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(>0? LIBRARY
Mich:- ‘ State

Ui'iivmalt'y'

 

 

 

This is to certify that the
dissertation entitled

INTERACTION BETWEEN DENDRITIC CELLS AND
CAMPYLOBACTER JEJUNI: ROLE OF TOLL-LIKE RECEPTOR
SIGNALING

presented by

VIJAY ANAND KARUPPANNAN RATHINAM

has been accepted towards fulfillment
of the requirements for the

PhD. degree in COMPARATIVE MEDICINE AND

INTEGRATIVE BIOLOGY

 

 

 

05/29/09

 

Date

MSU is an Affirmative Action/Equal Opportunity Employer

 

 

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5/08 K /PrqlAcc&Pres/C IRC/DaIeDue indd

INTERACTION BETWEEN DENDRITIC CELLS AND CAMPYLOBACTER
JEJUNI: ROLE OF TOLL-LIKE RECEPTOR SIGNALING
By

Vijay Anand Karuppannan Rathinam

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Comparative Medicine and Integrative Biology

2009

ABSTRACT

INTERACTION BETWEEN DENDRITIC CELLS AND CAMPYLOBACTER
JEJUNI: ROLE OF TOLL-LIKE RECEPTOR SIGNALING

By
Vijay Anand Karuppannan Rathinam

Campy/abacterjejuni is a clinically significant food-bome pathogen that
causes enteritis. Dendritic cells (00s) are central to initiating immune responses
to pathogens. One objective of this study was to understand the interaction of
murine DCs with C. jejuni and its impact on induction of T cell responses
mediating resistance. Following infection with C. jejuni, DCs were found to
efficiently kill C. jejuni and undergo activation by up regulating the surface
expression of maturation markers and by secreting IL-12, lL-6 and TNF-a.
Notably, C. jejuni-infected DCs induced Th1-differentiation of naive CD4+ T cells.
Next, we investigated the role of toll-like receptor (TLR) signaling in mediating
these responses. Upregulation of maturation markers was significantly impaired
in both TLR2-I- and TLR4-/- 005 relative to wild type (WT) DCs after C. jejuni
challenge. In contrast, TLR4 deficiency, but not TLR2 deficiency, profoundly
impaired the cytokine responses following C. jejuni infection. Because TLR4
utilizes both My088 and TRIF adapters for signal transduction, we investigated
the role of MyD88 and TRIF in these responses. The expression of maturation
markers and cytokines in response to C. jejuni was greatly reduced in the
absence of either MyD88 or TRIF. Furthermore, C. jejuni infection induced IRF-3

phosphorylation and IFN-B secretion by 005 in a TLR4-TRIF dependent fashion,

 

further demonstrating activation of this pathway by C. jejuni. lmportantly, TLR2,
TLR4, My088, and TRIF deficiencies all markedly impaired Th1-priming ability of
C. jejuni-infected DCs. Thus, our results show for the first time that cooperative
signaling through MyDB8-dependent and -independent (TRIF) arms of the TLR4
signaling represents a novel mechanism mediating C. jejuni-induced

inflammatory activation of DCs.

Copyright by
Vijay Anand Karuppannan Rathinam

2009

ACKNOWLEDGEMENTS

I would like to acknowledge first and foremost Dr. Linda S. Mansfield for
being a great advisor and allowing me to pursue my research interests in
immunology as well as for providing all the guidance and resources I needed. I
am also thankful to her for the freedom I enjoyed in my research.

I thank my committee member Dr. Kathleen Hoag for her critical inputs in
my research and for helping me in establishing the dendritic cell culture method
and other assays I have used. I would also like to sincerely thank my committee
members Dr. John Linz, Dr. Katheryn Meek, and Dr. Jon Patterson for their
guidance and support.

Members of the Mansfield lab made the working environment very friendly
and fun-filled. Julia Bell was of great help with statistical consultations and
critically reviewing my manuscripts. It has been a pleasure working with Jamie
Kopper, Jessica St Charles, JP Jerome, and Andy Flies and I would miss them
and all the fun moments we had in the last few years. I also thank other members
of the Mansfield lab such as Alice Murphy, Jenna Gettings, Jennifer Olmstead,
Amanda Stanton, Eric Smith, Alex Adrian, Bianca Buffa, and Anne Plovanich-
Jones for all their help.

I thank Vilma Yuzbasiyan-Gurkan for her support and for providing me with
the travel funds to attend various conferences. Victoria Hoelzer-Maddox and
Margaret Nicholas were of great administrative help. Louis King helped me with
the flow cytometry. I thank Andrea Amalfitano and Daniel Appledom for breeding

and providing the TLR2, MyD88 and TRIF KO mice I used and for all the exciting

 

 

 

immunology discussions.

I also thank Dr. Puliyur MohanKumar, Dr. Sheeba MohanKumar, and my
friends Ravi, Senthil, Madhu, Sugaleshini, Sarguru, and Madhan for all their help
and support.

I am grateful to my family for their love and support. My wife Sivapriya
Kailasan Vanaja has been a tremendous support and without her

encouragement, cooperation and help, I could not have completed this study.

vi

PUBLICATIONS

Chapters 2 and 3 of this dissertation have been published in Microbes and

Infection and Infection and Immunity, respectively, as follows:

Rathinam, V. A., K. A. Hoag, and L. S. Mansfield. 2008. Dendritic cells from
C57BU6 mice undergo activation and induce Th1-effector cell responses against
Campylobacterjejuni. Microbes Infect 10:1316-1 324.

Rathinam, V. A., D. M. Appledorn, K. A. Hoag, A. Amalfitano, and L. 8.
Mansfield. 2009. Campy/abacterjejuni-induced activation of dendritic cells
involves cooperative signaling through TLR4-My088 and TLR4—TRIF axes. Infect
Immun 77(6): 2499-2507.

vii

 

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................. ix
LIST OF FIGURES .............................................................................................. .x
CHAPTER 1. Introduction ........................................................................ 1
Camp ylobacter jejuni ........................................................................ 2
Host defense responses to C. jejuni ................................................... 15
Dendritic ells ................................................................................. 21
Toll-like receptors: Pathogen sensing and initiation of immune
responses ..................................................................................... 26
Rationale for this study .................................................................... 31
CHAPTER 2. Dendritic cells from C57BU6 mice undergo activation and
induce Th1-effector cell responses against Campy/abacterjejuni .................... 35
Abstract ........................................................................................ 36
Introduction ................................................................................... 37
Materials and methods .................................................................... 39
Results ........................................................................................ 44
Discussion .................................................................................... 49
Figures ......................................................................................... 55
CHAPTER 3. Campy/obacterjejunI-induced activation of dendritic cells
involves cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes ..... 68
Abstract ....................................................................................... 69
Introduction .................................................................................. 70
Materials and methods .................................................................... 73
Results ........................................................................................ 79
Discussion .................................................................................... 85
Figures ........................................................................................ 91
CHAPTER 4. Summary and future directions ............................................. 100
Summary ................................................................................... 101
Future directions .......................................................................... 1 13
APPENDIX ....................................................................................... 1 19
BIBLIOGRAPHY ................................................................................. 120

viii

LIST OF TABLES

Chapter 1

Table 1. TLR ligands ......................................................................... 33

LIST OF FIGURES

 

Chapter 1
Figure 1. TLR2 and TLR4 signaling ..................................................... 34
Chapter 2
Figure 1. Internalization and killing of C. jejuni by BM-DCs ....................... 56
Figure 2. Maturation and cytokine production by BM-DCs
infected with C. jejuni ....................................................................... 58
Figure 3. Effect of viability of C. jejuni on activation of DCs ...................... 60

Figure 4. Requirement for contact and internalization of C. jejuni
for activation of DCs ........................................................................ 62

Figure 5. Cytokine responses of BM-DCs to various strains of C. jejuni ...... 65

Figure 6. Polarization of CD4+ T cells by C. jejuni-infected BM-DCs ............ 67
Chapter 3
Figure 1. TLR2 and TLR4 signaling are required for C. jejuni-induced
maturation of BM-DCs ....................................................................... 92
Figure 2. C. jejuni-induced cytokine responses of BM-DCs is mainly
dependent on TLR4 signaling ............................................................. 94
Figure 3. MyD88 and TRIF signaling dependent Upregulation of surface
markers and cytokine secretion by C. jejuni-infected BM-DCs ..................... 96
Figure 4. Activation of lRF-3 phosphorylation by C. jejuni-induced TLR4wTRIF
signaling ......................................................................................... 97
Figure 5. IFN-B production by C. jejuni-infected BM-DCs .......................... 98
Figure 6. TLR2, TLR4, MyD88, and TRIF signaling are necessary for maximal
Th1 polarization by BM-DCs infected with C. jejuni .................................. 99
Chapter 4

Figure 1. Proposed model for the role of TLR signaling in

C. jejuni-induced activation of DCs .................................................... 112

xi

CHAPTER 1

Introduction

CAMPYLOBA C TER JEJUNI

Campy/obecterjejuni is one of the leading causes of food-borne bacterial
enteritis in humans. C. jejuni is also the second leading cause of traveler’s
diarrhea after enterotoxigenic Escherichia coli (146). Although Campy/obacter
was described by Theodor Escherich as early as 1886 as a spiral bacterium in
the colonic tissues of children who died of what he termed ‘cholera infantum’
(46), it was not recognized as a widespread etiological agent of diarrheal illness
until the 1970’s (23). The crucial breakthrough for this discovery was the
successful isolation of Campy/obacter from the feces and blood of a woman
suffering from severe diarrhea and fever (42). Heretofore, the microaerophilic
and fastidious growth requirements of this organism had made it difficult to
isolate.

C. jejuni belongs to the epsilon subdivision of the Proteobacten'a and is a
member of the family Campy/obacteraceae (40). C. jejuni is a non-spore forming
Gram-negative and microaerophilic bacterium. It is 0.2 - 0.8 um wide and 0.5 — 5
um long with a spiral, curved or rod shape and is highly motile with a
characteristic cork-screw darting motility. C. jejuni has a single unsheathed polar
flagellum at one or both ends (40). C. jejuni possesses a relatively small genome
of size ~ 1.7 Mb (1.59 -— 1.78 Mb) with low G+C content (~ 30%). C. jejuni exists
as a commensal in the gastrointestinal tract of many food animals, especially

chickens, and is transmitted to humans by the consumption of contaminated

water, undercooked meat, and raw milk (23, 54, 94) and by contact with pet

animals and livestock.

Epidemiology of C. jejuni infection

C. jejuni and C. coli are responsible for about 95% of Campy/obacter
infections in humans (100); however, other species of Campy/cheater such as C.
fetus, C. upsaliensis, C. hyointestinalis, and C. Iari are also implicated in human
intestinal and extraintestinal diseases. The significance of the non-jejuni and non-
coli species of Campy/obecters as human pathogens is just beginning to be
understood (100).

C. jejuni is the most common food-borne enteric bacterium in many
developed countries and is the second most common in the US (26). More than 2
million people are estimated to be affected by diarrheal illness due to C. jejuni
infection in the US every year (1). Infections with campylobacters account for
approximately 17% of hospitalization associated with food-borne pathogens
(112). In addition, the economic burden due to campylobacter illnesses is high,
up to $8 billion in the US annually (24). The incidence of Campy/cheater infection
in immunocompromised individuals such as AIDS patients has been reported to
be 40 — 100 times higher than in the immunocompetant population (152). The
rate of C. jejuni infection in the US and developed countries is higher in children
less than 1 year old. The incidence of C. jejuni infection has been reported to be
more frequent in males than in females in all age groups except for the 20-29

year old group, where the trend is reversed (126). Moreover, seasonal variation

occurs in the incidence of C. jejuni infection; C. jejuni cases appear to rise during
the spring, reach peak levels in summer, and decline thereafter with the smallest
number of cases reported during the winter months. The vast majority of C. jejuni
infections occurs as sporadic isolated cases not as part of large outbreaks (126).
In developing countries, C. jejuni infection is hyperendemic among
younger children with 5 — 10 separate episodes of symptomatic infection in the
first few years of life owing to poor hygiene (25, 160, 161 ). However, the rate of
symptomatic infections of C. jejuni decreases as age increases, which has been
shown to correlate with the appearance of C. jejuni-specific IgA antibodies in

serum (19).

Intestinal manifestations of C. jejuni infection

Infection with C. jejuni causes a spectrum of diseases in humans (184).
The clinical outcome of C. jejuni infection is likely to depend on multiple factors
such as dose and genetic make up of the C. jejuni strain, immune status of the
host and the individual’s intestinal microbiota (15). Self-limiting intestinal illness is
the most common manifestation in the industrialized nations. As few as 800
colony forming units (cfu) of C. jejuni can cause illness in humans (17) and 100
cfu in IL-10”' mice (110). After an incubation period of 1 — 7 days (usually 24 - 48
h), patients exhibit one or more of the following clinical symptoms: headache,
fever, abdominal pain, myalgia, and diarrhea. Abdominal pain and watery to
bloody diarrhea are the most frequent symptoms of C. jejuni infection, but

vomiting is not very common (18). Diarrhea persists for 3 - 4 days, with frank

blood appearing in the feces during the later stages in approximately 15% of all
patients and in 30% of hospitalized patients. Patients may continue to experience
abdominal pain for several days even after the diarrhea has disappeared. Usually
the duration of intestinal illness is less than a week even without antibiotic
therapy (172). The relative contributions of innate and adaptive immunity in this
resolution of infection are not clear. Relapses, usually characterized by mild
illness, can occur in about 20% of the patients (35, 172). It has been reported
that C. jejuni continues to be excreted for 16 days on average after the onset of
diarrhea (172). In developing countries, infection with C. jejuni can be
asymptomatic, and mild non-inflammatory diarrhea is also not uncommon (103,

160).

Extraintestinal manifestations of C. jejuni infection

Self-limiting intestinal illness, as described above, is the most common
clinical presentation in C. jejuni infection. In contrast, patients with
immunodeficiencies such as hypogammaglobulinemia and AIDS experience
prolonged and recurrent intestinal disease often accompanied by bacteremia and
extraintestinal diseases (18, 35, 94). Pancreatitis, cholecystitis, hepatitis,
peritonitis, and meningitis have been reported to be caused by C. jejuni (35).
Bacteremia during pregnancy can lead to premature labor, perinatal sepsis,
neonatal meningitis, and abortion; however, such pregnancy-related

complications due to C. jejuni infection are rare (18).

Complications due to C. jejuni infection

The common post-infectious complications of C. jejuni infection include
reactive arthritis (ReA), Reiters Syndrome, and Guillain-Barré Syndrome (688)
(18). ReA affecting ankles, knees, wrists and other peripheral joints has been
reported to occur in about 1% — 5% of C. jejuni patients (136). ReA is
characterized by stiffness, pain, swelling, warmth, and redness of the involved
joints. Though the incidence of ReA after C. jejuni infection did not correlate with
the human leukocyte antigen (HLA) subtype 827, the possession of this HLA-
827 tissue antigen is implicated in the severity of ReA (18). The molecular basis
of the development of ReA following C. jejuni infection remains unknown.

688, an acute progressive peripheral neuropathy, is also a rare
autoimmune complication associated with C. jejuni infection. C. jejuni infection is
the most common antecedent infection associated with the development of GBS;
about 20% of 688 patients have had a previous C. jejuni infection (159).
Infections with cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae,
and Haemophilus influenzae have also been associated with 688 cases (94,
159). Notably, asymptomatic infection with C. jejuni can also lead to the
development of 63$ (98). CBS is estimated to occur in 1 in 1.000 C. jejuni
patients (75, 94). The onset of neurological symptoms of GBS mostly occurs
about 1 - 3 weeks after infection with C. jejuni (75). 688 is the leading cause of
acute flaccid paralysis in polio-free regions of the world. 688 is characterized by
paralysis, pain, muscular weakness and respiratory insufficiency in some cases,

which warrants the use of ventilators (94).

One possible mechanism widely believed to underlie the development of
638 following C. jejuni infection is molecular mimicry between ganglioside
structures and lipooligosaccharide (LOS). The vast majority of GBS patients have
been reported to have autoantibodies against gangliosides, which are sialic acid-
containing glycosphingolipids that are highly concentrated in myelin sheaths of
neuronal axons. Autoantibodies have been detected against more than 20 types
of human cell surface gangliosides but more frequently against LM1, GM1,
GM1b, GM2, GD1a, GalNAc-GD1a, GD1 b, 602, GD3, GT1a and GQ1b (75). It
is hypothesized that the antibodies directed against C. jejuni LOS cross react
with gangliosides of nerves, initiating a vicious cycle of inflammation that
ultimately results in demyelination and nervous tissue damage (75). However, a
clear understanding of pathogenic mechanism(s) that contribute to the initiation

of CBS is still lacking.

Pathogenesis of C. jejuni.

After oral ingestion, C. jejuni reaches its predilection sites such as terminal
ileum, cecum, and proximal colon, where it adheres to and colonizes the apical
surface of the epithelium. In human patients and animal models, invasion of
intestinal epithelial cells by C. jejuni has been suggested to play an important role
in its pathogenesis (35, 54). This key process depends on several bacterial
factors including motility, adhesion, and secreted proteins. C. jejuni exhibits a

chemotactic response towards the mucus—particularly the glycoprotein

mucin—Iining the intestinal epithelium (69, 97). Consistent with this observation,

C. jejuni cells were found to be associated with mucus near goblet cells in pigs
orally challenged with C. jejuni (109). Furthermore, the chemotactic property of
C. jejuni has been shown to be important for its colonization of the intestinal tract
in murine models (158). Previous studies showed that the motility of C. jejuni
requires a functional flagellum and FlaA, one of the flagellar proteins (174). The
adhesion of C. jejuni to epithelial cells has been shown to be mediated by
surface structures such as PEB1 (a homolog of gram-negative ATP-binding
transporters), CadF (a fibronectin binding protein), and leA (surface lipoprotein)
(79, 80, 118, 131, 132). More importantly, a flagellar export system similar to the
type III secretion apparatus of other gram-negative bacteria has been identified in
C. jejuni. This flagellar secretion system was found to be essential for secreting
Campy/abacter invasion antigens into host cells and thus facilitating invasion
(96). Furthermore, a homolog of a type IV secretion system was identified in a
large plasmid (pVir) present in some but not all of strains of C. jejuni. The ComB
and VirB11 proteins encoded by this plasmid have been shown to contribute to
invasion of INT407 intestinal epithelial cells (IEC) in vitro and to virulence in a
ferret diarrheal disease model (11). C. jejuni has been shown to breach the
mucosal epithelial layer and gain access to the lamina propria by two other
routes besides apical invasion of epithelial cells: 1) via paracellular entry by
disrupting epithelial tight junctions, and 2) via uptake by M cells (168), after which
C. jejuni can enter epithelial cells basolaterally. All of these interactions with
epithelial cells trigger intense inflammatory responses characterized by the

infiltration of the lamina propria and submucosa with neutrophils and

lymphocytes, resulting in tissue damage and pathology. However, the exact role
of C. jejuni and its components in eliciting acute inflammatory responses and in

inducing tissue damage remains to be elucidated.

Colonization of intestinal tract by C. jejuni

Colonization of the small and large intestines is a critical step in the
pathogenesis of C. jejuni infection. Development of new animal models of C.
jejuni in recent years allowed identification of important pathogen factors that
mediate the colonization process. These factors include flagellin, a membrane
protein peb1A, a member of a two-component regulatory system (RacR) (22),
capsule, Png a member of a glycosylation system (83), and a multidrug efflux

pump CmeABC (102); some of those factors are described in detail below.

Virulence factors of C. jejuni

After the genome sequencing of C. jejuni NTCC 11168, there has been a
renewal of interest in studying the pathogenesis of C. jejuni infection, which has
led to the delineation of some of its virulence mechanisms. The following

virulence factors of C. jejuni have been shown to play a key role in pathogenesis.

Flagella
C. jejuni has a single unsheathed flagellum at one or both ends of the cell.
Flagella aid in motility of C. jejuni allowing it to move through the GI tract lumen

and into the mucus layer and have been shown to play a role in intestinal

epithelial cell attachment and invasion in vitro (55). Besides contributing to
motility-related processes, the flagellar apparatus serves as a type III secretion
system (96). Toll-like receptor 5 (TLR5) is an innate receptor expressed by host
cells that recognizes bacterial flagellins and triggers inflammatory responses (4).
Owing to amino acid substitutions at the TLR5 recognition site, C. jejuni flagellins
studied to date are not ligands for TLR5, thus contributing to immune evasion by
this bacterium (6). However, it is not known whether other pattern recognition
receptors for flagellin detection such as Nod-like receptor C4 and neuronal

apoptosis inhibitory protein 5 (NAIP5) (155) are activated by flagellin of C. jejuni.

Toxins

Pathogenic bacteria produce a variety of cytotoxins that play multiple roles
in mediating virulence during pathogenesis, including colonization, invasion, and
killing of host cells (170). Various strains of C. jejuni have been shown to be
cytotoxic to various cell lines; hemolytic activities have also been demonstrated.
The C. jejuni genome encodes four cytotoxins including cytolethal distending
toxin (CDT), a putative hemolytic cytotoxin (leA), a putative integral membrane
protein with a hemolysin domain, and phospholipase A (PldA) (129). With the
exception of CDT, the role(s) of these toxins in the disease process, except for
CDT, have yet to be elucidated. It has been shown that pore-forming cytolysins
of bacteria exhibit a diverse spectrum of pathogenic effects on host cells
including hemolysis, cytokine induction, cytoskeletal dysfunction, and killing of

host cells (170). There are many reports on the cytotoxic effects of C. jejuni on

10

various cell lines (3, 47, 82, 133). However, no consensus has been reached on
cytotoxin production by C. jejuni since different cell lines and strains of C. jejuni
were used in these studies. The genome sequence of C. jejuni contains a gene
tIyA of 762 bp encoding a putative hemolysin of 253 amino acids with molecular
weight of 29 kDa. The predicted amino acid sequence has a high identity to
hemolysins of Helicobacter pylori (43% identity), Treponema hyodysenteriae
(40% identity), and Mycobacterium tuberculosis (37% identity) (129). The C.
jejuni genome also contains the pIdA gene (990 bp) encoding phospholipase A2
of 329 amino acids with a molecular weight of 38.8 kDa. The predicted amino
acid sequence of PldA of C. jejuni is highly homologous to phospholipase A of
Campy/obacter coli (74% identity), Yersinia enterocolitica (35% identity) and H.
pylori (26% identity) ( 129). Preliminary experiments indicated that PldA of C.
jejuni is essential for cecal colonization in chicken (191), but it has not been
characterized further in vitro or in vivo.

Cytolethal distending toxin (CDT) is a holotoxin secreted by C. jejuni,
which is composed of three subunits (Cth, CdtB and CdtC). It possesses
DNAse I activity and causes cell cycle arrest in the GZIM phase and cell
distension and has been implicated in invasion and immunomodulation (62). CDT
has been shown to induce lL-8 production by intestinal epithelial cell line INT407.
It has also been documented that CDT triggers apoptosis in cultured human
28SC monocytes (61 ). Fox et al. (2004) reported that CDT is required for
persistent colonization of C57BL/129 mice as a cdtB mutant of C. jejuni, unlike

wild type strain, was not recovered from C57BL/129 mice at 2 and 4 months after

11

oral inoculation. They also suggested that CDT elicits proinflammatory responses

in the host and contributes to immune evasion by C. jejuni (48).

Capsule

Using genetic and biochemical approaches, Karlyshev et al. (2000)
demonstrated that C. jejuni possesses capsular polysaccharides (87), until then
considered to be high molecular weight lipopolysaccharides. Significant
variations in the capsular polysaccharide structure have been observed among
various strains of C. jejuni; this variation is partly due to phase variation. The
capsular polysaccharide of C. jejuni strain 11168 used in our studies has 6-
methyl-D-glycero-a-L-glucoheptose, B-D—glucouronic acid modified with 2-amino-
2-deoxyglycerol, B-D—GalfNAc and B-D—ribose (154), and possesses a novel
modification on the GalfNAc (157, 183). Capsular polysaccharides of C. jejuni
have been shown to play an important role in the INT407 cell invasion model in
vitro. A capsular polysaccharide-deficient strain of C. jejuni (kpsM mutant)
displayed a significantly reduced adherence and invasion phenotype upon
challenge of cultured INT407 cells (12). Correspondingly, Bacon et al. (2001)
showed in a ferret diarrheal disease model that capsular polysaccharides of C.
jejuni contribute to virulence. None of the ferrets inoculated with the kps mutant
of C. jejuni developed diarrhea whereas the wild type 81 -1 76 capsule-sufficient
strain caused diarrhea in 50% of ferrets (12). Furthermore, capsular
polysaccharides contributed to bacterial resistance to host anti-microbial

mechanisms. C. jejuni capsular polysaccharides appeared to protect the bacterial

12

cells to a certain extent from the bactericidal effects of constitutively expressed
epithelial-antimicrobial peptides human B-defensin 1 (hBD) and lysozyme but not
hBD—Z and hBD -3 (190). More research is warranted to conclusively understand

the role of capsular polysaccharides in host-pathogen interactions and immune

evasion by C. jejuni.

Lipooligosaccharide (LOS)

Lipopolysaccharide (LPS) is a key molecule associated with virulence and
immunogenicity of Gram-negative bacteria. The general architecture of LPS is
that a lipid A moiety anchored to the cell wall is covalently attached to highly
conserved inner and outer cores of sugars (45). A polymer of repeating
saccharide subunits called the O-polysaccharide, or O-chain, is attached to the
outer core (45). C. jejuni lipid A is antigenically similar to that of enterobacterial
pathogens such as Escherichia coli and Salmonella (120). But, unlike other
enterobacterial lipid A, C. jejuni lipid A contains 2,3-diamino-2,3-dideoxy-D-
glucose (GIcN3N) instead of glucosamine residues. Also, C. jejuni LOS has only
a short non-repeating polysaccharide unit instead of the O-polysaccharide
typically found in LPS. The inner core region of C. jejuni LOS is comprised of an
unique tetrasaccharide (Glc—Hep—Hep—Kdo) and a trisaccharide that also occurs
in the inner core of other bacterial species (120). Sialic acid substitutions can
occur in the inner and outer core oligosaccharides of C. jejuni LOS, which lead to
the molecular mimicry between C. jejuni LOS and ganglioside structures in host

nerve tissues (75, 120). Besides playing a role in the development of 688 in

13

infected patients, LOS of C. jejuni induces strong proinflammatory cytokine

responses from human dendritic cells (67).

Genetic variation in C. jejuni strains

The gene content of C. jejuni strains is highly variable, which is partly due
to the exchange of DNA between bacterial cells as well as variation that occurs
within the genome itself (183). C. jejuni are naturally competent, allowing the
transfer of genomic DNA from other C. jejuni strains (179). Furthermore, the C.
jejuni genome possesses several homopolymeric tracts (129)— repetitive
sequences ofa single nucleotide—that can give rise to slip-strand mispairing
during replication. This mechanism can result in variation in surface antigenic
structures of C. jejuni, including flagella and LOS. Consequently, the
pathogenicity of C. jejuni strains varies significantly: a recent study in our
laboratory demonstrated that various strains of C. jejuni that were isolated from
different sources, such as chicken and cattle, and belong to six different multi-
Iocus sequence types, vary in their ability to colonize and cause disease in IL-10

deficient mice (1 5).

14

HOST DEFENSE RESPONSES TO C. JEJUNI

Interaction of C. jejuni with innate cells

Intestinal epithelial cells (IE C)

Epithelial cells lining the mucosal tracts form the first line of defense
against invading pathogens. The innate responses from epithelial cells not only
provide a barrier to the invading pathogenic microbes but also educate the
antigen-presenting cells to induce appropriate adaptive responses (33). A
relatively well-characterized aspect of C. jejuni-host interplay is the C. jejuni-IEC
interaction. C. jejuni has been shown to invade IEC by both actin- and
microfilament-dependent pathways (16, 124). It has also been shown that lipid
rafts or caveolae on the cell membrane, which are enriched for signaling
molecules such as receptor tyrosine kinases, play a role in C. jejuni
internalization into IEC (175). C. jejuni is capable of surviving inside IEC for a
prolonged period of time, and Watson et al. (2007) demonstrated a possible
mechanism underlying this survival. They found that after internalization, C.
jejuni-containing vacuoles deviate from the canonical endocytic pathway, thereby
avoiding fusion with Iysosomes and bacterial degradation. The unique pathway
of entry of C. jejuni involving lipid rafts or caveolae is suggested to be central to
the escape of C. jejuni-containing vacuoles from being delivered to Iysosomes
(1 75).

Cultured IEC lines such as INT407 and T84 and human colonic tissue

explants infected with C. jejuni secrete lL-8. This cytokine is a chemotactic factor

15

that recruits neutrophils, macrophages, and other immune cells to the site of
infection (176). Two mechanisms have been suggested by which C. jejuni may
activate lL-8 production by epithelial cells: 1) adhesion to and invasion of cells by
C. jejuni (60), “and 2) direct action of CDT on epithelial cells (62). It has been
shown that signaling by ERK kinase—a member of the mitogen-activated protein
(MAP) kinases—is essential for lL-8 production by intestinal epithelial cells
infected with C. jejuni in vitro (176). C. jejuni infection of epithelial cells was
shown to activate nuclear translocation of NF-KB, a transcription factor that
mediates the production of various chemokines and cytokines involved in innate
immunity (113). Consistent with this finding, NF-KB signaling has been shown to
be essential for the development of Th1-associated antibody responses against
C. jejuni in CS7BL/129 mice (48).

The chemokine responses of intestinal epithelial cells after C. jejuni
infection have also been analyzed. C. jejuni was shown to up-regulate the
expression of macrophage inflammatory protein 1, monocyte chemoattractant
protein 1, and gamma interferon-inducible protein 10 in INT407 cells within 4 h of
infection (68). Intestinal epithelial cells also secrete antimicrobial peptides such
as human beta defensins 2 and 3 in response to C. jejuni infection in vitro; these
molecules have been suggested to play a role in limiting the infection (188).
Swine intestinal epithelial cells, IPEC-1, secreted lL-1B, TNF-a, lL-6, IL-8 and IL-
18 after C. jejuni infection (G. Parthasarathy, K. Jones, L. Cunningham et al.
unpublished data). In a swine rectal challenge model, C. jejuni induced

Upregulation of several proinflammatory cytokines such as lL-8, lL-6, lL-18 and

16

TNF-a and iNOS in the distal colon and lymphoglandular complexes from 1 — 48

h after infection (K. Jones, unpublished data).

Neutrophils

Neutrophilic exudates are a hallmark of C. jejuni-induced intestinal
disease in both human beings and animal models (48, 108, 149). However, little
is known about the interaction between C. jejuni and neutrophils. Using a green
fluorescent protein-tagged C. jejuni strain, Mixter et al. (2003) reported that after
intraperitoneal inoculation of BALB/c mice, C. jejuni was mostly associated with
CD11b+ Gr1” neutrophils recovered by peritoneal lavage (114). This finding
suggests that murine neutrophils can recognize and internalize C. jejuni in vivo.
Furthermore, fluorescence associated with intracellular bacteria in these cell
types decreased over time indicating the failure of C. jejuni to survive within
neutrophils (114). Consistent with this finding, another study showed that C.
jejuni strains elicited production of antimicrobial molecules such as oxidative
metabolites by neutrophils to varying degrees. Furthermore, opsonization of C.
jejuni with human serum increased phagocytosis by neutrophils, which was
accompanied by enhanced intracellular production of these oxidative metabolites
(167). These studies indicated that complement opsonization of C. jejuni

facilitates killing of intracellular bacteria by phagocytes such as neutrophils.

17

Macrophages and monocytes

Macrophages have been suggested to play an important role in resistance
to C. jejuni as depletion of macrophages resulted in mortality in more than 50%
of NMRI mice infected with C. jejuni (14). Consistent with this finding, cultured
macrophages, unlike IEC, have been demonstrated to be efficient in killing
internalized C. jejuni (70, 173, 175). In line with these findings, infection of
cultured murine macrophages with C. jejuni induced more than a 100-fold
increase in the expression of inducible nitric oxide synthase (N082) (70), which
is involved in the synthesis of an importantanti-microbial molecule, nitric oxide, in
phagocytic cells. Furthermore, wild type macrophages capable of producing high
levels of inducible nitric oxide were more efficient in killing C. jejuni than N082-
deficient macrophages (70). Also, C. jejuni triggered proinflammatory responses
from murine macrophages and the human monocytic cell line THP—1,
characterized by the production of cytokines such as lL-6 and TNF—a (84).
Furthermore, cytokine responses of THP-1 cells were found to be independent of
C. jejuni viability and presence of cytolethal distending toxin (84).

Like other enteric pathogens such as Salmonella and Shigella, C. jejuni
induced apoptosis of infected THP-1 macrophages (148). But C. jejuni-induced
apoptosis appeared to be delayed because significant levels of cell death were
only observed 24 h after infection. Furthermore, maximal apoptosis induction by
C. jejuni required Campy/obacter invasion antigen (CiaB) and proteinase K- and
heat-resistant bacterial component(s) but was independent of caspase-1 and

caspase-9 activation in host cells (148). In contrast, another study reported that

18

C. jejuni CiaB was dispensable for triggering apoptosis of infected 288C human
monocytic cells (61). Therefore, the mechanisms underlying C. jejuni-induced
apoptosis remain unclear. Even though apoptosis of C. jejuni-infected
macrophages has been suggested to favor the host by eliminating a potential
niche for C. jejuni in macrophages (148), the true significance of apoptosis-

induction by C. jejuni is not known.

Humoral and cell-mediated immunity against C. jejuni

The two different outcomes of C. jejuni infection in immunocompetant and
immunocompromised patients show that host immune responses are an
important determinant of the outcome of C. jejuni infection. Furthermore, oral
inoculation of SCID mice—that lack adaptive immune responses—with C. jejuni
resulted in persistence of C. jejuni in the intestine and severe inflammation in the
cecum and colon (27). This finding suggests that acquired immunity is essential
to protect mice of this genotype from C. jejuni-induced intestinal disease and to
clear C. jejuni colonization. Humoral immune responses to C. jejuni have been
characterized in human infections and mouse models. C. jejuni elicits a strong
antibody response in humans: serum lgA levels peak at 7 to 10 days after the
onset of symptoms but decline rapidly thereafter, whereas serum IgG levels peak
after 3 to 4 weeks of infection and persist long term (76). Furthermore, anti-C.
jejuni lgA antibodies have been detected in a variety of samples from infected
hosts including milk, saliva, urine and feces (99, 144). The surface exposed

components of C. jejuni such as PEB1, capsular polysaccharide antigens, and

19

cytolethal distending toxin were found to be immunogenic, with C. jejuni flagellin
being the immunodominant antigen (76). Consistent with human infections,
specific lgA, IgM, and lgG antibody responses were elicited against C. jejuni in
murine models of infection. IL-10"’+ and lL-10"‘ mice of C57BU6 (108), C3H and
non-obese diabetic (NOD) genetic backgrounds (110) and C57BL/129 mice (48)
challenged orally with C. jejuni exhibited a predominantly Th1-type associated
lgGZb antibody response. However, such robust antibody responses did not
protect the mice from C. jejuni-induced disease in the context of IL-10 deficiency
(108). In summary, the role of specific immunoglobulins in protection against C.
jejuni remains controversial.

The higher incidence and persistent nature of C. jejuni infections in
individuals with AIDS indicates that cell-mediated immunity plays an important
protective role against C. jejuni infection. However, little is known about cell-
mediated immunity to C. jejuni infection compared to infection with other enteric
bacterial pathogens such as Salmonella and Listeria. It is yet to be determined
which component of the cell-mediated immune response i.e., CD4 T cells, CD8 T
cells, NK cells or a combination of these, is activated by C. jejuni infection and

mediates protection against it.

20

DENDRITIC CELLS

Dendritic cells (DCs) are antigen-presenting cells that act as sentinels in
body tissues for the recognition of foreign infectious agents. DCs are present as
immature cells in peripheral tissues and are efficient in pathogen recognition and
uptake because they are equipped with pathogen recognition receptors (PRRs)
(89). Upon capturing microbial pathogens, DCs become mature, migrate to
draining lymph nodes, and present antigens to naive CD4+ T cells (32, 72, 89,
140). Extensive research in the last decade has demonstrated that DCs play an
instructive role in the development of appropriate T cell responses to pathogens
(74). Depending upon the nature of the pathogen and pathogen-associated
molecular patterns (PAMPs), DCs provide different kinds of signals, particularly
cytokine signals, to naive CD4+T cells, which then drive them towards a Th1,
Th2, TM? or T regulatory phenotype (86). Furthermore, mucosal 003 have the
ability to discriminate and induce tolerance towards self-antigens and intestinal
microflora (106, 121 ). On the other hand, pathogens have evolved ‘a multitude of
strategies’ (116) to evade and subvert DC functions, which include the
suppression of DC maturation and migration and hampering of cytokine
production (1 1 1 , 1 16, 164). Thus, given the facts that DCs are central to the
induction of antigen specific acquired immune responses and that infectious
agents have the ability to interfere with DC functions, it is of paramount

importance to understand DC-C. jejuni interactions.

21

Dendritic cell development

The development of DCs is one of the most complex phenomena in
Iimmunobiology because of the existence of multiple subsets of DCs and the
flexibility of DC developmental pathways. DCs and related cell types, monocytes
and macrophages, develop from progenitor cells in the bone marrow through
intricate pathways. The latest findings by Liu et al. (2009) provide evidence for
various stages in DC development in vivo and also for the stage at which DC and
monocyte developmental pathways diverge (104). According to their model, a
myeloid progenitor (MP) population gives rise to macrophage and DC precursors
(MDP) that differentiate into common DC precursors (CDP) and monocytes. The
point of divergence between DCs and monocytes thus appears to be at the MDP
stage. CDP differentiates into plasmacytoid DCs (pDCs) and conventional DCs
precursors (pre-cDC), which later give rise to cDCs. Furthermore, the terminal
differentiation of cDCs in the peripheral lymphoid tissues is controlled by

regulatory T cells in a FMS-like tyrosine kinase 3 (Flt3)—dependent manner (104).

Pathogen sensing by DCs

DCs are equipped with a set of germ-line encoded receptors known as
pattern recognition receptors (PRRs) that recognize highly conserved
components associated with pathogens. PRR expressed by DCs include Toll-like
receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors
(NLRs), and C-type lectin-Iike receptors (CLRs) (4). Various combinations of

these PRR combined with their intracellular and surface locations allow DCs to

22

recognize and respond to almost any kind of pathogen: viral, bacterial, fungal, or
parasitic. Highly orchestrated signaling cascades triggered by the PRR induce
inflammatory and innate immune responses that regulate antigen presentation
and the ensuing adaptive immune responses (4, 74). Pathogen sensing by DCs
and the signaling pathways involved are described in detail later in the chapter

with the main focus on TLR signaling.

Antigen presentation by DCs

Antigens in the cytosol and phagocytic vesicles are processed in two
different ways (77). In the first pathway, endogenous proteins in the cytosol—for
example, viral proteins synthesized by cellular machinery—are degraded by the
proteosome complex of the cell into peptides that are translocated into the lumen
of the endoplasmic reticulum (ER) by transporters associated with antigen
processing (TAP), a heterodimer of TAP1 and TAP2. Then, the peptide binds to
the partly folded MHC class I molecule—which is present as a complex with TAP
and a chaperone molecule in the ER—resulting in complete folding of the MHC
class I molecule that is subsequently released from the complex. This MHC-I
complex with the peptide is then translocated onto the cell surface through the
Golgi apparatus and presented to CD8+ T cells. In the second pathway, following
internalization, pathogens and their products are subjected to limited proteolysis
in phagosomes by proteases such as cathepsins B, D, S and L to generate short
peptides. The peptides thus generated are loaded onto the peptide groove in the

MHC class II molecule after the release of the CLIP fragment from the MHC

23

class II molecule by HLA-DM (an MHC class Il-like molecule in the lysosome).
The MHC-II molecules with the antigenic peptides are exported to the cell
surface and presented to CD4+ T cells (77).

Besides these classical pathways of antigen presentation, a new pathway
of cross presentation has been identified. Cross-presentation is a mechanism by
which DCs present exogenous antigens derived from the extracellular milieu in
the context of an MHC-l molecule instead of the conventional MHC-ll (143).
Cross presentation represents a novel mechanism of generating effector CDB T
cell responses by DCs to exogenous antigens, such as bacterial pathogens.
Recently it has also been shown that cross presentation of apoptotic cells by

D05 is critical for the induction of tolerance against self-antigens (143).

DC-driven polarization of naive CD4*T cells

A large body of evidence clearly shows that DCs are the key cell type that
determines the nature of effector CD4“ T cell responses such as Th1, Th2,
regulatory T cells, or the recently described Th17 responses (86, 116, 138). This
capacity of DCs is primarily due to their ability to differentially recognize various
pathogens and convey distinct combinations of signals to naive T cells. Potent
activation of T cell responses by DCs requires delivery of three signals to naive
CD4’ T cells: signals 1 and 2 are MHC-ll plus antigen complex and costimulatory
molecules, respectively. lmportantly, DCs deliver a third instructive signal, mostly
cytokines, which are largely responsible for inducing the effector T cell phenotype

(86). In general, upon recognition of intracellular bacteria such as Salmonella

24

lyphimurium, DCs express lL-12, lL-18, or CD70 promoting IFNay-secreting Th1
cells. Infection with parasitic helminths induces DCs to up-regulate molecules like
OX40L, which primes Th2-differentiation of naive CD4+ T cells (86). Furthermore,
intensive research during the last few years demonstrated that DCs are/also
capable of inducing Th17 cells, a novel subset of effector T helper cells which
play an important role in mucosal immunity and autoimmune responses (9). In
response to ATP produced by bacteria in the GI tract, a CD70"'9"CD11c'°‘” subset
of DCs in the lamina propria secretes IL-6, lL-23 and TGF-B; the combination of
these cytokines leads to development of lL-17- producing Th17 cells from naive

CD4” T cells (9).

25

TOLL-LIKE RECEPTORS: PATHOGEN SENSING AND INITIATION OF
IMMUNE RESPONSES
Toll-like receptors (TLRs) are a type of pattern recognition receptor that

recognize conserved molecular patterns associated with a broad range of
pathogens and initiate protective immune and inflammatory responses against
them. Various cell types, particularly professional antigen-presenting cells, such
as dendritic cells, macrophages, and B cells, express TLRs (74). TLRs are type I
integral membrane glycoproteins with extracellular and cytoplasmic domains.
Extracellular domains are characterized by varying numbers of Ieucine-rich-
repeat motifs, 24-29 amino acids in length. The cytoplasmic signaling domain is
homologous to that of the IL-1 receptor (IL-1 R) and contains a Toll/IL-1 R (TIR)
domain (91 ). Each TLR identifies a different microbial structure (Table 1); for
example, TLR2 recognizes peptidoglycan and lipopeptides, TLR4 recognizes
lipopolysaccharide (LPS), and TLR5 recognizes flagellin. Upon detecting
pathogens, TLRs initiate intracellular signaling cascades through adapter
molecules, which ultimately result in the production of proinflammatory cytokines
and other immune responses (91). A growing body of evidence suggests that
TLR2 and TLR4 signaling pathways play an essential role in the detection of
invading bacterial pathogens and in mounting protective immune responses

against them (63, 88, 162).

26

TLR4 and TLR2 signaling

MyD88-dependent pathway

Upon binding LPS, TLR4 undergoes dimerization leading to the
recruitment of an adapter protein MyD88 via another molecule TIRAP. Similarly,
recognition of specific ligands by TLR2 in combination with TLR1 or TLR6
recruits MyD88 via TIRAP. MyD88 activates lL-1R-associated kinase-4 (IRAK4),
which then recruits IRAK1 (4). The downstream target molecule for IRAK1 is
TRAF6, which is then polyubiquitinylated and activated by ch13 and Uev1A,
leading to the activation of TAK1. In combination with TAB1, TABZ and TAB3,
TAK1 activates the IKK complex (lKKa + IKKB + lKKy/NEMO), which catalyzes
the phosphorylation of IKB proteins. Under normal conditions, IKB sequesters NF-
KB in the cytoplasm thus preventing its activation and nuclear translocation. The
phosphorylation of “(3 leads to its degradation, releasing NF-kB that translocates
to the nucleus to activate transcription of a number of inflammatory genes. The
IKK complex also activates MAP kinases leading to the activation of transcription
factors such as AP-1 and c-Jun. Additionally, the My088-IRAK4-TRAF6 complex
phosphorylates the IRF5 transcription factor, which then translocates to the
nucleus to activate genes involved in proinflammatory responses. Thus, MyD88
signaling triggers robust Inflammatory responses through the activation of various

transcription factors (91).

27

MyD88-independent pathway

TLR4 also signals through a MyD88-independent pathway, which is
mediated by TRIF, another adapter molecule (4). Following ligand binding, the
TLR4 cytoplasmic domain recruits the adapter protein TRIF through TRAM.
Medzhitov and colleagues recently demonstrated that MyD88 and TRIF signaling
are sequentially activated by TLR4 (85). According to their model, activation of
TLR4 by LPS first triggers TIRAP-My088 signaling from the cell membrane.
Then TLR4-LPS is endocytosed, leading to displacement of TlRAP-MyD88 from
TLR4 and termination of MyD88 signaling. TRIF is then recruited to the
cytoplasmic domain of TLR4 in the endosomes via TRAM. leading to a second
phase of signaling from TLR4. TRIF recruits TRAF3, which forms a complex with
TBK1 and IKKi. This complex phosphorylates a transcription factor unique to the
TRIF pathway, lRF-3, which mediates the expression of type I IFN and IFN-
inducible genes. TRIF also interacts with TRAF6 and RIP1, which mediate NF-KB
and MAP kinase activation as described above in the MyD88-dependent pathway
(90). Thus, TLR4-TRIF and TLR4-My088 pathways activate a number of
overlapping cellular responses, but activation of type I interferon responses is

unique to the TRIF arm of TLR4 signaling.

TLR4, TLR2, MyD88, and TRIF signaling in host defense

As sensors of invading pathogens, TLRs expressed by DCs have been

shown to play an instructive role in the development of adaptive T-cell immunity.

28

In murine models of Bordetella pertussis infection, TLR4 signaling was shown to
activate antigen-specific regulatory T cells by inducing lL-10 production from
DCs, which confers resistance by limiting inflammatory pathology (63). Similarly,
TLR2 was described to have a protective role in Listeria monocytogenes infection
in mice; infected TLR2-deficient mice had increased bacterial burdens and
reduced survival rates (162). Furthermore, the maturation of and TNF-a
production by BM-DCs following Francisella tularensis infection were found to be
mediated via TLR2 (88). Host defense responses to various pathogens have
been shown to be dependent on MyD88. For instance, MyDB8-deficient mice
infected with Leishmania major failed to produce IL-12 and developed a Th2-type
response instead of a protective Th1-type response and were therefore severely
compromised in their resistance to infection (122). Furthermore, the production of
TNF-a, lL-12 and nitric oxide and the expression of co-stimulatory molecules by
BM-DCs and macrophages following L. monocytogenes infection were almost
abolished in the absence of MyD88 (162). Correspondingly, MyDB8-deficient
mice were highly susceptible to L. monocytogenes infection (162). In contrast to
My088, the role of TRIF signaling in mediating protection against bacterial
pathogens is just beginning to be understood. Recent studies have shown that
TRIF is essential for pulmonary host defense against Gram-negative bacteria
such as Pseudomonas aeruginosa and Escherichia coli (78, 137). However, the
role of TLR2, TLR4, MyD88, and TRIF signaling in host defense against C. jejuni
infection remains largely unknown and thus formed a large part of the focus of

my work.

29

TLR recognition of C. jejuni

C. jejuni has been reported to possess multiple Iipoproteins, including
CjaA, HisJ, and Omp18 as well as leA. Even though none of these have been
demonstrated to be recognized by TLR2, the presence of multiple lipoproteins
suggests that C. jejuni has potential ligands that can activate TLR2 signaling.
Indeed, Friss et al. (2009) recently reported that TLR2 signaling was necessary
for C. jejuni-induced lL-6 production by cultured IEC (50). C. jejuni flagellin is not
recognized by TLR5, and Andersen-Nissan et al. (2005) reported that this
immune evasion is due to mutation in the TLR5 recognition site of C. jejuni
flagellin (6). Furthermore, owing to the low frequency of CpG motifs in its
genomic DNA, C. jejuni is a poor inducer of TLR9 signaling (38). Accordingly, C.
jejuni DNA was found to be dispensable for triggering lL-8 secretion from IEC in
vitro (187). It has also been shown that My088 signaling is necessary for the
innate inflammatory responses of intestinal epithelial cells and antigen-presenting

cells following infection with C. jejuni (177, 187).

30

RATIONALE FOR THIS STUDY

Our understanding of the interaction between C. jejuni and DCs, a key cell
type that bridges innate and adaptive immunity, is very limited. Hu et al. (2006)
reported that human monocyte-derived DCs recognize C. jejuni, undergo
maturation, and secrete proinflammatory cytokines (67). However, the ability of
C. jejuni-infected DCs to activate T cell responses—a primary function of DCs-is
not known. Furthermore, the TLRs that recognize C. jejuni and mediate DC
responses against C. jejuni remain unknown. Also, the relative contribution of
MyD88—dependent and -independent pathways to the activation of DCs has yet
to be determined. The main objective of this study was to address these
knowledge gaps.

This study was designed with the following specific aims to investigate the
interplay of C. jejuni with murine DCs and its impact on the development of
subsequent T helper responses and to understand the mechanism of activation

of C. jejuni-infected murine DCs.

Specific Aim 1: To characterize the interaction of C. jejuni with murine bone-
marrow derived-DCs (BM-DCs) with respect to internalization and processing of
C. jejuni and maturation of DCs.

Hypothesis: Murine BM-DCs take up C. jejuni, process it, and undergo

maturation in vitro.

31

Specific Aim 2: To determine whether the C. jejuni-stimulated murine BM-DCs
produce T helper (Th) cell-polarizing signals and induce Th polarization in vitro.
Hypothesis: C. jejuni-stimulated murine BM-DCs produce Th1-polarizing

signal(s) and thereby induce Th1-type immune response in vitro.

Specific Aim 3: To determine the role of TLR2, TLR4, MyD88, and TRIF in the
activation of BM-DCs by C. jejuni.
Hypothesis: C. jejuni-induced activation of bone marrow-derived DCs
(BM-DCs) is mediated by TLR2 and TLR4, and that MyD88 and/or TRIF

signaling is necessary for this activation.

The findings for specific aims 1 and 2 are described in chapter 2. Chapter
2 also describes the bacterial factors that contribute to C. jejuni-induced
activation of DCs. The findings from specific aim 3 are covered in chapter 3.
Finally, chapter 4 summarizes the findings reported in chapters 2 and 3 and

discusses future investigations suggested by this study.

32

Table 1. TLR ligands

 

 

 

 

 

 

 

 

 

 

 

 

TLRs Ligands
Peptidoglycan of Gram-positive bacteria; porins of
Neisseria; lipoarabinomannan of Mycobacteria;
TLR2
phospholipomannan of Candida albicans; hemagglutinin
protein of measles virus
TLR2 and TLR1 Triacyl Iipoprotein
Diacyl Iipoprotein; zymosan; lipoteichoic acids from group
TLR2 and TLR6
B Streptococcus
TLR3 Double-stranded RNA from viruses
Lipid A of LPS; DnaK from Francisella tularensis;
TLR4 fusion protein from respiratory syncytial virus; envelope
protein from mammary tumor virus
TLR5 Flagellin
TLR7 Single-stranded RNA
TLR8 Single-stranded RNA
TLR9 CpG DNA, hemozoin
Profilin like protein, uropathogenic E. coli
TLR1 1

 

components

 

33

 

 

 

Activation of Type I interferon
Inflammatory genes response

Figure 1. TLR2 and TLR4 signaling

34

CHAPTER 2

Dendritic cells from C57BU6 mice undergo activation and induce Th1-

effector cell responses against Campylobacterjejuni

Rathinam, V. A., K. A. Hoag, and L. 8. Mansfield. 2008. Dendritic cells from
C57BU6 mice. undergo activation and induce Th1-effector cell responses against
Campy/obacterjejuni. Microbes Infect 10: 1 316-1 324.

35

ABSTRACT

Food-borne Campy/obacterjejuni is an important cause of enteritis. We
showed that CS7BL/6 mice and congenic interleukin (lL)-10"' mice serve as
models of C. jejuni colonization and enteritis, respectively. Thus, C57BU6 mice
are resistant to C. jejuni-induced disease. Because dendritic cells (DCs) are
central to regulating adaptive immune responses, we investigated the interaction
of C. jejuni with murine bone marrow-derived DCs (BM-DCs) to assess bacterial
killing, DC activation, and the ability of C. jejuni-infected BM-DCs to stimulate
Campy/abacter-specific T cell responses in vitro. BM-DCs challenged with C.
jejuni efficiently internalized and killed C. jejuni 11168 and significantly up-
regulated surface MHC-II, CD40, CD80 and CD86 demonstrating a mature
phenotype. Infected BM-DCs secreted significant amounts of tumor necrosis
factor-a, lL-6 and lL-12p70. Formalin-killed C. jejuni also induced maturation of
BM—DCs with similar cytokine production but at a significantly lower magnitude
than live bacteria. Maximal activation of murine BM-DCs required internalization
of C. jejuni; attachment alone was not sufficient to elicit significant responses.
Also, various strains of C. jejuni elicited different magnitudes of cytokine
production from BM-DCs. Finally, in a coculture system, C. jejuni-infected BM-
DCs induced high-level interferon-y production from CD4“ T cells indicating Th1
polarization. Thus, DCs from resistant C57BU6 mice initiate T cell responses

against C. jejuni.

36

INTRODUCTION

Campylobacterjejuni is an common bacterial agent of enteritis worldwide.
Campylobacteriosis usually presents as self-limiting intestinal illness with the
clinical spectrum ranging from mild watery to severe bloody diarrhea. However,
persistent and systemic illnesses can develop in immunocompromised patients
(1 71 ).

C. jejuni invades the intestinal mucosa (108, 166) and resides inside
intestinal epithelial cells (IEC) (166) and in the lamina propria causing pathology
predominantly in colon (166) and, in mice the cecum (108). DCs densely
populate the lamina propria, are in close proximity to the epithelium, and can
form transepithelial dendrites to sample the lumen for microbes (123). We
observed C. jejuni associated with mononuclear cells morphologically similar to
DOS in the lamina propria and submucosa of infected C57BU6 IL-10"' mice with
enteritis (108). The strategic location of DCs in the subepithelial area and the
invasive nature of C. jejuni (108, 166) together with our recent observations
suggest that DCs in the lamina propria are likely candidates for capture and
processing of C. jejuni for antigen presentation. Furthermore, Johanesen and
Dwinell demonstrated that infection of cultured IEC with C. jejuni upregulates
secretion of the DC chemokine CCL20, suggesting this as a mechanism of
increased DC recruitment into the intestinal mucosa (81 ). Therefore, it is crucial
to understand DC-C. jejuni interactions and the role of DCs in stimulating host

defense against C. jejuni.

37

Human monocyte-derived DCs were recently shown to undergo
maturation and secrete proinflammatory cytokines subsequent to C. jejuni 81-176
challenge in vitro (67); but to our knowledge, the resultant antigen-presenting
capacity of DCs for CD4+ T cell polarization has not been investigated. We
hypothesized that murine bone marrow-derived DCs (BM-DCs) internalize C.
jejuni, which induces BM-DCs to undergo activation and initiate a Th1-type
immune response. In this study, we investigated activation of BM-DCs by C.
jejuni, mechanisms mediating the activation and the ability of C. jejuni-infected

BM-DCs to stimulate T cell responses.

38

MATERIALS AND METHODS
Mice
CS7BU6J mice purchased from The Jackson Laboratory (Bar Harbor, ME)
were bred, maintained, and monitored as described (108), and used at 8—12
weeks of age. Animal protocols were approved by MSU Institutional Animal Care

& Use Committee conforming to NIH guidelines.

Bacteria and inoculum preparation

C. jejuni strains were grown on Bolton agar (BA) plates at 37 °C in 10%
C02. Bacterial growth was resuspended in R10.2 medium (RPMI 1640 Dutch
modification [Sigma-Aldrich, St. Louis, MO] with 2 mM L-glutamine [lnvitrogen,
Grand Island, NY] and 10% fetal bovine serum [FBS; lnvitrogenl) to an optical
density of 0.12 at 560 nm (~2 x 108 colony forming units [CFU]/ml). Where
needed, bacteria were inactivated by treatment with 2% formaldehyde in Hank’s
Balanced Salt Solution (HBSS) for 30 min at 37 °C. CFU in the inocula and

bacterial killing were confirmed by limiting dilution on BA plates.

Generation of BM-DCs

BM-DCs were generated as described with minor modifications (21, 105,
186). Bone-marrow cells from femurs and tibiae were cultured in bacteriological-
grade Petri-dishes in 10 ml (2.5 x 105 cells/ml) of R10 medium (R10.2 medium
containing 50 uM 2-mercaptoethanol, 100 U/ml penicillin, 100 uglml streptomycin

[lnvitrogen] and 20 ng/ml murine granulocyte-macrophage colony-stimulating

39

factor [Peprotech, Rocky Hill, NJ]). Non-adherent cells in the culture were
discarded on days 3 and 6. On day 3, 10 ml of fresh medium was added; on days
6 and 8, 10 ml of medium was exchanged for fresh medium. DCs were collected
and used on day 9. The proportion of DCs in the culture was consistently >90%

as determined by flow cytometric analysis of CD110 and MHC-ll markers.

Gentamicin killing assay

This assay was performed as described with minor modifications (173).
Bacteria diluted in R10.2 medium were added to 2 x 105 BM-DCs per well in a
24-well plate (BD Falcon, Bradford, MA) at multiplicity of infection (MOI) of 10 or
100 bacteria per BM-DC. After 1 h of incubation, non-phagocytosed bacteria
were removed by washing the cells and resuspending in R10 medium containing
250 ”9”!“ gentamicin but without penicillin and streptomycin. After 1 h, cells were
washed and resuspended in R10 medium without antibiotics. Viable intracellular
bacteria were enumerated at 2, 4 and 8 h post-infection (p.i.) by lysing the cells
with 0.5 ml of 0.1% Triton X-100 in phosphate-buffered saline for 15 min and
spreading serial dilutions of lysate on BA plates. Colonies were counted 72 h

after incubation at 37°C with 10% C02.

Assessment of maturation and cytokine secretion by C. jejuni-infected BM-

DCs
BM-DCs (2 x 105 cells/ml) were treated with c. jejuni (MOI 10:1 [in

cytokine experiments only] or 100:1 ), R10.2 medium (negative control) or 0.1

40

uglml Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS;
positive control; Sigma-Aldrich). In some experiments, to inhibit internalization of

C. jejuni, BM-DCs were preincubated with cytochalasin D (1 jig/ml) and

nocodazole (20 uM; Sigma-Aldrich) or DMSO (solvent control, 0.1%) for 1 h and
treated as described above. In other experiments, BM-DCs were also treated
with formalin-killed bacteria. One h after infection, gentamicin (250 pglml) was
added to all wells to kill extracellular bacteria. DCs were analyzed by flow
cytometry at 24 h pi For cytokine analysis, supernatants were collected at 4, 24

and 48 h p.i. unless otherwise indicated.

Flow cytometry

Antibodies were from BD Pharmingen, San Diego, CA unless otherwise
noted, including R-PE-conjugated anti-CD11c (HL3), F lTC-conjugated anti-MHC-
ll (l-A") (AF6-120.1), FlTC-conjugated anti-CD40 (3/23), FITC-conjugated anti-
C080 (16-10A1), PE-Cy5-conjugated anti-CD80 (Biolegend, San Diego, CA),
F ITO-conjugated anti-CD86 (GL1), PE-Cy5-conjugated anti-CD86 (Biolegend)
and FITC-conjugated anti-CD4 (RM4—5) along with corresponding isotype
controls. Cells were incubated with Fc block (anti-mouse Fchl/lll) for 10 min at 4
°C. Fluorochrome-conjugated antibodies were added to cells and incubated for
30 min at 4 °C. The cells were washed twice with HBSS containing 1% FBS and
0.1% sodium azide and analyzed with a FACS Vantage flow cytometer (BD

Biosciences).

41

DC-CD4’T cell coculture

CD4+ T cells were negatively selected from splenocytes using the B0”
IMag Mouse CD4 T Lymphocyte Enrichment Set-DM (BD Pharrningen) according
to manufacturer’s instructions. The proportion of CD4+ T cells was enriched to >
90% as measured by flow cytometry. BM-DCs in complete DMEM containing 25
mM HEPES, 10% FBS, 2 mM L-glutamine, 50 uM 2-mercaptoethanol, 1 mM
sodium pyruvate, and 1% MEM non-essential amino acids seeded in 24—well
plates (105 cells/well) were treated with one of the following: C. jejuni (MOI of
100:1), R10.2 medium, S. Iyphimurium LPS (0.1 ug/ml; inducer of IFN-y
producing Th1 cells), or zymosan (10 ug/ml; inducer of IL-4 production from Th
cells; Sigma-Aldrich). One h after infection, gentamicin (250 jig/ml) was added to
all wells. At 24 h p.i., CD4+ T cells in complete DMEM supplemented with 100
U/ml penicillin and 100 pg/ml streptomycin were added to DCs at a DC-to-T cell
ratio of 1:10. After coculturing for 72 h, supernatants were collected for

measurement of IFva, IL-4 and IL-10.

ELISA

Cytokines in culture supernatants were measured using Ready-SET-Go!
ELISA Sets (eBioscience, San Diego, CA) according to manufacturer’s
instructions. Assay detection limits (in pg/ml) were lL-12, 15; lL-10, 30; lL-4, 4; IL-

6, 4; TNF-a, 8; IFN-y, 0.8.

42

Statistical analysis
One-way or two-way analysis of variance followed by the Holm-Sidak
correction for multiple comparisons was performed using SigmaStat 3.1 (Systat

Software, San Jose, CA). P values < 0.05 were considered significant.

43

RESULTS

BM-DCs internalize and kill C. jejuni

Within 2 h p.i., BM-DCs internalized C. jejuni in a dose-dependent manner
(Fig. 1A). Approximately 5 i 0.8 x 103 and 7.38 i 0.2 x 104 viable intracellular
bacteria were recovered at M0ls of 10:1 and 100:1, respectively, 2 h pi (Fig 1A),
which decreased rapidly to 1 a 0.18 x 103 and 1.3 a 0.0076 x 104 for M0ls of
10:1 and 100:1, respectively, by 4 h p.i.; no viable bacteria were recovered at 8 h
p.i. at either MOI. In contrast, CFU of C. jejuni inoculated into R10 medium

without antibiotics and DCs increased by several-fold at 8 h p.i. (Fig. 1B).

BM-DCs undergo maturation following C. jejuni infection

In experiments to test BM-DC maturation, mean fluorescence intensities
(MFI) indicating the levels of expression of MHC-II, CD40, C080 and CD86 were
significantly higher by 2.1-, 1.9-, 3.7-, and 2.5-fold, respectively, in C. jejuni-
infected BM-DCs compared to uninfected cells (Fig. 2A). Furthermore, infection
with C. jejuni resulted in a significant (P < 0.05) increase in the proportion of BM-
DCs expressing these markers (Fig. 2A). In all cases, these levels were

comparable to those induced by LPS.

C. jejuni-infected BM-DCs secrete IL-12p70 and proinflammatory cytokines
C. jejuni induced a dose-dependent increase in lL-12p70 secretion at 24
and 48 h p.i. (Fig. 2B). Peak levels of lL-12p70 secretion were observed with the

higher dose of C. jejuni at 24 h p.i. In contrast, the lower dose of C. jejuni induced

44

markedly lower amounts of lL-12p70 at 24 and 48 h p.i. C. jejuni also induced
dose-dependent production of lL-6 and TNF-a by BM-DCs at 4, 24 and 48 h p.i.
(Fig. 2B). At 4 h p.i., moderate production of lL-6 was induced by the higher dose
of C. jejuni, which increased at later time points (24 and 48 h). At the lower dose
of bacteria, a similar trend was observed in IL-6 secretion but at a significantly
lower magnitude (Fig. 2B). BM-DCs challenged with C. jejuni did not produce
detectable quantities of lL-10 at any of the three time points studied (data not

shown).

C. jejuni viability is required for maximal activation of BM-DCs

Infection with killed C. jejuni also resulted in upregulation of maturation
markers; however, live C. jejuni induced significantly higher (P < 0.05) levels of
expression of CD80 and CD86 than formalin-killed C. jejuni. Similarly, a
significantly higher proportion (P < 0.05) of live C. jejuni-exposed DCs expressed
CD40 and CD86 than DCs treated with formalin-killed C. jejuni (Fig. 3A).
However, the level of MHC-II expression and proportion of DCs expressing MHC-
II or CD80 were not significantly different (P > 0.05) between live- and formalin-
killed C. jejuni-exposed DCs (Fig. 3A).

Bacterial viability also affected cytokine secretion. At 24 and 48 h p.i., live
C. jejuni-exposed DCs secreted 4.7- and 3.2-fold higher levels of lL-12 and 4.5-
and 4.2-fold higher levels of IL-6 than DCs exposed to formalin-killed C. jejuni
(Fig. 33). Live C. jejuni elicited significantly greater (P < 0.05) TNF-a production

from DCs than formalin-killed C. jejuni at all time points. Like live C. jejuni,

45

formalin-killed C. jejuni did not elicit detectable IL-10 secretion from DCs at 4, 24

or 48 h p.i. (data not shown).

Full activation of BM-DCs requires contact of C. jejuni with DCs and
bacterial internalization

C. jejuni entry into IEC has been suggested to involve both actin filaments
and microtubules (125). Therefore, we used a combination of cytochalasin D (1
uglml) and nocodazole (20 pM), inhibitors of actin and microtubule
polymerization respectively, to inhibit phagocytosis of C. jejuni by DCs. This
treatment of DCs resulted in about 80% inhibition of uptake of C. jejuni as
assessed by the gentamicin killing assay, which was markedly higher than that
observed with either inhibitor alone (Fig. 1C).

Preincubation with inhibitors alone caused a significant increase in the
MFI of MHC-ll and CD80 and in the proportion of CD40+ and C086" DCs as
shown previously (115). Comparison of inhibitor-treated and non-treated DCs
infected with C. jejuni showed that pretreatment with cytochalasin D and
nocodazole abolished the increase in MHC-ll expression level and markedly
reduced CD40, C080 and CD86 expression levels and the proportion of DCs
expressing CD40, CD80 and CD86 (Fig. 4A-B). Blocking DC phagocytosis of C.
jejuni with cytochalasin D and nocodazole completely inhibited IL-12 secretion
and significantly (P < 0.05) reduced TNF-oi secretion, but did not inhibit IL'6

production. The effect of inhibitors on Salmonella LPS-induced expression of

46

surface markers and cytokines was comparable to that observed for C. jejuni-
induced responses.

To test whether activation of DCs depends on direct contact with C. jejuni
or whether a diffusible bacterial product is sufficient to stimulate DC responses,
C. jejuni and DCs were separated by a 0.2 uM transwell insert, which allowed
passage of bacterial products across the membrane but not intact bacteria. The
presence of a membrane between DCs and C. jejuni resulted in a significant (P <
0.05) reduction in MHC-ll and CD80 expression levels and in the number of
CD40“, 0080*, and CD86+ DCs. Furthermore, membrane presence abolished
the increase in CD86 expression level and in the proportion of DCs expressing
MHC-II after C. jejuni challenge (Fig. 4C-D). lmportantly, the prevention of direct
contact between C. jejuni and DOS resulted in abrogation of lL-12, TNF-a and IL-
6 production (Fig. 4E). However, the presence of membrane only minimally
affected DC expression of surface markers, TNF-a and lL-6 in response to
Salmonella LPS. Conversely, Salmonella LPS-induced lL-12 secretion was

significantly reduced by the presence of transwells (Fig. 4C-E).

Various strains of C. jejuni differ in their ability to elicit cytokine responses

from BM-DCs .
We investigated whether different strains of C. jejuni elicit different
cytokine responses from BM-DCs. lL-12 production by DCs exposed to various

strains ranged from 107.9 i 1.3 to 310.5 :1: 8.6 pg/ml at 24 h p.i. and 70.2 i 2.4 to

231.7 i 7.3 pg/ml at 48 h p.i. (Fig. 5A). At both time-points, strain 33560 induced

47

the highest levels whereas strain NW induced the lowest levels of IL-12. Peak
levels of lL-12 secretion were observed at 24 h p.i. with all strains, consistent
with previous experiments with-strain 11168. All C. jejuni strains elicited TNF-a
and IL-6 secretion; maximum levels of TNF-a and lL-6 were observed with strain
33560 and minimum levels were detected with strains NW or D0835 (Fig. 5B-C).
The highest levels of TNF-a and IL-6 were detected at 24 and 48 h p.i.
respectively for all strains. Strain 33560 induced a minimal amount of lL-10 (36.6
:I: 1.6 pg/ml) only at 24 h pi; no other strains induced detectable amounts of lL-10

(data not shown).

C. jejuni-infected BM-DCs induce Th1 polarization of CD4”T cells

Because Th-polarizing ability of C. jejuni-infected DCs is not known, an in
vitro DC-CD4+ T cell coculture system was employed to assess this function.
Negative control groups (T cells + medium alone, T cells + medium alone-treated
DCs, and DOS + C. jejuni) did not produce detectable quantities of IFN-y (Fig.
6A). However, co-culture of C. jejuni-infected BM-DCs with CD4+ T cells induced
marked production of IFNdy that was significantly higher (P < 0.05) than observed
with all negative controls and comparable to that induced by S. typhimurium LPS-
treated BM-DCs. In contrast, C. jejuni-infected BM-DCs did not elicit detectable
lL-4 (Fig. 6B) or lL-10 (data not shown) secretion by CD4+ T cells. It is evident
from this experiment that BM-DCs infected with C. jejuni induce Th1 polarization

of CD4+ T cells in vitro.

48

DISCUSSION

Pathogen-host cell interactions in C. jejuni infection remain largely
unknown. We demonstrate here that C. jejuni induces phenotypic and functional
activation of DCs from resistant C57BL/6 mice. C. jejuni viability and
internalization were necessary for maximal activation of DCs. Furthermore,
murine DCs primed C. jejuni-specific Th1-effector responses in vitro, consistent
with this murine model where C. jejuni was limited to colonization of the
gastrointestinal tract (108). Together these findings support a role for DCs in
regulating innate and adaptive immune responses during C. jejuni infection.

Our data show that murine DCs efficiently internalize and kill C. jejuni
within 8 h p.i. and that C. jejuni internalization is mediated by both actin filaments
and microtubules as suggested previously (125). Wassenaar et al. (173) reported
failure of C. jejuni to survive inside activated human macrophages and suggested
that “infra-phagocytic survival is not a common phenomenon” in C. jejuni
infection. While C. jejuni persists within cultured IEC (175), our data and that of
Hu et al. (67) suggest that near complete killing of C. jejuni by DCs likely rules
out the possibility that DCs serve as transporters of C. jejuni for extra-intestinal
dissemination.

In our study, immature murine DCs infected with C. jejuni undergo
maturation by upregulating surface expression of MHC-ll, CD40, and
costimulatory molecules. This implies that C. jejuni-infected murine DCs are
capable of engaging both T-cell receptors and CD28 molecules on naive Th

cells, thereby delivering essential signals for induction of anti-microbial T cell

49

responses. In our studies, most of the unstimulated immature DCs (> 70 %)
expressed CD80 but only a small fraction of unstimulated DCs (< 20—35 %)
expressed C086. As a result of this basal difference, the magnitude of change
due to C. jejuni stimulation and/or other treatment conditions, like presence of
transwells, is distinct for CD80 and C086. That is, the magnitude of increase in
proportion of cells expressing CD80 following C. jejuni infection is lower than that
observed with CD86 (1.1—1.2-fold increase for C080 vs. 2.5—3-fold increase for
C086 in our studies). Thus, we show for the first time the effect of C. jejuni
viability on DC maturation. When compared to live C. jejuni, killed C. jejuni
induced comparable levels of MHC-ll expression but significantly lower levels of
costimulatory molecule expression, which may be due in part to very low
amounts of TNF-a—a known stimulus inducing DC maturation—elicited by
nonviable C. jejuni.

We observed a C. jejuni dose- and time-dependent secretion of IL-12 by
DCs. IL-12 secreted by DCs enhances CD4+ T cell proliferation and IFN-y
production. Furthermore, recent studies demonstrated that lL-12 mediates DC
activation of natural killer (NK) cells, contributing to initial resistance to bacterial
pathogens (57, 92). This ability to elicit lL-12 suggests that C. jejuni may also
stimulate NK cells, leading to mucosal resistance.

C. jejuni-infected DCs secreted TNF-a and lL-6 suggesting that DCs may
initiate or amplify the proinflammatory reaction to C. jejuni enhancing recruitment
of neutrophils and macrophages to the intestinal mucosa. Indeed, neutrophilic

exudates are a prominent feature of C. jejuni-induced disease in humans and

50

animals (48, 108, 149). DC-mediated recruitment of neutrophils to intestinal
mucosa may contribute to C. jejuni killing as recently suggested in Aspergillus
fumigatus infection (52). Additionally, killed C. jejuni elicited significantly lower
levels of lL-12 and proinflammatory cytokines than live C. jejuni. Our data taken
together with previous reports (60, 67, 84) demonstrate that, in contrast to IEC,
phagocytes such as DCs and THP-1 monocytic cells do not require live C. jejuni
to secrete proinflammatory cytokines. However, C. jejuni viability is necessary for
maximal induction of cytokine secretion by murine DCs, unlike human DCs and
monocytes. We also observed that the maturation and cytokine responses of
DCs from C3H/He0uJ mice to C. jejuni were similar to that observed with DCs
from C57BU6 mice (V. Rathinam and L. Mansfield, unpublished data).

Blocking phagocytosis of C. jejuni resulted in inhibition of IL-12 and TNF-a
but not lL-6 production by murine DCs, consistent with previous reports on
Neisseria meningitidis and Streptococcus pneumoniae (31, 165). Helicobacter
pylori-induced 0C secretion of lL-12 but not lL-8 also depended on phagocytosis
(57). C. jejuni internalization was also essential for optimal surface expression of
DC maturation markers. Transwell experiments showed that diffusible products
secreted by C. jejuni induced only partial upregulation of maturation markers and
did not elicit secretion of proinflammatory cytokines. Collectively, these findings
show that internalization of C. jejuni was necessary for complete maturation of
DCs and secretion of lL-12 and TNF-a but was dispensable for lL-6 production. A
possible explanation for this requirement is that, following C. jejuni uptake,

additional intracellular signaling pathways are activated from phagosomes that

51

are required for complete activation of DCs. As shown previously, our data also
suggest that the Salmonella LPS-induced responses of DCs depend on
internalization of LPS (34). To the best of our knowledge, this is the first report
describing the requirements of C. jejuni contact and internalization for DC
activation.

We show that murine 0C secretion of lL-12, TNF-a, and lL-6 varies
among C. jejuni strains. This result is likely due to variation in antigenic surface
structures among these strains of C. jejuni (130). Similar variation in induced
levels of lL-8 and CCL20 secretion was observed in IEC challenged with different
clinical isolates of C. jejuni (60, 81). Moreover, separate studies in our lab
showed that these C. jejuni strains exhibited varying colonization and disease
phenotypes in C57BU6 lL-10"' mice (Bell et al., unpublished data). No detectable
IL-10 production was observed from C. jejuni 11168-infected DCs. We suspect
that lL-10 secretion by C. jejuni-infected DCs may require, along with a microbial
stimulus, ligation of CD40 on DCs with CD40L that occurs during encounter with
NK cells and T cells (147).

We show for the first time that C. jejuni-infected DCs elicit high levels of
IFN-y secretion from CD4+ T cells demonstrating a Th1 polarization. This finding
correlates with the secretion of lL-12 from C. jejuni-infected DCs observed here
and is further strengthened by in vivo studies demonstrating Th1-associated
lgG2b antibody responses in IL-10+’+ and lL-10"' mice of C57BU6 (108), C3H
and non-obese diabetic (NOD) genetic backgrounds (108, 110) and in

C57BL/129 mice (48) challenged orally with C. jejuni. Consistent with our data,

52

two earlier studies documented elevated plasma levels of IFN-y in a naturally
infected human patient and in C57BU6 mice intraperitoneally inoculated with C.
jejuni (2, 13). IFN-y secreted by effector CD4+ T cells could activate infected
macrophages to augment microbicidal activities for killing of ingested C. jejuni.
Wassenaar et al. (173) and Iovine et al. (71) showed that IFN-1y treated human or
murine macrophages were enhanced for killing clinical isolates of C. jejuni.
Similarly, DC-induction of IFN-y from CD4+ T cells has been reported for
infections with various pathogens including H. pylori and A. fumigatus (51, 57).
Thus, adaptive immunity plays a role in clearing C. jejuni infection in
mouse models (27). Th1 effector responses initiated by DCs may contribute to
mucosal resistance to C. jejuni infection in C57BU6 mice. However, excessive
innate or T-cell mediated inflammatory responses in the intestine triggered by
DCs in the absence of immunoregulatory elements, like lL-10, presumably
contribute to immune pathology as evident in our CS7BL/6 lL-10"' enteritis model
(108). Further investigations are needed to establish these mechanisms in vivo.
Currently, we are investigating the role of toll-like receptor signaling in the

activation of DCs in response to C. jejuni.

ACKNOWLEDGEMENTS
We thank Alice Murphy for breeding mice and Dr. Julia Bell for critical
manuscript review. This project was funded in part with federal funds from NIAID,

NIH, Department of Health and Human Services, under Contract No. N01-Al-

53

30058 and Grant No. K26 RR023080-01. Dr. Rathinam was supported by funds

from the MSU-CVM.

54

FIGURES

Figure 1. (A) Internalization and killing of C. jejuni by BM-DCs. BM-DCs (2 x
105) in a 24-well plate were infected with C. jejuni at MOls of 10:1 (2.1 x 10‘5
CFU; A) and 100:1 (1.91 x 107 CFU; A). After 1 h of infection, non-phagocytosed
bacteria were removed or killed by washing followed by gentamicin treatment.
Subsequently BM-DCs were lysed at 2, 4, and 8 h p.i. and lysates cultured on BA
plates to enumerate viable intracellular bacteria. (B) Viability of C. jejuni in R10.2
medium without BM-DCs. R10.2 medium without BM-DCs (as a control) was
inoculated with 2.1 x 106 CFU or 1.91 x 107 CFU of C. jejuni and the bacterial
viability was assessed at 2, 4, and 8 h post-inoculation. Data are from three wells
in an experiment and are expressed as mean i SEM. One experiment
representative of three independent experiments is shown. (C) Inhibition of BM-
DC internalization of C. jejuni by cytochalasin 0 (CCD) and nocodazole (NCD).
BM-DCs, left untreated or preincubated with 1 jig/ml CCD, 20 uM NCD, 1 jig/ml
CCD and 20 0M NCD or DMSO (0.1%, vehicle control)°for 1 h, were inoculated
with C. jejuni (MOI of 100:1). The internalized bacteria were determined at 2 h
p.i. by using gentamicin killing assay as described above. lnternalized bacteria in
each treatment group were presented as the % of that observed in DC + C. jejuni
group (without inhibitors). Data are from two wells in an experiment and are
expressed as mean :2 SEM. One experiment representative of two independent

experiments is shown.

55

 

+ C. jejuni1.91 x 107 CFU
I + C.jejuni2.1 x 106 CFU

LogtoCFU
O -* N 00 A 01 O)

 

 

 

0 2 4 6 8 10

Time post-infection (h)

 

+ c. jejuni1.91 x 107 CFU
. —A— c. jejuni2.1 x 106 CFU

L0910CFU

 

 

 

T —r T

0 2 4 6 8 10

Time post-infection (h)
C

 

DC+Q

DC + DMSO + Cj

DC + CCD + Cj

DC + NCD + Cj

DC + CCD + NCD + C]

 

 

0 50 100 150

% Relative internalization

Figure 1. Internalization and killing of C. jejuni by BM-DCs

56

Figure 2. Maturation and cytokine production by BM-DCs infected with C.
jejuni. Data are from three wells in an experiment and are expressed as mean i
SEM. One experiment representative of two independent experiments is shown.
(A) Maturation: BM-DCs (2 x 105 cells/ml) seeded in 6-well plates (B0 Falcon)
were infected with C. jejuni at a MOI of 100:1, treated with LPS (0.1 ug/ml) or
R10.2 medium alone. After 24 h, cells were double-stained with R-PE-conjugated
anti-CD11c and FITC-conjugated anti-MHC-ll (l-Ab), anti-CD40, anti-CD80 or
anti-CD86 antibodies or appropriate isotype controls and subjected to flow
cytometric analysis. The mean fluorescence intensities of MHC-ll, CD40, CD80,
and C086 markers on CD11c+ cells and the percentage of C011c" cells
expressing these maturation markers are shown. Asterisks indicate P < 0.05 for
1) C. jejuni-infected DCs or LPS-treated DCs vs. medium alone-treated DCs. (B)
Cytokine production: BM-DCs (2 x 105 cells per well of a 24-well plate) in R10
medium without antibiotics were treated with C. jejuni at MOI of 10:1 and 100:1
or LPS (0.1 ug/ml) or R10.2 medium alone. The levels of lL-12, lL-6 and TNF-a
cytokines in the culture supernatants at 4, 24 and 48 h p.i. were measured by
cytokine-specific sandwich ELISA. Data from LPS treatment group was included

in the statistical analysis but the significance levels are not shown in figure.

57

>

Mean fluorescence intensity

on

 

 

m Medium alone
- C. jejuni
:1 LPS

 

 

._L
O
O

N-hODCD
000

O

% of CD11c+ cells

0

 

MHC-IICD40 CD80 CD86

 

 

 

 

 

500 lL-12 6
5
_ 4
E. 3
C
2
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0
50 lL-6
40 ll: lT—n
E 30
E: Medium alone
20 [:31 C. jejuni10:1
10 . . - C. jejuni100:1
P ITI l:l LPS

 

 

Figure 2. Maturation and cytokine production by BM-DCs infected with C. jejuni

58

Figure 3. Effect of viability of C. jejuni on activation of DCs. BM-DCs were
treated with approximately equal numbers of live or formalin-killed C. jejuni (MOI
of 100:1), LPS (0.1 ug/ml), or R10.2 medium alone. After 24 h, the expression of
cell surface markers was analyzed by flow cytometry. (A) Mean fluorescence
intensities of MHC-ll, C040, C080, and C086 markers on CD11c+ cells and the
percentage of CD11c+ cells expressing these maturation markers are shown. (B)
The levels of lL-12, lL-6 and TNF-ot cytokines in the culture supernatants at 4, 24
and 48 h p.i. were measured by cytokine-specific sandwich ELISA. Asterisks
indicate significance P < 0.05. Data are from three wells in an experiment and
are expressed as mean i SEM. One experiment representative of two

independent experiments is shown.

59

Mean fluorescence intensity

w

pg/ml

Medium alone
Live C. jejuni

‘EEIU

 

    
   

   

  

 

         

 

Formalin-killed C. jejuni

 

LPS
10000 120
8000 . £100 .
5 8 80 g? F
6000 Z ‘2. a g ¢
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2 r o 40 1 2 fit
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160
140
120
100
80 g,
60 ‘ c
40
20

 

   

 

ng/ml

 

4h 24h 48h

Time post-infection

Figure 3. Effect of viability of C. jejuni on activation of DCs

60

Figure 4. Requirement for contact and internalization of C. jejuni for
activation of DCs. BM-DCs, preincubated with 1 pg/ml CCD and 20 uM NCD or
DMSO (0.1% vehicle control) for 1 h, were treated with C. jejuni (MOI of 100:1),
LPS (0.1 ug/ml) or R10.2 medium alone. In experiments with transwells, BM-DCs
were added to wells of cell culture plates and 0.2 pM Anopore membrane cell
culture inserts were placed in wells. Then, C. jejuni at an MOI of 100:1, R10.2
medium alone or 0.1 ug/ml of S. typhimurium LPS was added to the top
Chamber. After 24 h of incubation, maturation and cytokine responses of BM-DCs
were analyzed as described before. (A-D) The mean fluorescence intensities of
MHC-II, C040, C080, and CD86 markers on CD11c+ cells and the percentage of
CD11c+ cells expressing these maturation markers are shown. For the
experiment with actin and microtubule inhibitors, MFI and percentage of CD11c
cells positive for markers in the medium alone group were considered basal
values and the increase in these parameters in the other two treatment groups
was expressed as fold increase over basal value. (E) The levels of cytokines
secreted at 24 p.i. Data are from three wells in an experiment and are expressed
as mean i SEM. One experiment representative of two independent experiments

is shown.

61

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Figure 5. Cytokine responses of BM-DCs to various strains of C. jejuni. BM-
DCs were infected with the one of following strains of C. jejuni: 11168, D2600,
00835, NW, D0121 and 33560 or treated with LPS (0.1 ug/ml) or R10.2 medium
alone. At 24 and 48 h p.i., the concentrations of IL-12 (A), TNF—a (B) and IL-6 (C)
and in the culture supernatants were measured by sandwich ELISA. Data are
from three wells in an experiment and are expressed as mean i SEM. One
experiment representative of two independent experiments is shown. DCs
treated with all C. jejuni strains or LPS were significantly different (P < 0.05) from

medium alone-treated DCs within each time point.

64

 

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Figure 5. Cytokine responses of BM-DCs to various strains of C. jejuni

Figure 6. Polarization of CD4’T cells by C. jejuni-infected BM-DCs. BM-DCs
treated with C. jejuni (MOI of 100:1) or LPS (0.1 pg/ml) or R10.2 medium alone
or zymosan (10 pig/ml) were cocultured with CD4+ T cells isolated from spleen for
72 h. The levels of IFN-y (A) and lL-4 (B) in the supernatants were determined by
a sandwich ELISA. Asterisks indicate P < 0.05: (A) T + DC + LPS was
significantly different from all the negative control groups; (B) T + DC + zymosan
was significantly different from all other treatments. Data are from three wells in
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67

CHAPTER 3

Campylobacterjejuni-induced activation of dendritic cells involves

cooperative signaling through TLR4uMyD88 and TLR4-TRIF axes

Rathinam, V. A., D. M. Appledorn, K. A. Hoag, A. Amalfitano, and L. S.
Mansfield. 2009. Campylobacterjejuni-induced activation of dendritic cells
involves cooperative signaling through TLR4-MyD88 and TLR4—TRIF axes. Infect
Immun 77(6): 2499-2507.

68

ABSTRACT

Campylobacterjejuni is an important cause of human enteritis and has
been linked to the development of autoimmune diseases. Recently, we showed
that infection of murine dendritic cells (DCs) with C. jejuni resulted in DC
activation and induction of Campylobacter-specific Th1-effector responses. Toll-
like receptor (TLR) signaling through myeloid differentiation factor-88 (MyD88)
and/or Toll-IL-1 R domain-containing adaptor-inducing IFN-B (TRIF) is critical in
inducing immunity against pathogens. In this study, we investigated the role of
TLR2, TLR4, MyD88, and TRIF signaling in C. jejuni-induced inflammatory
activation of DCs. DC upregulation of MHC-ll and costimulatory molecules after
C. jejuni challenge was profoundly impaired by TLR2, TLR4, MyD88, and TRIF
deficiencies. Similarly, C. jejuni-induced secretion of lL-12, IL-6, and TNF—a was
significantly inhibited in TLR2"', TLR4"', MyD88"', and TRIFJ' DCs compared to
wild-type DCs; however, the magnitude of inhibition was greater in MyD88”‘,
TRIF'", and TLR4"' 003 than in TLR24' DCs. Furthermore, C. jejuni induced
interferon regulatory factor-3 (IRF-3) phosphorylation and IFN-[3 secretion by
DCs in a TLR4-TRlF-dependent fashion, further demonstrating activation of this
pathway by C. jejuni. lmportantly, TLR2, TLR4, MyD88, and TRIF deficiencies all
markedly impaired Th1-priming ability of C. jejuni-infected DCs. Thus, our results
show that cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes
represents a novel mechanism mediating C. jejuni-induced inflammatory
responses of 003. To our knowledge, such a mechanism has not been

demonstrated previously for an intact bacterium.

69

lNTRODUCTION

Campylobacterjejuni is an important human pathogen that causes food-
bome diarrheal illness worldwide (26). C. jejuni is also one of the main causative
agents of traveler’s diarrhea (44). The most common presentation of C. jejuni
infection is self-limiting enteritis characterized by mild watery to bloody diarrhea,
while extra-intestinal, severe and persistent diseases, including septicemia and
meningitis, can occur in immunocompromised patients. Most importantly,
autoimmune neuropathies, including Guillain—Barré syndrome, have been
associated with antecedent C. jejuni infections (172).

It is evident that C. jejuni infection results in varying disease outcomes,
although the immunological contribution to this phenomenon is poorly
understood. We have recently shown that CS7BL/6 lL-10+l+ and congenic lL-10"'
mice can be used as C. jejuni colonization and enteritis models, respectively
(108). lmmunohistochemical examination of C. jejuni-infected intestinal tissues in
our mouse models (108, 110) and from human patients (166) revealed that C.
jejuni colonizes epithelial crypts of the intestine and invades the lamina propria.
Dendritic cells (DCs) are the primary cell type involved in immunosurveillance of
mucosal tissues and play an instructive role in the development of appropriate T
cell responses to pathogens (116). DCs underlying the epithelial barrier are likely
to encounter C. jejuni either in the lamina propria, as it transcytoses the barrier,
or intra-luminally when DCs extend their processes across the epithelium to
sample the lumen (123). We found that following C. jejuni infection, DCs from

CS7BL/6 mice undergo maturation, secrete lL-12 and proinflammatory cytokines,

70

and induce Campylobacter-specific Th1 effector cell responses (141 ). However,
the molecular mechanisms underlying the DC-mediated induction of adaptive
immune responses to C. jejuni are not known.

DCs express a broad repertoire of Toll-like receptors (TLRs) for sensing
and regulating expression of critical signals—including costimulatory molecules
and cytokines—in response to pathogens, thereby controlling the development of
adaptive immune responses (74). TLR2 and TLR4 have been shown to be critical
for host innate and acquired immune responses to a variety of bacterial
pathogens (63, 162). Furthermore, a relatively higher proportion of 005 in the
colonic lamina propria expresses TLR4 (117). Previous studies have shown that
C. jejuni possesses predicted ligands for TLR2 and TLR4 such as surface
Iipoprotein (JIpA) and lipooligosaccharide (LOS), respectively (80, 120).
Moreover, the role of TLR2 and TLR4 in the recognition of C. jejuni becomes
more important considering that TLR5, another crucial TLR that recognizes
bacterial fiagellin, has been shown to play a very limited role in C. jejuni detection
(6). Finally, C. jejuni DNA that can signal through TLR9 was found to be
dispensable for eliciting lL-8 secretion from intestinal epithelial cells (IEC)(187).

Following ligand binding, TLRs initiate intracellular signaling cascades
through cytoplasmic adaptor molecules, which ultimately result in activation of
various transcription factors, including NF-KB (4). MyD88 is an important adaptor,
mediating signals from most TLRs. Several lines of evidence suggest a critical
role for MyD88 signaling in host resistance to microbial infections (122). It has

been recently demonstrated that MyD88 signaling is essential for lL-8 production

71

by cultured IEC after infection with C. jejuni (187). Another adaptor molecule,
TRIF, is known to mediate MyD88-independent signaling from TLR4 and TLR3 in
response to lipopolysaccharide (LPS) and double stranded RNA, respectively (4),
but its contribution to immune responses against bacterial pathogens is just
beginning to be understood. Recent studies have shown that TRIF is essential
for pulmonary host defense against Gram-negative pathogens such as
Pseudomonas aeruginosa and Escherichia coli (78, 137). However, the
contribution of My088 and TRIF signaling to DC responses against enteric
pathogens such as C. jejuni has not been investigated.

Based on the accumulating evidence on the critical role of TLR signaling
in microbial infections and on the presence of potential TLR2 and TLR4 agonists
in C. jejuni, we hypothesized that C. jejuni-induced activation of bone marrow-
derived DCs (BM-DCs) is mediated by TLR2 and TLR4, and that MyD88 and/or
TRIF signaling is necessary for this activation. In this study, we show that
maturation, lL-12 secretion, and Th1-priming ability of C. jejuni-infected BM-DCs
depend on TLR2 signaling through My088 and TLR4 signaling through both
MyD88 and TRIF adaptor molecules. To our knowledge, this is the first report
demonstrating cooperation between MyD88-dependent and -independent arms
of the TLR4 pathway in mediating immune responses against an intact bacterial
pathogen. We also delineate the type I interferon induction occurring in response

to C. jejuni and signaling mechanisms involved in this pathway.

72

MATERIALS AND METHODS

Mice
C57BU6J, TLR2'I' mice on the C57BU6 background, and C3H/HeJ (TLR4'
l') mice purchased from the Jackson laboratory were bred and maintained as
described previously (7, 108). C3H/HeOuJ (TLR4+/+) mice were purchased from
the Jackson laboratory. MyD88'I' and TRIF’I' mice on the CS7BL/6 background
were kindly provided by Dr. Shizuo Akira. Mice were used at 8—13 weeks of age.
Experiments using DCs from C57BU6 genetic background mice (WT, TLR2"',
MyD88"', and TRIF"') were conducted concurrently and separately from
experiments using DCs from C3H/HeOuJ TLR4”+ and CBH/HeJ TLR4"' mice.
Animal protocols were approved by the Michigan State University Institutional

Animal Care & Use Committee and conformed to NIH guidelines.

Bacterial inoculum preparation

C. jejuni 11168, originally isolated from the feces of a diarrheic patient,
was obtained from the American Type Culture Collection (Manassas, VA, USA).
C. jejuni 11168 inoculum was prepared as described (141). In all experiments
described in this study, BM-DCs were infected with C. jejuni at a multiplicity of
infection of 100 bacteria per BM-DC, which was previously determined to be the

optimal dose (141).

73

Generation of BM-DCs and assessment of maturation and cytokine

secretion by C. jejuni-infected BM-DCs

BM-DCs were generated from WT, TLR2"', TLR4"‘, MyD88'", and TRIF'"
mice as described previously (141). Briefly, 2.5 x 106 bone marrow cells from
femurs and tibiae were cultured in bacteriological-grade Petri-dishes in 10 ml of
R10 medium (RPMI 1640 medium Dutch Modification [Sigma-Aldrich, St. Louis,
MO] supplemented with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 50 uM
2-mercaptoethanol, 100 U/ml penicillin, 100 pg/ml streptomycin [lnvitrogen,
Grand Island, NY], and 20 ng/ml murine granulocyte-macrophage colony-
stimulating factor [Peprotech, Rocky Hill, NJ]). 005 were collected and used on
day 9. BM-DCs diluted at a density of 2 or 3 x 105 cells/ml of R10 medium
without antibiotics were placed in a 6-well plate for maturation experiments or a
24—well plate for cytokine experiments and were treated with C. jejuni, medium
alone (negative control), or 0.1 ug/ml Salmonella enterica serovar Typhimurium
LPS (positive control; Sigma-Aldrich). One h after infection, gentamicin (250
pg/ml) was added to all wells to kill extracellular bacteria. At 24 h post-infection
(p.i.), DCs were stained with fluorochrome-conjugated antibodies specific for cell
surface markers such as MHC-ll, CD80, CD86, and CD40 and analyzed by flow
cytometry as described (141). For cytokine analysis, culture supernatants were
collected at 24 h p.i. and stored in aliquots at -80°C until further analysis.
Previous studies showed that 24 h p.i. is the optimal time point to analyze

cytokine responses from C. jejuni-infected 005 (141).

74

TLR2 assay

HEK293 cells that are stably transfected with human TLR2, CD14 and NF-
kB-inducible reporter system, secreted alkaline phosphatase (HEK-Blue-2 cells,
lnvivoGen, San Diego, CA) were cultured according to the manufacturer’s
instructions. HEK-Blue—2 cells in 96-well cell culture plates (5 x 104 cells/well)
were treated with medium-alone, C. jejuni, or 100 ng/ml Pam3CSK4 (synthetic
Iipoprotein; lnvivoGen). After 1.5 h of infection, gentamicin (250 lug/ml) was
added to all wells and at 23 h p.i., 160 pl of supernatant was added to 40 pl of
QUANTl-Blue (lnvivoGen) to detect secreted alkaline phosphatase in the
supernatant. After 30 min of incubation, plates were read at 630 nm using a Bio-

Tek EL-800 Universal plate reader (Bio-Tek Instruments, Winooski, VT).

ELISA

Cytokines in culture supernatants were measured using Ready-SET-Go!
ELISA Sets (eBioscience, San Diego, CA) according to the manufacturer’s
instructions. Assay detection limits (in pg/ml) were lL-12, 15; lL-6, 4; TNF-ot, 8;
IFN-y, 0.8.

IFN-B ELISA was performed as described previously (178) with
modifications. Nunc-lmmuno MaxiSorp 96-well plates (Nalge Nunc lntemational,
Rochester, NY) were coated overnight at 4 °C with 100 pl/well of 1pg/ml anti-IFN-
[3 monoclonal antibody (7F-D3; Abcam, Cambridge, MA) followed by blocking for
1 h with assay diluent (eBioscience, San Diego, CA) and addition of 100 til/well

of IFN-B standard (PBL Interferon Source, Piscataway, NJ) or samples. After

75

overnight incubation at 4 °C and washing, 100 pl/well of 40 U/ml rabbit anti-lFN-B
polyclonal antibody (PBL Interferon Source) was added. After 1 h of incubation at
room temperature and washing, 100 pl of 1:10,000 dilution of 0.8 mg/ml goat
anti-rabbit IgG-horseradish peroxidase (Jackson lmmunoResearch, West Grove,
PA) was added per well and incubated for 1 h at room temperature. Plates were
washed and 100 pl of 3, 3', 5, 5’- tetramethylbenzidine (eBioscience, San Diego,
CA) was added per well. After 15 min, 100 pl of 2N sulphuric acid was added per
well to stop the reaction and the plate was read at 450 nm using a Bio-Tek EL-
800 Universal plate reader (Bio-Tek Instruments, Winooski, VT). The sensitivity

of the assay was 7 pg/ml.

Quantitative real-time PCR (Q-PCR)

Total RNA was extracted at 2.5 and 5 h post-treatment using RNeasy kit
(QIAGEN, Valencia, CA) according to the manufacturer's instructions. cDNA was
synthesized from 1 pg of total RNA using the iScript Select cDNA synthesis kit
(BioRad, Hercules, CA) according to the manufacturer's instructions. Q-PCR for
IFN-B (43) and B-actin (128) were performed as described previously in a final
volume of 25 pl containing 12.5 pl of 2x iQ SYBR green superrnix (BioRad), 2 pl
of 1:5 dilutions of cDNA and 0.3 or 0.625 pl of primers (0.25 pM) in a BioRad iQS
iCycler. Fold change in expression was calculated according to Pfaffl (135).
Cycle threshold values of IF N-B were normalized to that of B-actin. Transcript
levels (threshold cycle values) of B-actin were very similar between medium-

alone treated cells and C. jejuni-infected cells.

76

Western blotting

BM-DCs were lysed in lysis buffer (20mM Tris-HCI, pH 7.4, 1 mM EDTA,
150 mM NaCl) containing 1% Triton X-100 with phosphatase and protease
inhibitors. The protein concentration in the lysate was determined using the
bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL)
according to the manufacturer’s instructions. Equivalent amounts of protein
samples were run on 10% polyacrylamide gels and transferred onto
nitrocellulose membranes. Blots were then probed with anti-IRF3 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-phospho-IRF3 (Ser396; 4D4G; Cell
Signaling Technology, Inc. Danvers, MA), or anti-tubulin (Sigma-Aldrich)
antibodies. Blots were washed and subsequently probed with fluorescently
labeled anti-mouse (Rockland lmmunochemicals, Inc. Gilbertsville, PA) or anti-
rabbit (lnvitrogen) secondary antibodies. Blots were scanned using a Ll-COR

Odyssey scanner (Ll-COR Biotechnology, Lincoln, NE).

DC-CD4‘T cell coculture

DC-CD4+ T cell coculture experiments were performed as described
previously with minor modifications (141). Briefly, highly enriched populations of
CD4+ T cells (2 93%; data not shown) were purified from spleens of WT mice by
negative selection using the B0” IMag Mouse CD4 T Lymphocyte Enrichment
Set-DM (BD Pharmingen) that contains the following antibodies: anti-mouse

CD8a (clone 53-67); anti-mouse CD11b (clone M1/70); anti-mouse

77

CD45R/8220 (clone RAB-682); anti-mouse CD49b (clone HM a 2); anti-mouse
TER-119/erythroid cells (clone TER-119). BM-DCs seeded in 24wwell plates (105
cells/well) were treated with either C. jejuni or medium alone. One h after
infection, gentamicin (250 pg/ml) was added to all wells. At 24 h p.i., CD4+ T cells
were added to DOS at a DC—to-T cell ratio of 1:6. After coculturing for 72 h,

supernatants were collected for measurement of lFNq.

Statistical analysis
One-way or two-way analysis of variance followed by the HoIm-Sidak
correction for multiple comparisons was performed using SigmaStat 3.1 (Systat

Software, San Jose, CA). P values 3 0.05 were considered significant.

78

RESULTS

C. jejuni-induced BM-DC maturation and IL-12 production is dependent on
TLR2 and TLR4 signaling

To determine the role of TLR2 and TLR4 in mediating the DC maturation
and cytokine secretion in response to C. jejuni, BM-DCs cultured from wild type
(WT), TLR2"' and TLR4"' mice were infected with C. jejuni and responses were
assessed by flow cytometry and ELISA, respectively, at 24 h p.i. As we have
shown previously, infection with C. jejuni induced significant (P = 0.05)
upregulation of surface expression of maturation markers in WT DCs (141). In
contrast, the mean fluorescence intensities—indicating cell surface expression
levels—of MHC-ll, CD40, CD80, and CD86 markers and the proportion of cells
expressing these markers were significantly (P = 0.05) lower in TLR4"’ DCs
compared to WT DCs following C. jejuni challenge (Fig. 1A-B). TLR2 deficiency
also significantly (P = 0.05) reduced the upregulation of MHC-ll, CD80, and
CD86 subsequent to C. jejuni challenge (Fig. 1C-D). However, the C. jejuni-
induced increase in CD40 expression levels and in percentage of cells. with CD40
on their surface was comparable between TLR2"' DCs and wild type DCs, in
contrast to that observed in TLR4”' DCs. These results suggest a selective role
for TLR2 in C. jejuni-induced upregulation of costimulatory surface proteins. We
also observed that Salmonella LPS-induced responses were reduced in TLR2"'
DCs relative to wild type cells. This may be due to the contamination of this

particular commercial preparation of LPS with certain TLR2 ligands from bacteria

79

(4, 58) or LPS itself signaling through TLR2, besides the expected TLR4, as
reported previously (153).

We also found that TNF-a and IL-6 secretory responses (referred to as
secretion in the rest of the manuscript), were significantly (P = 0.05) impaired by
several fold in TLR4"’ DCs 24 h following C. jejuni infection (Fig. 2A). In contrast,
C. jejuni-induced TNF-a and lL-6 production was only minimally reduced in
TLR2‘" DCs when compared to WT DCs (Fig. ZB). Most importantly, C. jejuni-
induced lL-12 secretion was completely abolished in TLR4"‘ DCs but was only
halved in TLR2‘" DCs (Fig. 2A-B). Overall these data clearly show that the
contribution of TLR2 and TLR4 signaling to maturation of DCs after C. jejuni
infection was comparable, and TLR4 plays a major role in relation to TLR2 in
inducing cytokine responses.

Since TLR2 seemed to play a less significant role compared to TLR4 in
mediating DC cytokine responses to C. jejuni, we sought to confirm the role of
TLR2 in C. jejuni infection further. We used HEK—Blue-2 cell line, which is a
HEK293 cell line stably transfected with human TLR2, CD14, and a NF-KB-
inducible reporter system (secreted alkaline phosphatase) to assess the ability of
C. jejuni to activate NF-kB via TLR2. This system allows specific assessment of
TLR2 stimulation by monitoring NF-KB activation through the level of secreted
alkaline phosphatase in the culture supernatant. In this system, infection with C.
jejuni induced more than 12-fold increase in NF-kB activation over that observed

with medium-treated controls (Fig. 20). Furthermore, C. jejuni-induced NF-kB

80

activation was comparable to that observed with the positive control, Pam3CSK4.

These data also provide evidence that C. jejuni activates signaling through TLR2.

Maturation and cytokine responses of C. jejuni-infected BM-DCs depend on

both MyD88 and TRIF signaling

The finding that TLR4 recognition of C. jejuni is necessary for
inflammatory activation of DCs suggests a role for MyD88 and/or TRIF
cytoplasmic adaptors in this process because TLR4 has been shown to utilize
both of these adaptors for signal transduction (4, 180). To test this hypothesis,
BM-DCs cultured from WT, My088"', and TRlF'" mice were infected with C. jejuni
and immune responses assessed at 24 h p.i. Experiments using BM-DCs from
C57BU6 genetic background mice (WT, TLR24', MyD88"', and TRIFJ') were
conducted concurrently and data from these experiments were analyzed
together. However, data from C57BU6 WT and TLR2"' mice are presented with
data from C3H/HeOuJ TLR4”+ and C3H/HeJ TLR4"' mice for ease of comparison
of results for TLR2 and TLR4. Therefore, results for BM-DCs from C57BU6 WT
mice are presented in Figures 1, 2 and 3. Consistent with our findings on C.
jejuni-infected TLR4"' DCs, MyD88 and TRIF deficiencies markedly decreased or
abolished DC responses to C. jejuni. lmportantly, both MyD88 and TRIF were
required for optimal maturation and cytokine responses (Fig. 3A-B). We observed
a profound reduction in C. jejuni-induced increases in the expression levels of
MHC-ll, CD40, CD80, and CD86 in MyD88"' and TRIF"’ DCs (Fig. 3A). Similarly,

MyD88 and TRIF deficiencies markedly reduced the proportion of DCs

81

expressing MHC-ll, CD40, and CD86 in response to C. jejuni challenge (Fig. 3A).
Additionally, TNF-a and lL-6 secretion in response to C. jejuni was significantly
(P = 0.05) reduced in My088'l' and TRIF"' DCs (Fig. 33). or most importance, IL-
12 production was completely abolished in infected MyD88"' and TRIFJ' DCs.
Collectively, these findings demonstrate that MyD88 and TRIF signaling
pathways are nonredundant and cooperate in C. jejuni infection to induce

activation of DCs.

C. jejuni-induced TLR4—rTRIF signaling leads to IRF-3 activation and IFN-[3

secretion in BM-DCs

Signal transduction from TLR4 can occur through both My088 and TRIF
adaptors (65), but only the TRIF arm of the pathway has been shown to mediate
IFN-[3 production via the activation of transcription factors lRF-3 and/or lRF—7 (41,
181 ). Since TLR4-TRIF signaling is activated by C. jejuni, we hypothesized that
C. jejuni induces downstream IRF-3 activation and IFN-B production in DCs in a
TLR4— and TRlF- but not in a MyDBB-dependent manner. To test this hypothesis,
we performed western blots to analyze phosphorylation of lRF-3 1 h after C.
jejuni challenge. C. jejuni infection markedly induced phosphorylation of lRF-3 in
WT DCs and MyD88-deficient DCs (Fig. 4). In contrast, C. jejuni- induced lRF-3
phosphorylation was abolished in the absence of TLR4 and TRIF (Fig. 4).
Surprisingly, total lRF-3 protein in BM-DCs increased markedly after infection

with C. jejuni in a TLR4-, MyDB8-, and TRIF-independent manner.

82

Next, IFN-B mRNA expression in C. jejuni-infected BM-DCs was assessed
at 2.5 and 5 h p.i. by quantitative real-time PCR. At 2.5 h following C. jejuni
infection, IFN-B mRNA levels were upregulated approximately 280-fold over the
basal levels observed in uninfected cells; this upregulation decreased to less
than 100-fold over the basal levels at 5 h p.i. (Fig. 5A). This IFN-[3 mRNA
expression was positively correlated with a significant induction of secreted IFN-8
measured at 24 h after C. jejuni exposure (Fig. 5B). To determine the role of
TLR4, TRIF, and MyD88 in this response, we evaluated levels of secreted IFN-8
in BM-DCs from mice deficient in these factors. Notably, TLR4"' and TRIFJ‘ BM-
003 did not produce detectable amounts of IFN-8 following C. jejuni infection
(Fig. 5B). In contrast, IFN-B production was only marginally increased in MyD88"'
BM-DCs after C. jejuni challenge compared to WT BM-DCs. Taken together,
these findings demonstrate that the TLR4—TRIF signaling axis is necessary for C.

jejuni-stimulated lRF-3 phosphorylation and IFN-[3 production by BM-DCs.

TLR2, TLR4, My088 and TRIF signaling are all necessary for maximal Th1

cell priming by C. jejuni-infected BM-DCs

The role of TLR2, TLR4, MyD88, and TRIF pathways in the induction of
Th1 cell responses by C. jejuni-infected DCs was investigated using an in vitro
DC-T cell coculture system (141). Highly enriched CD4+ T cells from WT mice
were cocultured with c. jejuni-treated wr, TLR2"', TLR4"', MyDB8"', and TRIF"'
DCs; IFN4y levels in the culture supernatants following 72 h of coculturing were

assessed by ELISA. Consistent with our previous findings (141), WT DCs

83

infected with C. jejuni induced high levels of IFN-y from CD4+ T cells (Fig. 6). In
contrast, there was a marked decrease in lFva production from CD4+ T cells
cocultured with c. jejuni-infected TLR4"', My088"', and TRIF”' 003 (Fig. 6).
TLR2"' DCs infected with C. jejuni also induced significantly lower levels of IFN-y
relative to WT DCs infected with C. jejuni. These findings show that TLR2, TLR4,
MyD88, and TRIF signaling all contribute to maximal induction of Th1 cell

responses by C. jejuni-infected 003, although to varying degrees.

84

DISCUSSION

The role of TLR signaling in eliciting host immune responses against the
clinically significant enteric pathogen C. jejuni remains largely unknown. In this
study, we investigated the contribution of TLR2, TLR4, MyD88, and TRIF
signaling pathways to DC recognition and responses to a known virulent strain of
C. jejuni.

This study demonstrates for the first time that C. jejuni-induced phenotypic
maturation of DCs is mediated by both TLR2 and TLR4 signaling whereas
cytokine production by DCs in response to C. jejuni is predominantly dependent
on TLR4. Lipooligosaccharide (LOS) of C. jejuni is the most likely candidate to
stimulate TLR4 signaling in DCs since it possesses diphosphorylated hexa-acyl
lipid A that was reported to be essential for optimal stimulation of cellular
responses (120). Indeed, C. jejuni LOS has been shown to trigger
proinflammatory responses in cultured human DCs to an extent similar to that
observed with live bacteria (67). Furthermore, previous studies have shown that
C. jejuni evades TLR5 detection by having a modification in the TLR5 recognition
site in its flagellin (6). Taken together with these published findings, our data
demonstrates that TLR4 is the major pattern recognition receptor involved in DC
recognition of C. jejuni.

Furthermore, it has been recently reported that the maturation and
cytokine responses of DCs derived from WT and TLR4"' mice to a TLR2 agonist
(Pam3CSK4, a synthetic lipoprotein) were very similar (8). TLR9 ligand (CpG

DNA)—induced responses of immune cells from TLR4”' mice were comparable

85

to that of WT mice (56, 145). All this functional evidence indicates that signaling
through other TLRs, such as TLR2 and TLR9 that are involved in bacterial
recognition, is not altered in TLR4-deficient cells. Therefore, the defects
observed in the responses of TLR4-deficient DCs in our experiments can be
attributed to the lack of signaling through TLR4.

It is also clear from our results that in addition to TLR4 signaling, maximal
expression of MHC-II and lL-12 after C. jejuni infection requires TLR2 signaling.
This suggests that some of the DC responses to C. jejuni require cooperative
signaling through TLR2 and TLR4. TLR2-dependent responses described here
may be due to recognition of a surface exposed Iipoprotein of C. jejuni, leA. leA
of C. jejuni has been shown to induce activation of NF-KB and p38 MAP kinase in
a HEp-2 epithelial cell line, suggesting that the host innate immune system can
recognize leA and trigger inflammatory responses (80).

Our data, together with previous findings (177, 187), show that MyDB8
signaling is necessary to initiate the C. jejuni induced innate inflammatory
responses noted in a variety of host cells, such as IEC and antigen-presenting
cells. lmportantly, the severe impairment of expression of critical signals— MHC-II,
co-stimulatory molecules and lL-12- in C. jejuni-infected MyD88"' DCs implies
that MyD88 signaling plays an important role in the induction of anti-C. jejuni
mucosal immunity. Similarly, MyD88 signaling has been shown to be
indispensable for inducing DC proinfiammatory responses and for mounting
optimal adaptive responses in vivo to a related gastric pathogen Helicobacter

pylori (1 39).

86

In these studies, we also demonstrate a functional role for the TRIF
(MyD88-independent) signaling pathway during C. jejuni infection: DC responses
following C. jejuni infection, including maturation and cytokine secretion, require
TRIF signaling. The most significant finding is that this MyDBB-independent
pathway in DOS cooperates with the MyD88-dependent pathway to elicit optimal
anti-C. jejuni immune responses. Consistent with our results, emerging evidence
points to synergism between multiple TLR signaling pathways as a mechanism
critical for ensuring efficient activation of host defense responses against
pathogens (127). Although the exact mechanism underlying the cooperation
between MyD88 and TRIF signaling pathways is not known, it is possible that
these two signaling pathways, when activated by C. jejuni, converge at the level
of critical transcription factors such as NF-KB and/or lRF5, resulting in their
enhanced and sustained activation and nuclear translocation. Supporting this
notion, infection with C. jejuni has been shown to induce NF-KB activation in
cultured human DCs (67) and IEC (187). Additionally, Fox et al., (48) previously
demonstrated that NF-KB is essential for the development of Th1-associated
antibody responses against C. jejuni in a murine model. However, further
investigation is warranted to elucidate the exact mechanism operating in C. jejuni
infections.

This study reveals that C. jejuni infection of DCs triggers lRF-3
phosphorylation and IFN-[3 secretion in a TLR4- and TRIF-dependent manner.
These data are consistent with C. jejuni-mediated activation of the MyD88-

independent pathway downstream of TLR4. Surprisingly, we noticed that total

87

IRF-3 production was inducible by C. jejuni infection in BM-DCs independent of
TLR signaling. This result is in contrast to previous reports showing lRF-3
expression was not inducible (10, 142). This discrepancy might be due to
differences in the tissue/cell type or culture conditions used in this study. The
kinetics of C. jejuni-triggered IFN-[3 mRNA expression is consistent with the
previously reported responses following E. coli LPS stimulation (66). We expect
that the TLR4-TRIF dependent activation of lRF-3, shown in this study, is most

likely to mediate IFN-B induction after C. jejuni infection. The role of type I

interferons in bacterial infections is just beginning to be understood. IFN-B has
been shown to act in an autocrine or paracrine manner through a positive
feedback mechanism involving IRF7 to enhance maturation and cytokine
responses of D08 (30, 53, 119). Particularly, C. jejuni-induced IFN-B could
contribute to the substantial production of lL-12p70 by DCs following C. jejuni
exposure (141), as has been shown with E. coli LPS (53). Further investigations
are underway to address the precise role of IFN-B in C. jejuni infections.

0. jejuni-treated TLR2"', TLR4"‘, MyD88"', and TRIF"' DCs failed to induce
maximal IFNq production from CD4+T cells in an in vitro DC-T cell coculture
system. This is consistent with infection of MyD88-deficient mice that, unlike WT
mice, fail to clear intestinal colonization and control extra intestinal spread of C.
jejuni (177). In light of these results, our data suggest that TLR2 signaling
through MyD88 and TLR4 signaling through both MyD88 and TRIF molecules
both play a significant role in the development of DC mediated Th1-type mucosal

immunity and resistance against C. jejuni infection.

88

It should also be noted that although 0. jejuni-treated TLR44', MyD88"',
and TRIF"' 005 had no detectable production of lL-12—a critical instructive

signal from DCs promoting lFN-y-secreting Th1 cells—their ability to induce IFN-y
production from CD4+ T cells was preserved. These results show that C. jejuni-
infected DCs can prime Th1 differentiation in an lL-12 independent mechanism
for which TLR signaling is dispensable. Previous studies provide evidence for
additional signaling mechanisms that are responsible for lL-12-independent
priming of Th1 cells by DCs. These mechanisms include lL-18 (49), CD70 (151)
and Delta 4 notch-like ligand (150) signaling. These findings suggest the
possibility that such pathways may operate in C. jejuni-infected DCs. However,
additional experiments are required to support this hypothesis.

Human and experimental animal model studies suggest that adaptive
cellular immunity is required in successful defense against C. jejuni infection (27,
76). DCs are the key cell type involved in eliciting adaptive T cell responses, and
the expression of MHC-ll and costimulatory molecules along with concomitant
cytokine (IL-12, TNF-a and lL-6) signals are highly critical for this function. This
study demonstrates that MyD88-dependent and -independent (TRIF) pathways
downstream of TLR4 cooperate in C. jejuni infection to mediate functional
activation of DCs. lmportantly, TLR2, TLR4, MyD88, and TRIF signaling all
mediate maximal induction of C. jejuni-specific Th1 cell responses by DCs,
suggesting that each has an important role in the development of anti-C. jejuni
mucosal immunity. The findings described here provide novel insights into the

contribution of TLR signaling to the host defense responses in C. jejuni infection

89

and form a basis for further studies to dissect immunoregulation in C. jejuni

infection and to aid in rational vaccine development approaches.

ACKNOWLEDGEMENTS

We thank Jenna Gettings for breeding mice, Jennifer Olmstead for
technical assistance, Dr. Julia Bell for breeding mice and critical review of
manuscript, and Louis King for discussions on flow cytometry. We thank Dr.
Shizuo Akira for MyD88'" and TRIF"' mice.

This project was funded in part with federal funds from NIAID, NIH,
Department of Health and Human Services, under Contract No. NO1-Al-30058
and Grant No. K26 RR023080-01. Dr. Rathinam was supported by funds from

the Michigan State University-College of Veterinary Medicine.

90

FIGURES

Figure 1. TLR2 and TLR4 signaling are required for C. jejuni-induced
maturation of BM-DCs. WT, TLR2"' and TLR4"' BM-DCs were infected with C.
jejuni or treated with Salmonella serovar Typhimurium LPS (0.1 pg/ml) or
medium alone. After 24 h, cells were immunostained and analyzed by flow
cytometry. (A, C) Fold increase in the mean fluorescence intensities (MFI) of
MHC-ll, CD40, CD80, and CD86 markers on CD11c” cells is shown. (B, D) Fold
increase in the percentage of CD11c" cells expressing MHC-II, CD80, and CDB6
markers, and the percentage of CD11c“ cells expressing CD40 are shown. MFI
and percentage of CD11c cells positive for markers in the medium alone group in
each DC type was considered as the basal value and the increase in these
parameters in the other two treatment groups was expressed as fold increase
over the basal value. Asterisks indicate P s 0.05 for WT vs. TLR2"' or TLR4‘I'
BM-DCs. Data are from three wells in an experiment and are expressed as mean
3: SEM. One experiment representative of two independent experiments is

shown.

91

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Figure 2. C. jejuni-induced cytokine responses of BM-DCs is mainly
dependent on TLR4 signaling. wr, TLR2‘" and TLR4"‘ BM-DCs were treated
with C. jejuni, Salmonella serovar Typhimurium LPS (0.1 pg/ml), or medium
alone. (A, B) Cytokine levels in the culture supernatant at 24 h p.i. were analyzed
by ELISA. Asterisks indicate P = 0.05 for WT vs. TLR2”' or TLR4'I' BM-DCs. (C)
HEK-Blue-2 cells were treated with medium-alone, C. jejuni or Pam3CSK4 (100
ng/ml). After 23 h of incubation, the level of secreted alkaline phosphatase in the
culture supernatant was quantified by incubating the supernatant with QUANTI-
Blue and reading the plate at 630 nm. Asterisks indicate P s 0.05 for medium vs.
C. jejuni or Pam3CSK4. Data are from three wells in an experiment and are
expressed as mean 2 SEM. One experiment representative of two independent

experiments is shown.

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94

Figure 3. MyD88 and TRIF signaling dependent upregulation of surface
markers and cytokine secretion by C. jejuni-infected BM-DCs. BM-DCs
derived from WT, MyD884', and TRIF"' mice were treated with C. jejuni,
Salmonella serovar Typhimurium LPS (0.1 pg/ml) or medium alone. After 24 h,
surface expression of maturation markers (A) and secretion of cytokines (B) were
analyzed by flow cytometry and ELISA, respectively. Data for BM-DCs from

057BLI6 WT mice are the same as in Figure 1C-D and 2B. Asterisks indicate P s

0.05 for WT vs. TRIF/'or My088"' BM-DCs. Data are from three wells in an
experiment and are expressed as mean i SEM. One experiment representative

of two independent experiments is shown.

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Figure 4. Activation of IRF-3 phosphorylation by C. jejuni-induced TLR4—
TRIF signaling. WT, TLR44', MyD88"', and TRIF”' BM-DCs treated with c.
jejuni or medium alone were lysed at 1 h p.i. The lysates were electrophoresed
on 10% polyacrylamide gels and transferred to nitrocellulose membrane. Blots
were then probed with appropriate antibodies and scanned with Ll-COR Odyssey
scanner. One experiment that is representative of two independent experiments

is shown.

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expression. Total RNA was extracted from BM-DCs treated with C. jejuni,
Salmonella serovar Typhimurium LPS, or medium alone at 2.5 and 5 h post-
treatment. IFN-B mRNA levels were quantified by Q-PCR. IFN-[3 mRNA levels in
C. jejuni and LPS treatment groups were expressed relative to that observed in
the medium-alone treated control. (B) BM-DCs derived from WT, TLR4"',
MyD884', and TRIF" mice were treated with C. jejuni, Salmonella serovar
Typhimurium LPS (0.1 pg/ml) or medium alone. IFN-[3 levels in the culture
supernatant at 24 h p.i. were analyzed by ELISA. Asterisks indicate P s 0.05 for
WT vs. TRIFJ', My088"'. or TLR44' BM-DCs. Data are from three wells in an

experiment and are expressed as mean 2 SEM.

98

 

 

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Figure 6. TLR2, TLR4, MyDBB, and TRIF signaling are necessary for
maximal Th1 polarization by BM-DCs infected with c. jejuni. WT, TLR2'",
TLR44', MyD88"', and TRIF‘" BM-DCs were treated with c. jejuni or medium

alone and CD4+ T cells were added to each well at 24 h p.i. IFN-y level in the
culture supernatant after 72 h of coculturing was analyzed by ELISA. Asterisks
indicate P s 0.05 for WT vs. TLR2'I', TLR44'. MyD88/2 or TRIF" BM-DCs infected
with C. jejuni. Data are from three wells in an experiment and are expressed as

mean :I: SEM.

99

 

 

CHAPTER 4

 

Summary and future directions

100

 

SUMMARY

Following its recognition as a human pathogen in the 19703,
Campylobacterjejuni was rapidly recognized as one of the most common
bacterial causes of gastroenteritis in humans in both industrialized and
developing countries (23). The Centers for Disease Control and Prevention
estimates that more than 2.4 million people in the US (0.8% of the population)
are affected annually with campylobacteriosis, the disease caused by C. jejuni
(1). C. jejuni exists as a commensal in the intestinal tracts of food animals,
particularly chickens, and is transmitted to humans through contaminated meat,
milk, and water (23, 54, 94). The most common symptoms include fever,
abdominal cramps, and watery to bloody diarrhea. However, patients with
immunodeficiencies, including AIDS patients, develop protracted and serious
diseases, such as bacteremia, peritonitis, hepatitis and meningitis following C.
jejuni infection (23). Because of the molecular mimicry between certain
Campylobacter cell components and host tissues (185), a small proportion of
patients develop debilitating and life-threatening autoimmune complications such
as Guillain-Barré syndrome (GBS) and Reiter’s syndrome (23). GBS is an acute
progressive neuropathy characterized by bilateral ascending paralysis and
occurs in 1 in 1000 cases of C. jejuni.

The pathogenesis of C. jejuni and the host defense mechanisms
controlling the infection remain poorly understood. The early events associated
with pathogenesis of C. jejuni include adherence to and colonization of epithelial

cells of terminal ileum, cecum, and proximal colon. A growing body of evidence

101

 

 

 

indicates that these processes are complex and multifactorial: several surface
structures of C. jejuni including PEB1, outer membrane protein CadF, capsular
polysaccharides and leA (surface lipoprotein) contribute to adherence and
invasion of epithelial cells by C. jejuni (79, 80, 118, 132, 182).

Interaction of C. jejuni with intestinal epithelial cells has been shown to
elicit a variety of innate responses (60, 107, 113, 176). C. jejuni infection of
cultured intestinal epithelial cell lines (INT407 and T84) results in secretion of IL-
8 (60, 107) as well as the antimicrobial peptides human B—defensins 2 and 3
(188). It also stimulates the secretion of several other chemokines including
macrophage inflammatory proteins (MlP)-10t and MlP-30t, monocyte
chemoattractant protein 1, and gamma interferon-inducible protein 10 (68, 81).
Furthermore, C. jejuni infection triggers signaling through NF-tcB and MAP kinase
pathways, which mediate the production of various chemokines and cytokines
involved in innate immunity (29, 113, 176). While innate responses of intestinal
epithelial cells to C. jejuni and the signaling pathways involved have been
extensively investigated, less is known about the interplay between C. jejuni and
host immune cells, particularly dendritic cells (DCs), which are necessary to elicit
protective immunity.

DCs are professional antigen presenting cells involved in
immunosurveillance of body tissues. DCs densely populate the lamina propria of
the small and large intestine and are capable of forming transepithelial dendrites
by a CXgCR1-dependent mechanism to sample the intestinal lumen for

commensals or pathogens (123). An extensive body of evidence demonstrates

102

 

that DCs play a role in educating the immune system for the development of
appropriate T cell responses to pathogens (86). Conversely, pathogens have
evolved many mechanisms to subvert DC functions as a means of immune
evasion (36, 116). Given the facts that DCs are central to the induction of
antigen-specific acquired immune responses and that certain pathogens can
interfere with DC functions, it is pivotal to understand DC-C. jejuni interactions. C. r.
jejuni has been shown to elicit maturation and proinflammatory responses from in

vitro cultured human DCs (67). However, at this time, the role of DCs in inducing

 

adaptive immune responses against C. jejuni is not known. ’ i;
Recently, we showed that CS7BU6 wild type and congenic lL-10 deficient
mice can serve as murine models of C. jejuni colonization and enteritis,
respectively (108). In this model, while C. jejuni infection induced enteritis in IL-
10 deficient mice, wild type mice were resistant to C. jejuni-induced disease but
stably colonized. These models will allow exploration of the basis of protective
immune responses that result in either healing or disease mediated by
immunopathology after a primary C. jejuni infection, which otherwise would be
difficult to investigate in humans. To be able to focus on the role of specific
immune components in controlling C. jejuni infection, we first need to understand
the overall immune responses elicited by C. jejuni in these models. In this
context, a study describing the role of DCs in inducing the innate and adaptive
immune responses to C. jejuni in mouse models is warranted. The objective of
these studies was to gain insight into the interaction of murine DC with C. jejuni

and the effect of this interaction on development of Th cell responses in vitro. We

 

103

hypothesized that murine bone marrow-derived DCs (BM-DCs) internalize C.
jejuni, which then undergo activation and initiate a Th1-type immune response.

As a primary goal, we wanted to determine whether C. jejuni 11168 is
efficiently phagocytosed and killed by murine BM-DCs and whether it is capable
of surviving intracellularly. It remains controversial in the literature whether C.
jejuni escapes killing by phagocytic cells and translocates from the intestinal
tracts to systemic organs inside these cells. In this study, the intracellular survival
of the bacteria was assessed at 2, 4 and 8 h post-infection using a standard
gentamicin killing assay. We show that C. jejuni 11168 is efficiently internalized
and killed by murine DCs within 8 h post-infection. These findings are consistent
with those of Hu et al., (2006) (67) who reported that ~ 99% of phagocytosed C.
jejuni 81-176 were killed by human 003 within 24 h post-infection. Similar results
were obtained with activated human macrophages. In contrast, intracellular
pathogens such as S. typhimurium and Mycobacterium tuberculosis resist killing
by DOS and survive intracellularly for an extended period of time; this survival
ability was implicated in the systemic spread of these bacteria (20, 28).

Under steady state conditions, 005 are present in the immature state but
rapidly undergo functional transition to the mature state following exposure to
microbial products (86, 116). Mature DCs are characterized by enhanced
surface-display of MHC-ll-antigen complexes and costimulatory molecules, which
provide activating signals 1 and 2, respectively, to naive T cells (86).
Accumulating evidence has demonstrated that DCs play a major role in eliciting

anti-microbial T-cell immunity vs. tolerance towards self-antigens and

104

commensals; this role is suggested to depend mechanistically in part on their
maturation status after antigenic exposure (106). In the current study, flow
cytometric analysis revealed that immature murine DCs infected with C. jejuni
11168 undergo maturation by up-regulating the surface expression of MHC-ll,
CD40, 0080, and CD86 molecules. Similarly, an earlier study showed that
human DCs become mature following C. jejuni infection as demonstrated by the
increased expression of CD40, CD80, and CD86 (67). The increased DC
expression of MHC-ll and co-stimulatory molecules after challenge suggests that
C. jejuni-infected DCs would provide essential signals 1 and 2 to stimulate
efficient anti-C. jejuni T cell responses.

In order to stimulate appropriate CD4” T cell responses, antigen
presenting cells must provide instructive cytokine signals to na'l've Th cells (106).
Several lines of evidence indicate that cytokine signals from DCs have a major
influence on the differentiation of naive Th cells to effector cells (86, 116). DC-
and macrophage-derived lL-12 is crucial for the optimal generation of Th1-type
immune responses to a variety of pathogens (86, 116). In this study we observed
a C. jejuni 11168 dose- and time-dependent secretion of lL-12 by infected DCs.
lL-12 has been shown to mediate the cross-talk between DOS and innate natural
killer (NK) cells, inducing lFva production by NK cells (57, 92, 93, 169). This DC-
mediated NK cell activation occurs early and locally at the site of infection,
mediating the initial resistance to bacterial infections (92, 169). Hu et al., (2006)
also showed that human monocyte-derived DCs secreted lL-12 after challenge

with C. jejuni 81-176 (67). With such elicitation of lL-12, it is likely that C. jejuni

105

also stimulates NK cell activation in' the host, although this event has not been
studied. TNF-a and lL-6 are multifunctional proinflammatory mediators induced in
many microbial infections (95). In this study, murine DCs secreted TNF-a and IL-
6 when infected with C. jejuni 11168. TNF-a secreted by C. jejuni-infected DCs
may act in an autocrine or paracrine manner to enhance DC—maturation and
migration (163). Additionally, TNF-ot may act together with IFN-y in activating
local anti-microbial mechanisms in macrophages (101). Likewise, human DCs
were also shown to elicit robust proinflammatory responses by secreting TNF-a,
IL-1B, lL-6 and lL-8 following C. jejuni 81-176 infection (67). Similarly, a number
of in vitro studies demonstrated that C. jejuni induces proinflammatory cytokine
and chemokine production from a variety of cells such as intestinal epithelial
cells, macrophages, or monocytic cell lines (60, 68, 84, 107, 177).

IL-10 is an immunoregulatory cytokine critical in preventing exaggerated
immune and inflammatory responses against normal enteric flora and pathogens
(134). In our mouse model, lL-10"' mice manifested moderate to severe colitis
from 2—35 days following primary C. jejuni 11168 challenge, whereas wild type
C57BU6 mice were resistant to disease despite uniform stable colonization
(108). Therefore, it is evident that IL-10 plays an important immunoregulatory role
in C. jejuni infection in mice that protects against tissue damage due to
unchecked inflammatory responses. In the current study, no detectable amounts
of lL-10 production were observed from C. jejuni-infected DCs at all time points
examined. Our in vitro experiments suggest that DCs are unlikely to be the

primary source of lL-10 in mice infected with C. jejuni. A recent report by Perona-

106

Wright et al., (2006) also demonstrated that DCs may not necessarily be the
most important source of IL-10 required for immunoregulation in vivo. These
authors suggested that non-hematopoietic and innate cells such as epithelial
cells may be significant contributors of lL-10 (134).

Because immunity to C. jejuni has been suggested to be strain-specific
(54) and cytokines are integral components of cell-mediated immune resistance,
we next investigated whether different strains of C. jejuni, which were isolated
from various sources, elicit differential cytokine production from murine DCs. C.
jejuni strains 11168 and 33560 induced higher levels of lL-12, TNF-a and IL-6,
whereas other four strains elicited relatively lower amounts of these cytokines. In
particular, strain NW was the least potent inducer of these cytokines. These
findings indicate that DCs can be activated by various strains of C. jejuni to
secrete IL-12 and proinflammatory cytokines in a strain-specific manner and
support the conclusion that strain-to-strain variation influences host-pathogen
interactions in C. jejuni infection and thus the disease development (15).

DCs have the ability to express different cytokines and membrane-bound
molecules and play a decisive role in the differentiation of naive T cells to effector
populations (86). The Th-polarizing ability of C. jejuni 11168-infected BM-DCs
was evaluated in an in vitro DC-CD4" T cell coculture system; BM-DCs infected
with C. jejuni 11168 induced Th1 but not Th2 priming of CD4‘ T cells in vitro.

Having demonstrated that 005 recognize C. jejuni, undergo activation,
and induce Th1-effector differentiation of naive CD4“ T cells, we sought to

understand the mechanisms, both bacterial and host, responsible for these

107

responses. We investigated the roles of viability of C. jejuni, bacterial contact with
DCs, and bacterial internalization by DCs in inducing activation of DC maturation
and cytokine production. Formalin-killed C. jejuni also elicited maturation and
cytokine responses from DCs but at a significantly lower magnitude compared to
live bacteria. Inhibition of C. jejuni phagocytosis by DCs resulted in significant
impairment of expression of maturation markers, IL-12, and TNF-a but not lL-6.
This result suggests that recognition of C. jejuni by surface receptors on DCs is
sufficient to induce lL-6 secretion by DCs. Furthermore, when direct contact
between C. jejuni and DOS was prevented using transwell inserts, the maturation
and cytokine responses of DCs to C. jejuni were impaired. Overall, all the
bacterial factors analyzed in this study, such as C. jejuni viability, contact of C.
jejuni cells with D03, and internalization of C. jejuni by DCs, are necessary for
maximal activation of DCs in response to C. jejuni.

Next, the role of TLR signaling in mediating DC responses to C. jejuni was
investigated. TLRs are germ-line encoded pattern recognition receptors that
recognize various conserved molecules of pathogens, including LPS, flagellin,
and nucleic acids (4). Antigen-presenting cells, particularly DCs, express a wide
array of TLRs on their surfaces as well as intracellularly. Following recognition of
their ligands, TLRs induce innate inflammatory responses that regulate the
subsequent development of adaptive immune responses (74). TLR2 (in
association with TLR1 or TLR6), TLR4, TLR5, and TLR9 are involved in sensing
of bacterial pathogens. In this study, .I focused on the role of TLR2 and TLR4

recognition of C. jejuni for two main reasons: 1) C. jejuni possesses potential

108

ligands for these receptors, namely LOS and lipoproteins (80, 120); 2) it has
been reported that TLR5 and TLR9 recognition of C. jejuni is weak because of
the nature of their agonists, flagellin and DNA respectively, in C. jejuni (6, 37).
The surface expression of MHC-ll, 0080, and CD86 molecules was
substantially reduced in TLR2- or TLR4-deficient DCs compared to WT 003
following infection with C. jejuni. The production of lL-6, TNF-ot and IL-12 was
profoundly impaired in infected TLR4-deficient 003 whereas TLR2 deficiency
resulted in only minimal reduction in lL-6 and TNF-ot secretion and about 40-50%
inhibition in the lL-12 response to C. jejuni. Overall, it appears that signaling
through both of these receptors is necessary for optimal maturation of DCs
following C. jejuni infection, whereas cytokine responses of 005 to C. jejuni are
mainly dependent on TLR4. Such a major role of TLR4 in C. jejuni-induced
activation of DCs is consistent with the structure of lipid A of C. jejuni: lipid A of
C. jejuni is hexa-acylated and diphosphorylated and thus possesses the essential
configuration to stimulate TLR4 signaling (45, 120). In most cases, recognition of
LPS by TLR4 involves two additional host factors such as CD14 and MD-2,
which act as the co-receptors (4). It would be of interest to determine whether
TLR4 recognition of C. jejuni LOS is also dependent on CD14 and MD—2.
Surprisingly, both MyD88-dependent and -independent (TRIF) pathways
downstream of TLR4 were activated by C. jejuni, and signaling through both of
these pathways was found be required for maximal maturation and cytokine
responses to C. jejuni. Besides stimulating distinct transcription factors, both

MyD88 and TRIF have also been shown to activate the same set of transcription

109

factors, including NF-KB, that mediate the transcription of genes involved in
innate responses. As a result, TRIF and MyD88 pathways induce a variety of
overlapping cellular responses (4). However, TRIF also activates a unique
pathway leading to activation of transcription factor lRF-3 that induces
expression of type I interferon genes (181). Indeed, C. jejuni signaling through
TLR4-TRIF was found to trigger phosphorylation of downstream lRF-3 and
secretion of IFN-B. Stimulation of TLR4 by E. coli LPS triggers IFN-[3 production
by D05, and the secreted IFN-l3 augments activation of DCs through lRF-7. This

finding suggests that IFN-[3 secreted by C. jejuni-infected DCs may amplify their
maturation and IL-12 responses (53).

Impairment of C. jejuni-induced maturation and cytokine profiles in the
absence of TLR2, TLR4, My088, and TRIF was reflected in the Th1 cell
activating ability of C. jejuni-infected DCs. C. jejuni-infected DCs deficient in
these components elicited significantly reduced amounts of IFN-y from naive
CD4+ T cells, indicating that TLR-mediated delivery of costimulatory and cytokine
signals are essential for maximal Th1 polarization. Consistent with these findings,
Watson et al., (2007) (177) reported that mice deficient in MyD88 following oral
inoculation with C. jejuni were compromised in their ability to eliminate the
bacteria. MyD88”' mice harbored higher numbers of C. jejuni in intestinal tissues
than WT mice, and the extraintestinal spread of C. jejuni into systemic sites such
as spleen and liver was observed mainly in MyD88"' mice.

Based on the results of my studies, we propose the following model (Fig.

1) for the role of TLR signaling in C. jejuni-induced activation of DCs: TLR4

110

recognizes C. jejuni, most likely the LOS of C. jejuni, and activates both MyD88
and TRIF signaling that cooperate in inducing DC maturation and cytokine
responses. It is possible that these two signaling pathways activated by C. jejuni
converge at the level of transcription factors such as NFKB resulting in sustained
activation and nuclear translocation. Furthermore, activation of the TLR4-TRIF
pathway induces lRF-3 activation and IFN-B production in response to C. jejuni.
Besides stimulating TLR4 signaling, C. jejuni, possibly through its Iipoprotein
leA, activates TLR2 signaling, which further augments some of the responses of
DCs via My088.

In conclusion, this study provides important mechanistic insights into the
interaction between C. jejuni and DCs, which are the players in tailoring
pathogen-specific adaptive immune responses. Identification of TLR3 responsible
for C. jejuni recognition and DC activation in this study would advance current
understanding of host defense mechanisms that confer protective immunity to
infection with C. jejuni and form a basis for future investigations to dissect

immunoregulation during C. jejuni infection.

111

C. jejuni

 

Figure 1. Proposed model for the role of TLR signaling
in C. jejuni-induced activation of DCs.

112

FUTURE DIRECTIONS

Does C. jejuni activate DCs in vivo?

An important question that arises from this study is whether C. jejuni
induces the activation of DCs present in the intestinal lamina propria. The in vitro
findings of this study combined with our laboratory’s previous observation—that
C. jejuni-specific immunohistochemical staining was found associated with DC-
Iike cells in the intestinal mucosa (108)-—suggest the hypothesis that DCs in the
intestinal mucosa recognize. C. jejuni and undergo activation. The use of confocal
microscopy and immunohistochemistry will allow us to investigate this in vivo
phenomenon. Localization of C. jejuni in the 005 can be assessed by
performing dual immunostaining for CD11c (dendritic cell marker) and C. jejuni
antigens in the frozen sections of ileocecocolic junctions and colon tissues from
mice orally infected with C. jejuni. Furthermore, the phenotypic maturation of DCs
in tissues from C. jejuni-infected mice can be assessed by immunohistochemistry
using antibodies against CDBO, CD86 and CD40. An alternate approach is to use
multicolor flow cytometry: single cell suspensions obtained from fresh tissues of
control and infected mice can be stained with fluorochrome—conjugated anti-
CD11c and anti-C080, anti-CD86, or anti-CD40 antibodies and analyzed by flow
cytometry. However, this method is cumbersome and has practical difficulties
since the numbers of DCs that can be harvested from intestinal tissue may be

too low to obtain reliable results.

113

Distinct subsets of DCs exist in the intestinal lamina propria, which include
CD11c”CD11b"CD8a', CD11c+CD1 1 b'CD8a‘, and CD11c"CD11b'CDBa' 00$
(73). Additionally, a population of DCs characterized by the expression of a
chemokine receptor, CX3CR1, has been identified in the lamina propria (123).
These CX3CR1+ DCs directly sample the intestinal lumen for microbes by
forming transepithelial dendrites. It is important to understand which subset(s) of
lamina propria DCs participates in C. jejuni recognition in vivo. This also can be
analyzed by using a combination of antibodies specific for each subset of DCs in

immunohistochemistry and flow cytometry.

114

Does NOD1 recognition of C. jejuni contribute to TLR-independent

induction of Th1 responses by DCs?

A surprising finding from this study is that priming of Th1 cells by C. jejuni-
infected DCs was preserved to a certain extent in the absence of TLR signaling.
This indicates that C. jejuni stimulates TLR-independent pathway(s) in DOS,
which then contribute to the induction of Th1 cells by DCs. Signaling through
cytoplasmic nucleotide-binding oligomerization domain (NOD)—1 receptor may
play a role in this process. NOD1 belongs to a family of cytoplasmic pattern
recognition receptors and is mainly expressed in antigen-presenting cells such as
DCs and epithelial cells (156). NOD1 recognizes y—D-glutamyl-meso-
diaminopimelic acid (iE-DAP) derived from peptidoglycan (PGN) of Gram-
negative bacteria. Sensing of iE-DAP by NOD-1 results in induction of
inflammatory cytokine genes via activation of MAP kinases and NF-KB (156).
Zilbauer et al. (2007) recently reported that NOD1 recognition of C. jejuni is
required for maximal expression of lL-8 and human B-defensin, an antimicrobial
peptide, by Caco-2 intestinal epithelial cells. Furthermore, the knock—down of
NOD1 expression in IEC resulted in increased numbers of intracellular C. jejuni
(189). On the basis of these published reports, I hypothesize that cytosolic NOD1
receptor signaling triggered by C. jejuni in 003 contributes to Th1 polarization by
C. jejuni-infected DCs. Even though C. jejuni has been shown not to escape the
phagolysosome into the cytoplasm in phagocytic cells (175) such as

macrophages and dendritic cells, it is possible that derivative products of PGN

115

from C. jejuni being degraded in the phagolysosome can leak into cytoplasm and
stimulate NOD1 signaling as reported previously for Listeria monocytogenes
(59). The experimental approach to determine whether NOD1 can mediate Th1-
induction by C. jejuni-infected DCs in the absence of TLR signaling would be to
coculture C57BU6 wild type DCs, MyD88"' TRIF"' DCs, and MyD88"' TRIF"'
NOD1"' DCs infected with C. jejuni with naive CD4+ T cells and analyze the IFN-y

levels in the supernatants by the methods described in the previous chapters.

116

Identification of TLR ligands in C. jejuni

Having demonstrated that C. jejuni activates signaling through TLR2 and
TLR4, the next step is to identify the ligands in C. jejuni that trigger TLR2 and
TLR4 pathways. Furthermore, identification of C. jejuni components that induce
potent innate immune responses through TLR signaling would aid in the
development of vaccines, particularly subunit vaccines, against C. jejuni. As
described in chapter 3, LOS of C. jejuni is the most likely candidate to be
recognized by TLR4. Supporting evidence for this speculation comes from the
structure of lipid A of C. jejuni LOS. Lipid A is the primary immunostimulatory
component of LPS/LOS, and the number and length of acyl chains in lipid A are
critical for this function (5). C. jejuni LOS, like Escherichia coli LPS, possesses
diphosphorylated hexa-acyl lipid A that was reported to be essential for optimal
stimulation of cellular immune responses (120). Indeed, it has been shown that
C. jejuni LOS exhibits comparable endotoxic activity in biological test systems
relative to that observed with prototype enterobacterial LPS preparations (120).

The putative ligands for TLR2 signaling by C. jejuni are lipoproteins.
Literature shows that C. jejuni genome encodes for several lipoproteins, namely
CjaA, HisJ, Omp18, and leA of which two (JIpA; CjaA) have been characterized
(79, 80). However, to date none of these molecules has been demonstrated to be
recognized by TLR2. Here, the hypothesis to be tested is that LOS and
lipoproteins, particularly leA, of C. jejuni induce phenotypic and functional
activation of DCs. The experimental approach is to isolate the LOS from C. jejuni

using the Darveau-Hancock method (39) or by using a commercial LPS

117

extraction kit (lntron Biotechnology, Korea). Genes encoding lipoproteins would
be cloned into and expressed in E. coli as His-tagged proteins and purified using
nickel columns. DCs would be stimulated with various doses of purified LOS and
lipoproteins and be assessed by flow cytometry and ELISA for maturation and
cytokine production, respectively. A caveat in using purified components from
bacteria is that they are often contaminated with other undesirable components
that invalidate the results of experiments designed to examine the role of specific
bacterial mediators (4, 64). It has been well documented that LPS preparations
are often contaminated with lipoprotein and vice versa, which would confound the
results obtained with such preparations. However, this problem can be overcome
to certain extent by using both WT and TLR4"' DCs in experiments to test LOS
preparations and WT and TLR2"’ DCs in experiments to test Iipoprotein

preparations.

118

APPENDIX

Besides my research projects, I have contributed to the following

publications from our laboratory:

Bell, J. A., J. L. St. Charles, A. J. Murphy, V. A. Rathinam, A. E. Plovanich-
Jones, E. L. Stanley, J. E. Wolf, J. R. Gettings, T. S. Whittam, and L. S.
Mansfield. 2009. Multiple factors interact to produce responses resembling
spectrum of human disease in Campylobacterjejuni infected CS7BL/6 IL10"'
mice. BMC Microbiol 9(1):57.

Mansfield, L. S., J. S. Patterson, B. R. Fierro, A. J. Murphy, V. A. Rathinam, J. J.
Kopper, N. l. Barbu, T. J. Onifade, and J. A. Bell. 2008. Genetic background of
lL-10-l- mice alters host-pathogen interactions with Campylobacterjejuni and
influences disease phenotype. Microb Pathog 45:241-257.

Mansfield, L. S., J. A. Bell, D. L. Wilson, A. J. Murphy, H. M. Elsheikha, V. A.
Rathinam, B. R. Fierro, J. E. Linz, and V. B. Young. 2007. C57BU6 and
congenic interleukin-10-deficient mice can serve as models of Campylobacter
jejuni colonization and enteritis. Infect lmmun 75:1099-1115.

My contributions include oral inoculation of mice with C. jejuni, clinical

assessment of infected mice, blood collection, necropsy, and tissue harvesting.

119

 

 

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