The Effects of Helicobacter pylori Infection on Dendritic Cells

by

David Rizzuti

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology University of Toronto

© Copyright by David Rizzuti (2012)

ii

The Effects of Helicobacter pylori Infection on Dendritic Cells

David Rizzuti

Master of Science

Department of Physiology

University of Toronto

2012

Abstract

Helicobacter pylori infects the human gastric mucosa and induces a chronic gastritis that does

not eliminate the pathogen. H. pylori infection is the primary risk factor for gastric cancer

development. The host immune system is crucial for both bacterial elimination and tumor

surveillance. The interaction between H. pylori and dendritic cells (DC), key orchestrators of the

host immune response, was examined.

H. pylori activated the Signal Transducer and Activator of Transcription 3 (STAT3) pathway,

assayed using immunoblotting, in bone marrow-derived DCs. This blunted DC maturation,

determined by CD86 and MHC II expression, in an autocrine/paracrine IL-10-dependent

pathway. H. pylori infection stimulated the production of cytokines including IL-10, IL-12p40,

and TNF-α as determined using ELISA and multiplex analysis. DC co-culture with T cells did

not drive regulatory T cell development. These results demonstrate that H. pylori blunts DC

maturation, which may play an important role in promoting bacterial persistence and disease.

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Acknowledgments

There are many people that I would like to acknowledge for making this thesis possible.

My supervisor, Dr. Nicola Jones, thank you for your incredible mentorship and support. I could

not have asked for anything more from a supervisor; thank you for believing in me and giving

me the motivation to succeed when I needed it. Thank you for helping me grow, not just as a

scientist, but as a person. You were committed to helping me realize my potential and your

concern for my well-being is what made you truly stand out.

My supervisory committee members, Dr. Dana Philpott and Dr. Anthony Gramolini, thank you

for your feedback and guidance. You provided me with the ideal combination of support and

motivation to succeed in this project.

To my lab members, thank you for making this research such an enjoyable experience. Esther,

thank you for your support and advice, and teaching me the lab skills and techniques I needed to

succeed. Dana, thank you for being there with invaluable advice whenever I needed it.

Christiane, the morning and afternoon coffee breaks were essential and incredible; thank you for

helping me stay motivated. Mika, Ted, Dave, Emma, and Heidi, thanks for being awesome

members of the lab and making it an enjoyable environment. Ilya, thanks for being a great friend

and for all your advice and support in everything, from the experiments to the applications.

Ramzi, thanks for teaching me everything I know about flow cytometry, and for the priceless

advice. You were an excellent model for teaching and I follow your example when showing

others new protocols. Kelsey, thanks for keeping the ice cold. Everyone else in the SickKids Cell

Biology department, thanks for making this a great time.

My family, especially my parents Donna and Luciano, and brother Daniel, thank you for

supporting me throughout this endeavor and helping me through the tough times. To all my

friends, especially Vince, Dustin, Theo, Looch, and Champ, you guys were always there for me.

Last but most importantly, I want to thank the Lord for watching over me through every step of

this journey. Nothing would be possible without Him.

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Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Abbreviations ..................................................................................................................... vi

List of Figures ................................................................................................................................. x

Chapter 1 Introduction .................................................................................................................... 1

1.1 Helicobacter pylori ............................................................................................................. 1

1.1.1 Gastric Mucosal Infection and Colonization .......................................................... 1

1.1.2 Disease Pathogenesis .............................................................................................. 2

1.1.3 Virulence Factors .................................................................................................... 5

1.2 The Immune Response ...................................................................................................... 10

1.2.1 The Dendritic Cell ................................................................................................. 11

1.2.2 T Cell Phenotypes ................................................................................................. 14

1.2.3 Dendritic Cell Responses to H. pylori .................................................................. 17

1.2.4 H. pylori-altered DCs: potential role in disease .................................................... 22

1.2.5 Signal Transducer and Activator of Transcription 3 (STAT3) ............................. 23

1.3 Summary ........................................................................................................................... 26

Chapter 2 Materials and Methods ................................................................................................. 28

2.1 Bone Marrow-Derived Dendritic Cells ............................................................................. 28

2.2 Treatment/Infection ........................................................................................................... 28

2.3 Western Blotting ............................................................................................................... 29

2.4 Densitometric analysis ...................................................................................................... 30

2.5 Flow Cytometry ................................................................................................................ 30

2.6 ELISA Cytokine Analysis ................................................................................................. 30

2.7 Multiplex Bead-based Luminex Assay ............................................................................. 30

v

2.8 OT-II Transgenic Mouse CD4+ Splenocytes ................................................................... 31

2.9 STAT3 KO Mice ............................................................................................................... 31

2.10 DC:T Cell Coincubation .................................................................................................. 31

2.11 Statistical Analysis ........................................................................................................... 32

Chapter 3 Results .......................................................................................................................... 33

3.1 H. pylori infection induces STAT3 phosphorylation in dendritic cells ............................ 33

3.2 H. pylori upregulates expression of DC maturation markers ........................................... 35

3.3 Activated dendritic cells secrete a specific cytokine profile in response to H. pylori

infection ............................................................................................................................ 38

3.4 H. pylori induces more pronounced maturation in STAT3 KO dendritic cells ................ 41

3.5 IL-10 secretion by dendritic cells in response to H. pylori infection activates STAT3

to blunt maturation ............................................................................................................ 44

3.6 H. pylori-infected dendritic cells do not drive regulatory T cell development ................. 47

Chapter 4 Discussion .................................................................................................................... 50

Chapter 5 Future Directions .......................................................................................................... 57

References ..................................................................................................................................... 59

vi

List of Abbreviations

A. lwoffi: Acinetobacter lwoffi

APC: antigen-presenting cells

APCa: allophcocyanin

BMDC: bone marrow-derived dendritic cell

BSA: bovine serum albumin

cag PAI: cag pathogenicity island

CagA: cytotoxin-associated gene A

CD: cluster of differentiation

DAMP: danger-associated molecular pattern

DC: dendritic cell

DNA: deoxyribonucleic acid

E. coli: Escherichia coli

ELISA: enzyme-linked immunosorbent assay

EPIYA: glutamate-proline-isoleucine-tyrosine-alanine

ERK 1: extracellular signal-regulated kinase 1

ERK 2: extracellular signal-regulated kinase 2

FB: flow cytometry buffer

FITC: flourescein isothiocyanate

GEEC: GPI-AP-enriched early endosomal compartments

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GERD: gastroesophageal reflux disease

GI: gastrointestinal

GM-CSF: granulocyte macrophage colony-stimulating factor

GPI-AP: glycosylphosphatidylinositol-anchored proteins

H. pylori: Helicobacter pylori

IBD: inflammatory bowel disease

IFN: interferon

IL: interleukin

JAK: Janus kinase

JAM-A: junctional adhesion molecule

KO: knockout

LFA-1: lymphocyte function-associated antigen

LPS: lipopolysaccharide

MALT: mucosal-associated lymphoid tissue

MHC: major histocompatibility complex

MOI: multiplicity of infection

NLR: Nod-like receptor

OVA: ovalbumin peptide

PAMP: pathogen-associated molecular pattern

PBS: phosphate-buffered saline

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P-CagA: phosphorylated CagA

PD: programmed death

PE: phycoerythrin

PFA: paraformaldehyde

PRR: pattern recognition receptor

PSTAT3: phosphorylated STAT3

RIPA: radioimmunoprecipitation assay

RPMI: Roswell Park Memorial Institute

SDS: sodium dodecyl sulfate

SH2: Src-Homology 2

STAT3: signal transducer and activator of transcription 3

T4SS: type IV secretion system

TAA: tumor-associated antigen

TBST: tris-buffered saline supplemented with tween

Tc: cytotoxic T cell

TCR: T cell receptor

TGF: transforming growth factor

Th: helper T cell

Th1: type 1 helper T cell

Th17: Th17 helper T cell

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Th2: type 2 helper T cell

TLR: Toll-like receptor

TNF: tumor necrosis factor

Treg: regulatory T cell

VacA: vacuolating cytotoxin A

VEGF: vascular endothelial growth factor

WT: wildtype

ZO-1: zonula occludens 1

x

List of Figures

Figure 1.1-1: Gastric cancer model as proposed by Correa. ........................................................... 4

Figure 1.1-2: CagA signaling pathways. ......................................................................................... 6

Figure 1.2-1: The immunological synapse .................................................................................... 13

Figure 1.2-2: T cell differentiation ................................................................................................ 15

Figure 1.2-3: Potential dendritic cell - H. pylori interactions ....................................................... 18

Figure 1.2-4: STAT3 signaling pathway. ..................................................................................... 25

Figure 3.1-1: H. pylori activates the STAT3 pathway in dendritic cells ...................................... 34

Figure 3.2-1: H. pylori induces expression of dendritic cell maturation markers ........................ 36

Figure 3.3-1: Dendritic cells secrete a specific cytokine profile upon infection with H. pylori ... 40

Figure 3.4-1: STAT3 KO DCs express enhanced levels of maturation markers in response to H.

pylori infection .............................................................................................................................. 43

Figure 3.5-1: α-IL-10 antibody inhibits H. pylori-induced STAT3 phosphorylation in BMDCs to

promote enhanced expression of maturation markers .................................................................. 46

Figure 3.6-1: H. pylori infection does not induce dendritic cells to produce CD4+ FoxP3

+ T cells

....................................................................................................................................................... 49

Figure 4-1: Potential model of dendritic cell responses to H. pylori ............................................ 54

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Chapter 1 Introduction

1.1 Helicobacter pylori

1.1.1 Gastric Mucosal Infection and Colonization

Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped, Gram-negative bacterium that

selectively infects the gastric mucosa of almost half of the human population (Cover and Blaser,

2009; Kusters et al., 2006). Infection usually occurs early in childhood and colonization

generally persists for the lifetime of the host. Infected individuals can develop several disease

phenotypes, including simple gastritis, peptic ulcers, and gastric cancer (Wroblewski et al.,

2010). Indeed, H. pylori was recognized as a Type I carcinogen in 1994 and infection with this

pathogen remains the primary risk factor for gastric cancer development (Amieva and El-Omar,

2008; Kusters et al., 2006; Peek and Crabtree, 2006; Uemura et al., 2001).

H. pylori possess numerous adaptations that facilitate successful colonization of the human

gastric epithelium. The generation of cytosolic and cell-surface urease permits transient survival

in the highly acidic gastric lumen (Amieva and El-Omar, 2008). The urease enzyme functions to

convert urea into NH3 and CO2, which buffer the acidic environment of the gastric lumen.

Several polar flagella and a spiral shape facilitate mobility of the bacteria in the highly motile

gastric environment, which is constantly churning and secreting mucus. This mobility, combined

with a chemotaxis receptor (TlpB) that detects extracellular pH, allows H. pylori to orient and

remain close to the epithelial layer where the pH is almost neutral (Amieva and El-Omar, 2008;

Croxen et al., 2006). Several specialized adhesion molecules allow the bacteria to adhere to the

epithelium and further facilitate colonization and persistence. The outer-membrane protein BabA

binds to Lewis B blood group antigens which are expressed on gastric epithelial cells, while the

adhesin SabA binds to sialyl-Lewis-X, a glycoprotein expressed on the epithelial cell surface

(Amieva and El-Omar, 2008; Linden et al., 2002; Montecucco and Rappuoli, 2001). H. pylori

uses these diverse factors in concert to orient, localize, and adhere to the gastric epithelial

surface, which facilitates the translocation of bacterial proteins into epithelial cells and grants the

2

bacteria access to cellular nutrients that are necessary for survival. This creates a remarkable

niche that facilitates bacterial survival and contributes to life-long infection and persistence.

1.1.2 Disease Pathogenesis

Helicobacter pylori infection is associated with three main disease phenotypes. All infected

individuals will develop gastritis (a chronic mild inflammation in the gastric mucosa); 80-90% of

these individuals will remain asymptomatic and will not develop any serious gastrointestinal (GI)

complications as a result of this H. pylori infection. This phenotype has therefore been termed

the “simple gastritis phenotype” and is found in the overwhelming majority of infected

individuals (Amieva and El-Omar, 2008). Approximately ten percent of individuals infected with

H. pylori develop a “duodenal ulcer phenotype” characterized by an antral-predominant gastritis

accompanied by high levels of acid secretion (Cover and Blaser, 2009). Patients with this

phenotype, which is particularly prevalent in Western societies, are predisposed to the

development of duodenal ulcers (Kusters et al., 2006).

Approximately 1-2% of individuals infected with H. pylori will develop cancer, including gastric

adenocarcinoma and mucosal-associated lymphoid tissues (MALT) lymphomas (Wroblewski et

al., 2010). These are associated with the third disease phenotype, known as the “gastric cancer

phenotype”, which is characterized by decreased levels of acid production and a corpus-

predominant gastritis (El-Omar et al., 1997). The H. pylori-mediated chronic gastritis is thought

to be the primary stage in the transformation from a normal gastric mucosa through a series of

definite histological and functional stages which culminates in gastric adenocarcinoma, as

proposed by Correa (Correa, 1992, 2004) (Figure 1.1-2). Briefly, the transformation begins with

chronic gastritis that proceeds into atrophic gastritis and inflammatory destruction of the mucosa.

Several factors, including H. pylori infection, inflammatory responses, dietary and lifestyle

habits, and genetic susceptibility, are thought to contribute to this progression. Intestinal

metaplasia followed by dysplasia and finally transformation to adenocarcinoma are the later

stages of this transformation (Correa and Houghton, 2007). However the exact mechanisms of H.

pylori-mediated carcinogenesis have yet to be fully elucidated, and bacterial, host, and

environmental factors appear to play complex and often synergistic roles in mediating the cancer

risk associated with H. pylori infection (Amieva and El-Omar, 2008). The goal of this study is

not to directly examine the carcinogenic potential of H. pylori, rather to examine the interaction

3

between H. pylori and the host immune response. Subversion or modulation of the host immune

response may play an important role in bacterial persistence and simultaneously contribute to

disease pathogenesis by inhibiting immune responses to developing cancerous cells.

4

Figure 1.1-1: Gastric cancer model as proposed by Correa

H. pylori infection induces chronic gastritis in the mucosa. This inflammation, combined with

environmental and host genetic factors, induces atrophy of the gastric mucosa and loss of

glandular tissue. Low acidity can then contribute to the development of intestinal and colonic

metaplasia, followed by dysplasia and finally transformation into adenocarcinoma.

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1.1.3 Virulence Factors

Pathogenesis of H. pylori strains vary considerably largely due to variations in two major

bacterial factors: the cytotoxin-associated gene A (CagA) toxin, an injected protein that is

encoded by the cag pathogenicity island (cag PAI), and the vacuolating-cytotoxin A (VacA),

which is toxin is secreted by the bacteria and induces the formation of large vacuoles in cultured

epithelial cells (Akopyants et al., 1998; Terebiznik et al., 2006). The molecular and cellular

mechanisms of action of these two major H. pylori virulence factors have garnered much

attention and are described below.

1.1.3.1 cag PAI and CagA

The cag PAI is a 40-kb deoxyribonucleic acid (DNA) sequence in the H. pylori genome that

contains approximately 27 to 31 genes, the majority of which (at least 18) code for a bacterial

type IV secretion system (T4SS). This T4SS acts as a “molecular syringe” to transfer the

bacterial effector protein CagA into the host cell cytoplasm (Akopyants et al., 1998; Odenbreit et

al., 2000). CagA is a 120-140kDa protein, encoded by the cagA gene; CagA+ strains of H. pylori

confer a much higher risk for the development of gastric cancer to their hosts (Parsonnet et al.,

1997; Robinson et al., 2007). Once CagA is injected into the host cell, it localizes near the

plasma membrane, and becomes phosphorylated via host c-Src and Abl family kinases (Amieva

and El-Omar, 2008; Selbach et al., 2002; Tammer et al., 2007) (Figure 1.1-3). The

phosphorylation occurs on the tyrosine residue of glutamate-proline-isoleucine-tyrosine-alanine

(EPIYA) motifs, which are located near the C-terminus of the CagA protein (Naito et al., 2006).

Upon phosphorylation of the EPIYA motifs, the CagA protein will interact with multiple

intracellular effector molecules. Phosphorylated CagA (P-CagA) interacts with the tyrosine

phosphatase SHP-2, inducing activation of the extracellular signal-regulated kinases 1 and 2

(ERK 1/2), which culminates in morphological changes such as cell migration, elongation, and

scattering, collectively known as the “hummingbird phenotype” (Moese et al., 2004; Robinson et

al., 2007; Yamazaki et al., 2003).

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Figure 1.1-2: CagA signaling pathways

H. pylori uses the cag PAI-encoded T4SS to inject CagA into the host cell. Injected CagA has

the potential to localize near the plasma membrane and become phosphorylated by SRC or ABL

family kinases on EPIYA motifs. Phosporylated CagA can bind SHP-2, activating ERK

signaling pathways that result in the production of the “hummingbird phenotype” in epithelial

cells. Unphosphorylated CagA also has the potential to interact with cell signaling proteins such

as c-Met and Grb2, and junctional proteins such as ZO-1 and JAM-A to alter tight junctions.

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CagA has also been shown to exert phosphorylation-independent effects, where the N-terminus

targets the protein to epithelial cell junctions (Bagnoli et al., 2005). Unphosphorylated CagA

forms induce profound effects on the apical junctional complex, interacting with multiple

cellular proteins, including c-Met, E-cadherin, and Grb2. This results in mitogenic and

proinflammatory responses, ablation of cell polarity, disruption of adherens junctions, and

activation of matrix metalloproteases, which induces epithelial cell invasiveness (Amieva et al.,

2003; Oliveira et al., 2006; Wroblewski et al., 2010). Unphosporylated CagA can also alter the

epithelial cell tight junction, interacting with the junctional adhesion molecule A (JAM-A) and

zonula occludens 1 (ZO-1) to alter the development of tight junctions. In addition, our lab has

shown that CagA activates the signal transducer and activator of transcription 3 in a

phosphorylation independent manner (Bronte-Tinkew et al., 2009). Collectively, these studies

show that phosphorylation of CagA is not a requirement for induction of epithelial cell changes.

H. pylori CagA+ strains are associated with higher incidences of gastric cancer (Blaser et al.,

1995; Wu et al., 2003), peptic ulcers (Nomura et al., 2002), metaplasia, and inflammation

(Amieva and El-Omar, 2008; Brandt et al., 2005; Wirth et al., 1998) than CagA- strains. As

described above, CagA has the ability to induce profound morphological changes and increase

cell proliferation (Peek et al., 1997), and the potential to interfere with numerous cell functions

and pathways. The specific molecular mode of action of CagA in the colonization of the human

stomach, however, remains to be determined. Studies have found phosphorylated CagA in

gastric biopsies from infected individuals, indicating that it is indeed injected into the cell and

phosphorylated (Yamazaki et al., 2003). It is not currently known which pathways described

above are actually activated in human H. pylori infection; indeed, although CagA can induce

phosphorylation-independent effects in cultured epithelial cells, the presence of

unphosphorylated CagA has not yet been demonstrated within host cells. Additionally, the host

cells and proteins that are the actual targets of CagA insertion have not been delineated, and the

advantages gained by the bacteria from CagA insertion are ambiguous. The mechanisms by

which CagA induces carcinogenesis have not been fully elucidated; while animal models and

cell culture experiments have uncovered potential oncogenic molecular pathways and

epidemiological studies have linked CagA unequivocally with increased cancer risk, many

questions remain unanswered. Studies often produce conflicting data, owing to different

experimental techniques, strains, and models. While transgenic mice expressing CagA develop

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carcinomas (Ohnishi et al., 2008), this occurred without an inflammatory reaction; in human H.

pylori infection as well as Mongolian gerbil models (Shibata et al., 2006), CagA is associated

with increased inflammation, and this effect is associated with the development of cancer.

Further confounding all of these issues is the fact that the vast majority of patients infected with

CagA+ strains of H. pylori remain cancer-free and asymptomatic. Clearly much remains to be

discovered about CagA, its targets and mechanisms of action, and the factors that contribute to

CagA-associated disease progression in H. pylori -infected patients.

1.1.3.2 VacA

The second major H. pylori virulence factor is the vacuolating cytotoxin A (VacA) – an 88-kDa

secreted toxin that was initially found in vitro to induce the production of large vacuoles in

epithelial cells (Leunk et al., 1988). The presence of VacA has also been associated with an

increased risk for H. pylori-induced carcinogenesis (Robinson et al., 2007) VacA is encoded by

the vacA gene, and genetic diversity in this gene among strains results in variations in phenotype,

cancer risk, and toxic effects (Amieva and El-Omar, 2008; de Sablet et al., 2011).

The secreted VacA protein can be disassociated into p33 N-terminal and p55 C-terminal subunits

upon exposure to either basic or acidic conditions (Cover and Blanke, 2005), but assembly into a

large (6- or 7-subunit) oligomeric complex is necessary for the formation of an anion-selective

membrane channel which is crucial for the biological activity of VacA (Dhar et al., 2003). The

VacA membrane channel increases transcellular permeability and releases anions, urea,

bicarbonate, and other nutrients from the cell cytosol to support bacterial growth (Montecucco

and de Bernard, 2003). The complex is endocytosed into GPI-AP-enriched early endosomal

compartments (GEECs). Here the anion channel conducts chloride ions into the endosome

(Gauthier et al., 2007) resulting in osmotic swelling of the compartment, inducing the large

characteristic vacuoles.

In addition to the induction of vacuoles, VacA can induce multiple effects on different host cell

types. The p33 subunit targets the cellular mitochondria, forms pores in the inner membrane and

changes the inner membrane potential, and induces apoptosis (programmed cell death) (Cover

and Blanke, 2005; Cover et al., 2003; Galmiche et al., 2000). VacA induces the release of

cytochrome c from the mitochondria, resulting in the activation of caspase 3, culminating in the

induction of apoptosis (Galmiche et al., 2000; Willhite and Blanke, 2004; Yamasaki et al., 2006).

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A novel mechanism of VacA-induced apoptosis has been demonstrated, whereby the toxin

fragments the mitochondrial network via recruitment of dynamin-related protein 1 (Jain et al.,

2011). This activates the Bcl-2-associated X protein, resulting in apoptosis. VacA has also been

recently shown to induce autophagy in gastric epithelial cells (Terebiznik et al., 2009).

Autophagy is an important cellular mechanism that plays a role in the maintenance of cellular

homeostasis as well as innate immunity (Raju and Jones, 2010).

VacA has the potential to modulate the host immune system by interacting with several types of

immune cells, including CD4+ T cells. VacA can inhibit T cell proliferation, activation, and

antigen presentation, thus suppressing T-cell-mediated immunity (Basso et al., 2008;

Boncristiano et al., 2003; Gebert et al., 2003). A recent study implicated the protein kinase C

family in uptake of VacA into T cells (Sewald et al., 2011). VacA has also been shown to bind

CD18 (the β2 integrin subunit) on T cells and cause cellular vacuolization using the lymphocyte

function-associated antigen (LFA-1) (Sewald et al., 2008). VacA also affects immune antigen-

presenting cells (APCs), inhibiting Ii-dependent antigen presentation (Boncristiano et al., 2003;

Molinari et al., 1998). Furthermore it has been shown to inhibit B cell activation and

proliferation, and induce B cell apoptosis (Singh et al., 2006; Torres et al., 2007). VacA can also

exert proinflammatory effects, inducing release of TNF-α and IL-6 release from mast cells

(Cover and Blanke, 2005). Clearly VacA is a multifunctional toxin that can exert profound

effects on the host immune response. VacA induces multiple responses from diverse host cell

types in vitro; although the precise actions of VacA in vivo remain to be elucidated.

1.1.3.3 Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) is a structural component of the cell wall found in Gram-negative

bacteria such as H. pylori and E. coli that typically induces a strong inflammatory response. LPS

is recognized by the innate immune receptor TLR4 to induce production of proinflammatory

cytokines (Lu et al., 2008). However H. pylori LPS does not induce inflammatory responses in

epithelial cells, even at high concentrations, and may not be recognized by host TLR4 (Backhed

et al., 2003). Furthermore H. pylori LPS has been shown to possess 103 times less endotoxin

activity as compared to LPS from other Gram-negative bacteria such as E. coli (Cunningham et

al., 1996; Perez-Perez et al., 1995). It has also been shown to bind trefoil factor 1 in the gastric

mucosa to potentially promote colonization (Reeves et al., 2008). Therefore the low

10

immunogenicity of H. pylori LPS represents a potential mechanism by which the pathogen is

able to evade the host immune response.

1.2 The Immune Response

Chronic gastritis is a hallmark of H. pylori infection. Despite the presence of this low-level

inflammatory response, the infection persists for the lifetime of the host, which suggests that H.

pylori is able to evade or subvert the host immune response. The causative role that H. pylori

infection plays in the development of gastric cancer further emphasizes the bacteria’s potential

immune-moderating effects, as a lack of tumor immunosurveillance during infection may be

involved(Strioga et al., 2012). Therefore, the interaction between the bacterium and the host

immune response is an area of intense research interest.

The host immune response is composed of several components, including the innate immune

response and the adaptive immune response. The innate response is a rapid, generalized response

to distinct molecular patterns that are conserved among microorganisms (Murphy et al., 2012).

These patterns are known as Pathogen-Associated Molecular Patterns (PAMPs), and innate

immunity uses Pattern Recognition Receptors (PRRs) to recognize these PAMPs (Murphy et al.,

2012). These PRRs include the Toll-like Receptor (TLR) family, a group of receptors that

recognize components such as bacterial lipopolysaccharide or unmethylated DNA in

extracellular and endosomal compartments (Rad et al., 2009). Nod-like receptors (NLRs) are a

more recently-discovered type of PRR that recognize PAMPs in intracellular compartments such

as the cytosol; these include Nod1 and Nod2 which recognize a component of the bacterial cell

wall, peptidoglycan (Fritz et al., 2007; Girardin et al., 2003). These PAMPs are often highly

conserved among microorganisms and are not found in the normal host; therefore these PRRs

provide the host immune system with a quick and generalized way of discriminating “self” vs.

“non-self” (Murphy et al., 2012).

The adaptive immune response is a precise and targeted response to specific antigens. This

adaptive response includes the recruitment, activation, and proliferation of different classes of

immune cells, and thus is more delayed than the innate immune response (Murphy et al., 2012).

Briefly, antigen-presenting cells (APCs) utilize innate PRRs and other receptors to detect foreign

bodies, invaders, and Danger-Associated Molecular Patterns (DAMPs) such as cytokines and

peptides released from apoptotic, necrotic, or tumor cells (Murphy et al., 2012). These APCs,

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which include immune cells such as macrophages and dendritic cells (DCs), are the sentinels of

the immune system; once they detect something awry in the extracellular environment they

activate the suitable effector cells of the immune system (including T cells and B cells) to deal

with it appropriately (Murphy et al., 2012). Dendritic cells are particularly specialized APCs;

they express the broadest repertoire of PRRs and are capable of inducing powerful effector

immune responses to antigens as well as immunoregulatory and tolerant responses to self-

peptides and commensal organisms (Rad et al., 2009). Thus DCs are key moderators of the

adaptive immune response and represent an ideal target for H. pylori’s immune-modulating

effects. This thesis is focused on the interaction between H. pylori and this decisive immune cell.

1.2.1 The Dendritic Cell

As dendritic cells are the most potent APCs in the host immune system, their primary role is to

initiate adaptive immune responses by detecting, processing, and presenting antigens to T cells in

order to activate these effector cells appropriately (Lenahan and Avigan, 2006; Murphy et al.,

2012). DCs express high levels of the type I transmembrane protein CD11c, making it a suitable

marker for DC detection. Immature dendritic cells are found throughout the body at the sites of

antigen capture such as the gastric lamina propria. DCs use PRRs and processes such as

phagocytosis, macropinocytosis, and membrane ruffling to constantly sample the extracellular

environment, vigilantly searching for antigenic material (Murphy et al., 2012). Upon encounter

with an antigen, DCs internalize the antigen and undergo a maturation process to present

peptides of the antigen to T cells. The DCs enlarge, grow characteristic dendrites, and migrate to

draining lymph nodes, where the population of naïve T cells awaits their antigen presentation.

Within the DC, antigens are internalized into endosomes that fuse with lysosomes to degrade the

contents into antigenic peptides. The peptides are loaded onto Major Histocompatibility

Complex (MHC) molecules and the MHC-peptide complex is transported to the cell surface. The

MHC class I complex can then synapse with a T Cell Receptor (TCR) combined with a CD8

molecule on the surface of a CD8+ Cytotoxic T cell (Tc), while the MHC class II complex

synapses with a TCR combined with a CD4 molecule on the surface of a CD4+ Helper T cell

(Th) (Murphy et al., 2012). This MHC II-TCR interaction is the first signal needed for the DC to

activate the naïve Th cell, and the TCR must recognize the specific antigen loaded onto the MHC

II complex. The second signal is accomplished when the DC costimulatory complex of CD80/86

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and CD40 contacts the CD28 and CD40L receptors on the T cell. Together these interactions

form the immunological synapse (Figure 1.2-1). In order to achieve these appropriate

interactions, part of the DC maturation process involves the upregulation of CD40, CD80, and

CD86 expression on the cell surface. Thus immature DCs express low levels of cell surface

MHC II and CD40/80/86, and increase expression of these molecules upon maturation. When the

DC presents the MHC II-antigen complex as well as the costimulatory complex to the CD4+ T

cell with the appropriate TCR, it activates the T cell and induces its proliferation, producing an

army of effector cells that will specifically target the antigen that was presented by the DC

(Murphy et al., 2012).

In addition to their role in activating immune responses to pathogenic microorganisms, DCs also

play a very important role in tumor surveillance (Steinman and Banchereau, 2007). DCs

recognize tumor associated antigens and mature in the tumor microenvironment (Lin et al.,

2010). DCs have the ability to induce immune responses towards these tumor associated

antigens, activating T cells to target tumor cells (Lin et al., 2010). Therefore dendritic cells play a

key role in immune surveillance, as they alert the immune system to danger signals from foreign

invaders as well as native cells that have been transformed into malignant problems. DCs

activate appropriate immune responses to deal with the threat accordingly.

13

Figure 1.2-1: The immunological synapse

After encounter with an antigen, the DC undertakes a maturation process to present the antigenic

peptides on its cell surface in the context of MHC II to the TCR of a naïve CD4+ T cell (Signal

1). The DC also upregulates surface expression of the CD40/80/86 costimulatory complex,

which binds to the appropriate receptors (CD28 and CD40L) on the T cell (Signal 2). This

induces activation and proliferation of the T cell, commencing the adaptive immune response.

14

1.2.2 T Cell Phenotypes

DCs are not only important activators of the adaptive immune response; they also moderate

immune responses by dictating the phenotype of T cell that develops upon activation. As

described above, DCs can present antigen in the context of the MHC class I molecule to CD8+

cytotoxic T cells, priming them to proliferate and recognize virally infected cells (Murphy et al.,

2012). DCs also present antigen in the context of MHC class II molecules to naïve CD4+ helper

T cells, and it is through these CD4+ T cells that the DC’s true immunomodulating potential is

revealed. Different types of cytokines are released by DCs depending upon the type and context

of the antigen that the DC encounters. This cytokine secretion is the third signal that DCs

provide to the T cell to stimulate proliferation, differentiation, and initiation of adaptive T cell

responses. These cytokines will drive the development of different CD4+ T cell phenotypes, each

of which produces a different adaptive immune response (Figure 1.2-2).

15

Figure 1.2-2: T cell differentiation

The mature dendritic cell will secrete different cytokines to produce various CD4+ T cell

phenotypes. IL-12 drives the production of Th1 cells, which produce IFN-γ and activate

macrophages to eliminate virally infected cells. IL-4 appears to drive Th2 development, which

secrete more IL-4 and activate B cells to produce antibodies targeting extracellular pathogens

such as bacteria. IL-6 with IL-23 and TGF-β drive Th17 development; Th17 cells secrete the

inflammatory cytokine IL-17 and are found at the interfaces between the external and internal

environment such as the GI tract and the skin. The secretion of TGF-β and IL-10 drive Treg

development, while IL-2 is important for proliferation. Tregs secrete IL-10 and TGF-β, which

are immunosuppressive cytokines that inhibit activity of immune cells.

16

DCs can induce different inflammatory immune responses, which are dependent on the type of

cytokines secreted, to deal with pathogenic microorganisms and antigens, or they can induce

tolerogenic responses to prevent immune reactions to commensal bacteria and self-peptides. The

release of the cytokine interleukin-12 (IL-12) from the DC with appropriate antigen and

costimulatory presentation will drive the development of type 1 helper T cells (Th1) and the

cellular immune response (Murphy et al., 2012). Th1 cells secrete the cytokine interferon-gamma

(IFN-γ) and activate macrophages and cytotoxic T cells to kill virally infected cells (Murphy et

al., 2012). The cellular immune response is adept at dealing with intracellular pathogens such as

viruses. The cytokine IL-4 is crucial for the development of the Th2 helper T cell phenotype,

which drives the humoral immune response. Th2 cells activate B cells to secrete antibodies;

these antibodies bind to extracellular pathogens such as bacteria and activate the complement

system to kill the invading microorganism (Murphy et al., 2012).

The release of the cytokines IL-6, IL-23, and transforming growth factor beta (TGF-β) from the

DC stimulates the transcription factor RORγt in the naïve T cells and drives the Th17 helper T

cell phenotype (Dong, 2008; Manel et al., 2008). Th17 cells are proinflammatory and are found

at the interfaces between the external and internal environment, such as the lining of the GI tract

and the skin. They secrete the inflammatory cytokine IL-17, and recruit inflammatory immune

cells such as neutrophils to deal with extracellular pathogens such as bacteria and fungi (Ivanov

et al., 2006). Excessive activity of Th17 cells appears to play a significant role in the

pathogenesis of multiple inflammatory and immune-mediated diseases, including inflammatory

bowel disease (IBD), rheumatoid arthritis, and asthma (Tesmer et al., 2008).

Additionally DCs can drive development of an inducible regulatory T cell (Treg) phenotype to

prevent immune reactivity to auto-antigens and commensal bacteria. Treg development is

regulated by the forkhead transcription factor FoxP3. Tregs express FoxP3 in their nucleus and

CD25 on the cell surface (Chen et al., 2003). The exact cytokine profile driving Treg

development has not been fully elucidated, but the release of TGF-β as well as IL-10 from the

DC appears to be required (Chaudhry et al., 2011; Fu et al., 2004; Lan et al., 2006); while IL-2

(Malek, 2003) and more recently IL-18 (Oertli et al., 2012) have been implicated in Treg

proliferation and skewing. Tregs are immunoregulatory T cells that inhibit the proliferation,

activation, and activity of other immune cells including APCs and effector T cells, effectively

suppressing the immune response by contact-dependent and contact-independent mechanisms

17

(Curotto de Lafaille and Lafaille, 2009). These inducible Tregs produce immunosuppressive

cytokines such as TGF-β and IL-10 to inhibit immune cells (Corthay, 2009; O'Garra et al., 2004).

IL-10 particularly has potent immunoregulatory effects, blocking the maturation of DCs and

inhibiting the antigen-presenting functions of other APCs (Corinti et al., 2001; Enk et al., 1993).

Thus inducible Tregs can effectively suppress the immune response by inhibiting other immune

cells. Tregs play important roles in preventing autoimmune disease and shutting down the

immune response after the offending organism has been successfully eliminated (Mills and

McGuirk, 2004).

This ability to drive the differentiation of multiple distinct T cell phenotypes, each of which

produces a vastly different immune response, makes dendritic cells key orchestrators of the

immune system. DCs have the ability to profoundly modulate the immune response, initiating

both powerful effector responses towards threats (such as pathogens and tumor cells) and

tolerant regulatory responses towards self-peptides and commensal organisms.

1.2.3 Dendritic Cell Responses to H. pylori

H. pylori has the ability to evade or subvert the host immune response, inducing a chronic

gastritis that is insufficient to eliminate the pathogen and potentially inhibiting immune tumor

surveillance to play a causative role in the development of gastric cancer. As dendritic cells are

key modulators of the immune response, they are ideal targets for H. pylori’s immune-

manipulating effects. Dendritic cells are present in the gastric mucosa of H. pylori-infected

individuals, residing in the lamina propria just beneath the epithelial cell layer (Kao et al., 2006).

DCs extend long dendritic projects through epithelial-apical junctions in order to directly take up

bacteria (Necchi et al., 2009) (Figure 1.2-3). Furthermore H. pylori can be found in the lamina

propria as well as inside vacuoles in gastric epithelial cells (Dubois and Boren, 2007; Necchi et

al., 2007; Terebiznik et al., 2006) thus allowing exposure to DCs. Several studies have begun to

assess the effects of H. pylori on dendritic cell maturation, activation, and induction of T cell

responses. However these limited studies to date have produced conflicting results.

18

Figure 1.2-3: Potential dendritic cell - H. pylori interactions

H. pylori has the potential to interact with the host DC in multiple ways, and DCs have been

shown to drive differing T cell responses upon H. pylori infection or stimulation.

19

A study by Rad et al. found that H. pylori lysates induced maturation of murine bone marrow-

derived dendritic cells (BMDCs) as determined by MHC class II, CD80, and CD86 upregulation

(Rad et al., 2007). The BMDCs secreted IL-12 and IL-10 in response to the H. pylori lysates. A

later study by Rad et al. also implicated specific PRRs, including TLR2, TLR4, and TLR9, in the

DC recognition of H. pylori (Rad et al., 2009).

Kranzer et al. found that H. pylori induced maturation of human DCs as measured by

upregulation of CD80, CD83, CD86, and MHC II surface expression (Kranzer et al., 2004). H.

pylori also induced production of IL-6, IL-8, IL-10, and IL-12 in the DCs (Kranzer et al., 2004).

Importantly, in this study these effects were similar to the effects of stimulating the DCs with

lipopolysaccharide (LPS) from Escherichia coli (E. coli) bacteria, a potent activator of DC

maturation (Kranzer et al., 2004). The H. pylori-stimulated DCs produced less IL-12 than the

LPS-stimulated DCs, although the functional effect on T cell phenotype was not examined. In

further studies, this group showed that the virulence factors cag PAI and VacA were not required

for H. pylori-induced DC maturation, activation, and DC-mediated T cell activation (Kranzer et

al., 2005).

Guiney et al. utilized DCs derived from human peripheral blood monocytes and found that H.

pylori induced secretion of IL-12 with no production of IL-10 (Guiney et al., 2003). The authors

suggest that this would polarize a Th1 phenotype, although no functional assays were conducted.

Interestingly, in this study the H. pylori-stimulated DCs produced less IL-12 than DCs stimulated

with another bacteria, Salmonella enterica (Guiney et al., 2003).

A comprehensive study by Bimczok et al. found increased numbers of DCs in H. pylori-infected

patients as compared to uninfected controls (Bimczok et al., 2010). The DCs from H. pylori-

infected patients also had increased surface expression of the MHC II component HLA-DR, as

well as the maturation markers CD83 and CD86 (Bimczok et al., 2010). This study also

examined the effect of H. pylori infection on gastric DCs isolated from uninfected control

subjects, as well as peripheral monocyte-derived DCs. In this study H. pylori induced maturation

of the gastric DCs and the monocyte-derived DCs, as assessed by upregulation of CD80, CD83,

CD86, and CD11c cell surface expression (Bimczok et al., 2010). Interestingly, H. pylori

induced the gastric DCs to secrete IL-6, IL-8, and IL-10, but did not induce secretion of IL-12

(Bimczok et al., 2010). Despite the lack of secretion of this important Th1-stimulating cytokine,

20

the H. pylori-pulsed gastric DCs induced T cells to secrete IFN-γ, which is consistent with Th1

responses. Therefore the authors concluded that H. pylori activates human gastric DCs to induce

a predominant Th1 response (Bimczok et al., 2010).

Another study utilizing human monocyte-derived dendritic cells found that H. pylori upregulated

expression of the maturation markers CD40, CD80, CD86, and the MHC II in DCs (Chang et al.,

2012). This study demonstrated that H. pylori induced secretion of IL-10 and TNF-α from these

DCs, and specific PRRs such as TLR2 and TLR4 were implicated in this secretion (Chang et al.,

2012). Interestingly, the authors found that monocyte-derived DCs that were produced from

gastric cancer patients did not secrete IL-10 in response to H. pylori stimulation, and speculated

that this defective IL-10 response contributes to the gastric cancer seen in H. pylori patients

(Chang et al., 2012).

Kao et al. found that H. pylori induced secretion of IL-12 and IL-10 from murine BMDCs (Kao

et al., 2006). However they also demonstrated much less secretion of IL-12 and IL-10 in the H.

pylori-infected BMDCs compared to BMDCs stimulated with another gastritis-causing pathogen,

Acinetobacter lwoffi (A. lwoffi) (Kao et al., 2006). The authors found H. pylori-stimulated

BMDCs induced the release of IFN-γ and TNF-α from T cells, but to a lesser extent than A.

lwoffi-stimulated BMDCs. Furthermore they found that H. pylori could effectively inhibit the

release of IL-12 but not IL-10 from the BMDCs stimulated with A. lwoffi (Kao et al., 2006).

These results led the authors to suggest that H. pylori can have inhibitory effects on the dendritic

cell, causing defects in the host immune response (Kao et al., 2006). A more recent study by the

same group found that H. pylori induced BMDCs to polarize a Treg polarized response,

maintaining high levels of Tregs and decreasing Th17 development in a TGF-β and IL-10

dependent manner (Kao et al., 2010). In this study the authors suggest that this DC-induced Treg

polarization and Th17 suppression facilitates H. pylori persistence and immune escape (Kao et

al., 2010). In contrast, a study by Khamri et al. found that H. pylori stimulated human monocyte-

derived DCs to secrete IL-23 and IFN-γ, which induced the production of IL-17-secreting Th17

cells (Khamri et al., 2010). The authors found that the cag PAI and an intact T4SS were required

for this DC-mediated IL-17 production (Khamri et al., 2010).

Finally, a recent study showed that H. pylori induces the development of tolerogenic DCs which

stimulate naïve T cells to express Foxp3; these DCs were capable of preventing airway

21

hyperresponsiveness in a murine experimental model of asthma (Oertli et al., 2012). In this study

H. pylori did not induce maturation of BMDCs as assessed by CD40, CD80, and CD86 cell

surface expression but induced secretion of IL-10 from these immature DCs. Indeed the authors

showed that H. pylori inhibited LPS-induced maturation of DCs in a contact dependent manner.

H. pylori also inhibited LPS-induced secretion of IL-6 and IL-12 (Oertli et al., 2012). The

authors demonstrated that H. pylori induces the BMDCs to drive Foxp3+ Treg development and

that the release of TGF-β and IL-18 from the DC was necessary to drive this Treg conversion

(Oertli et al., 2012). Supporting these results are studies that show H. pylori-specific Tregs can

suppress memory T-cell responses to H. pylori in infected individuals (Lundgren et al., 2003).

This stimulation of immunosuppressive Tregs by H. pylori also decreases gastric tumor

immunosurveillance and immune response to tumor antigens (Groux, 2003). Tregs have been

shown to contribute to myeloma-related immune dysfunction, and the accumulation of Tregs in

various human carcinomas is associated with a poor prognosis (Braga et al., 2012; Ladoire et al.,

2011). Furthermore the balance between immunosuppressive Tregs and highly inflammatory

Th17 cells is perturbed in many human cancers, indicating that these classes play a key role in

the potential development or suppression of cancer (Braga et al., 2012).

Thus the studies to date assessing H. pylori and dendritic cells contain some conflicting results.

Methodological differences can explain some of these paradoxical results. Different strains of H.

pylori are used in these studies; these strains often differ in terms of pathogenicity owing to

differences in expression of virulence factors such as VacA, CagA, and the cag PAI itself.

Furthermore the “dendritic cells” used in the studies vary quite widely, as some studies use

murine bone marrow-derived dendritic cells, others use human peripheral monocyte-derived

DCs, which still others take pre-differentiated DCs from the gastric mucosa. Among the cultured

DCs, even slight differences in culture conditions can affect the phenotype of the DC before it

has even been exposed to the H. pylori. Finally, the use of different controls, and the comparison

of H. pylori’s effects to those of different bacteria can explain differences in the presentation of

some results. However despite these concessions, it is clear that the effects of H. pylori infection

on DCs remain unclear, and the mechanisms by which the bacteria evades or moderates the host

immune response are still being elucidated.

22

1.2.4 H. pylori-altered DCs: potential role in disease

Additional evidence for the ability of H. pylori to affect the host immune system can be found in

recent epidemiological studies which link H. pylori infection with protection against the

development of several autoimmune and immune-mediated diseases. As H. pylori has infected

and coevolved with human hosts for at least 50,000 years; the presence of this bacterium has

likely influenced the evolution and development of modern human physiology and immunology

(Atherton and Blaser, 2009). Yet H. pylori colonization is presently detected in fewer than 6% of

children in developed countries; this eradication of an immunological influence has been

epidemiologically correlated with an increase in the occurrence of several disease pathologies. H.

pylori eradication has been associated with an increase in gastroesophageal reflux disease

(GERD), Barrett’s oesophagus, and the development of esophageal adenocarcinoma (Atherton

and Blaser, 2009). Additionally H. pylori infection has been negatively associated with

esophageal eosinophilia, an allergic inflammatory condition (Dellon et al., 2011).

An inverse association between H. pylori colonization and asthma has also been described (Chen

and Blaser, 2007). Recently, a study attempting to elucidate the mechanisms behind this

correlation used mouse models of allergen-induced asthma (Arnold et al., 2011a). In this study,

mice were infected with H. pylori as newborns or as adults; neonatal mice infected with H. pylori

displayed a reduction in multiple asthma indicators. Significantly, this protection was associated

with both impaired maturation of DCs that infiltrated the lungs and accumulation of Tregs. This

protection was abrogated by antibiotic elimination of H. pylori and abolished by systemic Treg

depletion. Furthermore, adoptive transfer of purified Tregs was sufficient to confer protection to

uninfected recipient mice (Arnold et al., 2011a). More recently the same group found that H.

pylori causes DCs to drive Treg differentiation and induce immune tolerance (Oertli et al., 2012).

Interestingly the authors showed that H. pylori exposure was able to prevent DCs from inducing

asthma in a DC-mediated experimental model of asthma (Oertli et al., 2012). Furthermore Tregs

from H. pylori-infected mice were able to prevent asthma in this model.

H. pylori infection has also been found to be inversely correlated with the incidence of

Inflammatory Bowel Disease (IBD) (Luther et al., 2010). A recent study demonstrated that H.

pylori DNA had an increase in immunoregulatory DNA sequences and that incubation of DCs

with this DNA reduced proinflammatory cytokine expression as compared to E. coli DNA

23

(Luther et al., 2011). Furthermore, the H. pylori DNA was able to suppress the inflammatory

response caused by the E. coli DNA. In addition, administration of H. pylori DNA reduced

dextran sodium sulphate-induced colitis in a murine model while E. coli DNA did not. This

presents an intriguing mechanism for the negative correlation between H. pylori infection and

IBD, and demonstrates a new pathway by which H. pylori DNA can alter host immunity.

Collectively these studies further demonstrate that H. pylori can modulate the host immune

system. In some cases this negatively impacts the host, allowing for persistent infection and

decreasing immune tumor surveillance to cause cancer. However it also appears to play a

beneficial role in the prevention of other immune-mediated disorders and pathologies.

Importantly DCs may play a potential role in mediating these effects.

1.2.5 Signal Transducer and Activator of Transcription 3 (STAT3)

The mechanisms by which H. pylori evades and modulates the host immune response remain

unclear. One potential mechanism by which H. pylori may modulate the host immune response is

through activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway.

STAT3 is a member of the STAT family, signal transducer proteins that are activated by the

tyrosine phosphorylation cascade in response to ligand binding. STAT3 is a cytoplasmic protein

that acts as a signal messenger and transcription factor to induce cellular responses to cytokines

and growth factors. Importantly hyperactivation of STAT3 is found in many human cancers and

is correlated with both tumor progression and poor prognosis (Lin et al., 2010). Recent work

form our laboratory indicates that during H. pylori infection in vivo, STAT3 is activated in

gastric epithelial cells in a CagA-dependent manner. (Bronte-Tinkew et al., 2009). STAT3

activation was also found in a subset of infiltrating immune cells (Bronte-Tinkew et al., 2009).

Therefore H. pylori possesses the ability to manipulate and activate this crucial signaling cascade

whose abberrant activation is found in many human cancers.

1.2.5.1 STAT3 Signaling

STAT3 is activated via a tyrosine phosphorylation cascade in response to exctracellular ligand

binding. Ligands that activate the STAT3 pathway include cytokines such as IL-10 and IL-6 and

growth factors such as Vascular Endothelial Growth Factor (VEGF) (Lin et al., 2010). The

ligand (e.g. IL-6) binds to its receptor (IL-6R) on the cell surface (Figure 1.2-4). The IL-6R then

24

dimerizes with a gp130 subunit, resulting in the phosphorylation and activation of associated

Janus Kinase 2 (JAK2) enzymes (Kim et al., 2007). JAK2 is a tyrosine kinase which

phosphorylates tyrosine residues on the cytoplasmic tail of the gp130 subunit (Greenlund et al.,

1995). The phosphorylated tyrosines recruit cytoplasmic STAT3 monomers to the gp130

subunit. The STAT3 monomers are subsequently phosphorylated on their Y705 residues, and

dimerize through Src-Homology 2 domain (SH2) phosphotyrosine interactions. The

phosphorylated STAT3 dimer (PSTAT3) is then translocated into the nucleus via nuclear

localization signals (Reich and Liu, 2006). Inside the nucleus DNA binding domains permit

binding to promoter regions of target genes which are involved in cell maturation, growth,

proliferation, angiogenesis and immune function (Kim et al., 2007; Kisseleva et al., 2002; Levy

and Darnell, 2002). Importantly, STAT3 is implicated in tumorigenesis and cancer progression.

Abberant, constitutive STAT3 activation has been found in a majority of both hematopoeitic and

epithelial cancers as well as tumour cells (Ernst and Putoczki, 2011; Wang et al., 2004).

Constitutive STAT3 activation in diverse human cancers enhances tumor proliferation,

angiogenesis, metastasis, and survival, and inhibits tumor apoptosis as well as antitumor

immunity (Wang et al., 2004; Yu et al., 2009).

25

Figure 1.2-4: STAT3 signaling pathway

The extracellular signaling ligand (e.g. IL-6) binds to the receptor (IL-6αR) on the cell surface.

The receptor dimerizes with the gp130 subunit, activating Janus kinases (JAK2) associated with

the cytoplasmic tail. The JAK2 kinases initiate tyrosine phosphorylation of the gp130

cytoplasmic tail, recruiting STAT3 monomers to the phosphorylated tail. The JAK2 kinases then

phosphorylate the STAT3 monomers on the Y705 residue, allowing them to dimerize via Src-

Homology 2 (SH2) domains. The importin αβ complex then translocates the phosphorylated

STAT3 dimer (PSTAT3) into the nucleus, where it binds to and induces transcription of STAT3

response genes, which are involved in angiogenesis, cell proliferation, and immune moderation.

Modified from Ang (2010).

26

1.2.5.2 STAT3 Activation in Dendritic Cells

STAT3 activation in dendritic cells is a potential mechanism by which the DC response is

disrupted by H. pylori, leading to immune disruption and potentially carcinogenesis. Current

evidence suggests that DC maturation is inhibited by STAT3 activation, while inhibition of

STAT3 in dendritic cells promotes DC maturation (Lin et al., 2010; Melillo et al., 2010). STAT3

activation is often associated with tolerogenic functions (Barton, 2006; Cheng et al., 2003;

Nefedova et al., 2005). Indeed many tumors secrete high levels of the growth factor VEGF; this

VEGF activates STAT3 in DCs, preventing their maturation and thus decreasing the host

immune tumor surveillance (Lin et al., 2010). Iwata-Kajihara et al. demonstrated that STAT3-

depleted DCs were more resistant to cancer-cell derived inhibitory factors (Iwata-Kajihara et al.,

2011). These STAT3-deficient BMDCs were more potent activators of T cells and had a high

capacity to induce Th1 responses (Iwata-Kajihara et al., 2011). STAT3 signaling is also

necessary for Th17 development (Harris et al., 2007). This highly inflammatory STAT3-

dependent Th17 response has been shown to be pro-carcinogenic (Wang et al., 2009). Therefore

it appears STAT3 activation in dendritic cells supports a pro-tumorigenic environment.

Furthermore STAT3 activation in DCs prevents their maturation and activation, which decreases

immune surveillance and represents a potential mechanism by which the host immune response

can be manipulated.

1.3 Summary

H. pylori is a gastric pathogen that has the ability to alter the host immune response. Infection

with H. pylori persists for the lifetime of the host despite the induction of chronic gastritis, a low-

level inflammatory immune response. The bacterium is a type I carcinogen and plays a causative

role in gastric cancer, potentially by inhibiting host tumor immunosurveillance. Epidemiologic

evidence indicates that H. pylori infection is negatively correlated with several autoimmune and

immune-mediated diseases, supporting a role for the bacteria in altering immune responses.

Dendritic cells are ideal targets for the pathogen’s immune-altering efforts as they play a key role

in modulating the host’s immune response. DCs can drive powerful effector responses to

pathogenic organisms and necrotic/tumor cells as well as tolerant regulatory responses to

commensal organsisms and self-antigens. Therefore the hypothesis that H. pylori may

manipulate this key immune orchestrator in order to evade the immune response and persist in

27

the host is both intriguing and plausible. Mechanistically the activation of STAT3 in dendritic

cells has been shown to prevent DC maturation and inhibit DC-driven effector T cell responses

(Iwata-Kajihara et al., 2011; Melillo et al., 2010). STAT3 activation may decrease tumor

immunosurveillance by DCs to contribute to cancer development (Lin et al., 2010).

H. pylori activates STAT3 in both epithelial cells and infiltrating immune cells (Bronte-Tinkew

et al., 2009). Thus, H. pylori could activate STAT3 in DCs to modify DC maturation/activation

and thereby alter immune responses to promote persistence and inhibit DC tumor

immunosurveillance.

Hypothesis

H. pylori activates signal transducer and activator of transcription 3 (STAT3) in dendritic cells to

promote chronic infection and disease.

Specific Aims

This hypothesis will be addressed using several specific aims.

Aim 1: Determine whether H. pylori activates STAT3 in bone marrow derived dendritic cells

and characterize the phenotype of DCs.

Aim 2: Determine the effect of STAT3 activation on DCs during H. pylori infection.

Aim 3: Determine the mechanism of STAT3 activation in DCs during H. pylori infection.

Aim 4: Determine how H. pylori infected dendritic cells modulate T cell development.

28

Chapter 2 Materials and Methods

2

2.1 Bone Marrow-Derived Dendritic Cells

Bone Marrow-Derived Dendritic Cells (BMDCs) were obtained and cultured from C57BL6

wildtype (WT) mice, 6-8 weeks old. BMDCs were also obtained from STAT3 CD11c-Cre

knockout (KO) mice or littermate controls (Melillo et al., 2010). Bone marrow was extracted

from the femur and tibia and seeded at 2 x 105cells/ml with DC media (Lutz and Rossner, 2007).

DC media contains: Roswell Park Memorial Institute (RPMI)-1640 media, 10% fetal bovine

serum, 1% L-glutamine, 1% penicillin/streptomycin, 1X sodium pyruvate, 1X non-essential

amino acids, and 0.1% 2-mercaptoethanol. Recombinant murine granulocyte macrophage

colony-stimulating factor (GM-CSF) was added at a concentration of 40ng/ml, to stimulate

dendritic cell growth (Lutz and Rossner, 2007). DCs were seeded on bacteriological petri dishes

with 10ml DC media supplemented with GM-CSF for 3 days at 37°C in 5% CO2. On day 3, an

additional 10ml of DC media supplemented with GM-CSF was added to each plate. On day 6

and day 8 10ml of the solution from each plate was removed, centrifuged to pellet floating cells,

and resuspended with 10ml of fresh DC media supplemented with GM-CSF per plate. On day

10, the floating, non-adherent cell population (immature DCs) was harvested, counted, and

plated in 6-well tissue culture plates in antibiotic-free DC media to be used for

treatment/infection.

2.2 Treatment/Infection

H. pylori strain 7.13 was grown on Columbia blood agar plates (supplemented with 5% sheep

blood) under microaerophilic conditions (5% O2, 10% CO2, 85% N2) for 72h (Zheng and Jones,

2003). Bacteria were then collected using sterile inoculation loops and transferred into a bacterial

flask containing Brucella broth supplemented with 10% fetal bovine serum for 24 hours. Bacteria

were then harvested by centrifugation, resuspended in antibiotic-free DC media, and added to

immature BMDCs at a multiplicity of infection (MOI) of 10:1. Treated cells were incubated at

37°C for 20 hours. The optimal infection time was previously determined using a series of time-

course experiments (Ang, 2010). A set of untreated DCs served as control cells. A set of DCs

29

was treated with IL-6 [100ng/ml] to serve as a positive control for STAT3 activation, and a

negative control for DC maturation (Niemand et al., 2003). The optimal dosage of IL-6 was

determined by a dose-response experiment performed previously (Ang, 2010). An additional set

of DCs was treated with E. coli lipopolysaccharide (LPS; Sigma Aldrich) [1ng/ml] to serve as a

positive control for DC maturation (Foti et al., 1999). Treatment of DCs with a neutralizing rat-

anti-mouse IL-10 antibody (eBioscience, San Diego, CA, USA) [0.5µg/ml or 1µg/ml] was

conducted to examine the effects of IL-10 on the DC maturation and STAT3 activation. Rat-anti-

IgG antibody (BioLegend, San Diego, CA, USA) [1µg/ml] served as a control antibody to ensure

the specificity of the IL-10 antibody-mediated effects. After 20 hours of incubation the cells

were harvested for analysis. Western blotting was used to examine STAT3 phosphorylation and

activation. Flow cytometry using αCD11c, αCD86, and αMHC class II fluorophores was used to

examine the maturation status of the dendritic cells. ELISA assays and multiplex bead assays

will be used to examine the cytokine release profile of the dendritic cells.

2.3 Western Blotting

After 20h of treatment/infection, cells were harvested by vigorous pipetting and washed with

phosphate-buffered saline (PBS; Sigma Aldrich). Cells were lysed using 100µl of a

radioimmunoprecipitation assay (RIPA) buffer cocktail supplemented with protease inhibitors on

ice. Lysates were centrifuged, supernatants collected and stored at -20°C (Bronte-Tinkew et al.,

2009). Lysates (supplemented with 10% laemelli buffer) were loaded onto 12% SDS-

polyacrylamide gels and run at 110V at room temperature for 1.5h. Separated proteins were

transferred onto nitrocellulose membrane (BioTrance, NT; Pall Corp., All Arbor, MI, USA) at

30V for 8h at 4°C (Bronte-Tinkew et al., 2009). Membranes were then blocked with 5% skim

milk made using Tris-buffered saline supplemented with tween (TBST) for 30min, followed by

overnight incubation at 4°C using rabbit-anti-PSTAT3 antibody (1:200; Cell Signalling MA,

USA) and mouse-anti-actin antibody (1:10,000) in TBST milk. Blots were then incubated using

corresponding horseradish peroxidase-conjugated secondary antibodies (1:2,000 and 1:10,000

for the PSTAT3 and actin primary antibodies, respectively) for 1h at room temperature (Bronte-

Tinkew et al., 2009). Bands were then visualized by chemiluminescence using Kodak Biomax

MR film or FluorChem E Imager (Proteinsimple, Santa Clara, CA, USA).

30

2.4 Densitometric analysis

Densitometry was performed utilizing the FlourChem FCII software. Densities of PSTAT3 and

actin bands were measured for each treatment and expressed as a ratio of PSTAT3/actin, and

then normalized to the control treatment.

2.5 Flow Cytometry

After 20h of treatment/infection, BMDCs were harvested by vigorous pipetting and centrifuged

at 350g for 10min. Supernatants were collected and stored at -20°C to examine cytokine release

profile. Cells were rinsed using flow cytometry buffer (FB) (PBS supplemented with 1% bovine

serum albumin (BSA)), and then incubated with anti-CD16/32 (FC receptor block; eBioscience,

San Diego CA USA) [25µg/µl] for 15min on ice, to block non-specific antibody binding (Oliver

et al., 1999). DCs were then incubated with flourescein isothiocyanate (FITC)-labelled αCD11c,

phycoerythrin (PE)-labelled αCD86, and allophcocyanin (APCa)-labelled αMHC class II

antibodies (all from eBioscience) for 30min. DCs were then washed twice with FB before

fixation using 1x paraformadehyde (PFA, made with PBS) for 20min. Cells were subsequently

washed with FB and analyzed using a LSR-II flow cytometer (Becton Dickson, San Jose, CA,

USA). Flow cytometry data was then analyzed utilizing FlowJo software (Tree Star Inc,

Ashland, OR, USA).

2.6 ELISA Cytokine Analysis

BMDCs were infected for 20h as described above. Supernatants were collected and stored at -

80°C. Supernatants were assayed for IL-6, IL-10, IL-12p40 and TNF-α using

specific ELISAs as per the manufacturer’s instructions (R&D systems, MN, USA).

2.7 Multiplex Bead-based Luminex Assay

Supernatants from BMDCs were assayed for IL-2, IL-4, IL-5, IL-10, IL-12, IL-17, and IFN-γ

using the Th1/Th2 Cytokine Mouse 6-Plex Panel kit plus the IL-17 Mouse Singleplex Bead

Luminex Assay Kit as per the manufacturer’s instructions (Invitrogen).

31

2.8 OT-II Transgenic Mouse CD4+ Splenocytes

OT-II transgenic mice were kindly donated by the laboratory of Dr. Tania Watts or purchased

from Jackson Laboratory. These transgenic mice express the mouse alpha-chain and beta-chain T

cell receptor that pairs with the CD4 coreceptor and is specific for chicken ovalbumin 323-339 in

the context of the MHC class II molecule (Barnden et al., 1998). Consequently all CD4+

splenocytes (i.e. T cells) harvested from these animals will be activated when presented with the

ovalbumin peptide by APCs (such as DCs) with appropriate co-receptors and cytokine

stimulation (Barnden et al., 1998). This circumvents the need for antigen specificity when

working with a model of T cell activation by APCs.

2.9 STAT3 KO Mice

STAT3 KO mice were kindly donated by the laboratory of Dr. Tak Mak or Dr. Christian

Schindler. These DC-specific STAT3 KO mice were generated using a Cre-flox system. Briefly,

Stat3flox/flox

mice, in a 129/C57BL/6J mixed background were crossed seven generations onto

C57BL/6J background, and then with CD11c-BAC-Cre transgenic mice (Melillo et al., 2010).

STAT3 expression was examined via immunoblotting to confirm knockout.

2.10 DC:T Cell Coincubation

Sets of DCs were loaded with 10ng/ml or 100ng/ml ovalbumin peptide 323-339 (OVA) with or

without H. pylori for 20h. DCs were then counted and incubated with splenocytes from OT-II

transgenic mice (at a ratio of 1:2) which were harvested and magnetically sorted for the CD25-

CD4+ population according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch

Gladbach, Germany), in DC media with or without recombinant murine IL-2 [10ng/ml] and

recombinant human TGF-β1 [10ng/ml] (Oertli et al., 2012). The coculture was incubated for 72h

at 37°C in 5% CO2. Cells were then harvested, supernatants collected to examine cytokine

release using ELISA-based assays, and flow cytometry performed to examine T cell phenotype

using αCD4 and αFoxP3 fluorophores (Oertli et al., 2012).

32

2.11 Statistical Analysis

All experiments were performed at least 3 times, and statistical significance (P values) was

calculated using a one-way ANOVA to compare the means ± SE for independent treatment

groups. A P value of less than 0.05 was deemed statistically significant.

33

Chapter 3 Results

3

3.1 H. pylori infection induces STAT3 phosphorylation in dendritic cells

To determine whether H. pylori induces activation of the STAT3 pathway in DCs, expression of

phosphorylated STAT3 (PSTAT3) was measured in dendritic cell lysates using western blotting.

Cultured BMDCs were incubated with H. pylori strain 7.13 at an MOI of 10:1, treated with IL-6

(a known activator of STAT3), or treated with E. coli LPS (a known inducer of DC maturation)

for 20h. Immunoblot analysis of these cell lysates revealed phosphorylation of STAT3 in DCs

treated with H. pylori and IL-6 compared to uninfected control and LPS-treated cells (Figure

3.1-1A, B).

34

CIL

-6LPS

HP 7

.13

0

2

4

6

8

****

B

PS

TA

T3:A

cti

n

no

rmali

zed

to

C

ns

Figure 3.1-1: H. pylori activates the STAT3 pathway in dendritic cells

A) Lysates from DCs incubated with H. pylori strain 7.13 at an MOI of 10:1, IL-6 [100ng/ml], or

E. coli LPS [1ng/ml] for 20h were subjected to immunoblot analysis to examine changes in

STAT3 phosphorylation. B) Graph represents densitometric analysis of PSTAT3 bands

normalized to actin bands, normalized to control treated cells. Columns, mean; bars, SEM; *,

P<0.05; ***, P<0.001 using a one-way ANOVA (n=6).

35

3.2 H. pylori upregulates expression of DC maturation markers

The effect of H. pylori infection on dendritic cell maturation was next assessed. BMDCs were

untreated (C) or treated with IL-6 [100ng/ml], E. coli LPS [1ng/ml], or Helicobacter pylori strain

7.13 at a MOI of 10:1 and incubated for 20h. Flow cytometry was performed using a BD LSR-II

flow cytometer to quantify and analyze the expression of the DC maturation markers CD86 and

MHC class II. Expression of the DC-specific marker CD11c was used to select a pure dendritic

cell population (Figure 3.2-1A). H. pylori infection increased the proportion of MHC IIhi

CD86hi

BMDCs to a similar extent as treatment with E. coli LPS, whereas treatment with the STAT3

activator IL-6 did not increase the expression of these maturation markers compared to untreated

(control) cells (Figure 3.2-1B, C).

36

A

CIL

-6LPS

HP

0

10

20

30

40***

% M

HC

IIh

i CD

86

hi

DC

s

Figure 3.2-1: H. pylori induces expression of dendritic cell maturation markers

A) Live dendritic cells were selected based on their size (FSC), complexity (SSC), and

expression of the DC-specific marker CD11c. B) DCs were incubated with H. pylori strain 7.13

at an MOI of 10:1, IL-6 [100ng/ml], or E. coli LPS [1ng/ml] for 20h. Flow cytometry analysis

C

37

was used to quantify the population expressing high levels of the maturation markers CD86 and

MHC class II. Cells in the gated population in the upper right quadrant of each graph represent

the DCs with high expression of both markers (i.e. mature DCs). C) Quantification of gated

CD86hi

MHC IIhi

DCs. Columns, mean; bars, SEM; ***, P<0.001 using a one-way ANOVA

(n=6).

38

3.3 Activated dendritic cells secrete a specific cytokine profile in response to H. pylori infection

As the cytokines released from the DC dictate the CD4+ helper T cell phenotype that develops,

the cytokine release profile from H. pylori-infected DCs was examined. The culture supernatants

from control, H. pylori, LPS, and IL-6 treated BMDCs were subjected to Luminex multiplex

assays which profiled levels of the following cytokines: IL-2, IL-4, IL-5, IL-10, IL-12, IL-17,

and IFN-γ. Treatment with H. pylori increased the levels of IL-10 and IL-12 in the BMDC

culture supernatants (Figure 3.3-1A). H. pylori infection also increased the ratio of IL-10:IL-12

released by the BMDC. In contrast treatment with proinflammatory LPS increased the IL12:IL-

10 ratio (Figure 3.3-1B). These results were confirmed with ELISA-based assays which revealed

significant increases in IL-10 and TNF-α release from BMDCs in response to H. pylori infection,

as well as non-significant IL-12p40 increases (Figure 3-3C).

39

40

Figure 3.3-1: Dendritic cells secrete a specific cytokine profile upon infection with H. pylori

A) Luminex milliplex assay was used to assess cytokine secretion for Th1/Th2 cytokines (IL-2,

IL-4, IL-5, IL-10, IL-12, IL-17 and IFN-γ in cell supernatants obtained from BMDCs incubated

with either E. coli LPS (1μg/ml), IL-6 (100ng/ml), or WT H. pylori strain 7.13 (MOI 25:1) for

24h. Columns, means; bars, SE; *, P < 0.05; **, P < 0.01 using one-way ANOVA (n=3). B)

Graphs depict ratios of mean IL-10:IL-12 and IL-12:IL-10. C) ELISA was used to detect

cytokine secretion for IL-10, IL-12, IL-6 and TNF-α in cell supernatants obtained from BMDCs

incubated with either E. coli LPS (1μg/ml), IL-6 (100ng/ml), or WT H. pylori strain 7.13 (MOI

25:1) for 24h. Increases in IL-12p40, IL-10, TNF-α, and IL-6 were detected with ELISA assays.

Increases in IL-10 and TNF-α secretion were significant over control. Columns, means; bars, SE;

*, P < 0.05; **, P < 0.01 using one-way ANOVA (n=3). Study conducted by M. Ang.

41

3.4 H. pylori induces more pronounced maturation in STAT3 KO dendritic cells

I next elucidated the effect of H. pylori-induced STAT3 activation in dendritic cells by utilizing

BMDCs derived from dendritic cell-specific STAT3 KO mice (kindly provided by the laboratory

of Dr. Tak Mak). A CD11c-Cre STAT3-flox system was used to generate Cre+ flox

+ BMDCs

that lack STAT3 expression. Cre- sex-matched littermates provided control STAT3

+ BMDCs.

Cre+ (STAT3 KO) and Cre

- (WT) BMDCs were treated with IL-6 [100ng/ml], E. coli LPS

[1ng/ml], no treatment (C), or Helicobacter pylori strains 7.13 and 60190 at a MOI of 10:1 and

incubated for 20h. Sets of STAT3 KO and WT DCs were then lysed and immunoblotting

performed to determine STAT3 expression and activation. STAT3 KO (Cre+) DCs lacked

STAT3 expression (Figure 3.4-1A). In parallel, DCs from each condition were stained with

αCD11c, αCD86, and αMHC class II fluorophores and flow cytometry analysis was performed

to quantify the expression. H. pylori infection induced significantly more maturation as assessed

by expression of maturation markers in the STAT3 KO dendritic cells as compared to the WT

DCs (Figure 3.4-1B, C).

42

B

A

43

Contr

olIL

-6LPS

H. p

ylori

Contr

olIL

-6LPS

H. p

ylori

0

20

40

60

80 ** ***

WT

KO

*C

Treatment

% M

HC

II+

CD

86+

D

Cs

Figure 3.4-1: STAT3 KO DCs express enhanced levels of maturation markers in response

to H. pylori infection

A) Lysates from STAT3 KO (Cre+) and WT (littermate Cre

- control) DCs incubated with H.

pylori strain 7.13 at an MOI of 10:1, IL-6 [100ng/ml], or E. coli LPS [1ng/ml] for 20h were

subjected to immunoblotting analysis to determine changes in STAT3 phosphorylation. B)

STAT3 KO and WT DCs were incubated with H. pylori strain 7.13 at an MOI of 10:1, IL-6

[100ng/ml], or E. coli LPS [1ng/ml] for 20h. Flow cytometry analysis was used to quantify the

population expressing high levels of the maturation markers CD86 and MHC class II. Cells in

the gated population in the upper right quadrant of each graph represent the DCs with high

expression of both markers (i.e. mature DCs). C) Quantification of gated CD86hi

MHC IIhi

DCs.

Columns, mean; bars, SEM; *, P<0.05; **, P<0.01; ***, P<0.001 using a one-way ANOVA

(n=3).

44

3.5 IL-10 secretion by dendritic cells in response to H. pylori infection activates STAT3 to blunt maturation

To determine whether the IL-10 released by the dendritic cells in response to H. pylori infection

was responsible for STAT3 phosphorylation, BMDCs were incubated with a neutralizing α-IL-

10 antibody [0.5 and 1µg/ml], in the presence or absence of H. pylori to examine the effects of

the IL-10 released by the dendritic cells. An isotype control IgG antibody was used to ensure the

specificity of the IL-10 antibody. BMDCs were lysed and immunoblotting performed to examine

phosphorylation of STAT3. Treatment with the α-IL-10 antibody inhibited the STAT3

phosphorylation induced by H. pylori in the BMDCs (Figure 3.5-1A, B). Importantly, α-IL-10

treatment also enhanced expression of the maturation markers MHC class II and CD86 in

BMDCs in response to H. pylori infection as compared to BMDCs treated with H. pylori alone

(Figure3-5C). Treatment with the isotype control IgG antibody affected neither the STAT3

phosphorylation induced in the BMDCs by H. pylori (Figure 3.5-1A, B) nor the maturation of

the BMDCs in response to the H. pylori (Figure 3.5-1C), demonstrating that this was an IL-10-

specific effect.

45

C HP C H

PHP C H

P

0

1

2

3

4

5

n=4

B

****

***No antibody

IL-10 (1g/ml)

IL-10 (0.5g/ml)

IgG (1g/ml)

PS

TA

T3:A

cti

n

no

rmali

zed

to

C

C HP C H

PHP C H

P

0

10

20

30

40

50 ***

ns

n=4

IL-10 (1g/ml)

IL-10 (0.5g/ml)

No antibody

IgG (1g/ml)

C

% M

HC

II+

CD

86+

D

Cs

46

Figure 3.5-1: α-IL-10 antibody inhibits H. pylori-induced STAT3 phosphorylation in

BMDCs to promote enhanced expression of maturation markers

A) Lysates from BMDCs treated with functional grade α-IL-10 antibody [0.5 or 1µg/ml] or

isotype control IgG antibody [1µg/ml], in the presence or absence of H. pylori, were subjected to

immunoblotting analysis to determine STAT3 phosphorylation levels. B) Densitometric analysis

was used to quantify the changes in STAT3 phosphorylation levels. Columns, mean; bars, SEM;

**, P<0.01; ***, P<0.001 using a one-way ANOVA (n=4). C) BMDCs were treated with

functional grade α-IL-10 antibody [0.5 or 1µg/ml] or isotype control IgG antibody [1µg/ml], in

the presence or absence of H. pylori at an MOI of 10:1 for 20h. Flow cytometry analysis was

used to quantify the population expressing high levels of the maturation markers CD86 and

MHC class II. Graph represents quantification of the gated CD86hi

MHC IIhi

DCs (i.e. mature

DCs). Columns, mean; bars, SEM; ***, P<0.001 using a one-way ANOVA (n=4).

47

3.6 H. pylori-infected dendritic cells do not drive regulatory T cell development

The functional effect of H. pylori infection on dendritic cell activation and phenotype was next

examined. STAT3 KO and WT BMDCs were incubated with H. pylori for 20h prior to 6h

treatment with penicillin-streptomycin to kill any remaining extracellular bacteria. These

BMDCs were then loaded with differing concentrations of the ovalbumin peptide (OVA;

residues 323-339) for 6h. BMDCs were then co-incubated with naïve CD4+ CD25

- splenocytes

from OT-II transgenic mice at a ratio of 1:2. Recombinant murine IL-2 [10ng/ml] (R&D

Systems, Minneapolis, MN, USA) and recombinant human TGF-β1 [10ng/ml] (PeproTech,

Rocky Hill, NJ, USA) were added to drive regulatory T cell development (Kryczek et al., 2007;

Malek, 2003). Cells were cultured for 72h before they were harvested, supernatants collected,

and cells were stained with αCD4 and αFoxP3 fluorophores to examine regulatory T cell (Treg)

development. Loading BMDCs with a low dose of the ovalbumin antigen (10ng/ml) combined

with addition of the recombinant cytokines IL-2 and TGF-β1 was sufficient to drive CD4+

FoxP3+ Treg development (Figure 3.6-1A). Furthermore STAT3 KO BMDCs induced

development of lower levels of CD4+ FoxP3

+ Tregs (Figure 3.6-1B). However both STAT3 KO

and WT BMDCs treated with H. pylori did not induce production of CD4+ FoxP3

+ Tregs

(Figure 3.6-1C, D).

48

A

B

49

Figure 3.6-1: H. pylori infection does not induce dendritic cells to produce CD4+ FoxP3

+ T

cells

A) Immature BMDCs were loaded with ovalbumin peptide (residues 323-339) [10ng/ml or

100ng/ml] for 6h. DCs were then harvested, counted, and cocultured at a ratio of 1:2 with naïve

CD4+ CD25

- splenocytes from a sex-matched OT-II transgenic mouse in the presence or absence

of recombinant murine IL-2 [10ng/ml] and recombinant human TGF-β1 [10ng/ml] for 72h. Cells

were then harvested and stained with αCD4 and αFoxP3 fluorophores, then subjected to flow

cytometry analysis. Upper-right quadrant of each graph depicts proportion of CD4+ FoxP3

+ T

cells. B) STAT3 KO and WT BMDCs were subjected to the treatment described in A). C)

BMDCs were incubated with H. pylori strain 7.13 at an MOI of 10:1 for 20h and then treated

with 200 U penicillin/streptomycin [200U/0.2mg/ml] (to kill bacteria) and subjected to the same

treatment as in A). D) Graph depicts quantification of the gated CD4+ FoxP3

+ T cells (i.e. Tregs).

Columns, mean; bars, SEM (n=3).

C

D

50

Chapter 4 Discussion

4

The ability to alter and evade the host immune response is crucial for the survival and persistence

of H. pylori in the host gastric mucosa. As dendritic cells are key orchestrators of the host

immune response, they are ideal targets for the pathogen’s immune-manipulating efforts. The

interaction between H. pylori and dendritic cells is beginning to be elucidated, but the

mechanisms by which H. pylori affects DC function are still being determined.

This study demonstrated for the first time that H. pylori activated the STAT3 pathway in

dendritic cells. This STAT3 activation serves to blunt DC maturation, as H. pylori infection

induced more maturation (as assessed by expression of the maturation markers MHC II and

CD86) in STAT3 KO DCs as compared to control DCs. This is consistent with current literature

indicating that STAT3 is a negative regulator of DC maturation and function (Iwata-Kajihara et

al., 2011; Lin et al., 2010; Melillo et al., 2010). The H. pylori-mediated STAT3 activation in

dendritic cells represents a potential mechanism by which H. pylori manipulates the immune

system to persist and promote carcinogenesis. STAT3 activation in DCs is often associated with

tolerogenic functions (Barton, 2006; Cheng et al., 2003; Nefedova et al., 2005); therefore this H.

pylori-induced STAT3 activation could potentially induce tolerant DCs that suppress immune

responses to the bacteria instead of inducing effector immune responses. This represents a novel

potential mechanism by which the bacterium evades the host immune response to persist in the

gastric mucosa and may also help to explain the causative role of H. pylori in gastric cancer

development. Current evidence indicates that STAT3 activation in DCs contributes to tumor

progression by preventing DC maturation and thus decreasing the host tumor

immunosurveillance (Lin et al., 2010). Indeed it was demonstrated that STAT3-depleted DCs

were more resistant to cancer-cell derived inhibitory factors (Iwata-Kajihara et al., 2011).

Therefore the activation of STAT3 in DCs by H. pylori may potentially inhibit the DC responses

to tumors that develop in the gastric mucosa while simultaneously inhibiting the DC responses to

the bacterium.

51

In this study IL-6 was used as a control to drive STAT3 activation in the dendritic cells, and

while H. pylori induced STAT3 activation, it was not as pronounced at the IL-6-mediated

STAT3 activation (Figure 3.1-1). As IL-6 is a ligand that binds to its receptor to induce STAT3

activation, a strong induction of STAT3 phosphorylation upon treatment with this pure ligand is

expected. However live H. pylori has numerous virulence factors, secreted proteins, and

adhesion molecules, and interacts with the DC in multiple ways. Therefore it is not surprising

that the potency of the DC STAT3 activation by this live organism is slightly lower than that of

the pure ligand IL-6.

In response to H. pylori infection DC maturation is blunted and the dendritic cells secrete a

specific cytokine profile, including IL-10, the IL-12p40 subunit, TNF-α, and an increased

IL10:IL-12p40 ratio. IL-10 is a multifunctional cytokine that acts as an autocrine and paracrine

inhibitor of DC maturation and function (Corinti et al., 2001). IL-10 also activates the STAT3

pathway (Lin et al., 2010). Therefore an IL-10-neutralizing antibody was used to determine if the

H. pylori-mediated IL-10 secretion was acting as an autocrine/paracrine activator of STAT3.

Treatment with the neutralizing antibody completed abrogated the H. pylori-mediated STAT3

activation in the DCs. Furthermore anti-IL-10 treatment resulted in increased maturation of DCs

in response to H. pylori infection, indicating that this IL-10 autocrine/paracrine activation of

STAT3 is responsible for the blunted DC maturation.

IL-10 has been shown to possess immunoregulatory potential and induce the differentiation of

FoxP3+ Tregs (Lan et al., 2006). Furthermore a recent study demonstrated that H. pylori-infected

murine BMDCs drove the development of FoxP3+ Tregs that had potent immunoregulatory

capacities (Oertli et al., 2012). Therefore we co-incubated our H. pylori-infected dendritic cells

with naïve CD4+ T cells and examined the ensuing FoxP3 expression in an effort to replicate

their results. However, we found that H. pylori-infected DCs did not drive FoxP3 expression in T

cells. Methodological differences can potentially explain the differing results, as the BMDCs

were grown using slightly differing protocols. Furthermore Oertli et al. utilized the pre-mouse

clinical isolate strain PMSS1, while our study used the 7.13 mouse-adapted strain. Importantly

the 7.13 strain has been shown to be highly carcinogenic and injects significant amounts of the

CagA toxin into cells (Franco et al., 2008). Interestingly, recent evidence indicates that tolerant

responses (mediated by Tregs) rather than immunity (mediated by effector T cells) are protective

against H. pylori-induced gastric cancer precursor lesions (Arnold et al., 2011b). Therefore it is

52

entirely plausible that a carcinogenic strain of H. pylori (7.13) would not drive Treg development

while a non-carcinogenic strain will induce tolerant Treg responses.

Indeed the full complement of cytokines released by DCs in response to H. pylori infection likely

play an important role in pathogenesis. TNF-α is a pleiotropic cytokine which, while primarily

known to be proinflammatory, has also been shown to play immunoregulatory roles. In fact

dendritic-cell derived TNF-α has been shown to be necessary for the development of IL-10-

producing CD4+ T cells (Hirata et al., 2010). Furthermore TNF-α was found to upregulate

Programmed Death-1 (PD-1) levels on monocytes to cause IL-10 production, which effectively

inhibited T cell expansion and function (Said et al., 2010). These studies demonstrate that TNF-α

has clear immunoregulatory potential, therefore it is plausible that H. pylori-induced DC

secretion of TNF-α may act in an immunoregulatory manner, complementing the IL-10 secretion

and STAT3 activation to dampen the immune response and promote persistence.

The IL-12 cytokine is composed of a p40 (40 kDa) and a p35 (35kDa) subunit; the biologically

active form consists of two covalently linked chains of p40 and p35 and is known as IL-12p70

(Oppmann et al., 2000). The assays used in this study detected the secretion of the IL-12p40

subunit from the DC in response to H. pylori infection. Importantly the p40 subunit is shared by

another member of the IL-12 cytokine family: IL-23. IL-23 is composed of the p40 subunit and

another 19 kDa subunit, p19 (Oppmann et al., 2000), and can mediate pro-carcinogenic immune

responses (Kortylewski et al., 2009). IL-23 induces the development of Th17 cells (Iwakura and

Ishigame, 2006). Interestingly Th17 cells are proinflammatory and STAT3 signaling has been

shown to play a role in Th17 development (Harris et al., 2007). The highly inflammatory

STAT3-dependent Th17 response has been shown to be pro-carcinogenic (Wang et al., 2009).

Therefore the potential implications of H. pylori-mediated DC secretion of IL-23 for disease

pathogenesis are intriguing and require further study.

Taken together, these results present a potential model of H. pylori persistence and pathogenesis.

H. pylori induce the secretion of IL-10 from the dendritic cell, which acts in an autocrine and

paracrine fashion to activate STAT3 in DCs, blunting maturation. This inhibits the development

of proper effector T cell and other immune responses, facilitating bacterial persistence. However

the STAT3 activation in the DCs may also decreases DC tumor immunosurveillance. Therefore

the manipulation of the DC by H. pylori is a double-edged sword: it induces the secretion of

53

proinflammatory mediators which drive carcinogenesis, and also inhibits DC tumor

immunosurveillance to prevent recognition and DC-mediated immune elimination of the

developing tumor cells.

54

Figure 4-1: Potential model of dendritic cell responses to H. pylori

H. pylori induces immature dendritic cells to mature and secrete IL-10, which acts in an

autocrine and paracrine fashion to activate STAT3 in DCs, blunting maturation. H. pylori also

induces secretion of IL-12p40, which may drive Th17 development to promote chronic infection

and carcinogenesis. H. pylori infected dendritic cells do not appear to promote regulatory T cell

development.

55

While this study provides a reductionist examination of the interaction between H. pylori and the

dendritic cell, it is important to recognize the limitations of an in vitro model of host-pathogen

interaction. In the infected host the bacterium does not encounter DCs in isolation; rather it

interacts with a plethora of host cells including the gastric epithelial cell and likely other immune

cells such as macrophages, neutrophils, and natural killer cells (Alvarez-Arellano et al., 2007;

Jones et al., 1999; Lewis et al., 2011; Yun et al., 2005). These interactions will likely influence

the DC and immune response to the bacteria. Indeed preliminary data from our laboratory

suggests that exosomes (intercellular communicative vesicles) secreted by H. pylori-infected

epithelial cells can influence DC maturation (data not shown). Therefore while the in vitro

interaction between H. pylori and cultured murine dendritic cells is an appropriate method for

studying this pathogen, it is important to remember that these interactions take place within the

context of the full host-pathogen relationship. It has been shown that gastric stromal factors can

condition the DC response to H. pylori, suppressing H. pylori-stimulated DC activation

(Bimczok et al., 2011). This demonstrates that while in vitro assays are useful for examining the

interaction between pathogens and isolated cell types, the true in vivo host-pathogen interaction

is likely conditioned by numerous factors and may significantly differ from the in vitro results in

the laboratory. Therefore this STAT3 activation in DCs may be validated in human H. pylori

infection. Gastric tissue sections from H. pylori-infected patients can be assayed for the presence

of STAT3+ CD11c

+ dendritic cells and compared to tissue sections from uninfected control

subjects. This would validate these in vitro findings in human H. pylori infection and support the

hypothesis that STAT3 activation in DCs is a mechanism by which this pathogen manipulates

the host immune response.

While the generation of murine bone marrow-derived dendritic cells provides a good tool to

study the interaction between H. pylori and immature DCs, there are other methods for

generating cultured dendritic cells. One alternative DC culture method involves the

differentiation of harvested peripheral blood monocytes into DCs (Guiney et al., 2003). The use

of DCs derived from human peripheral blood monocytes would allow the validation of H. pylori-

induced STAT3 activation in human DCs. Additionally gastric dendritic cells can be harvested

from the gastric lamina propria of subjects and infected with H. pylori (Bimczok et al., 2010).

This would allow for the study of host-generated DCs, which may be more pertinent to human

56

infection than DCs cultured from precursors in a Petri dish. However despite these limitations

bone marrow-derived dendritic cells remain an inexpensive, non-invasive, valuable tool to study

the interaction between H. pylori and DCs. Other models can be used to validate findings and

explore their relevance in infection.

There are various strains of H. pylori that differ on many levels, such as pathogenicity and

presence of virulence factors. Some strains express a functional cag PAI and inject the CagA

toxin, others express the cag PAI and construct a functional T4SS but do not produce CagA,

while still others do not express the cag PAI at all. Strains also differ in expression of VacA and

the outer membrane protein OipA. Strains that express the intact T4SS and inject the CagA toxin

are more carcinogenic than strains that do not express these factors, while the presence of VacA

and OipA is also associated with pathological outcomes (Franco et al., 2008). This study used the

laboratory mouse-adapted H. pylori strain 7.13, which injects large amounts of CagA into host

cells (Franco et al., 2008). The necessity of the virulence factors for STAT3 activation in the

DCs is not examined in this study, nor is the relevance for other H. pylori strains. Despite these

limitations, this study describes a novel interaction H. pylori and the dendritic cell, an important

moderator of the host immune response.

In conclusion, these results demonstrated that H. pylori infection induces the activation of the

STAT3 signaling pathway in dendritic cells. Importantly IL-10 secreted by the DC acted as an

autocrine/paracrine activator of STAT3 to blunt DC maturation. Infected DCs also secreted a

specific cytokine profile in response to H. pylori infection, which does not appear to drive Treg

development. Overall this study describes a novel mechanism by which H. pylori alters the host

immune response to facilitate persistence, a mechanism that may also contribute to the

carcinogenic potential of this pathogen.

57

Chapter 5 Future Directions

The results of this study provide some exciting insights into the interaction between H. pylori

and dendritic cells. They also provide multiple future directions to further examine the effect of

H. pylori infection on the host immune response. The cytokines released from the STAT3 KO

BMDCs infected with H. pylori still need to be fully elucidated and should provide clues as to

the specific effect that the STAT3 activation has on the DC cytokine release profile. Particularly

it will help to elucidate whether STAT3 activation induces a change in the ratio of the pro-

versus anti-inflammatory cytokines that are released. Furthermore the presence of other

cytokines such as IL-18 and TGF-β in the H. pylori-treated DC supernatants was not examined

and could be explored in the future. This is pertinent since the Oertli paper found IL-18 to be

critical for the induction of Foxp3+ Treg development by H. pylori-treated DCs (Oertli et al.,

2012). Similarly TGF-β is a critical cytokine in the development of Tregs as well as Th17 cells,

so the presence of this cytokine should be examined to shed light on the T cell responses being

driven by the H. pylori-infected DCs. The cytokines released from the DC:T cell cocultures will

also help elucidate the phenotype of T cell which is being produced by the dendritic cell in

response to H. pylori infection.

The mechanisms by which H. pylori induces IL-10 secretion that activates STAT3 will be

interesting to elucidate. Preliminary data indicates that STAT3 activation in the dendritic cells

occurs independently of the presence of the CagA toxin (data not shown). Furthermore a heat-

stable molecule appears to play a role (data not shown). Previous studies have implicated TLR2,

TLR4, and TLR9 in recognition of H. pylori by DCs, which lead to secretion of proinflammatory

cytokines (Rad et al., 2009). Therefore the use of genetically engineered KO mice that lack

expression of these innate immune PRRs will help elucidate the role that they play in the IL-10

secretion and STAT3 activation shown by DCs in response to H. pylori infection. Interestingly

the intracellular PRR Nod1 has been shown to recognize H. pylori peptidoglycan that is injected

into the host cell by the cag PAI-encoded T4SS (Grubman et al., 2010; Viala et al., 2004).

Furthermore Nod1 has been shown to work synergistically with TLR2 and TLR4 to induce the

secretion of IL-10 from DCs (Fritz et al., 2007). Therefore it is possible that Nod1 signaling in

58

response to H. pylori plays a role in the dendritic cell secretion of IL-10, which leads to the

autocrine/paracrine activation of STAT3. The use of Nod1-specific KO mice will help to

elucidate the role of this H. pylori-sensing innate immune receptor.

Furthermore correlation of STAT3 activation in DCs with human disease progression will

strongly support the role of this pathway in H. pylori pathogenesis. To this end we have access to

paraffin-embedded gastric tissues sections from H. pylori patients at the different precancerous

stages, including gastritis, atrophy, metaplasia, dysplasia, and adenocarcinoma, as well as control

human tissue sections. These sections will be subjected to immunohistochemical and

immunofluorescent staining to examine the expression of phosphorylated (activated) STAT3 in

CD11c+ dendritic cells. If increases in the number of CD11c

+ PSTAT3

+ dendritic cells are seen

in the progression from gastritis through atrophy, metaplasia, dysplasia, and carcinoma, it would

strongly support the hypothesis that STAT3 activation in the DC plays an important role in H.

pylori carcinogenesis.

Overall this study has demonstrated a novel mechanism by which H. pylori manipulates the

dendritic cell, a key orchestrator of the immune response, to promote immune evasion and

bacterial persistence. These future studies will further elucidate the mechanisms by which H.

pylori induces the STAT3 activation in the DC, and help to delineate the role of this activation in

the pathogenesis of the bacteria.

59

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