presence of 43Gap26, which protected against decreased ganglionic neuron

55

density (92 ± 2%, 89 ± 3% and 108 ± 8% for ADP, ADPβS and BzATP with 43Gap26
respectively, compared to buffer control, Figure 2.1F). These results strongly suggest
that glial cells are responsible for driving neuron death because Cx43 hemichannels are
confined to enteric glial cells within the ENS4.

Glial Cx43 hemichannels are required for in vivo neuron death during inflammation
To more accurately test the role of glial Cx43 hemichannels in inflammatory neuropathy,
we combined in vivo DNBS-colitis with a targeted gene deletion approach to
conditionally ablate Cx43 in GFAP-expressing glial cells following the administration of
tamoxifen4 (inducible and conditional Cx43 knockout; Cx43i-cKO; Figure 2.2). In
agreement with our previous work, the density of HuC/D-immunoreactive myenteric
neurons was reduced by 24% (2057 ± 68 vs. 1565 ± 139 neurons mm-2) at the peak of
DNBS colitis in control mice (littermates treated with tamoxifen but lacking cre
recombinase in glia, Cre-; Figure 2.2A). However, mice with a conditional ablation of
Cx43 hemichannels in glia (Cre+) were resistant to the neurotoxic effects of in vivo
inflammation (2057 ± 68 neurons mm-2 in Cre-/Saline controls vs. 1850 ± 117 neurons
mm-2 in Cre+/DNBS, Figure 2.2A). This effect is not likely due to an alteration in the
inflammation driven by DNBS because Cx43i-cKO (Cre+) mice exhibited the same
pattern of weight loss and macroscopic damage scores as littermate controls (Figure
2.2B-C). Further, the ablation of Cx43 in glia did not affect normal neuron packing
density in healthy animals (2057 ± 68 neurons mm-2 in Cre-/Saline animals vs. 2149 ±
101 neurons mm-2 in Cre+/Saline; Figure 2.2A). Collectively, these data show that the

56

Figure 2.2: Genetic ablation of glial Cx43 limits neuropathy without modifying
overt inflammation. (A) Mean packing density of myenteric neurons following DNBScolitis in transgenic mice with an inducible ablation of Cx43 in glial cells (Cx43i-cKO,
Cre+) and their littermate controls (Cre-). Weight loss pattern and macroscopic damage
for experimental groups shown in (B) and (C), respectively (n = 5-7 animals, *P≤0.05,
ANOVA).
57

expression of glial Cx43 hemichannels is a requirement for inflammatory neuropathy in
the ENS.

Stimulation of enteric glial P2Y1Rs elicits Cx43-dependent ATP release
One possible mechanistic explanation for glial-driven neuron death is that glial Cx43
hemichannel opening modulates P2X7R activation threshold by augmenting levels of
extracellular ATP. In support, astroglial Cx43 hemichannels are highly permeable to
ATP22,23 and neurotoxic activation of P2X7Rs requires a conformational change that
only high concentrations of ATP are capable of inducing by occupying all four ATP
binding sites24. We tested if purinergic activation of enteric glia drives Cx43-dependent
ATP release by stimulating glial P2Y1Rs while monitoring extracellular ATP release with
ATP-sensitive microelectrodes14. In these experiments, we either directly stimulated
glial P2Y1Rs with the non-hydrolysable agonist ADPβS or indirectly generated
endogenous ADP by activating neuronal P2X7R-dependent ATP release with the
agonist BzATP. We found that stimulating glial P2Y1Rs with ADPβS elicits robust ATP
release from enteric glia (Figure 2.3A-B). P2Y1R-driven glial ATP release was
completely dependent upon Cx43 because ATP release was absent in the presence of
the Cx43 mimetic peptide, 43Gap26 and in tissue from Cx43i-cKO (Cre+) mice (Figure
2.3A-B). Likewise, stimulation of P2X7R-dependent neuron-glia communication
generated high levels of extracellular ATP (Figure 2.3C-D). Interestingly, panx1dependent ATP release from enteric neurons accounted for only approximately 1/3 of
total ATP release while Cx43-dependent release from glia accounted for the majority of

58

Figure 2.3: Stimulation of enteric glial P2Y1Rs elicits Cx43-dependent ATP
release. (A,C) Representative traces and (B,D) quantified measurements of ATP
release from the mouse myenteric plexus obtained with ATP-selective electrodes. Glial
P2Y1Rs were directly stimulated with the P2Y1R agonist, ADPβS (A,B; 100μM), or by
eliciting neuron-to-glia communication with the neuronal P2X7R agonist, BzATP (C,D;
300μM), in the presence or absence of the Cx43 mimetic peptide, 43gap26 (100μM),
the pannexin-1 mimetic peptide, 10Panx (100μM), or in tissue from Cx43i-cKO mice
following the selective ablation of glial Cx43 (Cre+ KO or Cre- littermate controls). n=3
animals.

59

ATP release. These results indicate that enteric glia have the potential to release large
quantities of ATP through Cx43 hemichannels following stimulation of P2Y1Rs.

Oxidative stress coincides with neuron death during in vivo inflammation and is required
for P2X7R-driven neuron death in situ
Next, we asked whether the neurotoxic opening of glial Cx43 channels requires
potentiation of channel function by other factors associated with inflammation. We
initially focused on oxidative stressors because Cx43 hemichannel opening is
potentiated by oxidative stress25 and oxidative stress is considered a key mechanism in
the pathogenesis of gut inflammation1,26,27. In support, we observed high levels of
oxidative stress in myenteric ganglia during DNBS-colitis (Figure 2.4A,C). Measures of
oxidative stress included neuronal thiol oxidation ratios (ratio of oxidized/reduced
neuronal glutathione) (Figure 2.4A) and ganglionic concentrations of superoxide
measured by fluorescence of the superoxide-specific fluorescent indicator
dihydroethidium (DHE) (Figure 2.4C). We confirmed that our measures truly reflected
oxidative stress by administration of the antioxidant N-acetyl cysteine (NAC, 5g/L) in
vivo prior to and during the induction of DNBS-colitis (Figure 2.4A). Treatment with
NAC prevented increased oxidative stress during DNBS-colitis (Figure 2.4A) without
altering the acute inflammatory insult, as there was no change in macroscopic damage
score with NAC treatment (Figure 2.4B). Importantly, we investigated the mechanistic
significance of oxidative stress and found that treatment with NAC prevented P2X7Rdriven neuron death in situ (Figure 2.4D-E). Because our results above show that glial

60

Figure 2.4: Oxidative stress coincides with neuron death during in vivo
inflammation and is required for P2X7R-driven neuron death in situ. (A) Thiol
oxidation measurements (ratio of oxidized/reduced glutathione) in myenteric neurons
from healthy (saline) or inflamed (DNBS-colitis) animals drinking normal water (H2O) or
water containing the antioxidant N-Acetyl Cysteine (NAC, 5g/L) (n=5 ganglia, **** P <
0.001, ANOVA). (B) Macroscopic damage score for animals treated in (A). (C)
Ganglionic fluorescence of the superoxide indicator dihydroethidium (DHE, 2 μM) in the
myenteric plexus of healthy (saline) or inflamed (DNBS-colitis) mice (n = 5-6 animals, *
P < 0.05, unpaired t-test). (D) Representative myenteric ganglia from in situ
preparations treated with BzATP in the presence or absence of the antioxidant NAC.

61

Figure 2.4 (cont’d)
Neurons are labeled with the neuronal marker HuC/D (scale bar = 30 μM). (E) Mean
packing density of HuC/D-immunoreactive neurons in myenteric ganglia following in situ
activation of P2X7Rs with BzATP (300 μM) in the presence of NAC (n=4 animals, * P <
0.05, ANOVA).

62

pathways mediate P2X7R-driven neuron death, these results indicate that oxidative
stress contributes to the activation of neurotoxic glial mechanisms.

Enteric glia contribute to oxidative stress via local nitric oxide (NO) production during
inflammation
Glial cells can directly contribute to the local generation of oxidative stressors by upregulating the activity of iNOS28,29; an enzyme that produces large amounts of NO
during inflammation30. We assessed the glial contribution to local NO levels within
enteric ganglia from healthy and inflamed animals by measuring cellular NO
concentrations with the NO-sensitive fluorescent dye DAF-FM (Figure 2.5A). DAF-FM
fluorescence reliably reflected intracellular NO concentrations as treatment with the NO
donor PAPA NONOate elevated fluorescence while treatment with the pan-NOS
inhibitor L-NAME decreased fluorescence (Figure 2.5A). NO content was equally
distributed between neurons and glia in non-inflamed animals and this distribution was
not altered up to 6h following DNBS-colitis (Figure 2.5B-C). However, we observed an
elevation of glial NO content during the peak of the inflammatory response 48hrs after
initiation of DNBS-colitis (Figure 2.5B-C). Further, increases in immunoreactivity for
nitrosylated proteins, another measure of NO concentrations, correlated with changes in
glial NO content post DNBS-colitis treatment (Figure 2.5D). Nitrosylated protein
immunoreactivity primarily co-localized with GFAP immunoreactivity within myenteric
ganglia (Figure 2.5E), suggesting that glial cells are the main sites of nitrosylation in the
myenteric plexus during inflammation. Importantly, the kinetics of glial NO production

63

Figure 2.5: Enteric glia contribute to oxidative stress by producing nitric oxide
(NO) during inflammation. (A-C) In situ NO imaging with the NO sensitive dye, DAF-

64

Figure 2.5 (cont’d)
FM. (A) Representative traces of mean glial NO responses following treatment with the
NO donor PAPA NONOate (solid green line, 100 μM) or pan-nitric oxide synthase
(NOS) inhibitor L-NAME (dashed black line, 100 μM). (B) Representative images of
DAF-FM fluorescence in myenteric ganglia from healthy (saline) or inflamed (DNBScolitis) mice. Arrows point to representative glial cells and representative neurons (or
the lack thereof) are denoted by asterisks (scale bar, 30 μM). (C) Quantification of DAFFM fluorescence in observable myenteric neurons and glia in the healthy (saline) or
inflamed (DNBS) colon at 6h (left) and 48h (right) after the initiation of DNBS-colitis (n =
5-10 animals, *P < 0.05, t-test compared to glia-saline). (D) Percentage of nitrotyrosine
immunoreactive (nT +ve) ganglia in the myenteric plexus of saline DNBS-treated
animals at 6h (left) and 48h (right) after the initiation of DNBS-colitis (n = 3-5 animals, *
P < 0.05, unpaired t-test). (E) Representative myenteric ganglion showing
immunoreactivity for nitrosylated proteins (nTYR; magenta). Enteric glia are labeled with
the glial cell marker GFAP (green) and yellow arrowheads highlight areas of colocalization (scale bar, 20 μM).

65

and protein nitrosylation coincide with the appearance of neuron death during colitis in
mice9 and guinea pigs31.

NO potentiates glial ATP release and promotes enteric neuron death through a Cx43dependent mechanism
Given that nitrosylation of Cx43 hemichannels is associated with increased Cx43
channel opening in astrocytes25, we hypothesized that NO would potentiate the Cx43dependent release of ATP driven by glial P2Y1R activation. In support, we found that
the Cx43-dependent release of ATP from glia stimulated by the P2Y1R agonist ADPβS
was potentiated in the presence of the NO donor PAPA NONOate (Figure 2.6A). This
suggests that NO production during intestinal inflammation can contribute to the
activation of the neurotoxic Cx43 pathway in enteric glia.
In situ, we observed an equal extent of neuron death in whole-mounts of
myenteric plexus incubated with the NO donor PAPA NONOate as in preparations
exposed to the neuronal P2X7R agonist BzATP (24 ± 5% vs. 21 ± 4%, Figure 2.6C),
further supporting a for pathogenic role for NO in the context of neuron death.
Importantly, the neurotoxic effects of PAPA NONOate and BzATP were not additive.
This finding suggests that NO and P2X7R agonists drive neuron death through a
common mechanism. Indeed, BzATP and PAPA NONOate-driven neuron death were
both entirely dependent upon Cx43 hemichannel opening because the neurotoxic
effects of these compounds were abolished in the presence of 43Gap26. Likewise,
neurons were protected against BzATP-induced death in tissue from iNOS null (iNOS-/-)
mice and by blocking NO production from iNOS with the inhibitor, 1400W (Figure 2.6C).

66

Collectively, these data show that the production of NO by iNOS during inflammation is
essential for the activation of neurotoxic pathways mediated through glial Cx43
hemichannels and strongly suggest that NO contributes to neuropathy by potentiating
glial ATP release via Cx43 hemichannels.

Mechanisms of neuron death downstream of glial P2Y1R activation involve glial iNOS
and neuronal P2X7Rs
To determine the sequence of signaling events downstream of glial P2Y1R activation
that lead to neuron death, we activated glial P2Y1Rs in situ with the specific agonist
ADP (100 μM) in the presence of drugs to inhibit either iNOS or P2X7Rs. The inhibition
of glial iNOS with 1400W completely abolished the neurotoxic effect of glial P2Y1R
activation (118 ± 13% vs. 72 ± 7%; Figure 2.6B). Likewise, the inhibition of neuronal
P2X7Rs with A740003 protected against neuron death driven by glial P2Y1R activation
(90 ± 5%, vs. 72 ± 7%; Figure 2.6B). Taken together, these results suggest a cyclical
signaling mechanism where neuronal P2X7R-panx1-mediated ATP release activates
glial P2Y1Rs and intracellular signaling mechanisms that lead to glial NO production
and the potentiation of ATP release from glial Cx43 hemichannels which causes neuron
death through actions on neuronal P2X7Rs.

NO positively modulates glial Cx43 hemichannels and negatively modulates neuronal
panx1 hemichannels
NO could directly modulate a number of receptors and channels involved in the P2X7Rpanx1 neuron death pathway including glial Cx43 hemichannels and/or neuronal panx1

67

Figure 2.6: Nitric oxide (NO) potentiates ATP release from glia and promotes
neuron death in situ through a mechanism that involves glial Cx43 hemichannels.
(A) ATP release from myenteric ganglia following stimulation of glial P2Y1Rs with
ADPβS (100 μM) alone or in the presence of the NO donor PAPA NONOate (n=4, * P <
0.05, unpaired t-test). (B) Mean packing density of Hu-immunoreactive myenteric

68

Figure 2.6 (cont’d)
neurons following direct glial P2Y1R stimulation with ADP and inhibition of iNOS
(1400W; 10 μM) or P2X7Rs (A740003; 10 μM). Inhibition of iNOS or P2X7Rs protects
against P2Y1R-driven neuron death (*P≤0.01, ANOVA as compared to ADP, n=3-4
animals). (C) Mean packing density of myenteric neurons following application of
BzATP or NO-modifying drugs in wild type (bottom bars) or iNOS-knockout mice (iNOS/-,

top two green bars). Inhibition (1400W; 10 μM) or ablation (iNOS-/-) of iNOS protects

against P2X7R-driven neuron death. The NO donor, PAPA NONOate (100 μM), drives
neuron death to an equal extent as BzATP but the combination is not additive. Like
BzATP, PAPA NONOate–driven neuron death requires iNOS (blocked by 1400W and in
iNOS-/- mice) and Cx43 hemichannel opening (blocked by 43Gap26).
*P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, ANOVA as compared to BzATP, n=3-11
animals.

69

hemichannels and P2X7Rs32. To determine whether the neurotoxic effect of NO we
observe is due to the potentiation of neuronal (P2X7R-panx1) or glial (Cx43) signaling
mechanisms, we measured glial activity induced indirectly by the activation of neuronal
P2X7R-panx1 or directly via the activation of glial P2Y1Rs in the presence of the NO
donor PAPA NONOate (100 μM). We previously demonstrated that enteric glia can be
used as endogenous “sniffer cells” to measure the activity of the neuronal P2X7R-panx1
pathway9 and that glial network activity in response to the P2Y1R agonist ADP reflects
glial Cx43 activity4. Our results show that glial Ca2+ responses downstream of neuronal
P2X7R-panx1 activation are significantly blunted in the presence of PAPA NONOate
(72% decrease in peak ΔF/F vs. control, Figure 2.7A-B). Glial Ca2+ responses
downstream of neuronal P2X7R activation are entirely dependent on the neuronal
release of ATP through panx19 and blunted glial responses suggest that NO negatively
regulates P2X7R-panx1-dependent neuron-to-glia communication. This results is in
agreement with other studies showing that NO inhibits panx1 channel activity32,33. An
alternate explanation for this result is that NO decreased the ability of glial to respond to
neuronal activation. We tested this possibility by directly activating glial cells with ADP.
Instead of decreasing glial responsiveness, we find that NO significantly potentiates glial
Ca2+ responses to ADP (35% increase in peak ΔF/F vs. control, Figure 2.7C-D). This
outcome suggests that glial Cx43 hemichannel opening is facilitated by NO because
Ca2+ responses through the enteric glial network are mediated by Cx434. Our other data
supports this conclusion by showing that NO potentiates glial Cx43-dependent ATP
release (Figure 2.6A). Together, these results strongly support the conclusion that the

70

sensitization of glial release mechanisms, rather than neuronal signaling components, is
the primary cause of neuron death.

71

Figure 2.7: Nitric Oxide positively modulates glial Cx43 hemichannels and
negatively modulates neuronal panx1 hemichannels. (A-B) Representative Ca2+
imaging traces (A) and averaged peak ΔF/F responses (B) show that NO (NO donor
PAPA NONOate) blunts glial Ca2+ responses downstream of neuronal P2X7R-panx1
stimulation with BzATP (300 μM). (C-D) Representative Ca2+ imaging traces (C) and
averaged peak ΔF/F responses (D) of glial Ca2+ responses during direct glial P2Y1R
activation with ADP (100 μM) in the presence or absence of the NO donor PAPA
NONOate. Note that NO potentiates glial network responses driven by the direct agonist
ADP. *P<0.05, ****P<0.001, T-test compared to control, n= 51-139 individual cells in 3-7
ganglia.

72

Figure 2.8: Proposed model of the mechanisms involved in glial cell-driven
enteric neuron death during acute inflammation. The activation of neuronal P2X7Rs
leads to ATP release through panx1 channels that recruits activity in the surrounding
glia by activating glial P2Y1Rs. Intracellular signaling pathways downstream of P2Y1R
activation drive glial iNOS production and glial NO potentiates Cx43 hemichanneldependent ATP release. Glial NO may also feed back on neurons to block panx1
channel activity. Large quantities of ATP are released through glial Cx43 and act on
neuronal P2X7Rs to drive neuron death.

73

Discussion
Our observations provide the first evidence that enteric glial cells play an active role in
the death of enteric neurons during gut inflammation. Specifically, our data show that
mediators of inflammation such as NO potentiate the gating of glial Cx43 hemichannels
and subsequently, neuron death. Based on these data, we propose a model where
neuronal P2X7-panx1-mediated purine release during inflammation or neuron stress,
drives glial activation leading to pathogenic Cx43-dependent ATP release by glial cells
and enteric neuron death by activation of neuronal P2X7Rs (Figure 2.8).
Enteric glial cells are generally thought to support the function and survival of
enteric neurons and models of glial ablation support this conclusion as glial ablation
produces a rapid loss of enteric neurons5,6. Thus, a loss of the supportive roles of glial
cells is postulated as a potential mechanism for the development of ENS dysfunction in
inflammatory bowel disease2,5. In contrast, newer data show that the chronic activation
of astroglia in the context of neuroinflammation leads to the release of neurotoxic
mediators from glial cells7. Our results support a similar scenario in the ENS during
inflammation where a gain, rather than a loss, of glial cell function leads to the death of
enteric neurons. These findings highlight the dichotomous roles of enteric glial cells
during health and disease and suggest that either a loss of protective glial function or
gain of pathological glial functions can drive ENS dysfunction but presumably through
very different mechanisms.
Physiologically, enteric glial cells possess neuroprotective properties and are
integral in maintaining enteric neuron populations. Enteric glia secrete neuroprotective
compounds such as reduced glutathione and the prostaglandin derivative 15-deoxy-

74

PGJ23,34. Further, intraganglionic enteric glia express a number of receptors and
enzymes that can respond to and degrade neuroactive compounds, thus preventing
aberrant neuronal activation35,36. Finally, glial cells can directly participate in neuron
signaling by responding to and releasing neurotransmitters as previously shown for
Cx43 hemichannels in hippocampal astrocytes in the CNS37. In support of a functional
role for Cx43 hemichannels in the enteric nervous system, we observed that ablation of
Cx43 in enteric glial cells blunts glial network activity and disrupts neuronal regulations
of gastrointestinal transit under physiological conditions4.
Here, we find that glial Cx43 hemichannel opening is required for neuron death
during inflammation and that the selective ablation of glial Cx43 is neuroprotective in gut
pathology. We postulate that these observations reflect the necessity for Cx43
hemichannels in both physiological and pathophysiological functions of enteric glia,
where the pathological potential of glial Cx43 is evident when potentiated by
inflammatory mediators such as NO. Indeed, here, NO potentiated Cx43-mediated ATP
release from enteric glia, driving neuron death. Although inhibition of Cx43 was
neuroprotective, the therapeutic potential of CX43 inhibition may be limited given the
essential role of glial Cx43 in normal gut function. However, selective inhibition of
pathological channel opening by NO has the potential to disrupt pathology without
affecting physiological functions. Thus, drugs such as GW274150 that display excellent
iNOS selectivity and little toxicity38 may represent important new therapeutics in the
treatment of functional gastrointestinal disorders.
Cx43 hemichannels are subject to post-translational modifications including Snitrosylation25 by increased oxidant concentrations. Here, we show an increase in

75

nitrosylated protein immunoreactivity in enteric glia that correlates with increased glial
NO following in vivo inflammation. Importantly, the pattern of nitrosylation mirrors Cx43
immunoreactivity previously shown in enteric glia4. This strongly supports our
hypothesis that Cx43 is a primary candidate of post-translational nitrosylation as a result
of increased NO production during in vivo inflammation. We observed glial NO
production during in vivo inflammation using a fluorescent NO marker during the
initiation (6h) and peak (48h) of inflammation. Although we did not observe a significant
increase in glial NO content at 6h after initiating inflammation, this could be due to the
high variability between the inflammatory stages of individual ganglia at this early stage.
However, our nitrosylation data show a similar timeline of nitrosylation in glia so it is
likely that glial NO production occurs in response to active inflammation and is not
necessarily responsible for the initiation of inflammation.
One alternate interpretation of our findings is that NO production by glia and
oxidative stress in neurons directly affects the P2X7Rs and panx1 hemichannels on
enteric neurons; leading to neuron death independent of glial mechanisms9. Indeed, NO
modulates the activity of hemichannels including those composed of panx1 32,39,40 and
Cx4325,41. However, we do not believe that our data support this interpretation because
of several reasons. Firstly, we show that NO significantly blunts glial activity initiated by
the neuronal release of ATP through P2X7R-panx1. In contrast, NO potentiated Cx43dependent intercellular communication between enteric glia in response to direct
P2Y1R stimulation. Likewise, the neurotoxic effect of the NO donor was completely
abolished by the Cx43 mimetic peptide or the genetic ablation of glial iNOS. Further, our
data show that glial cell release of ATP through Cx43 is the primary contributor to

76

extracellular ATP levels following stimulation of either neuronal P2X7Rs or glial P2Y1Rs
while neuronal panx1 opening contributes only minor amounts of ATP. Finally, the
selective ablation of glial Cx43 alone was able to protect enteric neurons during
inflammation in vivo. We interpret these findings as indicating that the potentiation of
neurotoxic mechanisms is primarily occurring in glial cells although we recognize that a
combination of effects on neuronal and glial pathways could be required for neuron
death.
Our in vivo model of Cx43 ablation utilizes cre/lox technology and requires
expression of cre recombinase, driven by the GFAP promoter, in response to tamoxifen
administration. Although enteric glial cells are a heterogeneous population, the majority
of glia in the myenteric plexus are GFAP positive42 making this a suitable promoter for
our study. Importantly, our model is dependent on the presence of the GFAP promoter
and thus variability in GFAP protein expression with inflammation and time would not
likely have a major effect on cre activity and Cx43 excision42. In our experimental
paradigm, inflammation was induced after the ablation of Cx43 was induced. However,
enteric glia are subject to gliogenesis in response to injury and inflammation 43. Thus,
any newly generated glia following inflammation would have been exposed to tamoxifen
and would express functional Cx43 hemichannels. Notably, the timeline for
gliogenesis43 is substantially longer than that of our current inflammatory model31 so we
do not believe that glial turnover was a major confounding factor in these experiments.
GFAP is also expressed by astrocytes in the CNS44,45 and tamoxifen treatment in
our mouse model induces the ablation of Cx43 hemichannels in enteric glia and CNS
astrocytes. Disruption of astrocytic Cx43 gap junction and hemichannel function results

77

in CNS dysfunction and this may have exerted confounding effects in our disease
model. However, we do not observe unusual weight loss or gut inflammation in these
animals in the absence of inflammatory stimuli and we observed comparable weight
loss and inflammation to wild type animals during inflammation (Figure 2.2). Thus,
dysfunction in the brain-gut axis does not seem to be a significant contributor to disease
outcome in our in vivo model.
Purinergic activation of enteric glial cells is a central component of our proposed
signaling mechanism (Figure 2.8). We show glial purinergic responses are mediated
through P2Y1Rs that are primarily localized to enteric glial cells in the mouse myenteric
plexus. This supports previous work demonstrating glial P2Y1R expression and activity
in cultured glial cells18 and in whole mount preparations in mouse and guinea pig9,46,47.
Previous work suggests P2Y1R expression on enteric neurons17,48. However, we
demonstrate that activation with the P2Y1R agonist ADP elicits Ca2+ responses in glial
cells, a phenomenon observed in the CNS notwithstanding P2Y1R expression on both
neurons and astrocytes49. Given the present data, it is tempting to speculate that the
P2Y1R-mediated slow excitatory postsynaptic potentials recorded in enteric neurons 48
are actually downstream of glial activation and this will be an interesting hypothesis to
test in future experiments.
In conclusion, our findings have uncovered a novel role for enteric glia in the
pathogenesis of enteric neuropathies. Enteric neuropathy is increasingly recognized as
a key pathological finding in functional gastrointestinal disorders including irritable bowel
syndrome50 and slow transit constipation51. Inflammation is thought to produce
persistent gut dysfunction through effects on the ENS including enteric neuron death

78

and the functional remodeling of enteric circuitry. Indeed, our prior results show that an
acute inflammatory event triggers enteric neuron death and leads to gut motor
dysfunction that persists despite resolution of active inflammation 9. Our current
observations suggest that the activation of enteric glial cells is necessary for at least a
portion of these permanent effects on the ENS. Thus, new therapies that modulate the
pathophysiological functions of enteric glial cells could lead to the development of more
effective treatments for functional bowel disorders.

79

APPENDIX

80

Antibody

Source

Dilution

Catalog #

Rabbit anti-iNOS

Abcam, Cmabridge, MA

1:200

ab15323

Human anti HuC/D

1:200

A-21272

Chicken anti-GFAP

Molecular Probes, Grand
Island, NY
Abcam, Cambridge, MA

1:1000

ab4674

Rabbit anti-nitrotyrosine

Millipore, Billerica, MA

1:100

06-284

Rabbit anti-P2Y1R

Alomone Labs, Jerusalem,
Israel

1:200

APR-021

Table 2.1: Primary Antibodies used in Chapter 2.

81

Antibody

Source

Dilution

Catalog #

Alexa Fluor 488 Goat anti-rabbit

Invitrogen, Carlsbad,
CA

1:200

A-11034

Alexa Fluor 488 Goat anti-chicken

Invitrogen, Carlsbad,
CA

1:200

A-11039

Alexa Fluor 568 Goat anti-chicken

Invitrogen, Carlsbad,
CA

1:200

A-11041

Alexa Fluor 594-conjugated Streptavidin

Jackson
ImmunoResearch,
West Grove, PA

1:200

016-580-084

Table 2.2: Secondary Antibodies used in Chapter 2.

82

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83

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CHAPTER 3
The antioxidant glutathione protects against enteric neuron death in
situ, but its depletion is protective during colitis

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Abstract
Enteric glia play an important neuroprotective role in the enteric nervous system (ENS)
by producing neuroprotective compounds such as the antioxidant reduced glutathione
(GSH). The specific cellular pathways that regulate glial production of GSH, and how
these pathways are altered during, or contribute to, neuroinflammation in situ and in
vivo are not fully understood. We investigated this issue using immunohistochemistry to
localize GSH synthesis enzymes within the myenteric plexus and tested how the
inhibition of GSH synthesis with the selective inhibitor L-Buthionine Sulfoximine (BSO)
impacts neuronal survival and inflammation. Both enteric glia and neurons express the
cellular machinery necessary for GSH synthesis. Further, glial GSH synthesis is
necessary for neuronal survival in isolated preparations of the ENS. In vivo depletion of
GSH does not induce colitis but alters myenteric plexus neuronal phenotype and
survival. Importantly, global depletion of glutathione is protective against some macro
and microscopic measures of colonic inflammation. Together, our data highlights the
heterogeneous roles of GSH in the ENS and during GI inflammation.

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Introduction
The enteric nervous system (ENS) is a network of neurons and glial cells that
controls the reflexes and behaviors of the gastrointestinal (GI) tract. Inflammation,
among many other insults, can disrupt the control of GI reflexes by altering the function
and/or survival of enteric neurons and these changes contribute to common functional
GI motility disorders1-4. Recently, enteric glial cells have received increased attention for
their active roles in ENS function and mechanisms of neuron death 5,6. The activation of
glia contributes to neuron death via pathways that involve connexin-43 hemichannels
and oxidants such as nitric oxide7. However, glia also serve an important
neuroprotective role and the targeted ablation of glial cells results in enteric
neurodegeneration and gut inflammation8,9. The specific cellular mechanisms that
regulate the production and secretion of glial neuroprotective factors, and how these are
altered in (patho)physiology, are poorly understood.
Enteric glia synthesize and release a number of neuroprotective compounds
such as the prostaglandin derivative 15-deoxy-prostaglandin-J210, the neurotrophic
factor glial-derived neurotrophic factor (GDNF)11, the endogenous antioxidant reduced
glutathione 12 and the glutathione derivative s-nitrosoglutathione13. Reduced glutathione
(GSH) is the primary endogenous antioxidant in the body and promotes neuronal
survival in the central and peripheral nervous systems

14-18.

In the central nervous

system, astroglia are the primary cell type responsible for the production of GSH and
contain large quantities of GSH in vivo and in vitro19. Further, astrocytes have the ability
to secrete GSH, and glial-derived GSH can be uptaken by neurons or elicit its
antioxidant functions in the extracellular space20-22. Given the similarities between

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astrocytes and enteric glia, we hypothesized that enteric glia play a similar role in the
production and regulation of GSH in the ENS.
We tested our hypothesis by studying the cellular localization of glutathione
synthesis enzymes in the ENS and the impact of GSH depletion on neuron survival and
GI inflammation in situ and in vivo. Our data show that both enteric neurons and glia
express the cellular machinery necessary for the production of GSH. GSH production is
necessary for enteric neuron survival in situ but, surprisingly, in vivo depletion of GSH
was protective against some measures of GI inflammation. Together, our results
provide novel insight into the cellular regulation of a key antioxidant in the intestine.
GSH plays complex roles in the context of GI inflammation and its cytoprotective effects
may contribute to disease pathogenesis.

Material and Methods
Animals
All work was approved by the Michigan State University Institutional Animal Care and
Use Committee (IACUC). All experiments used male C57Bl/6 mice (Charles River
Laboratories, Hollister, CA) aged 6-10 weeks and maintained on a 12-hour light/dark
cycle with ad libitum access to food and water.

In Vivo Colitis
Colitis was induced in male C57Bl/6 as previously described

4,7.

An enema of 2,4-

dinitrobenzene sulfonic acid (DNBS, 5.5 mg/mouse in 0.1 mL 50:50 ethanol/saline) was
administered via a gavage needle inserted 3 cm into the colon. Experimental controls

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were given an enema of saline only. Animals were sacrificed 48 hours after
DNBS/saline treatment and macroscopic damage to the colon was assessed with a well
established scoring system 23. Tissue was then fixed overnight in Zamboni’s fixative for
use in immunohistochemical assays. Animal body weight and appearance were
recorded daily following the induction of colitis.

In Vivo Glutathione Depletion
Colonic glutathione was depleted as described by Watanabe et al.

24.

Briefly,

mice were given ad libitum access to drinking water containing 20 mM L-ButhionineSulfoximine (BSO) or normal drinking water (H2O) for a period of 14 days. Mice were
observed daily and changes in appearance, body weight and fluid consumption were
recorded.

In Situ Model of Neuroinflammation
Enteric neuron death was driven in live longitudinal muscle myenteric plexus
(LMMP) preparations in situ by the P2X7 receptor agonist 2’(3’)-O-(4benzoylbenzoyl)adenosine-5’-triphosphate triethylammonium salt (BzATP) (300 mM) 4,7.
Live LMMPs were prepared from segments of mouse distal colon tissue in sylgardcoated 35 mm2 dishes containing DMEM F12 media as described previously7. LMMPs
were incubated with BzATP (300 mM) or other drugs for 2 hours in 95% air: 5% CO 2 at
37°C. LMMPs were then rinsed three times with fresh Kreb’s Buffer and incubated for
an additional 2 hours in Kreb’s Buffer. LMMPs were fixed overnight at 4°C in Zamboni’s
fixative for IHC analysis.

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Whole-mount Immunohistochemistry (IHC)
Whole-mount LMMP preparations were prepared from colonic tissue fixed overnight in
Zamboni’s fixative at 4°C. Briefly, preparations were permeabilized by three 10 minute
washes in 0.1% Triton X-100 in phosphate buffered saline (PBS). Following a 45 minute
block in 4% normal goat or donkey serum, LMMP preparations were incubated in
primary antibody (see Table 3.1, appendix) either overnight at room temperature (RT)
or for 48 hours at 4°C. Finally, preparations were incubated in secondary antibodies
(see Table 2, appendix) for 2 hours at RT before mounting and imaging.
Images were acquired using the 40X objective (0.75 numerical aperture; Plan
Fluor, Nikon, Melville, NY) of an upright epifluorescence microscope (Nikon Eclipse Ni)
with a Retiga 2000R camera (QImaging, Surrey, BC, Canada) controlled by QCapture
Pro 7.0 (QImaging) software or by confocal imaging through the Plan-Apochromat 60X
oil immersion objective (1.42 numerical aperture) of an inverted Olympus Fluoview
FV1000 microscope (Olympus, Center Valley, PA).

Dual-Labeling with Primary Antibodies Raised in the Same Host
Dual-labeling with primary antibodies raised in the same host was achieved using a
Tyramide Signal Amplification Kit #14 (T20924) containing HRP conjugated-Goat AntiRabbit IgG and Alexa Fluor 568 Tyramide from Life Technologies (Carlsbad, CA), using
a modification of the protocol described by Gulbransen et al.25. The first 1° (rabbit antisynaptophysin) was used at a 1:2000 dilution and detected via TSA reaction while the
second primary (rabbit anti-GCLC) was detected via normal IHC staining protocols.
Preparations were incubated in the first 1° overnight at RT after endogenous

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peroxidases were quenched by a 60-minute pre-incubation in 3% H2O2 diluted in PBS.
Next, preparations were incubated with HRP-Goat Anti-Rabbit IgG for 2 hours at RT
followed by a 10 minute incubation in Alexa Fluor 568 Tyramide and an overnight
incubation with unlabeled Goat anti-Rabbit Fab fragments (1:50) at RT. Finally,
preparations were labeled with the second 1° and 2° pair as described above (wholemount IHC).

Quantification of Neuronal Thiol Oxidation
Cellular oxidative stress was quantified using a neuronal thiol oxidation ratiometric
analysis as previously described7,26. Reduced (-SH) and oxidized (GSSG) thiols were
labeled with 1μM Alexa Fluor 680 C2 maleimide and 1μM Alexa Fluor 546 C5 maleimide,
respectively (Life Technologies). Oxidized thiols were reduced to free thiols for labeling
using 5mM tris(2-carboxyethyl)phosphine hydrochloride (Sigma) in PBS. Images were
obtained by epifluorescence microscopy as described above and the ratio of 546maleimide/680-maleimide (SS/SH) calculated with ImageJ software
(http://imagej.nih.gov/ij/).

Chemicals/Drugs
BzATP, N-ethylmaleimide, tris (2-carboxyethyl)phosphine hydrochloride (TCEP), LButhione-sulfoximine (BSO) and γ-glutamyl-cysteine (γ-GC) were purchased from
Sigma-Aldrich (St. Louis, MO).

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Solutions and Media
Live tissue was collected in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12
(DMEM/F-12, Life Technologies) supplemented with 3 μM nicardipine and 1 μM
scopolamine. Normal goat/donkey serum contained 4% normal goat/donkey serum,
0.4% Triton X-100 and 1% bovine serum albumin in PBS.

Data and Statistical Analysis
Average ganglionic neuronal density was determined by calculating the number of
HuC/D-immunoreactive neurons per ganglionic area using the cell counter tool in
ImageJ, in at least 10 ganglia/animal. Average ganglionic fluorescence was quantified
using the measure tool in ImageJ. Thiol oxidation ratios were calculated using the
Region of Interest (ROI) tool in ImageJ. Fluorescence of 546-maleimide (oxidized thiols)
and 680-maleimide (reduced thiols) were measured in 15-20 neurons/ganglia in a
minimum 10 ganglia/animal. SS/SH (546/680) ratios were calculated per neuron and
averaged - first per ganglia and then per mouse.
Subpopulations of excitatory (calretinin positive) and inhibitory (nNOS positive)
neurons were calculated by expressing the number of calretinin or nNOS positive
neurons as a percent of HuC/D (total) neurons in a minimum of 10 ganglia/mouse.
Density of infiltrating neutrophils was calculated using the cell counter tool in ImageJ to
quantify the number of MPO-positive neutrophils in a 76,800 μm2 field of view
surrounding a single myenteric ganglion. Similarly, activation of MHC-II muscularis
macrophages was quantified by measuring average fluorescence (AFU) of a 76,800

96

μm2 field of view surrounding a single myenteric ganglion. Both measurements were
performed in a minimum of 10 ganglia/mouse.
Colonic macroscopic damage was quantified using the well characterized scoring
system described by Storr et al.23. The presence of fecal blood, diarrhea and/or
hemorrhage received 1 point/feature present. Adhesion of the colon to other organs and
peritoneal space was scored 0,1 or 2 based on the severity of adhesions. Colon length
and thickness were also scored with greater points for a shortened and/or thickened
colon. Data were analyzed by student’s t-test, One-Way or Two-Way ANOVA (with
Dunnett’s or Tukey’s post-hoc test) using Graphpad Prism 7 (Graphpad Software, San
Francisco, CA).

Results

Glutathione synthesis enzymes are expressed in myenteric ganglia
Enteric glia are thought to serve important neuroprotective roles in the enteric nervous
system because the ablation of glia leads to widespread neuron death 8,27. How glia
protect neurons in the gut is not clear. Reduced glutathione (GSH) is a neuroprotective
antioxidant that is secreted by enteric glia in culture12 and mice lacking the regulatory
glutathione peroxidase enzymes develop spontaneous colitis28. However, whether
enteric glia express the enzymes necessary for glutathione synthesis and contribute to
the protection against colitis in vivo is not known.
We tested whether glia express the machinery required for glutathione synthesis
in vivo by labeling for two key enzymes: glutamate-cysteine ligase and glutathione

97

synthetase. Glutathione is synthesized in a two-step pathway29. In the first and ratelimiting step, glutamate-cysteine ligase (GCL) catalyzes the formation of γglutamylcysteine (γ-GC) from glutamate and cysteine. γ-GC then combines with glycine
in a step catalyzed by glutathione synthetase (GS) to form reduced GSH (γ-glutamylcysteinyl-glycine). Our data show that the catalytic subunit of GCL (GCLC) is expressed
in mouse myenteric ganglia and co-localizes with the glial cell marker, glial fibrillary
acidic protein (GFAP; white arrows, Figure 3.1A). Further, GCLC is also expressed in
neuronal varicosities and its labeling co-localizes well with the pre-synaptic vesicle
marker synpatophysin (Figure 3.1B). The second synthesis enzyme, GS, is also
expressed by enteric glia but, interestingly, the majority of GS labeling was localized to
HuC/D-immunoreactive myenteric neurons (Figure 3.1C). This pattern of expression is
consistent with the known cellular distribution in the central nervous system, where
neurons express GSH synthesis enzymes and can synthesize GSH when supplied with
the dipeptide precursors, such as CysGly, from astrocytes21,30. These results show that
enteric glia express the enzymes necessary for GSH synthesis and further, suggest a
novel pathway where enteric neurons are capable of producing GSH.

In situ inhibition of GSH synthesis with L-Buthionine Sulfoximine decreases myenteric
neuronal density
Next, we tested the role of glial glutathione production in the maintenance of enteric
neuron survival by selectively blocking GCL activity with the specific inhibitor, LButhionine Sulfoximine (BSO, 100 μM)31. GCL is the rate-limiting enzyme in GSH
synthesis and the inhibition of GCL with BSO is sufficient to deplete GSH in vitro and in

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Figure 3.1: Expression of the glutathione synthesis enzymes, glutamate-cysteine
ligase and glutathione synthetase, in the enteric nervous system. Representative

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Figure 3.1 (cont’d)
mouse myenteric ganglia showing immunoreactivity for (A,B) the catalytic subunit of
glutamate cysteine ligase (GCLC; red) and (C) glutathione synthetase (GS, red). Enteric
neurons are labeled with the neuronal marker Hu C/D (blue), enteric glia with the glial
marker GFAP (green) and pre-synaptic vesicles with the vesicular marker
synaptophysin (blue). White arrowheads in the overlay image panel of A highlight areas
of co-localization between glutathione synthesis enzymes and GFAP. (scale bars = 20
μm).

100

vivo 18,24,32. In LMMP whole mount preparations, the inhibition of GCL decreased
myenteric neuron packing density by 14.1 ± 4% (1849 ± 75 neurons mm-2 in control vs.
1532 ± 62 neurons mm-2 in BSO-treated tissue; Figure 3.2A and 3.2B) within four
hours. Glutathione depletion drove neuron death to a similar extent as the activation of
P2X7 receptors (P2X7R) on enteric neurons with BzATP, a known mechanism of
enteric neuron death during inflammation (1532 ± 62 neurons mm-2 with BSO vs. 1559 ±
74 neurons mm-2 with BzATP) 4,7. Importantly, the effects of BSO and BzATP were not
additive (1334 ± 54 neurons mm-2), suggesting either that BSO and BzATP drive neuron
death through the same pathways or that they act through different neurotoxic
mechanisms in the same subpopulation of neurons.
We hypothesized that increased oxidative stress following BSO-inhibition of GCL
is the most likely mechanistic explanation for neuron death in our experiments.
However, BSO could also drive neuron death through direct neurotoxic effects on
enteric neurons. To differentiate between these two possibilities, we treated tissue with
γ-glutamyl-cysteine (γ-GC), the product of GCL, to “rescue” the depletion of GSH by
BSO. Treatment with γ-GC was protective against BSO-mediated neuron death (1889 ±
118 neurons mm-2 with γ-GC + BSO vs. 1532 ± 62 neurons mm-2 with BSO only, Figure
3.2A and 3.2B), indicating that BSO-mediated neuron death is primarily through direct
inhibition of GSH synthesis.
Treatment with BSO in vitro depletes cellular GSH, increases free radicals and
drives cellular oxidative stress 32,33. We measured neuronal oxidative stress in
myenteric preparations after in situ treatment with BSO and BzATP using a thiol
oxidation ratiometric analysis that quantifies the ratio of oxidized/reduced thiols.

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Figure 3.2: Glutathione (GSH) synthesis is required for enteric neuron survival.
(A) Representative images and (B) quantification of mean neuronal packing density in

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Figure 3.2 (cont’d)
mouse myenteric ganglia following inhibition of GSH synthesis with the GCLC inhibitor
L-Buthionine-sulfoximine (BSO, 100 μM). Whole mount preparations of myenteric
plexus were incubated with BSO in the presence, or absence, of the GCLC product γglutamyl-cysteine (γ-GC, 100 μM) or the P2X7 receptor (PRX7R) agonist BzATP (300
μM). Inhibition of BSO decreases neuronal density to the same extent as P2X7R
activation, while simultaneous treatment with γ-GC protects against BSO-mediated cell
death. (* P≤0.05, ** P≤0.005, **** P≤0.0001, One-Way ANOVA with Dunnett’s multiple
comparison test vs. control, n=4-16 myenteric preparations/group from 9 animals.)
Enteric neurons are labeled with HuC/D (blue) and enteric glia with GFAP (green) in all
panels. (scale bar = 30 μm). (C) Neuronal thiol oxidation measurements (ratio of
fluorescently labeled oxidized/reduced glutathione) from in situ myenteric preparations
treated with BSO (100 μM) in the presence or absence of BzATP (300 μm) (n=3-6
ganglia/group).

103

Surprisingly, in situ oxidative stress was not significantly increased despite the cell
death associated with BSO and BzATP treatment (Figure 3.2C). These results
demonstrate that in situ inhibition of glial GSH synthesis with BSO is neurotoxic to
myenteric neurons but does not alter neuronal thiol state.

In vivo inhibition of glutathione synthesis does not cause overt inflammation but is
protective against macroscopic measures of inflammation
To better understand the role of glial GSH during GI pathology, we treated mice ad
libitum with BSO in drinking water (20mM) for 14 days prior to induction of in vivo
inflammation with DNBS-colitis. This treatment paradigm decreases colonic GSH
content by 84% in adult mice 24, and liver GSH by 86% in pregnant rats 34, without any
toxic effects. In our experiments, BSO treatment produced no significant adverse effects
as evaluated by daily body weight measurements and observations of behavior and
appearance (Figure 3.3A). Half of BSO-treated mice developed preputial gland
abscesses around Day 7 of treatment (white and black arrows; Figure 3.3E). However,
all abscesses self-ruptured by Day 10 and mice were alert and in healthy condition prior
to DNBS treatment on Day 14. To determine whether there was an aversion to water
containing BSO, we measured daily fluid consumption in both treatment groups. Mice
drinking water containing BSO, consumed similar volumes of fluid daily, compared to
mice drinking normal H2O (Figure 3.3B). In fact, mice drinking BSO consumed slightly
more fluid (2.9mL H2O vs. 3.5 mL BSO in H2O /mouse/day).
Next, we induced inflammation with an enema of 2,4-dinitrobenzene sulfonic acid
(DNBS), while control mice received a saline enema. BSO consumption without

104

exposure to an inflammatory insult (Figure 3.3C, BSO-Saline, green solid line) did not
induce colonic inflammation as determined by weight loss (Body weight 2.6 ± 0.4 % and
2.4 ± 0.5% of control at days one and two, respectively; Figure 3.3C). Mice that
consumed normal drinking water prior to inflammation (H2O-DNBS, black dashed line)
lost significant body weight one and two days post-DNBS compared to H2O-Saline
controls (11.8 ± 1% at day one and 15.4 ± 0.9% at day two; Figure 3.3C). Similarly,
mice drinking BSO that received a DNBS enema (BSO-DNBS, green dashed line) also
lost significant body weight compared to H2O-Saline controls (8.5 ± 1.3% at day one
and 7.9 ± 2.4% at day two, Figure 3.3C). Importantly, BSO consumption prior to and
during inflammation was protective against inflammatory damage, as BSO-DNBS mice
lost significantly less weight than H2O-DNBS mice two-days post DNBS (7.9 ± 2.4% vs.
15.4 ± 0.9%; Figure 3.3C).
At sacrifice, mouse colons were collected and macroscopic damage score
assessed as an additional measure of inflammatory damage

23.

BSO consumption in

drinking water was not sufficient to increase macroscopic markers of inflammation
(Figure 3.3D; BSO-Saline). Macroscopic damage score was increased in DNBS-treated
mice, regardless of H2O or BSO consumption prior to DNBS treatment (gray bars,
Figure 3.3D). In agreement with the trend seen in body weight, BSO treatment prior to
DNBS was protective against increased colonic macroscopic damage (4.5 ± 0.7 and 2.5
± 0.5 in H2O-DNBS and BSO-DNBS respectively; Figure 3.3D). Collectively, these
results show whole body glutathione depletion does not induce overt colonic
inflammation. Rather, BSO treatment is protective against some key features of colitis in
mice.

105

Figure 3.3: Inhibition of glutathione synthesis alone is not sufficient to cause
overt inflammation but does alter susceptibility to inflammatory insults.
(A) Daily body weight of mice consuming either normal drinking water (H2O, black) or

106

Figure 3.3 (cont’d)
water containing the GCLC inhibitor L-Buthionine Sulfoximine (BSO at 20 mM , green)
for 2 weeks. (B) Average daily ad libitum fluid consumption for mice drinking normal
water (H2O) or water containing 20 mM BSO (*** p< 0.001, Student’s t-test). (C) Daily
percent weight loss and (D) macroscopic damage score of BSO treated mice (green
lines and gray bars) or their normal drinking water controls (H2O, black lines and bars)
after DNBS colitis (dashed lines in C, gray bars in D) or saline control treatment (solid
lines in C, black bars in D) (** P≤0.01, *** P≤0.005 and **** P≤0.0001 compared to H2O
Saline, # P≤0.05 compared to H2O DNBS via Two-way ANOVA with Tukey’s post-hoc
test; n= 6-9 animals/group). (E) External (white arrow) and internal (black arrow) view of
preputial gland abscess in BSO-treated mice on day of sacrifice.

107

Glutathione depletion decreases neutrophil infiltration during DNBS-colitis but does not
alter reactivity of MHC-II expressing macrophages or neuronal thiol oxidation levels
Next, we investigated how in vivo glutathione depletion affects microscopic markers of
inflammation. Increased immune cell infiltration is a key feature of many animal models
of colitis, including DNBS 1. In agreement with prior reports, we observed increased
infiltration of myeloperoxidase (MPO)-positive neutrophils into a 76,800 μm2 field-ofview area in the myenteric plexus in water drinking-inflamed animals (23.3 ± 10
neutrophils in H2O-DNBS vs. 0.1 ± 0.04 neutrophils in H2O-Saline, Figure 3.4A and
3.4B). The frequency of neutrophils within the myenteric plexus of healthy animals
drinking water supplemented with BSO was comparable to that of healthy animals
drinking water alone (0.2 ± 0.05 neutrophils in BSO-Saline, Figure 3.4A and 3.4B).
However, mice drinking BSO during the course of colitis exhibited significantly
attenuated myenteric plexus neutrophil infiltration compared to inflamed animals that did
not receive BSO (5.8 ± 2.5 neutrophils in BSO-DNBS vs. 23.3 ± 10 neutrophils in H2ODNBS; Figure 3.4A and 3.4B).
Next, we measured changes in major histocompatibility complex class II (MHC II)
immunoreactivity to quantify the reactivity of antigen-presenting cells in a 76,800 μm2
area surrounding individual myenteric ganglia. This population of cells predominantly
consists of muscularis macrophages which reside at the level of the myenteric plexus in
the colonic wall 35,36. Colonic inflammation increased the immunoreactivity of MHC IIexpressing muscularis macrophages in the myenteric plexus (Figure 3.4C). MHC-II
expression was also induced by glutathione depletion as both inflamed (DNBS) and

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Figure 3.4: Effects of glutathione depletion on neutrophil infiltration into the
myenteric plexus, MHC II-positive muscularis macrophage infiltration and
neuronal thiol oxidation levels during DNBS colitis in mice.
(A) Representative images and (B) quantification of the infiltration of myeloperoxidasepositive neutrophils (MPO, magenta) in myenteric ganglia of mice drinking normal water
(H2O) or water with 20mM BSO prior to induction of colitis with DNBS (gray bars) or
saline control (black bars). Enteric glia are labeled with GFAP (green). (*** P≤0.005
compared to H2O Saline, # P≤0.05 compared to H2O DNBS via Two-Way ANOVA with
Tukey’s post-hoc test; n=3-6 animals/group). (C) Fluorescence of major
histocompatibility complex class II (MHC-II)-immunoreactive muscularis macrophages in

109

Figure 3.4 (cont’d)
mouse myenteric plexus after DNBS-colitis (gray bar) or saline control animals (black),
drinking either normal H2O or water containing 20mM BSO. (Two-Way ANOVA with
Dunnett’s multiple comparison test, n=3 animals/group). (D) Neuronal ganglionic
oxidation measurements (ratio of fluorescently labeled oxidized/reduced glutathione)
(n=3-6 ganglia/group).

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control (Saline) animals that consumed BSO showed a non-significant increase in
ganglionic MHC II expression (Figure 3.4C). In agreement with our in situ data (Figure
3.2C), neuronal thiol oxidation state was not significantly altered following induction of
colonic inflammation (H2O-DNBS) or depletion of cellular glutathione in normal and
inflamed animals (BSO-Saline and BSO-DNBS; Figure 3.4D). Together, these results
show that BSO treatment attenuates the infiltration of neutrophils during DNBS-colitis
but does not significantly affect the infiltration or reactive state of MHC II-positive cells or
neuronal thiol oxidation levels.

In vivo inhibition of glutathione synthesis decreases neuronal density and alters
myenteric plexus neurochemical coding
Next, we assessed how in vivo depletion of glutathione affects myenteric neuron density
and neurochemical coding. In agreement with our prior published findings

4,7,

DNBS-

colitis decreased neuronal density by 19 ± 7% in animals drinking normal water (1751 ±
155 neurons mm-2 in H2O-Saline vs. 1421 ± 127 neurons mm-2 in H2O-DNBS animals;
Figure 3.5A and 3.5B). In vivo glutathione depletion with BSO decreased HuC/Dimmunoreactive neuron density to a similar extent as in vivo inflammation (1409 ± 78
neurons mm-2 in BSO-Saline animals). Consistent with our in situ results (Figure 3.2B),
glutathione depletion during DNBS administration does not potentiate neuron loss
during colitis (1647 ± 111 neurons mm-2 in BSO-DNBS mice; Figure 3.5A and 3.5B).
Glutathione depletion is known to reduce levels of inhibitory neurochemicals in
gastrointestinal tissue 37. However, how glutathione depletion specifically alters
neurochemical coding within myenteric ganglia is not known. We used

111

immunohistochemical markers for specific classes of myenteric neurons to investigate
how neurochemical coding is altered in myenteric ganglia after glutathione depletion.
Neither glutathione depletion nor inflammation altered the percentage of calretininpositive excitatory neurons in healthy animals (19 ± 1.5% in H2O-Saline, 21 ± 2.8% in
H2O-DNBS and 25.5 ± 1.7% in BSO-Saline; Figure 3.5C and 3.5D). In contrast,
glutathione depletion caused the percentage of calretinin-positive neurons to decrease
by approximately 10% during inflammation (25.5 ± 1.7 % in BSO-Saline vs. 14.4 ± 1.2
% in BSO-DNBS; Figure 3.5C and 3.5D). Conversely, glutathione depletion did not
significantly affect the percentage of nNOS-positive inhibitory neurons in healthy or
inflamed animals (33 ± 3% in H2O-Saline vs. 38 ± 2.5% in BSO-Saline vs. 39 ± 2% in
BSO-DNBS; Figure 3.5E and 3.5F). Together, these results show that glutathione is
necessary to maintain both the phenotype and survival of neurons in the myenteric
plexus of the colon.

Inflammation alters the expression of the glutathione synthesis enzymes, glutamatecysteine ligase and glutathione synthetase, in the ENS
Finally, we investigated how in vivo inflammation affects the localization and expression
of glutathione synthesis enzymes. DNBS-colitis did not alter the localization of either
GCLC or GS. GCLC expression remained localized to enteric glia and pre-synaptic
neuronal vesicles (Figure 3.6A) and GS expression remained localized to glial cells and
myenteric neuron cell bodies (Figure 3.6C). However, both in vivo inflammation and
BSO treatment altered the expression levels of GCLC and GS. In vivo

112

Figure 3.5: Inhibition of glutathione synthesis in vivo decreases enteric neuron
survival and alters myenteric plexus neurochemical coding.
(A) Representative images and (B) mean-packing density of HuC/D-immunoreactive
113

Figure 3.5 (cont’d)
myenteric neurons (blue) from mice consuming normal drinking water (H2O) or water
with 20mM BSO prior to DNBS (gray bars) or saline (black bars) enema administration.
Enteric glia are labeled with GFAP (green). (C, E) Representative images showing
excitatory (Calretinin, gray; C) and inhibitory (nNOS – neuronal nitric oxide synthase,
gray; E) neuronal sub-populations in myenteric ganglia. (D, F) Percentage of HuC/Dpositive neurons that are immunoreactive for Calretinin (D) and nNOS (F) in mice
drinking water (H2O) or water with 20mM BSO prior to DNBS (gray bars) or saline (black
bars) enema administration. Myenteric neurons are labeled with the pan-neuronal
marker Hu-C/D (blue), excitatory neurons with Calretinin (gray) and inhibitory neurons
with nNOS (gray). (n = 4-9 animals/ group, ***P≤0.05 Two-Way ANOVA with Tukey’s
post-hoc test), (scale bars = 20 μm).

114

inhibition of glutathione synthesis with the GCLC-inhibitor BSO (20 mM) decreased
ganglionic GCLC expression by 36% (Mean fluorescence: 267 ± 18 in H2O-Saline vs.
170 ± 46 in BSO-Saline; Figure 3.6B). This result supports well-established data
showing that cellular GSH concentrations regulate GCLC activity and expression via a
negative feedback loop 38,39. BSO-treated healthy animals also show a ~40% decrease
in expression of the second glutathione synthesis enzyme GS (Mean fluorescence: 284
± 19 in H2O-Saline animals vs. 168 ± 48 in BSO-Saline animals; Figure 3.6D).
In vivo inflammation doubled the ganglionic expression of GCLC in both normal
and BSO-treated animals (Mean fluorescence: 267 ± 18 vs. 555 ± 65 in H2O Saline vs.
DNBS, 170 ± 46 vs. 386 ± 79 in BSO Saline vs. DNBS; Figure 3.6A and 3.6B).
Similarly, GS expression increased ~200% following inflammation in both normal
animals and animals with depleted glutathione (Mean fluorescence: 285 ± 19 vs. 581 ±
46 in H2O Saline vs. DNBS animals, 169 ± 48 vs. 372 ± 83 in BSO Saline vs. DNBS
animals; Figure 3.6D). These results demonstrate a dynamic relationship between in
vivo inflammation and regulation of glutathione synthesis within the ENS.

115

Figure 3.6: Inflammation alters the expression of the glutathione synthesis
enzymes, glutamate-cysteine ligase and glutathione synthetase, in the enteric
nervous system. Representative images (A,C) and corresponding quantification (B,D)
of immunoreactivity for the catalytic subunit of glutamate-cysteine ligase (A,B; GCLC,
red), the rate-limiting enzyme in glutathione (GSH) synthesis, or glutathione synthetase
(C,D; GS, red) in mouse myenteric ganglia from healthy (saline) or inflamed (DNBS)
mice drinking either normal water (H2O) or water supplemented with BSO (20 mM;
BSO). (* P≤0.05 compared to H2O-Saline via Two-Way ANOVA with Tukey’s post-hoc
analysis), (n=7-9 animals/group).

116

Discussion
Our study investigates the role of the endogenous antioxidant reduced
glutathione (GSH) in the ENS and probes how enteric glial GSH contributes to
neuroprotection during in situ and in vivo neuroinflammation. Our findings show that
glutathione is necessary to support neuronal survival and phenotype in the gut.
However, the systemic inhibition of glutathione synthesis in vivo is neuroprotective
during colitis, possibly by limiting the infiltration of immune cells.
Glutathione and its derivatives are well known for their neuroprotective effects in
the ENS. Cultured enteric glia produce and secrete reduced glutathione, which is
neuroprotective to enteric neurons in the myenteric plexus12. In the mucosa, the glial
derived s-nitrosoglutathione (GSNO) helps maintain mucosal barrier function and
promotes tight junction formation13. However, no work to date has directly shown that
enteric glia express the enzymes necessary for glutathione synthesis. Our data here
show that the enteric glia express the molecular machinery necessary for glutathione
synthesis in vivo. These findings provide important support for previous work that
measured the release of glutathione and its derivatives from cultured enteric glia12,13.
The expression pattern of the glutathione synthesis enzymes in the ENS is
similar to that in the central nervous system, where astroglia are primarily responsible
for the production and secretion of neuroprotective GSH19,40. An unexplored hypothesis
in the ENS was the ability of enteric neurons to also produce GSH. In the brain, neurons
express the GSH synthesis enzymes, albeit at much lower levels and are capable of
producing limited quantities of GSH30. In addition, neurons rely heavily on astrocytes to
provide key precursor molecules necessary for GSH synthesis16,21. Our findings show a

117

similar relationship in the ENS where enteric neurons express GCLC and GS in
neuronal varicosities and cell bodies, respectively. This suggests that enteric neurons
have the ability to produce GSH and we hypothesize a novel neuroglia relationship
similar to that in the CNS in regards to GSH synthesis and release.
Our data show that glutathione is necessary to support neuronal survival
because the inhibition of glial GSH synthesis with the selective GCL inhibitor BSO
decreased myenteric neuron density in acutely isolated tissues. One possibility is that
BSO could produce direct neurotoxic effects on enteric neurons independent on its
inhibition of glutathione synthesis. However, this is unlikely because other reports of in
vitro BSO treatment show decreased GSH content in concurrence with cell death 18,33.
Further, treatment with GSH18 or its precursors was sufficient to protect against BSOmediated cell death14 (Figure 3.2B), suggesting that the cytotoxic effects of BSO are
directly related to its inhibition of GSH synthesis.
Glutathione depletion is reported to increase cellular oxidative stress and cellular
susceptibility to oxidative damage32. However, neuronal thiol oxidation (a measure of
cellular oxidative stress) was not increased in our live whole-mount preparations
following treatment with BSO, suggesting that in situ inhibition of GSH synthesis does
not affect cellular thiol state. A number of factors may explain this finding. Firstly, the
thiol oxidation ratiometric analysis quantifies the oxidation states of all cellular thiols
inclusive of, but not limited to, cellular glutathione. GSH depletion, while decreasing total
thiol levels, may not alter the state of other proteinaceous thiols. In our treatment
paradigm, live whole-mounts are incubated with BSO for 2h followed by a 2h incubation
in buffer only. BSO has been shown to dissociate from 1-3% of bound GCL enzyme/

118

hour when a BSO treatment solution is replaced with normal buffer 38. Thus, it is also
possible that dissociation of BSO during the second 2h incubation is sufficient to return
cellular thiol levels to normal, but not to reverse the cytotoxicity and cell death induced
by the 2h incubation with BSO.
GSH is synthesized in two-step process catalyzed by the rate-limiting GCL and
GS. Regulation of GCL and GS activity and expression are important mechanisms in
the regulation of cellular glutathione content41. In fact, GSH synthesis is controlled by a
negative feedback loop where GSH acts as a non-allosteric inhibitor on the rate-limiting
GCL enzyme38,39. Similarly, GCL transcription and protein expression are induced by
increased oxidative stress42,43. We observed this in our studies where increased
oxidative stress during colitis increased expression of both GCL and GS. Increased
expression of both enzymes is reported to significantly increase cellular GSH capacity
and is thus an expected result in response to GI inflammation44,45.
A primary goal of our study was to investigate the effects of in vivo inhibition of
GSH synthesis and how GSH depletion affects macro and microscopic damage during
inflammation. To this end, we administered 20 mM BSO in vivo via drinking water for 14
days prior to the induction of inflammation. This treatment paradigm has been
previously well characterized as a suitable animal model for tissue GSH depletion
without cytotoxicity24. In support, we observed few adverse effects in healthy BSOtreated animals throughout the treatment period or at the time of sacrifice. A proportion
of animals did develop preputial gland abscesses, a common condition seen in nonbreeder male mice46. If severe, this ailment requires surgical rupturing and antibiotic
treatment. However, our mice developed mild abscesses that self-ruptured and did not

119

significantly affect animal health and welfare. Specifically, depletion of GSH did not
decrease body weight, increase macroscopic colon damage or increase the infiltration
of neutrophils in otherwise healthy animals.
Reduced glutathione is the primary endogenous antioxidant in the body and is an
important cellular tool in protection against oxidative stress47. Typically, antioxidant
depletion increases cellular oxidative stress and increases cell susceptibility to
exogenous toxicants including oxidative stressors41. However, our results show that
decreased glutathione is protective during in vivo inflammation. Specifically, GSH
depletion prior to inflammation decreases colonic macroscopic damage score and
neutrophil infiltration, partially protects against colitis-associated weight loss and
prevents additional loss of myenteric neurons. One explanation of our findings is that
GSH supports glial cells in a pathogenic state and that decreasing glial GSH is
protective against glial activation and the subsequent neuron death. GSH serves a
similar unexpected function in central nervous system gliomas. In this disease,
increased glioma GSH synthesis is associated with the glutamate excitotoxicity
responsible for many glioma symptoms48. In support, inhibition of the
cysteine/glutamate System xc- transporter on glioma cells with sulphasalazine, a system
xc- inhibitor currently used to treat Crohn’s disease patients49, decreases cellular GSH
synthesis and protects against symptoms50. Thus, decreased glial synthesis of the
normally protective GSH may have therapeutic benefits in some pathogenic conditions.
The infiltration of immune cells is an important aspect of colitis pathology1. An
alternate hypothesis for our findings is that the protective effects of GSH depletion are
mediated via altered immune cell behavior. In some bacterial infections, decreased

120

neutrophil GSH content is associated with reduced neutrophil cytokine production and
decrease migration51. This aligns with our own finding showing decreased neutrophil
infiltration into the myenteric plexus of animals with depleted GSH prior to inflammation.
In conclusion, our findings show evidence for both enteric glial and neuronal
production of GSH in the ENS. Further, we demonstrate that in situ GSH synthesis is
necessary for neuroprotection and present a novel hypothesis where in vivo GSH
depletion is protective against inflammation. These observations present a potential
therapeutic target for improved GI pathology during inflammation.

121

APPENDIX

122

Antibody

Dilution

Source

Catalog No.

Chicken anti-GFAP

1:1000

Abcam, Cambridge, MA

ab4674

Biotinylated mouse anti-human

1:200

Invitrogen

A21272

Rabbit anti-GCLC

1:200

Abcam

ab190685

Rabbit anti-GS

1:200

Abcam

ab133592

Rabbit anti-MPO

1:200

Abcam

ab9353

Rat anti-MHC II

1:200

Novus

NBP2-21789

Goat anti-Calretinin

1:1000

Swant, Switzerland

CG1

Sheep anti-nNOS

1:500

Millipore, Darmstadt,

ab1529

HuC/D

Germany
Rabbit anti-synaptophysin

1:2000

Abcam

ab32127

Table 3.1: Primary Antibodies used in Chapter 3.
** GFAP, glial fibrillary acidic protein; GCLC, glutamate cysteine ligase catalytic subunit; GS, glutathione
synthetase; MPO, myeloperoxidase; MHC II, major histocompatibility complex II; nNOS, neuronal nitric
oxide synthase

123

Antibody

Source

Catalog No.

Streptavidin Dylight 405

Jackson

016-470-084

Goat anti-Chicken Alexa 488

Invitrogen

A-11039

Donkey anti-Goat Alexa 488

Jackson

705-545-003

Donkey anti-Sheep Alexa 488

Jackson

713-545-003

Goat anti-Rabbit Alexa 568

Invitrogen

A-11036

Streptavidin Alexa 594

Jackson

016-580-084

Table 3.2: Secondary Antibodies used in Chapter 3.

124

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125

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CHAPTER 4
The effect of disrupted T cell TGFβ signaling on myenteric plexus
structure and function

131

Abstract
Transforming growth factor beta (TGFβ) is a key anti-inflammatory cytokine that is
important for the regulation of immune homeostasis in a variety of tissues. Mice with
disrupted TGFβ signaling in CD4+ and CD8+ T cells (CD4-dnTGFβRII) develop severe
mucosal inflammation in the gastrointestinal (GI) tract. However, the alterations within
the myenteric plexus, which controls GI motility, are less understood. We used
immunohistochemistry and in vivo colonic motility assays to measure changes in female
CD4-dnTGFβRII mice. Disruption of T cell TGFβ signaling delayed colonic motility and
induced “reactive gliosis” in enteric glia, but did not alter myenteric plexus neuronal
density or neurochemical coding. Innate, but not adaptive, immune homeostasis was
altered in the myenteric plexus of CD4-dnTGFβRII mice. In a novel result, we find that
enteric neurons express the type II TGFβ receptor. Together, these findings
demonstrate that TGFβ signaling in CD4+ and CD8+ T cells regulates immune
homeostasis and function within the myenteric plexus.

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Introduction
Transforming growth factor beta (TGFβ) is a critical regulatory cytokine that
maintains immune homeostasis in a majority of tissues. TGFβ signaling requires
activation of the TFGβ receptor (TGFβR), a tetramer consisting of homodimers of the
Type I (TGFβRI) and type II (TGFβRII) receptor subtypes. Binding of TGFβ to TGFβRII
catalyzes the formation of the receptor tetramer, which then functions as a
serine/threonine kinase. TGFβ signaling ultimately results in the phosphorylation of
Smad proteins that then translocate to the nucleus, altering gene transcription and
resulting in the characteristic features associated with TGFβ signaling.
TGFβ consists of three isoforms (TGFβ1, 2, and 3) whose signaling is crucial for
normal fetal development. TGFβ2 knockout mice are embryonic lethal while TGFβ1 null
mice have a mixed phenotype where ~ 50% die by embryonic day 14 1,2. The remaining
50% surviving mice develop multi-organ autoimmune syndrome2. A similar phenotype is
also observed when TGFβ signaling is disrupted in CD4 and CD8 positive T cells by the
expression of a dominant-negative form of TGFβRII (dnTGFβRII)3. Specifically, CD4dnTGFβRII mice have prominent gastrointestinal (GI) inflammation characterized by
immune cell infiltration and disruption of the normal architecture of intestinal crypts3.
Here, we investigate how disrupted TGFβ signaling in CD4-dnTGFβRII mice affects GI
motility and immune homeostasis at the level of the enteric nervous system – the
intrinsic neural network that controls GI function.
Using female CD4-dnTGFβRII mice and their wild-type (WT) littermates, we
show that disrupted T cell TGFβ signaling alters distal colon function without changes to
myenteric plexus neuronal density or neurochemical coding. CD4-dnTGFβRII mice have

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altered myenteric plexus immune homeostasis with increased infiltration/ activation of
monocytes and macrophages, but no changes to B cell expression or immunoreactivity
of the key pro-inflammatory cytokine IL-1β. Disrupted TGFβ signaling in T cells resulted
in “reactive gliosis” of enteric glia, a hallmark phenotypical change observed in response
to inflammation or cellular injury. Finally, we show that enteric neurons express the
TGFβRII, the first demonstration of TGFβ receptor expression in the ENS. In
conclusion, we demonstrate that disrupted TGFβ signaling in CD4+ and CD8+ T cells is
sufficient to alter immune homeostasis and function within the myenteric plexus.

Materials and Methods

Animals
Experimental protocols were reviewed and approved by the Institutional Animal Care
and Use Committee (IACUC) at Michigan State University. Transgenic dominant
negative transforming growth factor beta receptor type II mice (dnTGFβRII) expressing
a truncated TGFβRII in CD4+ and CD8+ positive T cells were 1) generated in house by
crossing C57Bl/6 x CD4-dnTGFBRII+/- or 2) purchased as hemizygotes from The
Jackson Laboratory (Bar Harbor, ME) (B6.Cg-Tg(Cd4-TGFBR2)16Flv/J a.k.a. CD4dnTGFBRII; Stock No. 005551). Wild-type (WT) littermates were used as experimental
controls and all experiments used female mice aged 30-32 weeks. Mice were
maintained in a temperature controlled 12 h light: 12 h dark cycle environment with ad
libitum access to food and water.

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Distal Colon Motility (Colon Bead) Assay
Mice were lightly anesthetized using isoflurane and a 2mm glass bead inserted 2cm into
the distal colon. Mice were then individually housed and the time to bead expulsion
recorded as a measure of distal colon transit time.

Whole-mount Immunohistochemistry (IHC)
Whole-mount longitudinal muscle myenteric plexus (LMMP) preparations were
dissected from colon tissue fixed overnight in Zamboni’s fixative. LMMP preps were
permeabilized by 3x10 minute washes in 0.1% Triton X-100 in phosphate buffered
saline (PBS) followed by a 45 minute block at room temperature (RT) in 4% normal
serum containing 4% goat/donkey serum, 0.4% Triton X-100, and 1% bovine serum
albumin. Preparations were incubated in primary antibodies (Table 4.1, appendix)
overnight at RT, followed by a 2 hour incubation at RT in secondary antibodies (Table
4.2, appendix). Images were acquired using the 40X objective (Plan Fluor, 0.75
numerical aperture) or 20X objective (Plan Apo, 0.75 numerical aperture) of an upright
epifluorescence microscope (Nikon Eclipse Ni, Melville, NY, USA) with a Retiga 2000R
camera (QImaging, Surrey, BC, Canada) controlled by QCapture Pro 7.0 software
(QImaging) or by confocal imaging through the Plan-Apochromat 60X oil immersion
objective (1.42 numerical aperture) of an inverted Olympus Fluoview FV1000
microscope (Olympus, Center Valley, PA).

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Scoring of Colonic Macroscopic Damage
Colonic macroscopic damage was quantified using a well established scoring system as
described by Storr et al.4. Colon length and thickness were scored with greater points
for a shortened and/or thickened colon. The severity of adhesion of the colon to other
organs was scored as 0,1, or 2 based on the severity of adhesion and the presence of
fecal blood, diarrhea and/or hemorrhage scored at 1 point/feature.

Data and Statistical Analysis
Ganglionic neuronal density was calculated by quantifying the number of HuC/Dimmunoreactive neurons per GFAP-positive myenteric ganglia in at least 10 ganglia/
animal using the cell counter tool in ImageJ. Average ganglionic fluorescence was
calculated using the measure tool in ImageJ and expressed in arbitrary fluorescence
units (AFU). Subpopulations of calretinin-positive (excitatory) and nNOS-positive
(inhibitory) neurons were calculated by quantifying the number of calretinin/ nNOS
positive neurons as a percentage of total HuC/D myenteric neuronal population.
All data was analyzed by a student’s t-test using Graphpad Prism 7 software (La Jolla,
CA, USA). All data are presented as a mean ± the standard error of the mean (SEM).

Results
Mice with disrupted T cell TGFβRII signaling have increased distal colon transit but no
overt markers of colonic inflammation
Transforming growth factor beta (TGFβ) and its downstream signaling pathways have
long been recognized for their role in regulating a variety of cellular behaviors including

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cell proliferation and differentiation5,6. In addition, TGFβ signaling is necessary for the
proper maintenance of immune homeostasis7 and animals with disrupted TGFβ
signaling develop diseases with a key autoimmune component8-10. Here, we investigate
how disrupted TGFβ signaling in T cells affects immune homeostasis and inflammation
within the gastrointestinal (GI) tract and specifically, within the enteric nervous system
(ENS) - the neural network that controls GI function.
To this end, we used CD4-dnTGFβRII mice (see Methods) that express a
truncated dominant-negative form of the human TGFβ type II receptor (dnTGFβRII) in
CD4+ and CD8+ T-cells (Figure 4.1). This expression is sufficient to disrupt TGFβ
signaling in only T cells, while maintaining normal TGFβ signaling in other immune cells
such as B cells3. dnTGFβRII mice develop an inflammatory bowel disease (IBD)-like
phenotype characterized by immune cell infiltration at the level of the mucosa, and
disruption of normal intestinal crypt morphology3. However, how colonic motility and
gross macroscopic damage were altered in these mice was not investigated.
We investigated changes in colonic motility in dnTGFβRII by measuring distal
colon transit time using the colon bead assay, which records the time to expulsion of a
glass bead from the distal colon. Transgenic dnTGFβRII mice had increased distal
colon transport time (49 ± 9 seconds in WT vs. 154 ± 39 seconds in dnTGFβRII mice;
Figure 4.2A), suggesting that disruption of T cell TGFβ signaling impairs normal colonic
function. Next, we measured changes in gross colonic macroscopic damage using a
scoring system described by Storr et al.4. Disruption of TGFβ signaling in T cells did not
significantly increase total colonic macroscopic damage (0.18 ± 0.005 in WT vs. 1.33 ±
0.55 in dnTGFβRII mice; Figure 4.2B). Because of the anti-proliferative effects of TGFβ

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Figure 4.1: dnTGFβRII mice have disrupted TGFβ signaling in CD4 and CD8
positive T cells compared to wild-type (WT) mice. Schematic representing
downstream signaling after activation of TGFβRI(I) receptors in wild-type (WT) and
dominant negative transgenic mice (dnTGFβRII) which feature a truncated TGFβ type II
receptor in CD4+ and CD8+ T cells.

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Figure 4.2: dnTGFβRII mice have increased distal colon transport time but no
significant changes in macroscopic measures of inflammatory damage. (A) Distal
colon transport time, (B) total macroscopic damage score, (C) total colon length and (D)
distal colon thickness in wild-type (WT, black bars) and transgenic dnTGFβRII (gray
bars) mice. (n = 2-6 mice per group, analyzed by student’s t-test).

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signaling5,11,12, we hypothesized that disrupted T cell TGFβ signaling may alter
macroscopic features that are dependent on changes in cell proliferation, such as colon
length and thickness. Disrupted T cell TGFβ signaling did not significantly alter colon
length (7.5 ± 0.07 cm in WT vs. 6.7 ± 0.4 cm in dnTGFβRII mice; Figure 4.2C). Distal
colon bowel thickness was doubled in dnTGFβRII mice doubled compared to WT mice
(0.18 ± 0.005 mm in WT vs. 0.32 ± 0.05 mm in dnTGFβRII mice; Figure 4.2D).
However, this measure was variable and did not reach statistical significance. Together,
these results show that disrupted T cell TGFβ signaling alters distal colon motility
without driving gross macroscopic inflammation of the colon.

Expression of a dominant negative TGFβRII on CD4+ and CD8+ T cells does not alter
myenteric plexus neuronal density or neurochemical coding
Colonic motility is controlled by the myenteric plexus of the enteric nervous system
(ENS)13. Inflammation drives gut dysfunction, in part, by altering the survival,
phenotype, and functions of enteric neurons14,15. Therefore, we hypothesized that
changes in neuronal density and/or neurochemical composition contribute to altered
motility in dnTGFβRII mice. We investigated this using immunohistochemistry to
quantify the changes in myenteric plexus neuronal density and neurochemical coding
caused by disrupted T cell TGFβ signaling. The packing density of HuC/Dimmunoreactive myenteric neurons was not significantly different in dnTGFβRII mice
(1809 ± 141 neurons mm-2 in WT vs. 1954 ± 49 neurons mm-2 in dnTGFβRII mice;
Figure 4.3A’-A’’’). Similarly, disruption of T cell TGFβ signaling in our dnTGFβRII mice
did not significantly alter neurochemical coding in myenteric neurons (Figure 4.3B’-

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C’’’). Myenteric ganglia in WT and dnTGFβRII mice contained similar percentages of
calretinin-positive excitatory neurons (36 ± 8 % in WT vs. 37 ± 2 % in dnTGFβRII mice;
Figure 4.3B’-B’’’) and nNOS-positive inhibitory neurons (52 ± 4 % in WT vs. 46 ± 2 %
in dnTGFβRII mice; Figure 4.3C’-C’’’). These results show that disrupted T cell TGFβ
signaling causes motility dysfunction without significantly altering myenteric neuronal
survival or neurochemical composition.

Disrupted TGFβRII signaling induces reactive gliosis in the myenteric plexus enteric glia
Next, we investigated how disrupted T cell TGFβ signaling affects other aspects of
ganglionic structure and function in the myenteric plexus. In addition to enteric neurons,
myenteric ganglia also contain enteric glial cells, a unique class of peripheral glia that
are being increasingly recognized for their active roles in regulating neuronal survival
and function, and GI motility16,17. Like their central nervous system counterparts,
peripheral glia undergo cellular and morphological changes, a process known as
“reactive gliosis”, in response to environmental changes and stressors18. We used
immunohistochemistry to measure whether enteric glia became “reactive” in response
to disrupted TGFβ signaling in T cells. Disrupted TGFβ signaling in dnTGFβRII mice
increased ganglionic expression of the glial cell marker glial fibrillary acidic protein
(GFAP) in enteric glia (Average fluorescence: 663 ± 55 AFU in WT vs. 918 ± 61 in
dnTGFβRII; Figure 4.4A’-A’’’). Increased GFAP fluorescence is a hallmark of reactive
gliosis in the central and peripheral nervous systems19,20 and thus, these results
highlight a relationship between T cell TGFβ signaling and glial reactivity state.

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Figure 4.3: Disrupted TGFβRII signaling does not alter myenteric neuronal
density or neurochemical coding. (A’-A’’’) Representative images (A’-A’’) and mean
packing density (A’’’) of Hu C/D-immunoreactive neurons (magenta) in myenteric
ganglia in WT (black bars) and dnTGFβRII (gray bars) mice. (B’-C’’’) Representative
images (B’-B’’ and C’-C’’) and quantification (B’’’ and C’’’) of calretinin-positive
excitatory neurons (B’-B’’, gray) and nNOS-positive inhibitory neurons (C’-C’’, gray) in
myenteric ganglia. Total neuronal populations are labeled with the pan-neuronal marker
Hu C/D (magenta). (n=10 ganglia in 5-9 animals/group, analyzed by student’s t-test,
scale bar = 30 μm).

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Figure 4.4: Disrupted TGFβRII signaling induces reactive gliosis in the myenteric
plexus. Representative images (A’ and A’’) and quantification (A’’’) of ganglionic glial
fibrillary acidic protein (GFAP; green) fluorescence in WT (black bar) and dnTGFβRII
(gray bar) mice. (n=3 animal/group, *P<0.05 by student’s t-test, scale bar = 30 μm).

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dnTGFβRII mice have increased activation of MHC-II positive muscularis macrophages
but no change in ganglionic IL-1β expression
T cells play an important role in maintaining immune homeostasis as the appropriate
activation and differentiation of T cells is a key step in our body’s immune response 21,22.
Specifically, T cell mediated cytokine release is instrumental in determining the type and
extent of immune response that is initiated after detection of an antigen. Thus, we next
investigated whether disruption of T cell TGFβ signaling affected the expression and
activation of immune cells in the ENS and the cytokine expression profile within the
myenteric plexus.
We measured changes in the immunoreactivity of major histocompatibility
complex class II (MHC-II)-expressing antigen presenting cells in a total of ten regions of
interest (area = 307,200 μm2) surrounding 2-3 GFAP-positive myenteric ganglia per
mouse. This population of cells includes resident muscularis macrophages that inhabit
the muscle layer adjacent to the myenteric plexus23, but may also include infiltrating
macrophages from outside the plexus24. Macrophages at the level of the myenteric
plexus of transgenic dnTGFβRII mice had increased MHC-II immunoreactivity (Average
fluorescence: 80 ± 28 AFU in WT vs. 375 ± 86 AFU in dnTGFβRII mice; Figure 4.5A’A’’’). However, it is unknown whether this increased expression is due to increased
infiltration of macrophages from outside the plexus or increased activation of resident
muscularis macrophages.
TGFβ plays a key role in regulating the production of key pro- and antiinflammatory cytokines7. Thus, we next investigated how disrupted T cell TGFβ
signaling affects myenteric plexus expression of the pro-inflammatory cytokine

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interleukin 1β (IL-1β). WT mice with normal TGFβ signaling express IL-1β primarily in
enteric neurons (Figure 4.5B’). Disrupted T cell TGFβ signaling in transgenic
dnTGFβRII mice did not significantly alter ganglionic IL-1β expression levels or
localization (Average fluorescence: 228 ± 40 AFU in WT vs. 206 ± 53 AFU in
dnTGFβRII mice; Figure 4.5B’-B’’’).
We also used immunohistochemistry to investigate changes in the infiltration of
myeloperoxidase (MPO)-positive neutrophils in dnTGFβRII mice. However, neither WT
nor dnTGFβRII mice were immunoreactive for MPO within the myenteric plexus (data
not shown), suggesting that disrupted TGFβ does not significantly increase neutrophil
infiltration, a hallmark of colonic inflammation14. Together, these results show that
disrupted T cell TGFβ signaling increased myenteric plexus MHC-II expression but does
not alter the expression or localization of IL-1β or the infiltration of MPO-positive
neutrophils.

Mice with disrupted TGFβRII have dysregulated innate and adaptive immune responses
at the level of the myenteric plexus
Cluster of different (CD) markers are commonly used to identify particular subclasses of
immune cells. We used immunoreactivity for two such markers to investigate how
disrupted TGFβ signaling altered the infiltration of immune cell subtypes into the
myenteric plexus. We first measured changes in CD43 expression in a total of ten
regions of interest/mouse (area = 307,200 μm2), each containing 2-3 GFAP-positive
myenteric ganglia. CD43, the common lymphocyte antigen, is a surface protein
expressed on multiple immune cells and its expression is commonly used to identify

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Figure 4.5: dnTGFβRII mice have increased activation of MHC-II positive
muscularis macrophages but no changes in IL-1β immunoreactivity.
(A’-A’’) Expression and (A’’’) quantification of major histocompatibility complex class II
(MHC-II) fluorescence (magenta) in a 307,200 μm2 field of view surrounding myenteric
ganglia labeled with GFAP (green) in WT (A’, black bar) and dnTGFβRII (A’’; gray bar)
mice. (B’-B’’’) Ganglionic IL-1β expression (B’-B’’; magenta) and quantification (B’’’) in
WT (black bar) and transgenic (gray bar) mice. (n=3-9 animals/group, *P<0.05 by
student’s t-test). (scale bar = 60 μm for A’-A’’ and 30 μm for B’-B’’).

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populations of T and B lymphocytes25. WT mice had negligible CD43 expression in the
myenteric plexus (Figure 4.6A’). In dnTGFβRII mice, mean CD43 immunoreactivity
increased by almost 500% (Average fluorescence: 6 ± 3.4 AFU in WT vs. 29 ± 10 in
dnTGFβRII mice; p=0.2086; Figure 4.6A’-A’’’). However, this increase did not achieve
statistical significance (p=0.2086), likely due to the wide variability of expression levels
measured in individual animals (Figure 4.6A’’’).
Secondly, we investigated how disrupted TGFβ altered the expression and/or
infiltration of CD68-positive monocytes and macrophages into the myenteric plexus. WT
mice with normal TGFβ signaling had a small population of CD68-positive
monocytes/macrophages surrounding GFAP-positive enteric glia (Figure 4.6B’).
Disruption of T cell TGFβ signaling increased myenteric plexus CD68 immunoreactivity,
indicative of increased infiltration of CD68-positive macrophages and/or increased
activation of resident CD68-positive macrophages (Average fluorescence: 131 ± 16
AFU in WT vs. 213 ± 21 in in dnTGFβRII mice; Figure 4.6B’-B’’’). Further, in WT mice
CD68 expression was restricted to the extraganglionic space while dnTGFβRII mice had
CD68 positive cells both surrounding and within enteric ganglia. Importantly, this
increase in CD68 immunoreactivity supports our earlier result of increased macrophage
infiltration/activation as measured by immunoreactivity of MHC-II positive macrophages
(Figure 4.5A’’’). Taken together, our study of CD markers in the myenteric plexus
suggests that disrupted TGFβ signaling causes a variable adaptive immune response,
as measured by CD43 expression, but consistent innate immune activation in this
model.

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Figure 4.6: Mice with disrupted TGFβRII have dysregulated innate and adaptive
immune responses at the level of the myenteric plexus.
(A’-A’’’) Representative images (A’-A’’) and quantification (A’’’) of immune cell CD43
immunoreactivity (magenta) in a 307,200 μm2 area surrounding GFAP-positive
myenteric ganglia (green) in WT (black bar) and dnTGFβRII (gray bar) mice. (B’-B’’’)
Immunofluorescence (B’-B’’) and quantification (B’’’) of CD68-positive macrophages
(magenta) surrounding a single GFAP-positive myenteric ganglion (green) in WT (black
bar) and dnTGFβRII (gray bar) mice. (n=4-11 animals/group, *P<0.05 by student’s ttest). (scale bar = 60 μm for A’-A’’ and 30 μm for B’-B’’).

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The transforming growth factor beta receptor type II (TGFβRII) is expressed in enteric
neurons, but not enteric glial cells, in mouse distal colon myenteric plexus
TGFβ is a wide reaching cytokine and it is hypothesized that almost every cell type
expresses a subtype of the TGFβ receptor, and is sensitive to activation by one of three
isoforms of TGFβ. To this end, we investigated whether the TGFβ type II receptor
(TGFβRII) was expressed within the neural components of the ENS. We performed
immunohistochemistry using antibodies against the TGFβRII (magenta), enteric
neurons (HuC/D, blue) and enteric glial cells (GFAP, green). We found that TGFβRII is
expressed in the myenteric plexus of mice (Figure 4.7) and that receptor localization is
restricted to the cytoplasm and extracellular membrane of enteric neurons, as receptor
expression patterns co-localized well with the pan-neuronal marker HuC/D but not with
the glial marker GFAP (Figure 4.7). This finding supports the idea of indiscriminate
expression of the TGFβ receptor and presents the possibility for para-cellular TGFβdependent signaling between immune cells and the neural components of the enteric
nervous system.

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Figure 4.7: TGFβRII is expressed in murine myenteric neurons.
(A-D) A representative mouse myenteric ganglion showing immunoreactivity for
TGFβRII (magenta), Hu C/D (enteric neurons; blue) and GFAP (enteric glia; green). The
overlay image shows that TGFβRII is expressed in enteric neurons, but not enteric glial
cells. (scale bar = 40 µm).

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Discussion
Transforming growth factor beta (TGFβ) is an important signaling molecule with a wide
range of physiological functions. In early studies, TGFβ was primarily recognized for its
ability to maintain tissue homeostasis through the regulation of cell proliferation and
death12. In addition, TGFβ is also well recognized for its role in regulating immune
homeostasis7,26,27. In fact, disrupted TGFβ signaling produces diseases that feature a
key autoimmune component in many organ systems 3,8,28. In this manuscript, we
investigated how disruption of TGFβ signaling in CD4+ and CD8+ T cells affects immune
homeostasis and inflammation in the gut using CD4-dnTGFβRII mice, which express a
dominant negative form of the human type II TGFβ receptor driven by a CD4 promoter3.
dnTGFβRII mice with disrupted TGFβ signaling have increased distal colon transit
time (a measure of motility function) without changes in macroscopic colonic damage or
the density or neurochemical phenotype of myenteric neurons. This result was
unexpected as changes in motility are typically accompanied by increased macroscopic
colon damage and decreased neuronal density15,17. GI motility is controlled by a
neuromuscular circuit where enteric neuronal signaling ultimately synapses to a smooth
muscle cell, causing tissue contraction or relaxation. One possibility is that disrupted T
cell TGFβ signaling acts downstream in the motility pathway, acting directly on smooth
muscle cells or disrupting the culminating neuromuscular communication. Additionally, it
is possible that neuronal function, rather than survival or neurochemical coding, are
altered in these mice. In fact, Linden et al. show that the electrophysiological properties
of surviving neurons are altered following colitis, in the absence of persistent
macroscopic damage29. These hypotheses may explain the GI motility dysfunction

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present in these mice in the absence of macroscopic changes to the intestine or enteric
neurons.
TGFβ is classically recognized for its role as an anti-proliferative signaling
molecule30 and as a “master regulator” of tissue fibrosis. Increased TGFβ signaling is
associated with increased deposition of extracellular matrix proteins, and cellular and
tissue injury are associated with increased TGFβ mRNA and protein expression31.
Intestinal fibrosis is a serious and common complication associated with GI
inflammation and is a primary cause for bowel resections in Crohn’s disease
patients32,33. dnTGFβRII mice did not present with significant colon shortening or
thickening, indicating a lack of significant tissue fibrosis or cell proliferation. This
suggests that disrupted T cell TGFβ signaling may be sufficient to ameliorate the profibrotic effects of TGFβ and that the shift to a proinflammatory T cell phenotype in
dnTGFβRII mice might be insufficient to drive fibrosis and smooth muscle proliferation.
CD4+ T cells (T helper cells) play an important role in the regulation of our host
immune response. The interaction of CD4+ T cells with MHCII-expressing antigen
presenting cells activates the adaptive immune response and the resulting cytokine
release directly controls the type (Th1 vs. Th2 vs. Th17) of response generated34. In our
study, we investigated how disrupted T cell TGFβ signaling altered the infiltration and
activation of immune cells and the expression of key regulatory cytokines within the
myenteric plexus of the ENS. Aberrant T cell TGFβ signaling increased the expression
of major histocompatibility complex class II (MHC II) at the level of the myenteric plexus.
dnTGFβRII mice also had increased intraganglionic immunoreactivity for CD68-positive
monocyte and macrophages. These combined results suggest that disrupted T cell

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TGFβ signaling increases the activation and/or infiltration of macrophages into/at the
level of myenteric plexus. Unfortunately, the hallmark immunological markers used do
not allow us to determine if these changes are due to increased activation of resident
macrophages23 or increased infiltration of activated macrophages from outside of the
plexus.
Our findings are consistent with previous work showing a relationship between TGFβ
signaling and MHC-II expression in multiple cell types. Disruption of TGFβ signaling in
TGFβ1 null mice increased tissue MHC II expression35, while treatment with TGFβ
suppresses MHC II expression in vitro36,37. In further support, a MHC-II knockout
background was sufficient to rescue TGFβ-1 mice from their the characteristic
autoimmune features38. In light of these studies, increased MHC II expression in our
dnTGFβRII mice was not surprising and the validity of these results is further supported
by increased immunoreactivity of CD68, another marker for macrophages.
The anti-inflammatory effects of TGFβ are thought to be due in part, to their ability to
regulate the production of pro and anti-inflammatory cytokines7,27 such as interleukin 1beta (IL-1β). It is thought that the autoimmune features of disrupted TGFβ signaling are
due to disturbance of the sensitive immune balance by increased production of proinflammatory cytokines and/or decreased production of anti-inflammatory cytokines. In
contrast, we observed that both WT and dnTGFβRII mice had similar expression levels
and localization of the pro-inflammatory cytokine IL-1β within myenteric ganglia. One
explanation of these finding is that our experiments, which disrupt T cell TGFβ signaling
and shift T cells to a proinflammatory state, are not suitable for directly assessing the
effects of TGFβ on cytokine production in the ENS. In addition, studies investigating the

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specific relationship between TGFβ and IL-1β show that while TGFβ does alters IL-1β
dependent effects, it is through the production of an IL-1R antagonist39 or down
regulation of the IL-1 receptor40, rather than directly altering IL-1β levels. A worthy future
study would thus be to investigate the expression levels of cytokine receptors, as well
as investigating a broader panel of pro- and anti-inflammatory cytokines.
Infiltration of myeloperoxidase (MPO)-positive neutrophils into the myenteric plexus
is a hallmark of GI inflammation14 and represents the response of the innate immune
system to GI pathogens. dnTGFβRII mice did not show any changes in myenteric
plexus MPO expression and MPO-positive cells were undetectable in both WT and
dnTGFβRII mice via immunohistochemistry. Typically, the infiltration of macrophages is
associated with acute phase of inflammation and dissipates within 24 hours in chemical
models of colitis 14. Given the considerable age of our dnTGFβRII mice, it is likely that
any infiltrating neutrophils would no longer be present. Alternately, is it also possible
that this model of inflammation is simply not associated with the influx of neutrophils into
the myenteric plexus.
Reactive gliosis in the central nervous system is well associated with increased
cellular injury and inflammation. A hallmark molecular feature of said “reactivity” is
increased expression of the glial cell marker glial fibrillary acidic protein (GFAP)41.
Disrupting T cell TGFβ signaling in our dnTGFβRII mice transformed enteric glia to a
reactive phenotype as evidenced by increased glial branching and increased GFAP
expression (Figure 4.4). This demonstrates that disrupted TGFβ signaling in T cells can
impact ENS structure, and likely function. In fact, TGFβ is known to potentiate astrocyte
production of nitric oxide42, a signaling molecule that we have previously shown to be

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important for regulating enteric neuron survival and thus gut function 17. One hypothesis
is that this increased glial reactivity modulates glial communication and control of GI
function43, and may explain the observed changes in GI motility in the absence of
accompanying neuron death or changes in myenteric neurochemical coding.
Previous studies have primarily focused on understanding TGFβ signaling in
immune cells, due to its significant roles in maintaining immune homeostasis. However,
it is hypothesized that almost every cell type expresses some form of the TGFβ receptor
due to its other roles in regulating cell proliferation and apoptosis. We found that the
type II TGFβR is expressed in the myenteric plexus of the mouse and that antibodies
against TGFβRII co-localize well with the pan-neuronal marker HuC/D. One potential
area of future study would be to investigate the role TGFβ signaling in neural crest
derived cells (enteric neurons and glia), through the production of nestin-dnTGFβRII
mice. As our immunohistochemistry shows that TGFβRII expression is restricted to
enteric neurons, these transgenic mice would allow us to specifically investigate the role
of TGFβ signaling in enteric neurons.
In conclusion, we show that disruption of TGFβ signaling in CD4+ and CD8+ T cells
disrupts distal colon motility without accompanying changes in macroscopic colonic
damage or enteric neuronal density and neurochemical composition. On the
microscopic level, dnTGFβRII mice have increased infiltration and activation of MHC-II
and CD68 positive macrophages but not CD43 positive T and B lymphocytes or MPOpositive neutrophils. We show that altered TGFβ signaling in CD4+ and CD8+ T cells in
dnTGFβRII mice disturbs GI immune homeostasis and alters normal GI motility function.

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APPENDIX

156

Antibody

Dilution Source

Product No.

Chicken anti-GFAP

1:1000

Abcam

ab4674

Goat anti-calretinin

1:200

Millipore

AB1550

Mouse anti-HuC/D

1:200

Invitrogen

A21272

Rabbit anti-IL-1β

1:200

Abcam

ab9722

Rabbit anti-MPO

1:200

Abcam

ab9353

Rabbit anti-TGFβRII

1:500

Abcam

ab61213

Rat anti-CD43

1:200

AbD Serotec

MCA4702GA

Rat anti-CD68

1:200

AbD Serotec

MCA1957

Rat anti-MHC II

1:200

Novus

NBP2-21789

Sheep anti-nNOS

1:500

BD Labs

610332

Table 4.1: Primary Antibodies used in Chapter 4.

** GFAP = glial fibrillary acidic protein, IL-1β = interleukin 1 beta, MPO = myeloperoxidase,
TGFβRII = transforming growth factor beta receptor type II, CD43 = cluster of differentiation 43,
CD68 = cluster of differentiation 68, MHCII = major histocompatibility complex class II, nNOS =
neuronal nitric oxide synthase

157

Antibody

Dilution

Source

Product No.

Streptavidin Dylight 405

1:400

Jackson

016-470-084

Donkey anti-Goat Alexa 488

1:400

Jackson

705-545-003

Donkey anti-sheep Alexa 488

1:400

Jackson

713-545-003

Goat anti-chicken Alexa 488

1:400

Invitrogen

A-11039

Goat anti-rabbit Alexa 488

1:400

Invitrogen

A-11034

Goat anti-chicken Alexa 568

1:400

Invitrogen

A-11041

Goat anti-rabbit Alexa 568

1:400

Invitrogen

A-11036

Goat anti-rat Alexa 594

1:400

Jackson

112-585-003

Streptavidin Alexa 594

1:400

Jackson

016-580-084

Table 4.2: Secondary Antibodies used in Chapter 4.

158

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159

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CHAPTER 5
The effects of pro-inflammatory mediators on glial hemichannel
activity

164

Abstract
Connexin-43 hemichannels are expressed on enteric glia and are important for glial
communication and regulation of gastrointestinal (GI) function. Increased channel
opening is also associated with pathogenic glial activity in GI inflammation. Yet, how
pro-inflammatory mediators affect (basal or activated) Cx43 channel opening is
unknown. We used uptake of the dye ethidium bromide (EtBr) to measure channel
activity and found that the pro-inflammatory mediators IL-1β, IL-17, IFN-γ and NO
increase glial dye uptake under basal conditions. IL-1β potentiated ADP-stimulated dye
uptake in a Cx43-dependent manner. IL-17 and IFN-γ did not change EtBr uptake in
activated glia and were insensitive to the effects of the Cx43 mimetic peptide. NO
decreased dye uptake in activated glia. In summary, our results demonstrate the
differential effects of pro-inflammatory mediators on basal or activated Cx43
hemichannel opening in enteric glia.

165

Introduction
Connexin-43 gap junctions and hemichannels are important for cell-to-cell signaling and
communication and are a key conduit for transport between the intra and extracellular
spaces in many types of central and peripheral glia1,2. In the enteric nervous system
(ENS), Cx43 hemichannels are expressed by enteric glia and are necessary for the
maintenance of normal glia-to-glia communication3 and contribute to pathogenic glial
activation and neuron death during inflammation4. Cx43 activity and expression on
astrocytes can be modulated by a number of factors including reactive oxygen/nitrogen
species, pro- and anti-inflammatory cytokines, growth factors and neurotransmitters5-7.
However, it is unknown how pro-inflammatory cytokines and mediators affect the activity
of Cx43 hemichannel on enteric glia and whether cytokines/mediators affect channel
opening differently under basal or stimulated conditions.
Here, we tested the effects of key pro-inflammatory mediators on glial Cx43
hemichannel opening by measuring uptake of the cell impermeable intercalating agent
ethidium bromide (EtBr) 8,9. Our results show that the pro-inflammatory mediators IL-1β,
IL-17, IFN-γ and NO all increase glial dye uptake under basal conditions, albeit to
different extents. In the presence of the purinergic glial agonist ADP, which stimulates
Cx43 opening 4, NO decreased dye uptake. IL-1β potentiated dye uptake in stimulated
glia and this effect was blunted by the Cx43 mimetic peptide 43Gap26. IL-17 and IFN-γ
did not alter dye uptake in activated glia and, surprisingly, diminished the inhibitory
effects of the Cx43 mimetic peptide. Together, our findings demonstrate differential
effects of pro-inflammatory cytokines and mediators on enteric glial Cx43 hemichannel
opening under basal or activated conditions.

166

Materials and Methods
Animals
Experimental protocols were reviewed and approved by the Institutional Animal Care
and Use Committee at Michigan State University. All experiments used either 1)
C57Bl/6 mice from Charles River (Wilmington, MA) or 2) wild-type (WT) mice on a
C57Bl/6 background bred in house as genetic WT littermates for transgenic mouse
lines. Mice used in experiments were male and aged 8-12 weeks. Mice were maintained
in a temperature controlled 12 h light: 12 h dark cycle environment with ad libitum
access to food and water.

Murine Model of Colitis
We induced colitis in mice using the 2,4-dinitrobenzene (DNBS) model of colitis, as
previously described4,10. Mice were briefly anesthetized with isoflurane and an enema of
DNBS (5.5mg/mouse in 0.1 mL of 50:50 ethanol/saline) was administered via a gavage
need inserted 3 cm into the colon. Control mice received an enema of saline. Groups of
mice were sacrificed at the peak of inflammation (48 hours after treatment) and after the
resolution of inflammation (3 weeks after treatment). Colonic macroscopic damage
score was assessed using the scoring matrix described by Storr et al.11. Colon tissue
was collected and either 1) fixed overnight in Zamboni’s fixative for analysis by
immunohistochemistry or 2) immediately frozen on dry ice and stored at -80°C for
multiplex protein analysis.

167

Whole-mount Immunohistochemistry (IHC) for Interleukin 1 beta (IL-1β)
Ganglionic expression of the pro-inflammatory cytokine IL-1β was detected by IHC in
whole-mount longitudinal muscle myenteric plexus (LMMP) preparations that were
dissected from colon tissue fixed overnight in Zamboni’s fixative, as previously
described3,4,10. Tissue preparations were permeabilized by three 10 minute washes in
0.1% Triton X-100 in phosphate buffered saline (PBS), followed by a 45 minute block in
4% normal goat serum at room temperature (RT). LMMP preps were incubated in rabbit
anti-IL-1β (Abcam, ab9722, 1:200) overnight at RT and then in goat anti-rabbit Alexa
568 (Invitrogen, A-11036, 1:200) for 2 hours at RT. Images were acquired using the 40X
objective (0.75 numerical aperture; Plan Fluor, Nikon, Melville, NY) of an upright
epifluorescence microscope (Nikon Eclipse Ni) with a Retiga 2000R camera (QImaging,
Surrey, BC, Canada) controlled by QCapture Pro 7.0 (QImaging) software and analyzed
using ImageJ software (https://imagej.nih.gov/ij/)

Multiplex Protein Analysis
Tissue samples were prepared for multiplex analysis from distal colon samples that
were frozen in dry ice immediately after collection and stored at -80°C until processing.
Tissue samples were homogenized in homogenizing buffer using a tissue grinder and
sonicator. Samples were then centrifuged at 10,000 g for 10 minutes at 4°C. The
supernatant was collected and transferred to a new tube and protein concentration
measured. For this assay, sample concentrations were adjusted so that total protein/
sample was the same across all treatment groups. Samples from healthy and inflamed

168

mice were analyzed to detect changes in pro-inflammatory cytokines by Eve
Technologies (Calgary, Alberta, Canada) using a Multiplex Assay.

Ethidium Bromide Dye Uptake Assay
Connexin-43 hemichannel activity in enteric glial cells was measured by ethidium
bromide (EtBr) dye uptake as previously described by McClain et al. in enteric glia 9 and
Orellana et al.12 in cultured astrocytes . Live whole-mount LMMP preparations were
dissected from the distal colon in 35mm2 sylgard-coated dishes and treated with an
enzyme mix containing 1 U/mLDispase and 150 U/mL of Collagenase Type II for 15
minutes at RT. Tissue was titurated and repined in fresh DMEM. Preparations were
then incubated in a two-step process. In the first step, preps were incubated for 30
minutes at 37°C in 5% CO2/95% air in either Kreb’s buffer only (Control) or proinflammatory cytokines and mediators (see Table 5.1, appendix) in the presence or
absence of the Cx43 mimetic peptide 43Gap26 (100 μM). After 30 minutes, the first
incubation mixture was removed and preps incubated with EtBr (5 μM in Kreb’s) in the
presence or absence of the purinergic glial agonist ADP (100 μM) for 10 minutes at
37°C. Extracellular EtBr was removed by three washes in fresh Kreb’s and EtBr
fluorescence immediately recorded using an upright Olympus BX51WI fixed-stage
microscope (Olympus, Center Valley, PA) fitted with a 40X water-immersion objective
(LUMPlan N, 0.8 n.a.) and a Lambda DG-4 Plus xenon light source (Sutter, Novato,
CA). Fluorescence of EtBr was detected using a TRITC filter set with a 535 nm, 20 nm
band-pass excitation filter and a 610 nm, 75 nm band-pass emission filter. We acquired
images of individual ganglia using Andor iQ3 software (Andor, Belfast, UK) and

169

identified ganglia based on morphology, based on our expertise and previous
experiments with cell-permeable dyes4.

Chemicals, Reagents and Solutions
All tissue was collected and dissected in DMEM/F12 media buffer (ThermoFisher,
Carlsbad, CA) supplemented with 3uM Nicardipine and 1uM Scopolamine. Kreb’s buffer
contained (in mM) 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 10 HEPES,
21.2 NaHCO3, 1 pyruvic acid and 8 glucose (pH adjusted to 7.4 with NaOH) with 3uM
Nicardipine and 1uM Scopolamine. 4% normal serum contains 4% goat serum, 0.4%
Triton X-100, and 1% bovine serum albumin. 100 mL of homogenizing buffer contains:
90 mL of dI H2O, 10 mL of 10x Tris-buffered Saline (TBS; 1X concentration = Tris/TrisHCl 25mM, NaCl 130 mM, KCl 2.7 mM), 0.5 mL of Tween 20, one tablet of protease
inhibitor cocktail (Sigma Aldrich, Cat No. S8820) and was pH adjusted to pH = 7.5.
Adenosine 5’-diphosphate sodium salt (ADP) and Ethidium Bromide (15585011) were
purchased from ThermoFisher and the Connexin-43 mimetic peptide 43Gap26 (Cat. No.
AS-62644) from AnaSpec (Fremont, CA).

Data and Statistical Analysis
Images of individual ganglia were exported from Andor iQ3 software as tiff image files
and analyzed using ImageJ software. Individual glial cells were highlighted using the
region of interest (ROI) tool and fluorescence measured in a minimum of 10 glia/
ganglia. Mean fluorescence was then averaged per ganglion and n values represent the
number of individual ganglia analyzed. EtBr measurements were made in an average of

170

15 ganglia/ animal in 3-6 animals per treatment group. Ganglionic immunoreactivity for
IL-1β was calculated using the measure tool in ImageJ. Data were analyzed using by
student’s t-test, One-Way or Two-Way ANOVA using Graphpad 7 software (La Jolla,
CA) and are presented as mean ± the standard error of the mean (SEM).

Results

Activation of glial P2Y1 receptors leads to connexin-43 (Cx43) hemichannel opening
Recent data show that the activation of enteric glial cells contributes to enteric
neurodegeneration during inflammation4. Increased extracellular purines activate glial
P2Y1 receptors during colitis10, producing robust glial calcium responses and increasing
connexin-43 (Cx43) hemichannel opening (Figure 5.1)4. Astroglial Cx43 activity is
altered in the central nervous system (CNS) by a number of signaling molecules
including cytokines, neurotransmitters and reactive oxygen/nitrogen compounds 5.
Ganglionic concentrations of a number these compounds are modified during colitis and
other gastrointestinal (GI) diseases13. However, it is not known how these proinflammatory cytokines and mediators specifically affect enteric glial Cx43 activity. We
used glial uptake of the fluorescent intercalating agent ethidium bromide (EtBr)9,14 to
investigate if/ how the activity of Cx43 on enteric glia is modulated by key proinflammatory cytokines and signaling molecules.

171

Figure 5.1: Purinergic glial activation leads to connexin-43 (Cx43) hemichannel opening. We measured Cx43
hemichannel opening by uptake of the intercalating agent ethidium bromide (EtBr) and blocked channel opening with the
mimetic peptide 43Gap26.

172

Pro-inflammatory cytokine levels are altered in the distal colon during GI inflammation
Disruption of the balance between pro- and anti-inflammatory cytokines is a hallmark of
inflammatory bowel disease (IBD) in both animal models and human disease 13,15,16. We
measured tissue concentrations of three (3) pro-inflammatory cytokines in the distal
colon following the induction of DNBS colitis in mice. Protein levels of interferon-γ (IFNγ) were negligible in healthy animals and were not significantly altered during colitis (0.0
pg/mL in Saline vs. 0.73 ± 0.43 pg/mL in DNBS animals; p=0.1054; Figure 5.2A).
Healthy animals had similar baseline colonic levels of the pro-inflammatory cytokines IL1β and IL-17 (IL-1β = 1.0 ± 0.13 pg/mL, IL-17 = 1.0 ± 0.19 pg/mL; Figure 5.2B and
5.2C), but IL-1β and IL-17 were differentially altered during DNBS-colitis. Inflammation
decreased IL-17 protein content by 43% (1.0 ± 0.19 pg/mL in Saline vs. 0.57 ± 0.09
pg/mL in DNBS; Figure 5.2B) and increased GI IL-1β protein content by over 400% (1.0
± 0.13 pg/mL in Saline vs. 4.4 ± 0.93 pg/mL in DNBS; Figure 5.2C).
We next used immunohistochemistry to investigate whether IL-1β expression
within myenteric plexus ganglia mirrored changes to whole tissue protein levels. In the
myenteric plexus, IL-1β signaling is primarily mediated through enteric glial cells, which
secrete IL-1β 17 and express IL-1β receptors18. IL-1β expression is associated with GI
inflammation19,20 and we hypothesized that ganglionic, and specifically enteric glial, IL1β content would be increased during inflammation. We induced colitis using DNBS and
measured immunoreactivity for IL-1β in myenteric ganglia from healthy and inflamed
animals at peak inflammation (48 hours) and following resolution (3 weeks). In support
of our hypothesis, ganglionic IL-1β expression was doubled at the peak of inflammation

173

Figure 5.2: Pro-inflammatory cytokine levels are altered in the distal colon during
GI inflammation. (A-C) Whole tissue protein levels of the pro-inflammatory cytokines
(A) Interferon-γ (IFN-γ), (B) Interleukin-17a (IL-17) and (C) Interleukin-1β (IL-1β) in
healthy (Saline; black bars) and inflamed (DNBS; gray bars) animals. (*P<0.05,
***P<0.005 by Student’s t-test, n= 11-12 animals/group). (D) Immunofluorescence of the
pro-inflammatory cytokine IL-1β in murine myenteric plexus at the peak (48hr) and
resolution (3wks) of inflammation (DNBS; gray bars) and in healthy controls (Saline;
black bars). (**P<0.01, ***P<0.005 vs. 48hr-Saline by Two-Way ANOVA with Dunnett’s
multiple comparison test, n=3-5 animals/group).

174

(Arbitrary Fluorescence: 43 ± 7.2 in 48hr saline vs. 95 ± 3 in 48hr-DNBS; Figure 5.2D)
and remained elevated following resolution of colitis (Arbitrary Fluorescence: 95 ± 3 in
48hr-DNBS vs. 95 ± 12 in 3 week DNBS animals; Figure 5.2D). Basal ganglionic
expression of IL-1β was not significantly altered in saline-treated animals 3 weeks after
saline enema (Arbitrary Fluorescence: 43 ± 7 in 48hr saline vs. 70 ± 5 in 3 week saline;
Figure 5.2D). Our results support the previously suggested role for IL-1β as a key proinflammatory cytokine in the pathogenesis of colitis21-23.

Pro-inflammatory mediators increase glial connexin-43 (Cx43) hemichannel opening in
the absence of purinergic glial stimulation
Cellular uptake of the intercalating agent ethidium bromide (EtBr) is a selective method
to measure Cx43 hemichannel opening in astroglia24-26. Cx43-dependent EtBr uptake in
enteric glia is increased following purinergic stimulation with the P2Y1 agonist ADP 9,
which also increases Cx43-dependent ATP release 4. EtBr uptake by enteric glia is
minimal in the absence of ADP9, demonstrating differential Cx43 activity in stimulated
and unstimulated myenteric plexus preparations. Thus, we investigated how proinflammatory cytokines and mediators altered Cx43 activity in both the presence and
absence of purinergic activators of enteric glia.
First, we tested the singular effects of pro-inflammatory cytokines and mediators
on Cx43-mediated dye uptake in the absence of purinergic stimulation. Cx43
hemichannel opening in unstimulated glia was increased by incubation with the proinflammatory cytokines IL-1β, IL-17 and IFN-γ and the enteric signaling molecule and
inflammatory mediator nitric oxide (NO)4,27 (Figure 5.3). However, the extent of channel

175

opening varied by cytokine/mediator. NO and IL-17 had modest effects and both
increased EtBr uptake by 14% (Arbitrary Fluorescence: 479 ± 15 in control vs. 540 ± 16
in NO vs. 547 ± 16 in IL-17 treated preparations; Figure 5.3A-B, 5.3D, 5.3F). IL-1β and
IFN-γ produced much more robust increases in channel opening. Glial cell EtBr
fluorescence was increased by ~38% in tissue treated with IFN-γ (Arbitrary
Fluorescence: 479 ± 15 in control vs. 661 ± 31 in IFN-γ treated preparations; Figure
5.3E-F). Incubation with IL-1β increased EtBr uptake by ~70% compared to control
(Arbitrary Fluorescence: 479 ± 15 in control vs. 798 ± 21 in IL-1β treated preparations;
Figure 5.3C and 5.3F). Thus, the pro-inflammatory cytokines/ mediators IL-1β, IL-17,
IFN-γ and NO can increase Cx43 hemichannel activity in otherwise unstimulated enteric
glial cells.

Stimulation of glial P2Y1 receptors increase glial EtBr uptake
Next, we investigated how pro-inflammatory cytokines/mediators affect Cx43 opening in
enteric glia where channel opening was induced by purinergic activation. We stimulated
Cx43 channel opening by treating LMMP preparations with the P2Y1 agonist ADP (100
μM) for 10 minutes in the presence of EtBr. In agreement with McClain et al. (2016) 9,
treatment with ADP increased EtBr uptake by ~27% (Arbitrary Fluorescence: 479 ± 15
in control vs. 610 ± 18 in ADP treated preparations; Figure 5.4C), demonstrating that
this treatment paradigm was a suitable method for activating enteric glia and stimulating
Cx43 hemichannel opening in subsequent experiments.

176

Figure 5.3: Effects of the pro-inflammatory mediators NO, IL-1β, IL-17 and IFN-γ on Cx43 hemichannel opening in
unstimulated enteric glia. (A-E) Representative images and (F) quantification of glial ethidium bromide (EtBr)
fluorescence in live LMMP preps treated with (A) buffer control, (B) 100 μM PAPANONOate (Nitric Oxide donor), (C) IL1β (10 ng/mL), (D) IL-17 (10 ng/mL) and (E) IFN-γ (10 ng/mL), in the absence of the purinergic agonist ADP. (Scale bar =
50 μm) (*P<0.05, ****P<0.0001 vs. Control via One-Way ANOVA with Dunnett’s post-hoc test) (n= a minimum of 10
individual glia, from 7-24 ganglia/treatment group).

177

Figure 5.4: Effects of Nitric Oxide (NO) on EtBr dye uptake in ADP-stimulated enteric glia. (A, B) Representative
images and (C) quantification of Cx-43 mediated EtBr dye uptake in enteric glia stimulated with the purinergic agonist
ADP (100 μM) in the presence or absence of the nitric oxide donor PAPANONOate (NO; A) and the Cx43 mimetic peptide
43Gap26 (B). Control preps were treated with buffer only. (Scale bar = 50 μm) (**P<0.01, ****P<0.0001 vs. Control,
##P<0.01, ##P<0.0001

vs. ADP via One-Way ANOVA with Dunnett’s post-hoc test.) (n=a minimum of 10 individual glia,

from 7-33 ganglia/mouse and 3-6 mice/treatment group).

178

Nitric Oxide (NO) decreases glial EtBr uptake through a Cx43-independent pathway
Increased NO is frequently associated with tissue inflammation, in part due to the
increased activities of inducible nitric oxide synthase (iNOS)28,29. Cx43 hemi-channels
are susceptible to NO-mediated post-translational modifications 6,14,30 and we have
previously shown that NO increases Cx43-mediated release of ATP from enteric glial
cells4. Unexpectedly, NO slightly (~10%) diminished EtBr uptake in stimulated enteric
glia (Arbitrary Fluorescence: 610 ± 18 in ADP vs. 547 ± 13 in NO + ADP treated preps;
Figure 5.4A and 5.4C). Co-treatment with the Cx43 hemichannel mimetic peptide
43Gap26 (100 μM) decreased glial EtBr uptake by an additional 12% and returned EtBr
fluorescence to basal levels (Arbitrary Fluorescence: 547 ± 13 in NO + ADP vs. 478 ± 6
in NO + ADP + 43Gap26 treated preps; Figure 5.4B-C). These data show that the NO
inhibits EtBr uptake.

IL-1β potentiation of glial EtBr uptake is sensitive to connexin-43 blockade with
43Gap26
Next, we investigated the effect of IL-1β on Cx43 opening in activated glia. IL-1β
concentrations were increased in distal colon tissue, and importantly in myenteric
plexus ganglia, following colitis (Figure 5.2C-D). Due to its expression within enteric
ganglia, IL-1β is spatially primed to alter Cx43 activity and opening. In the presence of
20 ng of IL-1β protein, EtBr uptake in stimulated enteric glia was increased by ~42%
(Arbitrary Fluorescence: 610 ± 18 in ADP vs. 869 ± 16 in IL-1β + ADP treated preps;
Figure 5.5A and 5.5C). Treatment with the Cx43 mimetic peptide 43Gap26 (100 μM)
decreased dye uptake to levels similar to that in tissue treated with ADP only (Arbitrary

179

Fluorescence: 610 ± 18 in ADP vs. 685 ± 15 in IL-1β + ADP + 43Gap26 treated preps;
Figure 5.5B-C). These results suggest that the IL-1β-mediated increase in EtBr dye
uptake is dependent on Cx43 hemichannel activity.

Interleukin 17 (IL-17) does not alter ADP-stimulated dye uptake and in the presence or
absence of the Cx43 mimetic peptide 43Gap26
Colonic concentrations of the Th17-associated pro-inflammatory cytokine IL-17 were
decreased during colitis. Thus, we hypothesized that IL-17 would not significantly alter
Cx43 opening during inflammation. EtBr uptake in activated glia was not altered in the
presence of the Th17-associated cytokine IL-17 (Arbitrary Fluorescence 610 ± 18 in
ADP vs. 640 ± 23 in IL-17 + ADP treated preps; Figure 5.6B-C). Further, the inhibitory
effects of the mimetic peptide 43Gap26 were lost in the presence of IL-17 (Arbitrary
Fluorescence: 640 ± 23 in IL-17 + ADP vs. 653 ± 21 in IL-17 + ADP + 43Gap26 treated
preps; Figure 5.6B-C). Together, these results show that IL-17 does not potentiate EtBr
uptake, and further alters the inhibitory effect of the Cx43 mimetic peptide.

Interferon-γ (IFN-γ) does not potentiate ADP-stimulated dye uptake and is insensitive to
blockade with the connexin-43 mimetic peptide
During inflammation, protein levels of IFN-γ in the distal colon were not significantly
changed and both healthy and inflamed animals had minimal colonic IFN-γ. Thus, we
hypothesized that IFN-γ would not significantly affect glial Cx43 activity, which we have
shown to be important in GI inflammation4. Treatment with 20 ng of IFN-γ did not

180

Figure 5.5: Effects of Interleukin 1-β (IL-1β) on dye uptake in ADP-stimulated enteric glia. (A, B) Representative
images and (C) quantification of Cx43-mediated EtBr dye uptake in enteric glia in LMMP preps treated with buffer
(Control) or the purinergic agonist ADP (100 μM) in the presence or absence of the pro-inflammatory cytokine (IL-1β; A)
and the Cx43 mimetic peptide 43Gap26 (B). (Scale bar = 50 μm) (**P<0.01, ****P<0.0001 vs. Control, ##P<0.01,
##P<0.0001

vs. ADP via One-Way ANOVA with Dunnett’s post-hoc test.) (n=a minimum of 10 individual glia, from 7-23

ganglia/mouse and 3-6 mice/treatment group).
181

Figure 5.6: Effect of Interleukin 17 (IL-17) on ADP-stimulated dye uptake in enteric glia. (A, B) Representative
images and (C) quantification of Cx-43 mediated EtBr dye uptake in enteric glia in treated with buffer (Control) or
stimulated with the purinergic agonist ADP (100 μM) in the presence or absence of (A) the pro-inflammatory cytokine IL17 and (B) the Cx43 mimetic peptide 43Gap26. (Scale bar = 50 μm) (****P<0.0001 vs. Control via One-Way ANOVA with
Dunnett’s post-hoc test.) (n=a minimum of 10 individual glia, from 4-20 ganglia/mouse and 3-6 mice/treatment group).

182

significantly change glial EtBr uptake in tissue stimulated with ADP (Arbitrary
Fluorescence: 610 ± 18 in ADP vs. 593 ± 12 in IFN-γ + ADP treated preps; Figure 5.7A
and 5.7C). The inhibitory effects of 43Gap26 were lost in the presence of IFN-γ
(Arbitrary Fluorescence: 593 ± 12 in IFN-γ + ADP vs. 592 ± 9 in IFN-γ + ADP +
43Gap26 treated preps. Consistent with the effects seen with IL-17, IFN-γ does not
potentiate EtBr uptake, and negates the inhibitory effects of the Cx43 mimetic peptide
43Gap26.

183

Figure 5.7:Effects of the pro-inflammatory cytokine Interferon-γ (IFN-γ) of Cx43-mediated dye uptake in
stimulated enteric glia. (A, B) Representative images and (C) quantification of Cx-43 mediated EtBr dye uptake in
enteric glia treated with the purinergic agonist ADP (100 μM) in the presence or absence of the pro-inflammatory cytokine
Interferon-γ (IFN-γ; A) and the Cx43 mimetic peptide 43Gap26 (B). Control preps were treated buffer only. (Scale bar =
50 μm) (****P<0.0001 vs. Control via One-Way ANOVA with Dunnett’s post-hoc test.) (n=a minimum of 10 individual glia,
from 7-22 ganglia/mouse and minimum 3-6 mice/treatment group).

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Discussion
Glial hemichannels composed of Cx43 are important mediators of intercellular signaling
during health and disease in the ENS. Here, we investigated how glial hemichannels
are affected by key pro-inflammatory mediators such as IL-1β, IL-17, IFN-γ and NO. Our
key results show that IL-1β, IL-17, IFN-γ and NO differentially affect the baseline and
activity-dependent uptake of EtBr through Cx43. All pro-inflammatory mediators tested
had the potential to increase the glial uptake of EtBr, albeit to differing extents.
However, their effects on activity dependent uptake of EtBr were vastly different. IL-1β
significantly increased Cx43-dependent dye uptake in activated glia, while IL-17 and
IFN-γ did not change EtBr uptake following glial activation and interfered with the
inhibitory effects of the Cx43 mimetic peptide 43Gap26. Surprisingly, nitric oxide
decreased activity-dependent EtBr uptake.
Immune homeostasis in healthy animals involves maintenance of the sensitive
balance between pro- and anti-inflammatory cytokines. As such, disruption of this
balance is a characteristic component in the pathology of inflammatory bowel disease
(IBD) and other autoimmune disorders13,31,32. At the peak of inflammation during colitis,
distal colon concentrations of the pro-inflammatory cytokines IL-1β, IL-17 and IFN-γ are
changed. Inflammation increased IL-1β protein concentrations while simultaneously
decreasing IL-17 levels. IFN-γ levels were slightly increased, however the effect did not
reach statistical significance (P=0.1). One possibility is that 48 hours post DNBS colitis
is too late to measure the peak colonic expression of IFN-γ protein.
The DNBS model used in these experiments produces a T h1 cytokine response33,34,
which is associated with increased production of the effector cytokines IFN-γ, TNF-α, IL-

185

2 and IL-10, and mimics some of the features of human Crohn’s disease. Thus,
although colonic IFN-γ levels were not increased to a statistically significant level, this
finding may have some functional relevance and be indicative of the T h1 immune
response caused by DNBS. IL-17 is traditionally associated with a Th17-type immune
response. Thus, an increase in IL-17 would not be expected following induction of Th1associated DNBS colitis and decreased IL-17 may represent an suppression of Th17 T
cells in inflamed mice. The final cytokine studied was IL-1β, which is not traditionally
associated with a particular (Th1, Th2 or Th17) type of immune response. Rather, it is
up-regulated in a variety of autoimmune diseases, including IBD, where it can contribute
broadly to T Cell activation20,35-37. Consistent with our findings, IL-1β is associated with
animal models of colitis18,22, and more importantly, with human disease38-40.
The pro-inflammatory mediators IL-1β, IL-17, IFN-γ and NO all increased Cx43
hemichannel opening under control conditions. Our finding supports the long-standing
hypothesis that Cx43 hemichannel activity is modulated by pro-inflammatory cytokines
during inflammation5,7. These mediators altered Cx43 opening to different extents, with
IL-1β and IFN-γ robustly increasing channel opening by more that 40% and NO and IL17 producing a more moderate 14% increase in dye uptake. One explanation for these
differential effects is that Cx43 hemichannels are alternately modulated by different proinflammatory mediators. Cx43 is subject to post-translational modification by NO where
cysteine residues undergo S-nitrosylation in the presence of increased NO30,41.
Conversely, the mechanisms of cytokine-induced hemichannel opening are not as
clearly understood. In fact, Bennett et al. (2012)42 propose that the pro-inflammatory
cytokines TNF-α and IL-1β induce increased Cx43 opening through action on “stressed”

186

astrocytes, rather than directly modifying the channel itself. We hypothesize that
differential mechanisms of action likely explains the variability in extent of channel
opening produced by treatment with IL-1β, IL-17, IFN-γ and NO.
Hemichannel activity is the CNS is differentially regulated under normal and
pathological (inflamed) conditions43,44. We found that the effects of IL-1β, IL-17, IFN-γ
and NO on Cx43 hemichannel opening were activity dependent. IL-1β increased dye
uptake by an additional 42%, compared to the 27% increase in channel opening
following purinergic stimulation with ADP. Interestingly, the combined effect of ADP + IL1β (i.e. a 69% increase in dye uptake compared to control) was similar to that achieved
with incubation with IL-1β only (70%; Figure 5.3C and 5.3F). This highlights the
presence of a potential maximal degree of channel opening (70% above basal opening)
since the effects of ADP and IL-1β were additive solely to a maximal level. Importantly,
incubation with the mimetic peptide 43Gap26 demonstrated that the effects of IL-1β
were Cx43 sensitive. These findings, in conjunction with glial expression of the IL-1
receptor18 and secretion of IL-1β17, present the possibility of that IL-1β can function as
an autocrine or paracrine signaling molecule, and modulate glial Cx43-dependent
communication3.
Consistent with our findings, Retamal et al.8 report that IL-1β increases Cx43mediated dye uptake in astrocytes8. Two potential mechanisms are that 1) IL-1β
increases total cellular Cx43 expression but not individual rate of channel opening
and/or 2) IL-1β increasing channel opening of individual hemichannels. IL-1β has been
shown to increase cell surface Cx43 hemichannel expression 45 but because of the short
treatment time in our experiments, we hypothesize IL-1β-mediated effects are not due

187

to a change in Cx43 expression. Rather, as proposed by Retamal et al.8, we
hypothesize that IL-1β alters cellular redox state46 which then alters Cx43 channel state
and opening6,14,44.
IL-17 and IFN-γ had no effect on EtBr dye uptake in stimulated enteric glia cells
(Figures 5.6 and 5.7). The effects of these cytokines specifically on Cx43
hemichannels have not been thoroughly investigated in the literature. IFN-γ treatment
alters Cx43 expression and the formation of functional astroglial gap junctions47,48.
Similarly, IL-17 treatment also leads to decreased Cx43 expression and decreased gap
junction formation in mixed astrocyte/microglia cultures48. However, cytokines have
different effects on Cx43 hemichannels vs. gap junctions8. Thus, the findings of these
studies cannot be easily translated from the study of Cx43 gap junctions in astroglia, to
that of Cx43 hemichannels in enteric glia.
An unexpected finding in our study was that NO decreased EtBr dye uptake in
activated enteric glia. This finding contradicts the well-established report that NO
increases Cx43 hemichannel through S-nitrosylation4,14,30.
In summary, our findings show that the pro-inflammatory cytokines/mediators IL-1β,
IL-17, IFN-γ and NO are capable of modulating glial uptake of the dye EtBr, as a
measure of enteric glial cell Cx43 activity. The differential effects of the proinflammatory cytokines/ mediators on Cx43 channel activity, under basal and activated
conditions, provide important insight into our understanding of activity-dependent
hemichannel function. Cx43-insensitive effects on dye uptake potentially highlight novel
mechanisms through which inflammatory mediators may alter cell permeability and
warrants further study.

188

APPENDIX

189

Cytokine/Compound

Concentration

Source

Catalog Number

Interleukin 1β (IL-1β)

10 ng/mL

R + D Catalog

401-ML-005

Interleukin 17 (IL-17)

10 ng/mL

eBioscience

14-8171-62

Interferon- γ (IFN-γ)

10 ng/mL

eBioscience

BMD326

PAPANONOate (NO

100 μM

Cayman Chemical

82140

Donor)

Table 5.1: Cytokines and Chemicals used in the Ethidium Bromide Dye Uptake
Assay.

190

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191

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CHAPTER 6
Summary and Future Directions

196

Summary
The enteric nervous system is an intrinsic neuronal network comprised of enteric
neurons and glial cells embedded within the walls of the GI tract, and provides local and
moment-to-moment control of gastrointestinal (GI) functions and motility1. Loss of
enteric neurons disrupts this network and is associated with motility dysfunction in a
host of disorders2-6. Despite the worldwide prevalence of motility dysfunction7, drug
treatments for motility disorders are limited and primarily focus on providing
symptomatic relief. The lack of improved therapeutics is due to the gaps in our
understanding of the key factors and cellular mechanisms that contribute to motility
dysfunction. This dissertation investigated the cellular mechanisms by which oxidative
stress and immune dysregulation, two factors associated with GI inflammation,
contribute to enteric neuropathy and motility dysfunction. In particular, we were
interested in the role of enteric glial cells in mediating these effects.
In Chapter 2, we investigated how glial nitric oxide (NO) production contributes to
enteric neuron death during inflammation, and found that the activation of enteric glia by
extracellular purines contributes to neuron death through the pathogenic release of
ATP8. This release is mediated by increased opening of glial Cx43 hemichannels, which
are gated by glial NO concentrations, as previously shown 9. Pharmacological and
genetic inhibition of glial inducible nitric oxide synthase (iNOS) activity was protective
against neuron death in situ, highlighting a potential novel therapeutic target for
prevention of enteric neuropathy in inflammation.
Chapter 3 explored the antioxidant arm of ENS regulation of oxidative balance.
Enteric glia are hypothesized to be the primary producers of the endogenous

197

antioxidant reduced glutathione (GSH) in the ENS

10.

In support of this, we show that

enteric glia express the enzymes necessary for GSH synthesis. In an additional finding,
these enzymes are also expressed by enteric neurons. In situ inhibition of GSH
synthesis caused enteric neuron death, supporting a neuroprotective role for GSH.
Surprisingly, in vivo depletion of whole body GSH was protective against some features
of GI inflammation, suggesting a novel cellular role for this key antioxidant.
Chapter 4 studied how disrupted immune homeostasis in circulating T cells
affected myenteric plexus structure and GI motility. CD4-dnTGFβRII mice with disrupted
T cell TGFβ signaling have disrupted distal colon motility, without alterations in
myenteric neuronal density or neurochemical coding. Myenteric glia were impacted in
our animal model, with conversion to a “reactive” or “activated” state in CD4-dnTGFβRII
mice. Disrupted TGFβ signaling was sufficient to dysregulate immune homeostasis
within the innate, but not adaptive, immune system at the level of the myenteric plexus.
Lastly, we show that enteric neurons express the TGFβ type II receptor, the first
demonstration of TGFβ receptor expression in the ENS.
In the final data chapter (Chapter 5), we measured how key pro-inflammatory
mediators and cytokines affect glial Cx43 hemichannel function. The pro-inflammatory
cytokines IL-1β, IL-17, IFN-γ and NO all increase glial uptake of the intercalating agent
ethidium bromide (EtBr) under baseline conditions, but have differential effects in glia
stimulated with ADP. IL-1β potentiates dye uptake in ADP-stimulated glia in a Cx43dependent manner while NO decreases dye uptake. IL-17 and IFN-γ did not alter dye
uptake in activated glia and surprisingly, diminished the inhibitory effects of the Cx43
mimetic peptide 43Gap26.

198

Together, these studies add to our knowledge of key glial mechanisms that
contribute to enteric neuropathy and improve our understanding of ENS neuron-glia
interactions. These findings identified novel therapeutic targets (discussed below),
which will allow for the development of new and improved drugs for treatment of GI
motility dysfunction.

Novel Findings
Enteric neurons express the cellular machinery necessary for glutathione synthesis
Enteric glial cells help maintain neuronal health through the synthesis and release of
reduced glutathione (GSH) 10,11. This is analogous to the central nervous system (CNS)
where astrocytic neuroprotection is mediated in part by GSH12. In addition, CNS
neurons are capable of producing small amounts of GSH when provided with the
necessary precursors from astrocytes13. To date, whether this mechanism occurs in the
ENS had not been investigated. We showed that enteric neurons express the catalytic
subunit of glutamate-cysteine ligase (GCLC) and glutathione synthesis (GS), the two
enzymes necessary for glutathione synthesis. With this novel finding, we propose a
mechanism where enteric neurons may contribute to their own neuroprotection through
GSH synthesis.

Protective role of in vivo depletion of glutathione during inflammation
Reduced glutathione (GSH) is the primary endogenous cellular antioxidant in the body
and is traditionally associated with its cytoprotective features. We hypothesized that
depletion of glutathione would promote the enteric neuropathy, tissue damage and

199

motility dysfunction associated with GI inflammation, given the important role of
oxidative stress in motility disorders. Instead, we found that GSH depletion is protective
against some features of in vivo colitis, suggesting a novel role for GSH in the
patho(physiology) of GI inflammation and motility dysfunction.

The TGF-beta type II receptor is expressed in enteric neurons
The anti-inflammatory and pro-fibrotic effects of the regulatory cytokine transforming
growth factor β (TGFβ) are primarily mediated through TGFβ signaling in immune cells.
Thus, the primary literature focuses on those signaling pathways and TGF-β signaling in
other cell types has not been as thoroughly studied. In the CNS, there is evidence for
neuronal expression of TGFβ isoforms and their receptors 14,15. Here, we show novel
expression of the type II TGFβ receptor in the enteric nervous system, specifically on
enteric neurons. This finding presents the possibility for differential effects of
immunological vs. neural TGFβ signaling in the ENS.

Study Limitations
DNBS Model of Colitis
Our studies primarily used the 2,4-dinitrobenzene (DNBS) model to induce murine
colitis. This chemical model is characterized by a Th1-mediated immune response and
colonic fibrosis; features observed in human Crohn’s disease16. Thus, it is important to
interpret findings within the context of the features of this animal model and consider
that they may not be broadly applicable to all GI inflammatory conditions. The validity of

200

our findings would benefit from repetition of our some studies in other chemical and
genetic models of colitis17-19.

Use of Pharmacological Modulators
The studies in this dissertation used a number of drugs to modulate the behavior and/or
concentrations of enzymes, channels and signaling molecules of interest. Specifically,
studies in Chapter 3 and 5 relied heavily on the pharmacological properties of BSO
(GSH synthesis inhibitor) and 43Gap26 (Cx43 mimetic peptide). Thus, the conclusions
of these chapters are dependent on the assumption that these inhibitors perform as
intended and have little to no off target effects.

Sex of Experimental Animals
Recent research findings have increasingly emphasized the role of sex as a biological
variable in animal models. Studies in this dissertation were done in a single sex of
research animal (male mice for Chapters 2,3 and 5 and female mice for Chapter 4).
This deficiency should be considered when interpreting these data and may limit the
translation of findings to broader populations.

Future Directions
This dissertation presented a number of key findings that warrant further investigation in
future studies.

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Cell-specific Inhibition of Glial NO Production
Findings in Chapter 2 show a pathogenic role for glial iNOS activity and NO production
in enteric neuropathy during inflammation. This suggests that glial iNOS is a potential
therapeutic target for the prevention of cell death during inflammation. Preliminary
studies have begun to test this hypothesis and would be continued in future work.
Working with Dr. Fredric Manfredsson (Michigan State University), we designed an
AAV-FLEX-iNOS viral construct that will theoretically target iNOS expression in enteric
glia.
In this viral construct, microRNA (miRNA) against iNOS is packaged into an adenoassociated virus (AAV) containing the flip excision (FLEX) system and a GFP reporter.
The FLEX construct restricts expression of miRNA to cre-recombinase expressing cells.
Thus, injecting this virus into Sox10-CreERT2 mice will result in inhibition of iNOS in
enteric glia only. Target specificity of our miRNA will be confirmed by the use of a
control scrambled miRNA packaged in the same manner. Pilot studies using crepositive animals and cre-negative controls, both injected with control and iNOS mRNA,
shows non-specific labeling for GFP in enteric neurons (Figure 6.1), highlighting the
need for further troubleshooting and future work.

202

Figure 6.1: Myenteric ganglia in Sox10-CreERT2 mice and Cre- controls.
Immunohistochemistry (IHC) for GFP (green) and the glial cell marker (S100β) in Cre+
and Cre- mice injected with iNOS-AAV or scrambl-AAV

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Cell-specific Inhibition of Glial GSH Production
Novel findings in chapter 3 highlight a potential heterogenous role for the antioxidant
GSH. Our treatment paradigm involved whole body depletion of cellular GSH via
consumption of the GCL inhibitor L-Buthionine Sulfoximine in drinking water. This
treatment is sufficient to significantly deplete GSH in major organ systems including the
liver, kidney, heart, lung, intestines and in plasma and blood cells

20.

Thus, it is

challenging to elucidate what mechanisms are contributing to the protective effects of
GSH depletion during inflammation.
To specifically investigate the role of enteric glial GSH production during in vivo
inflammation, I would propose the development of a glial specific GCL inhibitor. This
transgenic mouse would be generated by crossing Sox10::CreERT2 mice with a floxed
GCL (GCLf/f) mouse. In this transgenic animal, the excision of GCL (and hence
inhibition of glial GSH synthesis) would by inducible and cre-recombinase activated by
treating animals with tamoxifen citrate in chow. Induction of colitis in these animals
would allow us to specifically determine role of enteric glial GSH production during in
vivo inflammation. Nestin::Cre/GCLf/f mice would lack GCL in both enteric neurons and
glia, and their use in the DNBS colitis model would allow distinction between the roles of
glial-derived and neuronal-derived GSH in the ENS.

The Role of Neuronal TGF-beta Signaling in Colonic Inflammation
We show that the type II TGFβ receptor is expressed in enteric neurons, but not enteric
glia, in the myenteric plexus. In the CNS, neurons express TGFβ receptors
disrupted TGFβ signaling is associated with increased neurodegeneration

204

14,15

21.

and

An

important future study would be to investigate if TGFβ signaling plays a similar role in
enteric neurons. Nestin::Cre transgenic mice would express cre-recombinase in neural
crest derived cells including enteric neurons and glia. Crossing these mice with B6;129Tgfbr2tm1Karl/J (TGFβRIIf/f; from Jackson Labs) would generate Nestin::Cre/TGFβf/f mice.
Cre expression would be constitutive (as in our CD4-dnTGFβRII mice) and would allow
us to determine the effect of disrupted TGFβ signaling in enteric neurons. Although
Nestin::Cre mice would have cre expression in both enteric neurons and glia, we have
no evidence of TGFβRII expression in enteric glia.

Modulation of Cx43 Channel Opening by Pro-Inflammatory Cytokines and Mediators
In Chapter 5, we investigated the effect of key pro-inflammatory cytokines on Cx43
hemichannel opening and EtBr dye uptake. These studies used a small sample of key
cytokines associated with GI inflammation. A key follow-up study would be to
investigate the effects of a wider battery of cytokines. In particular, it would be
interesting to determine if Th1/Th2/Th17 families of cytokines produce any differential
effects on dye uptake. Secondly, these studies relied on the Cx43 mimetic peptide to
determine whether observed effects were Cx43-dependent or independent. Repeating
studies in transgenic Cx43 null mice (Sox10::CreERT2/Cx43f/f) would validate the findings
produced by pharmacological methods.

Mechanisms of Glial GSH Release and Regulation
Our in situ experiments in Chapter 3 demonstrate that glial GSH synthesis is necessary
for enteric neuron survival in situ. However, key questions regarding glial release and

205

metabolism of GSH still remain unanswered. Cx43-mediated glutathione release has
been reported in astrocytes 22,23 so a key experiment would question whether enteric
glial GSH is released by similar mechanisms. Secondly, the metabolism of GSH is an
important mechanism for modulation of cellular glutathione content 12.
Immunohistochemistry for key GSH metabolism enzymes, such as glutathione
peroxidase and glutathione reductase, within the enteric nervous system, would add
considerably to our knowledge of GSH regulation in the ENS.

The data presented in this dissertation begins to address the important role of
enteric glia cells, and glial regulation of oxidative stress and immune mediators, in
regulating enteric neuropathy during inflammation. In combination with proposed future
experiments, these findings will aid in the identification of novel therapeutic targets to
treat GI motility dysfunction.

206

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