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The Effect of Helicobacter pylori on Innate Immunity
by
Michelle Ang
A thesis submitted in conformity with the requirements
for the degree of Masters of Science
Graduate Department of Physiology
University of Toronto
© Copyright by Michelle Ang (2010)
ii
The Effect of Helicobacter pylori on Innate Immunity
Michelle Ang
Master of Science
Department of Physiology
University of Toronto
2010
Abstract
The innate immune system is important in both acute and chronic infection. In this thesis, I
investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system,
and 2) autophagy, recently identified as an important component of innate immunity. I
determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and
inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile
may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition
I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin
aggregation which has been implicated in mediating autophagy in response to mitochondrial
damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in
promoting its chronic infection and disease.
iii
Acknowledgments
The many people who have helped me through the graduate process are so numerous that to
name them all would double this thesis’s length. However, I would like to acknowledge several
key people to whom I am greatly indebted.
To my lab members, thank you for your unwavering support, and for the amazing environment in
which I was privileged enough to work. Thank you Esther. Your sense of fun and humour were
integral to the great atmosphere that exists in the lab. Thank you for helping with me with all my
mice, for all the great advice, and all the amazing talks about food. To Deepa, thank you for all
the help with the autophagy project. I will miss our daily chats about cricket, world news and
sports, and I thank you for letting me rant when experiments experienced catastrophic failure!
Dana, for making the lab fun, for teaching me most of the basics when I first started, and for
allowing me to work on your great paper.
To my supervisor, Dr. Nicola Jones, for the tremendous mentorship, helping me reach levels in
research I could not have done on my own. Thank you for caring not only about the results, but
about making science enjoyable for me as a person. It is rare to find a supervisor who cares not
only about the data, but about the well being and stress levels of the person and I thank you for
helping me through the difficult times in my Masters career.
Thank you to my committee members, Dr. Dana Philpott and Dr. Michael Wheeler. You have
both given me great feedback and guidance, and I thank you for your mentorship throughout this
process.
I’d like to thank the Philpott lab, specifically Joao and Thiru, who listened to millions of
questions and provided countless hours of support in my FACS data.
To rock climbing! For being the best outlet for experimental aggression I could have ever hoped
for.
To my family, thank you for being there for me when I needed you. To my Ahmah, who will
never read a word of this language, but whose unconditional love and support I cherish with all
my heart.
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To my wonderful friends. You all know who you are. I love you all dearly, and am inspired by
all your successes. I am lucky to have such lovely and intelligent people in my life.
To my Greg, without whom I would never have completed this stage in my life. You have been
my number one supporter, having more confidence in me than I ever could have in myself. I
cannot express how much your love and support has meant to me. Thank you for holding my
hand through the tough times in the lab, for encouraging me, and for saying all the things I
needed to hear to find the will to finish. I could not have done this without you. Thank you, and I
love you.
And to God, for knowing I can turn to you when I need to.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ............................................................................................................................ v
List of Abbreviations .................................................................................................................... vii
List of Tables ................................................................................................................................. xi
List of Figures ............................................................................................................................... xii
Chapter 1 Introduction .................................................................................................................... 1
1 Helicobacter pylori .................................................................................................................... 1
1.1 Infection and Colonization .................................................................................................. 1
1.2 H. pylori Associated Diseases ............................................................................................. 2
1.3 H. pylori Virulence Factors ................................................................................................. 2
1.3.1 Cytotoxin Associated Gene A (CagA) .................................................................... 2
1.3.2 Vacuolating Cytotoxin A (VacA) ........................................................................... 4
1.3.3 Lipopolysaccharide (LPS) .................................................................................... 13
2 Innate Immunity ....................................................................................................................... 13
2.1 Dendritic Cells (DCs) ....................................................................................................... 14
2.2 Signal Transducer and Activator of Transcription 3 (STAT3) ......................................... 18
2.2.1 STAT3 Signalling Cascade ................................................................................... 18
2.2.2 STAT3, DCs and Cancer ...................................................................................... 21
2.2.3 STAT3 and H. pylori ............................................................................................ 21
2.3 Dendritic Cells and H. pylori ............................................................................................ 21
2.4 Autophagy ......................................................................................................................... 26
2.4.1 H. pylori and Autophagy ....................................................................................... 31
3 Summary .................................................................................................................................. 31
Chapter 2 Materials and Methods ................................................................................................. 33
vi
CHAPTER 3 MATERIALS AND METHODS ....................................................................... 33
CHAPTER 4 MATERIALS AND METHODS ....................................................................... 35
Chapter 3 H. pylori Activates the STAT3 Pathway in DCs, Causing DC Maturation but
Altering DC Cytokine Release ................................................................................................. 39
ABSTRACT ............................................................................................................................. 39
INTRODUCTION.................................................................................................................... 39
RESULTS ................................................................................................................................ 41
H. pylori infection induces tyrosine phosphorylation of STAT3 ..................................... 41
H. pylori induces expression of DC maturation markers .................................................. 44
H. pylori stimulated DCs induce an unusual cytokine profile .......................................... 47
H. pylori induced DC maturation and cytokine release is dependent on activation of
the STAT3 pathway. ............................................................................................. 52
S3I-201 does not affect DC viability. ............................................................................... 55
DISCUSSION .......................................................................................................................... 58
Chapter 4 Helicobacter pylori’s VacA Toxin Induces Reactive Oxygen Species Production
and Alters Parkin Distribution ................................................................................................. 61
INTRODUCTION.................................................................................................................... 61
RESULTS ................................................................................................................................ 62
H. pylori’s VacA toxin induces ROS production ............................................................. 62
VacA induced autophagosomes do not colocalize with the mitochondria ....................... 68
H. pylori’s VacA toxin causes Parkin redistribution that is not dependent on VacA
induced autophagy ................................................................................................ 71
VacA induced Parkin aggregation does not colocalize with the mitochondria. ............... 74
DISCUSSION .......................................................................................................................... 77
Chapter 5 Discussion and Future Directions ................................................................................ 79
FUTURE DIRECTIONS ......................................................................................................... 81
References ..................................................................................................................................... 83
vii
List of Abbreviations
APC Antigen presenting cell
Atg Autophagy gene
BMDC Bone Marrow derived Dendritic Cells
CagA Cytotoxin Associated Gene A
cagPAI cag Pathogenicity Island
CD Cluster of Differentiation
CKO Conditional knock-out
DC Dendritic Cell
DNA Deoxyribonucleic acid
DPI diphenylene iodonium
ELISA Enzyme-Linked Immunosorbent Assay
EPIYA Glu-Pro-Ile-Tyr-Ala
FACS Fluorescence Activated Cell Sorting
FBS Fetal Bovine Serum
FITC Flourescein isothiocyanate
GAS Gamma-IFN–stimulated activation sequence
GEEC GPI-AP-enriched early endosomal compartments
GFP Green Fluorescent Protein
GM-CSF Granulocyte-macrophage colony-stimulating factor
viii
HK Heat Killed WT bacteria
H. pylori Helicobacter pylori
IBD Inflammatory Bowel Disease
IFN Interferon
IL Interleukin
ISRE IFNα-stimulated gene response element
JAK Janus Kinase
JAM Junctional adhesion molecule
LBP LPS binding protein
LC3 Mictotubule-associated Light Chain 3
LPS Lipopolysaccharide
MAP Mitogen-Activated Protein
MHC Major Histocompatibility Complex
Min Minute
MOI Multiplicities of Infection
MPT Mitochondrial Permeability Transition
mTOR Mammalian Target of Rapamycin
MyD88 Myeloid Differentiation primary response gene 88
NAC N-acetylcysteine
NADPH Nicotinamide adenine dinucleotide phosphate
ix
NAP Neutrophil Activating Protein
NBT Nitroblue tetrazolium
NO Nitric Oxide
NF-κB Nuclear factor kappa-light-chain enhancer of activated B-cells
PAS Pre-Autophagosomal Structures
PAMP Pathogen-Associated Molecular Patterns
PE Posphatidylethanolamine
PerCP-Cy5.5 Peridinin chlorophyll protein conjugated to cyanine
PI3K Phosphatidylinositol-3-kinases
PSTAT3 Phosphorylated STAT3
RFP Red Fluorescent Protein
RNA Ribonucleic acid
ROS Reactive Oxygen Species
RPMI Roswell Park Memorial Institute
SH2 Src-Homology 2
siRNA Small interfering RNA
SNARE Soluble NSF Attachment Receptor
SNAP Soluble N-ethylmaleimide-sensitive attachment protein
STAT Signal Transducer and Activator of Transcription
TBST Tris-buffered saline with tween
x
Th T-helper
TFSS Type IV Secretion System
TGF Tumour Growth Factor
TLR Toll-Like Receptor
TNF-α Tumour Necrosis Factor-alpha
Treg T-regulatory cells
VacA Vacoulating Cytotoxin A
WT Wild-type
YFP Yellow Fluorescent protein
xi
List of Tables
Table 1: Host, genetic bacterial and environmental factors in the pathogenesis of H. pylori
induced gastric cancer.
xii
List of Figures
Figure 1-1: H. pylori CagA signalling pathway
Figure 1-2: VacA induces cellular vacuolation.
Figure 1-3: Multiple actions of H. pylori’s VacA Toxin
Figure 1-4: Model of DC maturation
Figure 1-5: The STAT3 activation pathway
Figure 1-6: From Bronte-Tinkew et al. Nuclear STAT3 detected in epithelial and inflammatory
cells of H. pylori CagA+ infected Mongolian gerbil tissue
Figure 1-7: Summary of the possible immune responses to H. pylori
Figure 1-8: Overview of the autophagic process in mammalian cells
Figure 3-1: H. pylori activates the STAT3 activation pathway in DCs
Figure 3-2: H. pylori infection upregulates surface expression of DC maturation markers
Figure 3-3: Luminex assay of H. pylori induced by DC secretion
Figure 3-4: ELISA determination of H. pylori induced DC cytokine secretion
Figure 3-5: H.pylori induced DC maturation and cytokine secretion is dependent on activation of
the STAT3 pathway
Figure 3-6: S3I-201 does not affect BMDC viability
Figure 4-1: VacA pure toxin induces ROS production in AGS cells.
Figure 4-2: VacA induced ROS production is not responsible for VacA induced autophagy
Figure 4-3: VacA induced autophagy does not colocalize with mitochondrial antibodies
xiii
Figure 4-4: VacA pure toxin induces redistribution of Parkin that is independent of its induction
of autophagy
Figure 4-5: VacA induced Parkin aggregation does not target to the mitochondria
1
Chapter 1 Introduction
1 Helicobacter pylori
1.1 Infection and Colonization
Helicobacter pylori (H. pylori) is a gram-negative, spiral shaped bacterium that infects the
stomach of approximately half of the world’s human population(Kusters et al., 2006). H. pylori
is acquired early in life and persists for the lifetime of the host. Infection is associated with the
development of chronic gastritis and peptic ulcers (Cover and Blaser, 2009). In addition, H.
pylori is classified as a type 1 carcinogen and is the single most important risk factor in the
development of gastric cancer, the second most common cancer worldwide (Amieva and El
Omar, 2008;Correa and Houghton, 2007).
The bacterium is highly adapted to colonize in the human stomach where various factors
including gastric acidity, nutrient availability, innate and adaptive immunity limit most other
bacterial colonization (Cover and Blaser, 2009). H. pylori achieves gastric colonization through a
number of factors such as utilizing flagella which act as propellers to travel through the viscous
mucosa, and through the enzyme urease, which converts urea into NH3 and CO2, thereby
increasing the pH to buffer the environment surrounding the bacteria (Dhar et al.,
2003;Montecucco and Rappuoli, 2001;Portal-Celhay and Perez-Perez, 2006). H. pylori also
utilizes specialized adherence mechanisms ensuring a persistent and chronic infection. Utilizing
the outer-membrane protein BabA, H. pylori strongly adheres to the Lewis B blood group
antigens expressed on gastric epithelial cells and within the gastric mucus (Linden et al.,
2002;Montecucco and Rappuoli, 2001). Another bacterial adhesion SabA also mediates
interaction with Lewis X receptors, maintaining a weaker interaction that is upregulated during
inflammation (Linden et al., 2002). Using these factors, H. pylori adheres to gastric epithelial
cells allowing the bacterium to maintain close proximity to the epithelial cell layer, allowing for
the translocation of bacterial effector proteins and access to cellular nutrients thereby forming an
environmentally favourable niche for bacterial survival.
2
1.2 H. pylori Associated Diseases
Infection with H. pylori is associated with the development of several disease phenotypes. All
individuals infected with H. pylori develop chronic gastritis, an inflammatory response that is
insufficient to eradicate the organism from the host. Additionally, approximately 10-20% of
infected individuals will go on to develop peptic ulcers, associated with antral predominant
gastritis and increased acid secretion (Kusters et al., 2006).
H. pylori infection is also the strongest risk factor for the development of gastric cancers
including lymphoma, MALT lymphoma and adenocarcinoma. Gastric cancer occurs in 1-2% of
the infected population (Cover and Blaser, 2009;Lochhead and El Omar, 2007). Correa proposed
a model of H. pylori-mediated gastric carcinogenesis in which there is a sequential array of
pathological changes that convert normal gastric mucosa to a cancerous environment beginning
with the development of chronic active gastritis, followed by atrophy of the gastric tissue and
inflammatory damage. This causes progression to intestinal metaplasia, dysplasia and eventually
the development of gastric cancer (Correa, 2003;Correa, 2004;Correa and Houghton, 2007).
However, the exact mechanisms leading to the development of gastric cancer remain unclear.
Bacterial, host and environmental factors (Table 1) are thought to contribute to an increased risk
for the development of gastric cancer. Investigating the role of bacterial factors in altering host
immune responses is the focus of my research.
1.3 H. pylori Virulence Factors
1.3.1 Cytotoxin Associated Gene A (CagA)
The most well characterized virulence factor is the cytotoxin-associated gene A (CagA) found
within the (cag) pathogenicity island (PAI). The cag PAI encodes a type IV secretion system
(TFSS) similar to those found in Agrobacterium tumefaciens, Bordetella pertussis and
Legionella pneumophila. The TFSS acts as a molecular syringe to inject CagA proteins into host
cells (Bourzac and Guillemin, 2005;Odenbreit et al., 2000). Infection with strains of H. pylori
containing CagA confers a higher risk for the development of gastric cancer (Parsonnet et al.,
1997). Furthermore transgenic mice expressing the CagA virulence factor develop
gastrointestinal and hematopoietic neoplasms, confirming that CagA is tumourigenic (Ohnishi et
al., 2008). The exact mechanisms by which CagA promotes cancer remain unknown. Once
3
Table 1: Host, genetic bacterial and environmental factors in the pathogenesis of H. pylori
induced gastric cancer.
Host genetic factors Bacterial virulence
factors
Environmental
factors
Gastric cancer
phenotype
IL-1B-511*T
IL-1-RN*2*2
IL-10 ATA haplotype
IL-8-251*A
TLR4+896*G
MBL2 HYD
haplotype
cagPAI, CagA
VacA
Smoking
Poor Diet
Corpus-predominant
gastritis
Multifocal atrophic
gastritis
High gastrin +
Hypochlorhydria
Low pepsinogen I and
pepsinogenI/II ratio
Increased risk of
gastric cancer
Modified from (Amieva and El Omar, 2008).
4
CagA is translocated into host cells via the TFSS, it localizes to the plasma membrane and
undergoes phosphorylation by the SRC family of kinases (Hatakeyama, 2004) (Figure 1-1). This
phosphorylation occurs within a repeated specific 5 amino acid motif (EPIYA – Glu-Pro-Ile-Tyr-
Ala) on the tyrosine residue (Parsonnet et al., 1997). The presence and number or EPIYA repeats
may contribute to severity of disease outcome (Dhar et al., 2003). Upon phosphorylation, CagA
binds to SRC Homology 2 (SH2) domain containing protein tyrosine phosphatase SHP2,
deregulating this oncoprotein, activating phosphatase activity and forming a complex that
initiates mitogenic signalling, cell scattering, elongation and rearrangement of the actin
cytoskeleton (Kusters et al., 2006). CagA also has actions independent of its EPIYA tyrosine
phosphorylation. For example, unphosphorylated CagA can target the c-Met receptor as well as
bind to Grb2 (Churin et al., 2003;Mimuro et al., 2002). CagA also interacts in a phosphorylation
independent manner with ZO-1 and junctional adhesion molecule (JAM) thereby altering tight
junctions (Amieva et al., 2003;Hatakeyama, 2006).
1.3.2 Vacuolating Cytotoxin A (VacA)
Another important bacterial virulence factor is the vacuolating cytotoxin A (VacA) so named for
its ability to induce vacuoles in cells. VacA is a toxin secreted by H. pylori. VacA released from
H. pylori is cleaved into two fragments, a 33 kDa N-terminal fragment (p33) and a 55 kDa c-
terminal fragment which exist as a large ogliomeric complex when purified (Dhar et al., 2003).
Exposure to acidic and alkaline conditions causes these oligomeric components to disassociate
into the monomeric subunits (Cover and Blanke, 2005). The p55 fragment has an important role
in the binding of VacA to host cells (Cover and Blanke, 2005;Galmiche et al., 2000). Once
bound to the cell, VacA is internalized by a pinocytic mechanism into GPI-AP-enriched early
endosomal compartments (GEECs) (Gauthier et al., 2007). Upon VacA’s internalization into
GEECs, it forms anion-selective channels by assembling into hexameric, ring like structures,
allowing chloride ions to pass through, resulting in swelling in endosomal membranes and
vacuole formation (Figure 1-2). These channels release bicarbonate and organic anions from the
cell cytosol to support bacterial growth (Cover and Blanke, 2005;Dhar et al., 2003;Szabo et al.,
1999). These large, intracellular vacuoles, with acidic intralumenal environments are enriched in
markers for late (but not early) endosomal compartments, such as Rab7, LAMP1 and Lgp110
(Papini et al., 1994).
5
6
Figure 1-1: H. pyori CagA signalling pathway.
CagA is injected into host cells upon H. pylori infection via the bacterial TFSS. Once injected,
CagA is localized near the cytoplasmic side of the plasma membrane until it is recruited by SRC
kineases. SRC kineases phosphorylate CagA on EPIYA motifs. Phosphorylated CagA then binds
SHP-2, activating SHP-2’s phosphatase activity and forming a complex that initiates mitogenic
signalling, cell scattering, elongation and rearrangement of the actin cytoskeleton.
Phosphorylation independent CagA interacts with c-Met and Grb2, as well as interacting with
ZO-1 and JAM to alter tight junctions.
Stomach Lumen
Oligomerization andMembrane interaction
Endocytic entry into cell
H+ (V-ATPase)
H2ONH3
NH4+
Cl- channel activity
Vacuole swelling
7
8
Figure 1-2: VacA induces cellular vacuolation.
Upon VacA release by H. pylori, VacA binds to the cell surface, where it oligomerizes and is
internalized via the GEEC pathway. Internalized VacA forms anion-selective channels which
allow chloride conductance, causing increased intraluminal chloride concentration. As a result,
increased ATPase activity occurs, increasing proton pumping which reduces intraluminal pH.
Membrane-permeant weak bases diffuse inwards and are trapped within these compartments.
Compartments undergo osmotic swelling, resulting in cellular vacuolation. Modified from Cover
and Blanke (2005)
9
The p33 fragment of VacA localizes specifically to the mitochondria where it affects
mitochondrial membrane permeability (Figure 1-3). VacA decreases the levels of 3, 3'-
dihexyloxacarbocyanine iodide, an indicator of mitochondrial membrane potential, followed by a
decrease in cellular ATP indicating mitochondrial and cellular damage (Kimura et al., 1999). The
decrease in mitochondrial membrane potential coincides with decreased ATP levels and O2
consumption which suggests that VacA damages the mitochondrial inner membrane (Kimura et
al., 1999). These mitochondrial effects are dependent on VacA’s ability to form anion-selective
channels described previously (Willhite and Blanke, 2004b). VacA also modulates the function
of several immune cells which will be discussed below.
In T-cells, VacA can inhibit T-cell proliferation and viability, by downregulating the IL-2 α-
receptor and inhibiting production of stimulatory IL-2 (Gebert et al., 2003;Sundrud et al., 2004).
VacA activates the MAP kinase pathway (Boncristiano et al., 2003;Sundrud et al., 2004) and the
NF-κB pathway in T-cells to promote proinflammatory effects through the induction of genes
involved in the classical NF- κB pathway (Takeshima et al., 2009). VacA inhibits the activation
of Jurkat T cells (a human T cell lymphoma/leukemia cell line) by inhibiting the activation of
NFAT, a key transcription factor required for the expression of genes involved in T cell
activation (Boncristiano et al., 2003;Gebert et al., 2003). Finally, it has been demonstrated that
VacA can suppress the proliferation of primary human CD8+ T cells and CD4
+ T-cells even
when strongly stimulated by potent T-cell activators (Torres et al., 2007).
VacA also acts on APCs, interfering with major histocompatibility complex (MHC) class II
mediated antigen presentation inhibiting the li-dependent pathway of antigen presentation. In
addition, VacA blocks antigen presentation on APCs to T-cells (Boncristiano et al.,
2003;Molinari et al., 1998).
B-cells have also been shown to be affected by the VacA toxin. VacA induces the apoptosis of
B-cells via the translocation of the apoptosis-inducing factor to the nucleus (Singh et al., 2006).
Similar to T-cells, VacA inhibits the activation and proliferation of B-cells, even when
stimulated by potent B-cell activators (Torres et al., 2007).
10
In macrophages, phagosome maturation is arrested by VacA, preventing the killing of H. pylori
by phagocytes (Zheng and Jones, 2003). In clinical isolates, there were lower observed levels of
bacterial multiplication in macrophages in strains that lacked the VacA gene (Wang et al., 2009).
There is some evidence to indicate that VacA has some pro-inflammatory effects, as increasing
levels of TNF-α and IL-6 due to VacA in mast cells was observed, however these effects need
further study (Cover and Blanke, 2005).
Surface bound VacA functioning as anadhesion
NucleusPro-in�ammatorysignalling
VacA
Localization tomitochondria
Cell death
Reduction of ∆ΨmRelease of cytochrome c
T-cell
Inhibition of T-cellactivation andproliferation
Vacuolation
Membranechannel activity
11
12
Figure 1-3: Multiple actions of H. pylori’s VacA toxin.
VacA toxin has many different effects within the cell. VacA can localize to the mitochondria,
causing a decrease in mitochondrial membrane potential, causing release of cytochrome c. VacA
can also be internalized and cause pro-inflammatory signalling, as well as interfere with the
activation and proliferation of T-cells. In addition, VacA induces formation of large intracellular
vacuoles as described previously. Modified from Cover and Blanke (2005).
13
1.3.3 Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS) is an important structural component of the outer membrane of gram-
negative bacteria containing three parts: lipid A, a core oligosaccharide and an O side chain (Lu
et al., 2008). LPS contains pathogen-associated molecular patterns (PAMPs) which are
recognized by host cells to initiate innate responses. Lipid A is the main PAMP in LPS.
Normally, LPS stimulation of mammalian cells occurs through a series of interacting proteins
such as LPS binding protein (LBP), which facilitates interaction of LPS to CD14, a
glycosylphosphatidylinositol-anchored protein (Lu et al., 2008). CD14 interacts with Toll-like
receptor (TLR) 4, which upon LPS recognition, undergoes oligomerization, recruiting
downstream adaptors, initiating activation of the NF-κB signalling pathway, increasing
proinflammatory cytokine production through a myeloid differentiation primary response gene
88 (MyD88) dependant or independent pathway (Lu et al., 2008).
LPS from H. pylori possesses some unusual characteristics that differ from other gram-negative
bacteria’s LPS as it appears to be less potent at driving a pro-inflammatory response. H. pylori
LPS fails to induce inflammatory responses in epithelial cells at high concentrations (Backhed et
al., 2003). It is also known to be 1000 times less effective than E. coli LPS in inducing pro-
inflammatory cytokine production in macrophages/monocytes, as well as being less effective
than E. coli endotoxin in binding to LBP (Cunningham et al., 1996;Gobert et al., 2004). H.
pylori LPS signalling may also differ from the conventional pathway. It has also been reported
that TLR-2 not TLR-4 is the major TLR activated by LPS during H. pylori infection giving H.
pylori LPS a different signalling pathway from normal LPS which may attribute to its differing
characteristics (Smith, Jr. et al., 2003;Uno et al., 2007). Other studies suggest that H. pylori
strains act as antagonists for human TLR-4 (Lepper et al., 2005).
2 Innate Immunity
As H. pylori causes a chronic infection, the ability of the bacterium to evade host immune
responses including potentially escaping tumour immune surveillance is an area of great interest
and the focus of my thesis. The host immune system consists of both innate and adaptive
immunity. Innate immunity is a fast acting response, reacting quickly in bacterial infection with
the aim of eliminating the bacteria. The adaptive immune response is generally more delayed and
14
its responses are shaped by the innate immune response. An additional component of the host
immune response, which has recently been recognized, is the autophagy pathway. Below I will
summarize current knowledge with respect to the key areas of focus of my research in H. pylori
and innate immunity: dendritic cells and autophagy.
2.1 Dendritic Cells (DCs)
Dendritic cells (DCs) are a type of antigen presenting cell (APC) whose primary role is to
uptake, process and present antigens to T-cells, initiating and activating cytokine release and
immune responses (Lenahan and Avigan, 2006;Zou, 2005). Immature DCs are adept at antigen
capture, and circulate throughout the body until they encounter antigens. Following antigen
uptake, they process and convert proteins to peptides and present these processed peptides to T-
cells on MHC molecules (Steinman and Banchereau, 2007) (Figure 1-4). During this antigen
processing phase, these DCs mature and differentiate, increasing surface expression of MHC
class II, CD80 and CD86, which are costimulatory molecules necessary for T cell activation
(Lenahan and Avigan, 2006). Activated T-cells can have many responses whose direction is
dictated by APCs. Under the control of DCs, T-cells can acquire the capacity to produce
powerful cytokines to initiate a Th1 proinflammatory response, a Th2 antibody forming
response, a Th17 autoimmune response and even a T-regulatory (Treg) immunotolerance
response (Ferrero, 2005).
To stimulate T-cells, peptides must be processed by MHC molecules which will present the
peptide on the surface of the DC. The peptide-binding proteins are of two types, MHC class I
and MHC class II. MHC class I molecules assist in the processing of intracellular antigens and
will present them to cytotoxic T-cells which, once activated, can directly kill a target
(Banchereau and Steinman, 1998). MHC class II complexes recognize and process extracellular
antigens and present them to T-helper cells which can have profound immune-regulatory effects.
A normal inflammatory response initially consists of the recruitment of neutrophils, followed by
T cells, plasma cells, and APCs which is accompanied by epithelial cell damage (Goodwin et al.,
1986). The failure of the recruitment and maturation of DCs has been observed at tumour sites,
indicating that a dysfunctional DC response may lead to carcinogenesis (Lenahan and Avigan,
2006). Additionally it has been observed that there is an increase in the number of immature DCs
in patients with cancer, indicating that maturation is an important factor in DC function (Lenahan
15
and Avigan, 2006). In many cancer types the signal transducer and activator of transcription 3
(STAT3) pathway is upregulated, which has been shown to inhibit DC maturation for oncogenic
survival and proliferation (Lunz, III et al., 2007;Wang et al., 2004).
16
17
Figure 1-4: Model of DC activation.
Immature DCs circulating in the lamina propria come into contact with antigens released by
extracellular factors or the epithelial cell layer. Immature DCs sample these antigens and
increase their surface expression in many DC maturation markers such as CD86, CD80 and
MHC class II. Immature DCs are now mature DCs and APCs, where they present the processed
antigen to other immune cells such as T-cells to orchestrate the appropriate immune response.
Activation of the STAT3 pathway can inhibit the maturation process.
18
2.2 Signal Transducer and Activator of Transcription 3 (STAT3)
2.2.1 STAT3 Signalling Cascade
STAT3 is a transcription factor whose upregulation is thought to be involved in oncogenesis.
The STAT3 pathway can be activated by a number of different cytokines (such as IL-6), growth
factors and peptides, all signalling through gp130, a signal transducer receptor subunit that can
be activated by several members of the IL-6 family of cytokines (Hirano et al., 1997;Kim et al.,
2007a) (Figure 1-5). IL-6 activation of STAT3 occurs through the binding of the cytokine to its
receptor IL-6R, resulting in heterodimerization with the gp130 subunit (Kim et al., 2007a).This
heterodimer activates the receptor associated Janus Kinase (JAK) kinases which in turn
phosphorylate tyrosine residues on the cytoplasmic tail of the gp130 receptor, recruiting STAT3
to the phosphorylated residues (Greenlund et al., 1995). STAT3 monomers will then be
phosphorylated themselves on tyrosine 705 residues, and then dimerize through reciprocal Src-
homology-2 (SH2) domain phospho-tyrosine interactions. STAT3 dimers will be translocated
into the nucleus, using nuclear localization signals mediated by the importinα3/α6 complex,
which when interacting with importinβ allows the dimer to traffic through nuclear pore
complexes that span the nuclear membranes (Reich and Liu, 2006;Yang and Stark, 2008). Here,
central DNA binding domains located on the STAT3 monomers allow the dimer to bind directly
to response elements in the promoter regions of target genes to trigger induction of transcription
of several genes involved in growth and proliferation (Liu et al., 2005;Reich and Liu, 2006). The
STAT family of transcriptional regulators include an IFNα-stimulated gene response element
(ISRE) and the Gamma-IFN–stimulated activation sequence (GAS: TTTCCNGGAAA) (Horvath
and Darnell, 1997). This interaction induces the transcription of multiple genes involved in
apoptosis (surviving, Bcl-nL, HSP27), cell cycle regulation (cyclin D1, c-myc, c-fos),
angiogenesis (vascular endothelial growth factor), cell invasion, metastasis and immune function
(Kim et al., 2007a;Kisseleva et al., 2002;Levy and Darnell, Jr., 2002). STAT3 dimers can also be
phosphorylated on the serine 727 residue. Phosphorylation of this residue results in dimers with
increased transcriptional activity for recognition sites involved in the mitogen-activated protein
(MAP) kinase pathway (Horvath and Darnell, 1997).
19
20
Figure 1-5: The STAT3 activation pathway.
IL-6 molecules bind to the IL-6-αR. This leads to heterodimerization with the gp130 subunit.
This dimer activates associated JAK kinases which phosphorylate residues on the cytoplasmic
tail of the gp130 receptor. Cytoplasmic STAT3 monomers are recruited to these phosphorylated
sites and form dimers via their reciprocal SH2 phospho-tyrosine interactions. Dimeric STAT3
translocates to the nucleus via importins-α3 and α6 through nuclear pore complexes and leads to
the transcription of genes involved in survival, proliferation and angiogenesis, potentially leading
to carcinogenesis.
21
2.2.2 STAT3, DCs and Cancer
Evidence suggests that STAT3 is involved in carcinogenesis, as this pathway is constitutively
activated in 50-90% of human cancers (Kortylewski and Yu, 2007). Disrupting constitutive
STAT3 activation in tumours results in the induction of apoptosis by increasing proinflammatory
cytokine and chemokine expression, indicating that STAT3 activation may play a role in the
reduction of proinflammatory responses, and thus increase immune evasion (Wang et al., 2004).
Inhibition of STAT3 results in a production of inflammatory mediators that can activate DCs
(Wang et al., 2004). Additionally, disruption of STAT3 activates innate immune cells in vivo,
and also effects adaptive immunity from DCs as when STAT3 is activated, DCs do not undergo
maturation (Wang et al., 2004). Similarly, blocking STAT3 in tumours leads to the activation of
T cells, promoting DC maturation, demonstrating the ability of STAT3 to inhibit DC mediated
immune responses (Wang et al., 2004).
2.2.3 STAT3 and H. pylori
We have shown that H. pylori activates the STAT3 pathway in gastric epithelial cells in vitro and
in vivo in a CagA dependant mechanism, thereby favoring the development of gastric cancer
(Bronte-Tinkew et al., 2009). In the Mongolian gerbil, STAT3 activation was detected in gastric
epithelial cells upon infection with CagA+ H. pylori (Bronte-Tinkew et al., 2009). In addition,
STAT3 activation was detected in infiltrating immune cells during infection of Mongolian
gerbils (Figure 1-6) suggesting that activation of this pathway may alter host immune responses.
2.3 Dendritic Cells and H. pylori
As DCs are key modulators of immune responses, the ability of H. pylori to alter DC function is
of great interest. How H. pylori alters DC function is controversial. DCs are capable of entering
H. pylori infected gastric epithelium, where they uptake bacteria directly through the extension
of long luminal projections by DCs through epithelial apical-junctions (Necchi et al.,
2009;Wilson and Crabtree, 2007) (Figure 1-7). H. pylori induces the secretion of many different
cytokines such as IL-6, IL-8, IL-10, IL-12, IL-1, and tumour necrosis factor (TNF) (Kranzer et
al., 2005;Rad et al., 2007).
Control
H. pylori infected
22
23
Figure 1-6: From Bronte-Tinkew et al. Nuclear STAT3 detected in epithelial and
inflammatory cells of H. pylori CagA+ infected Mongolian gerbil tissue.
A) Mongolian gerbil stomach sections were stained via immunohistochemistry with STAT3
(red) and DAPI (blue) antibodies. Nuclear STAT3 is detected in the inflammatory cell infiltrate
from H. pylori infected stomach sections from gerbils (red) (Bronte-Tinkew et al., 2009).
Ureaseother HP factors
IL-12IL-10
IL-23
IL-10TGF-β
IL-10TGF-β
IL-4IL-5IL-10
Th1
Th2
Th17
Treg
B-cell
IL-12
IFN-γTNF-αIL-2
24
25
Figure 1-7: Summary of the possible immune responses to H. pylori. Dashed lines represent
the components of the pathway that are more speculative. H. pylori has several effects on the
immune response, effecting the release of several cytokines that alter Th1, Th2 and Treg
responses. Modified from Wilson and Crabtree (2007).
26
The effects of this cytokine secretion are unclear. Some studies demonstrate that H. pylori can
induce DC maturation, upregulating DC surface expression of CD80, CD83 and CD86 (Kranzer
et al., 2004;Rad et al., 2007). Conversely, other studies show that H. pylori inhibits DC
activation via modulation of IL-12 secretion, potentially leading to immune evasion (Kao et al.,
2006;Obonyo et al., 2006). In our studies with the Mongolian gerbil, activation of STAT3 was
detected in infiltrating immune cells. Therefore, I hypothesized that H. pylori infection would
activate STAT3 in dendritic cells, thereby altering host immune responses. Determining the
effect of H. pylori infection on STAT3 activation in dendritic cells is the focus of chapter three
of my thesis.
2.4 Autophagy
Another important host response which modulates immunity and infection is autophagy.
Autophagy is a process that involves the sequestering and transit of cytoplasmic components or
organelles to the lysosome for degradation. These broken down substrates are then used to
produce amino acids for protein and energy synthesis (Meijer and Codogno, 2006). There are
three types of autophagy: Microautophagy, macroautophagy and chaperone-mediated autophagy.
Microautophagy is responsible for engulfing large structures, sequestering cytosolic components
directly by lysosomes through invaginations in their membranes (Klionsky and Emr, 2000).
Chaperone-mediated autophagy can only degrade soluble proteins, and involves direct
translocation of unfolded substrate proteins across the lysosome membrane (Klionsky and Emr,
2000). Macroautophagy (henceforth referred to as autophagy), sequesters and engulf large
structures within a unique double-membraned vesicle in selective and non-selective processes
(Klionsky and Emr, 2000). Subsequently, these cargo are delivered to the lysosome where the
cargo is degraded or recycled (Levine and Klionsky, 2004).
Upon induction of autophagy, the process begins with the formation of double-membrane
compartments called autophagosomes. The origin of these membrane compartments is unclear. It
is thought that membrane compartments may originate from the ER, however evidence also
demonstrates that the pre-autophagosomal structure (PAS) contains Atg9, and in addition to
localizing with the PAS, membrane components can also be visualized with Mitoflour, a dye
used to identify mitochondrial membrane components, suggesting that the mitochondria may
provide components for the autophagosome (Klionsky and Emr, 2000;Reggiori et al., 2005).
27
The formation of the PAS and autophagosome involves several Atg genes, with Atg9 being the
only transmembrane protein in the core machinery that is conserved among species (Xie and
Klionsky, 2007) (Figure 1-8). Two ubiquitin-like conjugation reactions are needed to form the
Atg12-Atg5 complex. The carboxy COOH terminal of Atg12 is activated by linking first to
Atg7, an E1-like enzyme, which passes the Atg12 to Atg10, an E2-like enzyme, which is then
attached to the internal lysine residue 130 of Atg5, later recruiting Atg16 to form an Atg12-Atg5-
Atg16 multimer complex (Klionsky and Emr, 2000). This complex is present on the PAS,
however dissipates upon autophagosome formation. Autophagosomal elongation is mediated by
mictotubule-associated light chain (LC)-3 (Atg8 in yeast). The second reaction of LC3 lipidation
which joins LC3 to phosphatidylethanolamine (PE) first involves Atg4 which cleaves LC3’s
carboxy terminal leaving a conserved glycine residue (Klionsky and Emr, 2000). Cleaved LC3
next binds to Atg7, then Atg3 and finally to autophagosomal membrane protein PE, where it
becomes membrane-bound LC3-II (Klionsky and Emr, 2000). This complex is present in the
initial stage of autophagosome formation, and remains on the autophagosome throughout
formation and fusion with the lysosome and is often used as a marker of autophagy in
experimentation (Yorimitsu and Klionsky, 2005).
Complete autophagosomes are then delivered to lysosomes in a manner dependant on
microtubules and maintenance of proper acidification(Klionsky and Emr, 2000). Intermediate
autophagosomes fuse with lysosomes and acquire cathepsins and acid phosphatases to become
mature autolysosomes, where the inner autophagosomal membrane is degraded.
Autophagy plays an important role in immunity, as autophagic machinery is often used in
defence against microbes, delivering microorganisms to lysosomes for degradation (a process
called xenophagy) and the delivery of microbial nucleic acids and antigens to
endosomal/lysosomal compartments to activate innate or adaptive immunity (Levine and
Kroemer, 2008). Several bacteria are degraded by xenophagy, including Mycobacterium
tubercuolosis, Shigella flexneri, Salmonella enterica, and Listeria monocytogenes (Huang and
Klionsky, 2007;Levine and Kroemer, 2008). Atg5 has been shown to be crucial in the delivery of
viral nucleic acids from the Sendat virus to endosomal TLR-7 receptors which then activate DCs
(Lee et al., 2007). Autophagy is also involved in the delivery of synthesized microbial antigens
to MHC class II APCs which subsequently activate CD4+ T cells (Paludan et al., 2005).
Formation ofSequestration Cresent
Lysosome
Lysosomal Hydrolases
Docking& Fusion
Atg5-Atg12-Atg16 complex
LC3 II
28
29
Figure 1-8: Overview of the autophagic process in mammalian cells. Cytosolic components
and organelles are either specifically or randomly targeted by the PAS. A sequesteration crescent
forms around these components initially requiring the Atg5-Atg12-Atg16 complex (red dots),
followed by LC3 lipidation into the LC3II form (yellow dots). The Atg5-Atg12-Atg16 complex
disassociates prior to autophagosome completion, while LC3II remains on the autophagosome
throughout. Lysosomes are trafficked to completed autophagosomes, where they fuse. The inner
membrane of the autophagosome is degraded, followed by degradation of autophagosomal
contents by lysosomal hydrolases.
30
Autophagy also has been shown to have a role in tumourigenesis as failure to clear the
intracellular debris normally eliminated by autophagy can lead to tissue damage, cell death and
chronic inflammation that is tumour promoting (White et al., 2010). Atg6/beclin1 is a
haploinsufficient tumour suppressor in mice (Qu et al., 2003;Yue et al., 2003). Atg6/beclin1 has
been shown to be monoalleically deleted in ovarian and breast cancer (Qu et al., 2003).
Inactivating mutations in Atg5 were found to be infrequently present in colorectal cancer (Jung
et al., 2008). Losses of other autophagy mediators such as p63 and Atg4c were also shown to
enhance tumour formation (Marino et al., 2007;Mathew et al., 2009).
Basal levels of autophagy are responsible for the clearance of damaged organelles, such as the
mitochondria. The importance of mitochondrial degradation is expected, given the fact that
mitochondrial homeostasis in a cell is crucial. Specific autophagy targeting the mitochondria
(mitophagy) has been a growing field of interest, as mitochondrial instability, such as changes in
Mitochondrial Permeability Transition (MPT) can play a role in the initiation of mitophagy.
Mitochondrial production of ROS is an important factor for the overall ROS level within a cell.
Mitochondria ROS production produces superoxide and H2O2 through its respiratory chain, and
accumulation of these ROS can lead to lipid peroxidation, apoptosis and necrosis, mitochondrial
dysfunction and DNA damage (Murphy, 2009). Additionally, the ROS produced by the
mitochondria have been reported to regulate autophagy and autophagic cell death, as autophagy
is induced by rotenone, H2O2, and superoxide (Chen and Gibson, 2008). ROS production can
also lead to 10-20 fold higher mutation rates in mitochondrial DNA versus nuclear DNA (Yakes
and Van Houten, 1997) which may also induce mitophagy. Failure to clear the cell of damaged
mitochondria can lead to cellular damage, thus leading to cell death (Kim et al., 2007b).
Oxidative damage due to ROS production plays an important role in tumourigenesis as increased
ROS levels are often associated with cancerous cells. Increased ROS levels can lead to DNA
damage, implicated in mutating tumour suppressor p53 (Morgan et al., 2003) causing
carcinogenesis. Additionally, ROS production is involved in Ras-Raf-MEK signalling. This
pathway is important in mediating protection against apoptotic cell death induced by increased
oxidative stress, directly enhancing the activation of Ras, and augmenting ERK1/2, leading to
oncogenesis (Pan et al., 2009). Lipid peroxidation leading to ROS production has also been
shown to cause DNA damage due to the production of high levels of oxygen radicals (Marnett,
31
2000). Thus there is a well-established link between ROS generation and tumourigenesis which
may prove to play a role in H. pylori mediated tumourigenesis.
2.4.1 H. pylori and Autophagy
Recently, our lab has shown that H. pylori can induce autophagy in human gastric epithelial
(AGS) cells, and that its VacA toxin is sufficient to induce this autophagy (Terebiznik et al.,
2009). Induction of autophagy was dependant on VacA’s channel-forming ability, and limited
the effects of VacA (Terebiznik et al., 2009). However, the exact mechanisms by which VacA
induces autophagy remain unknown. I hypothesized that VacA mediated damage to
mitochondria may be sufficient to induce autophagy. Determining the mechanisms responsible
for VacA mediated autophagy is the focus of chapter four of my thesis.
3 Summary
In summary H. pylori evades host responses to cause a chronic infection and promote
carcinogenesis in a subpopulation of those infected. Despite intense research in the field, the
mechanisms by which H. pylori evades the host immune response are still unclear. In my thesis,
I will explore two distinct avenues by which H. pylori may alter or hide from the innate immune
response. Firstly, I will explore the interaction of H. pylori on a key orchestrator of the immune
response: the dendritic cell. Through interactions with this key APC, H. pylori may significantly
alter the outcome of the innate immune response. Secondly, I will explore the role of H. pylori
mediated autophagy to try and determine the mechanisms responsible for the induction of this
response. Changes in the autophagic pathway may modify the host cell immune response,
providing another mechanism by with H. pylori alters the innate immune response.
Overall hypothesis
The overall hypothesis of my thesis is that H. pylori effects host innate immune responses to
promote chronic infection and disease.
Specific aims
The specific aims of my thesis are:
32
1. To determine whether H. pylori activates the STAT3 pathway in DCs thus altering the host
immune response and providing a mechanism for H. pylori’s immune evasion
2. To determine the mechanisms by which H. pylori’s VacA toxin induces autophagy
33
Chapter 2 Materials and Methods
CHAPTER 3 MATERIALS AND METHODS
Bone Marrow Derived DCs
Bone marrow-derived DCs (BMDCs) were obtained and cultured from wild-type (WT) C57BL/6
mice. Bone marrow was extracted from the femur and tibia and seeded at 2 x 106/plate in
bacteriological petri dishes with 10ml RPMI (Roswell Park Memorial Institute)-1640 with L-
glutamine and 25mM HEPES supplemented with 1X nonessential amino acids, 50 mM 2-β-
mercaptoethanol, 1mM Sodium pyruvate, 100U/ml of penicillin/streptomycin (all from
Invitrogen, Burlington, Ontario, Canada), and 10% fetal bovine serum (FBS) (Gibco BRL Life
Technologies, Gaithersburg, MD, USA) supplemented with 100 ng/ml of granulocyte-
macrophage colony-stimulating factor (GM-CSF). Cells were incubated at 37°C for 3 days
before an additional 10ml of RPMI mixture was added. On day 6, cells were harvested and
magnetically sorted for the CD11c+ surface marker (Miltenyi Biotec, Bergisch Gladbach,
Germany) to ensure a pure DC population. Sorted cells were then plated in 6-well tissue culture
plates and used for stimulation.
H. pylori Strains and Infection Conditions
H. pylori gerbil adapted strain 7.13 or the corresponding isogenic cagA- mutant were grown on
blood agar plates containing 5% sheep blood under microaerophilic conditions (5% O2, 10%
CO2, 85% N2). Isogenic mutant strains were grown on Columbia blood agar plates supplemented
with 10% FBS and 20μg/ml kanamycin under similar conditions. After a monolayer of bacteria
were grown on the plates, bacteria were then transferred and grown in Brucella broth with 10%
FBS for 18 h, harvested by centrifugation, and added to gastric cells at a multiplicities of
infection (MOI) of 25:1. In some experiments, H. pylori was heat killed by boiling for 20 min in
sealed sterile Eppendorf tubes. Heat killing was confirmed by lack of growth on Columbia blood
agar plates. Where indicated, 30 min before infection, cells were incubated with the STAT3
inhibitor S3I-201at 25μM (Calbiochem, La Jolla, CA, USA). For controls, cells were incubated
with IL-6 (50ng/ml; Cell Signalling, Danvers, MA, USA) for 30 min, or 24 hours; or E. coli LPS
(1μg/ml; Sigma Aldrich) for 24h.
34
Cell Lysates and Western Blotting
After 4h or 24h of infection, cells were washed and scraped in 75μl of RIPA buffer containing
phosphatase and protease inhibitors (all from Sigma Aldrich). Cell suspensions were centrifuged
and supernatants stored at -80°C. Protein concentrations were measured using the Biorad Dc
protein assay. Lysates in laemelli buffer were loaded onto 10% SDS-polyacrylamide separating
gels and run at 110V for 1.5h at room temperature. Proteins were then transferred onto a
nitrocellulose membrane (BioTrace NT; Pall Corp., All Arbor, MI, USA) at 100V for 1.5 hours
at 4°C. Membranes were blocked with Tris-buffered saline with tween (TBST), 5% skim milk
followed by incubation with either rabbit anti-phospho-STAT3 antibody (1:250; Cell Signalling
MA, USA) or mouse anti-STAT3 antibody (1:500, Cell Signalling) in TBST milk. Blots were
then incubated with corresponding secondary horseradish peroxidase-conjugated antibody
(Jackson Laboratories, PA, USA) for 1h at room temperature. Bands were visualized by
chemiluminescence using Kodak Biomax MR film.
Densitometric analysis
Densitometric analysis was performed using FlourChem FCII software. All blots from each
independent experiment were used. Densities of phosphorylated STAT3 (PSTAT3) and STAT3
bands were measured for each treatment and expressed as a ratio (PSTAT3/STAT3).
Fluorescence Activated Cell Sorting (FACS) – Flow Cytometry
BMDCs infected for 24h were utilized for flow-cytometric analysis. Cells were scraped gently
on ice in PBS and spun at 350g for 10 min. Cells were resuspended in FACS buffer (PBS
containing 0.05% of sodium azide). Cells were incubated with CD16/32 (Fc receptor block)
(1μl/106 cells; eBioscience, San Diego CA, USA) for 15 minutes at 4°C prior to staining to block
nonspecific staining. Subsequently, cells were washed with FACS buffer and incubated with the
following antibodies: Flourescein isothiocyanate (FITC)-labeled antibody to CD11c,
phycoerythrin (PE)-labeled antibodies to CD86 and CD80, peridinin chlorophyll protein-cyanine
(PerCP-Cy5.5)-conjugated antibody to CD11b (all from eBioscience), biotin-labeled antibodies
specific for MHC class II followed by streptavidin conjugated to allophycocyanin (APC) (Becton
Dickson, San Jose, CA, USA). After staining, cells were washed twice with FACS buffer and
35
analyzed by FACS (FACSCaliburflow cytometer; Becton Dickinson). FlowJo software was used
for the analysis of the results.
ELISA Cytokine Analysis
BMDCs were infected for 24h as described above. Supernatants were collected and flash frozen
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).
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).
MTT live/dead cell Assay
To determine whether cell viability was affected by the addition of S3I-201 or other agents, an in
vitro toxicology MTT assay (Sigma Aldrich) was performed. BMDCs were grown and infected
for 24h as described. Viable cells contain mitochondrial dehydrogenase, which cleaves the
tetrazolium substrate ring, yielding purple formazan crystals. The degree of mitochondrial
enzymatic activity was determined via the dissolution of formazan precipitate and
spectrophotometric analysis of the resulting solutions from each treatment.
CHAPTER 4 MATERIALS AND METHODS
Cell and Bacterial Culture
Human gastric epithelial cells (AGS) were grown in Ham’s F-12 media (Wisent St. Bruno, PQ,
Canada) supplemented with 10% FBS and incubated at 37°C in a CO2 environment.
H. pylori strain 60190 from frozen stock was inoculated on blood agar plates with 5% sheep
blood (Oxoid Biotech, Canada). Isogenic mutants lacking the vacA gene were inoculated on
Brucella agar plates supplemented with 10% fetal bovine serum (FBS) (Gibco BRL Life
technologies) and 20μg/ml kanamycin (Invitrogen). All cultures were incubated at 37°C in a
microaerophilic environment for 72h. Cultured H. pylori strains were then grown at 37°C
36
overnight in Brucella broth (Sigma-Aldrich) under microaerophilic conditions with shaking at
120 rpm. On the day of infection, bacteria were pelleted and resuspended in Ham’s F12 medium
with 10% serum.
Preparation of Concentrated Culture Supernatants
The culture supernatants of wt H. pylori 60190 and isogenic mutant vacA- were separated from
the bacterial pellet at 4000rpm for 20 min. Supernatants were then filtered through a 0.22μm
filter and concentrated 10 times in Ham’s F12 media supplemented with 10% FBS using a 30
kDa cut off Amicon Ultra centrifugal filter (Millipore, Billerica, MA, USA). The culture
supernatants are diluted a further 5 times in Ham’s F12 prior to addition to cells for intoxication
assays (Raju and Jones, 2010).
Purified VacA Toxin
VacA toxin was purified by chromatography (Willhite et al., 2003) (obtained from Dr. Steven
Blanke, University of Houston, Houston, Texas USA) and diluted 100 times in Hams F12,
adjusted to pH 2.0 using 1-Normal HCl to acid activate the toxin. Purified toxin was left at 37°C
for 30 min followed by neutralization using 1-Normal NaOH. Toxin is supplemented with 10%
FBS. Prior to addition of activated toxin to cells NH4Cl was added (5mM).
Antioxidants and Chemicals
Where indicated, cells were pre-treated with several antioxidants for 30 minutes. The following
antioxidants were used: Rotenone (50μM), α-tocopherol (100μM), diphenylene iodonium (DPI;
100 μM), n-acetylcysteine (NAC; 5mM) (all courtesy of the John Brumell’s laboratory,
University of Toronto, Toronto, Ontario, Canada), and Tempol (5mM; Sigma Aldrich).
Autophagy Inducers
Autophagy was induced by incubating AGS cells in Earle’s balanced salts solution (EBSS,
Gibco) or with rapamycin (25µg/ml) at 37°C for 4h.
Infection Conditions
37
Infection of AGS cells was performed in 6-well plates when 80-90% confluency was reached.
Cells were treated with culture supernatants, purified VacA toxin or inactive VacA toxin for 4h.
Where indicated, pretreatment with antioxidants was used for 30 min, followed by subsequent
VacA treatments. Upon completion, cells were either lysed for western blotting.
Immunoflourescence
AGS cells were grown on coverslips or 12-24-well plates. Cells were transfected using
FuGENE-6 (Roche Diagnostics Indianapolis, Indiana, USA) as a transfection agent for various
plasmids. Plasmids LC3-GFP, LC3-RFP (Obtained from John Brumell’s lab) Mito-RFP, and
Parkin-YFP (both kindly provided by Dr. Peter Kim, University of Toronto, Toronto, ON,
Canada) were used alone, or in combination as needed. Upon infection completion, cells were
washed with PBS and fixed in 4% paraformaldehyde for 20 min. Cells were then permeabilized
by incubation in 0.1% Triton X-100 (vol/vol) in PBS for 20 min, and blocked for 60 min with
5% milk in PBS (vol/vol). Permeabilized cells were incubated for 1 h with primary antibody at
room temperature, washed extensively with PBS buffer, and incubated for 1 h with secondary
antibodies. All steps were carried out at room temperature. The following primary antibodies
were diluted in 5% milk in PBS (vol/vol) as follows: HSP60 (1:40; Abcam, Cambridge, MA,
USA), and VacA (1:100; from the Blanke Lab), followed by washing in PBS and a Cy3 or Cy5
conjugated secondary antibody (dilution 1:1000). After washing in PBS, the cover slips are then
mounted onto microscope slides.
Parkin
Experiments examining Parkin used Parkin-YFP-transfected cells treated with VacA pure toxin
as described above. Rotenone (50µM) was used as positive control for Parkin redistribution.
Confocal Microscopy
Confocal image acquisition was performed in a Quorum Spinning Disk Confocal Microscope
consisting of a Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu
Back-Thinned EM-CCD camera and spinning disk confocal scan head, 4 laser lines (Spectral
Applied Research: 405 nm, 491 nm, 561 nm, 638 nm), an ASI motorized XY stage, and an
Improvision Piezo Focus Drive. The equipment is controlled by Volocity acquisition software
38
(Improvision) and powered by an Apple Power Mac G5 (Terebiznik et al., 2009). The number of
autophagic cells was enumerated in approximately 100 LC3-GFP transfected cells using the 63X
objective. Cells containing more than five visible autophagosomes were considered autophagic.
Quantification of Parkin redistribution was also counted in approximately 100 Parkin-YFP
transfected cells using the 63X objective. Cells containing visible Parkin puncta were counted as
Parkin-redistributed.
Immunoblotting
AGS cells in 6 well plates were treated for 4 h. Western blotting technique was similar as that
used previously in the chapter 3 methods, using GFP polyclonal antibody (1:1000) with a
secondary HRP-conjugated anti-rabbit antibody for detection of LC3-GFP.
Reactive Oxygen Species
AGS cells were grown in 12-well plates and treated for 4 h as described. Cells were then washed
in PBS 2X, and 300μl of 0.25% trypsin (Wisnet) was added until cells detached. Trypsin was
neutralized with 300μl Ham’s F12. Cells were spun at 2500rpm for 10min at room temperature.
Cells were washed with buffer containing PBS plus 1% bovine serum albumin (BSA; Sigma
Aldrich) and 0.05% azide. Cells were then incubated with 20mM 5-(and-6)-chloromethyl-2'7'-
dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Invitrogen), a dye that
remains nonfluorescent until the acetate groups are removed by intracellular esterases and
oxidation occurs within the cell. Radical formation was assessed via flow cytometry using a
FacsCalibur flow cytometer.
Statistics
All experiments were performed at least 3 times, and using values obtained, P values were
calculated using ANOVA to compare the means ± SE for independent treatment groups. A P
value of less than 0.05 was determined to be statistically significant.
39
Chapter 3 H. pylori Activates the STAT3 Pathway in DCs, Causing DC Maturation
but Altering DC Cytokine Release
ABSTRACT
H. pylori colonizes the stomach of nearly half the world’s human population and is an important
risk factor in the development of gastric cancer. Infection with H. pylori persists for the lifetime
of the host, utilizing mechanisms to evade the host immune response. The effect of H. pylori
infection on DCs, key orchestrators of the host immune response, is unclear. Recently, our lab
demonstrated that H. pylori activates the STAT3 pathway in epithelial cells. STAT3 activation in
DCs effects maturation, activation and can subvert tumour immune surveillance. Therefore, we
hypothesized that H. pylori activates the STAT3 pathway in DCs, altering maturation, thereby
providing a mechanism by which this bacteria evades host immune responses. BMDCs were
infected with H. pylori for 4 or 24 hours and activation of STAT3 was assessed by
immunoblotting. DC activation and maturation was assessed by flow cytometric analysis and
cytokine analysis. Infection of DCs with WT H. pylori resulted in robust PSTAT3 activation
compared to control, however this activation was independent of CagA. Activation of PSTAT3
was also seen with heat-killed wild-type bacteria, indicating a possible role for heat stable LPS.
STAT3 activation was associated with an induction of DC maturation markers and a cytokine
profile of increased IL-10: IL-12, providing a possible mechanism by which immune evasion
occurs. These findings suggest that H. pylori mediated STAT3 activation in DCs promotes an
immunoregulatory phenotype which may promote chronic infection and disease.
INTRODUCTION
Gastric cancer is the second leading cause of cancer-related deaths worldwide (Peek, Jr. and
Crabtree, 2006). As a type I carcinogen, infection with H. pylori is a leading cause of gastric
cancer and due to its long term and chronic infection, likely alters host immune response for
bacterial survival.
Dendritic cells are key players that orchestrate host immune responses. DCs are antigen
presenting cells that detect infection through recognition of pathogen associated molecules to
either induce or prevent the initiation of adaptive immune responses. In addition to responding to
40
infection, DCs are also involved in tumour surveillance. Classically immature DCs were thought
to promote tolerance while mature DCs promote T cell immunity. However, current evidence
suggests that DCs can exist in a variety of functional forms with different immunogenic
capacities (Banchereau and Steinman, 1998;Tan and O'Neill, 2005).
Based on their ability to regulate both host immune responses and tumour surveillance, the effect
of H. pylori infection on DCs has been an area of interest. However, controversy exists with
respect to the effect of H. pylori infection on DC function. Several studies suggest that H. pylori
can induce DC maturation, upregulating CD80, CD83 and CD86 surface expression by an as yet
unknown mechanism (Kranzer et al., 2004;Rad et al., 2007). The functional outcome of this
maturation is unclear. Some groups report an increase in DC IL-12 secretion (Guiney et al.,
2003) and a Th1 response, whereas other groups demonstrate inhibition of DC IL-12 secretion
when compared to other bacteria (Kao et al., 2006;Obonyo et al., 2006). Differences in cell lines
could contribute to the differences noted by these groups, as Guiney et al. performed
experiments on human peripheral blood monocytes treated with IL-4, which preferentially
polarizes cells toward an IL-12/Th1 response (Guiney et al., 2003), whereas Kao and Obonyo
used BMDCs cultured from mice with IL-4 treatment (Kao et al., 2006;Obonyo et al., 2006).
Conversely, others have demonstrated that H. pylori causes gastric epithelial cells to promote
thymic stromal lymphopoietin causing increased DC production of IL-4 and IL-13, causing a
Th2 response (Kido et al., 2010). Recently, it has been suggested that H. pylori induces DCs to
secrete factors causing a Treg polarized response through secretions of IL-10 and TGFβ (Kao et
al., 2009;Romero-Adrian et al., 2010).
Our laboratory has shown that H. pylori activates the STAT3 pathway in epithelial cells (Bronte-
Tinkew et al., 2009). STAT3 is constitutively activated in many cancer cells but is also increased
in infiltrating immune cells. Recent evidence indicates that STAT3 can suppress anti-tumour
immunity by inhibiting proinflammatory cytokine secretion and upregulating
immunosuppressive cytokines such as IL-10 and VEGF. STAT3 activation in DCs has been
shown to inhibit maturation, as blocking STAT3 increases expression of surface markers MHC
class II and CD40 (Wang et al., 2004). Blocking STAT3 also induces increased IL-12, IL-10,
TNFα and IL-6 promoting the development of Treg cells (Wang et al., 2004). Additionally, in
liver DCs, constitutively elevated IL-6 and STAT3 levels help inhibit liver DC maturation,
preventing robust inflammation in response to commensal bacteria (Lunz, III et al., 2007). These
41
studies suggest that, STAT3 contributes to the inhibition of DC maturation, thereby dampening
the initiation of robust immune responses by DCs. Therefore, with this body of evidence
implicating STAT3 as a modulator of DC function, we wanted to determine if the STAT3
pathway is activated during H. pylori infection and what effects it has on the DC phenotype.
RESULTS
H. pylori infection induces tyrosine phosphorylation of STAT3
To determine if H. pylori activates the STAT3 pathway in DCs, we measured phosphorylated
STAT3 (PSTAT3) in cell lysates using immunoblotting. Cultured immature BMDCs were
infected with H. pylori WT strain 7.13 or treated with IL-6, a known STAT3 activator, for 4h.
Immunoblotting analysis of lysates demonstrated robust activation of PSTAT3 with WT H.
pylori infection and IL-6 treatment in comparison with uninfected control cells (Figure 3-1A,
C). As we had previously shown that CagA is responsible for STAT3 activation in epithelial
cells we next determined if CagA was involved by utilizing the isogenic cagA mutant. Infection
with cagA- bacteria increased PSTAT3 in a manner similar to WT (Figure 3-1A) indicating that
phosphorylation of STAT3 is independent of the CagA virulence factor. Next we determined if
heat-stable proteins were required by employing heat killed (HK) bacteria. In comparison with
control cells, lysates from cells infected with HK bacteria showed increased PSTAT3 (Figure 3-
1B). However the degree of PSTAT3 was decreased in comparison with infection with the
wildtype strain, indicating that although heat-stable factors may contribute to STAT3 activation,
live WT bacteria are needed for full PSTAT3 activation.
Cont
rol
IL-6
WT
7.13
cagA
- 7.1
3
PSTAT3
STAT3
Cont
rol
IL-6
WT
7.13
HK 7
.13
PSTAT3
STAT3
* P < 0.05
A
B
C
42
43
Figure 3-1: H. pylori activates the STAT3 pathway in DCs.
A) Lysates from DCs incubated with IL-6 (100ng/ml, 30 min) or infected with H. pylori WT
strain 7.13, isogenic mutant cagA- or B) HK bacteria (MOI 25:1) for 4h were used for
immunoblotting to detect changes in PSTAT3 . C) Graph represents densitometric analysis of
PSTAT3 bands obtained for each protein signal normalized to native STAT3 bands. Fold
increase is given as a ratio of control PSTAT3/STAT3. Columns, mean; bars, SE, *, P<0.05,
using one-way ANOVA (n=6).
44
H. pylori induces expression of DC maturation markers
We next determined the effect of H. pylori infection on maturation of DCs. BMDCs were
infected with WT, HK H. pylori or incubated with IL-6 or E. coli LPS (positive control) for 24h.
Flow cytometric analysis was utilized to quantify expression of DC maturation markers CD86,
CD80, and MHC class II. Cells positive for both MHC class II and one other marker were
determined to be activated and mature. To ensure that a pure population of DCs was used, cells
were stained and gated for the CD11c+ and CD11b
+ surface markers (Figure 3-2A). Cells were
also stained for Gr1 to ensure the cells were not granulocytes (data not shown). Infection with
WT H. pylori increased the proportion of CD86+, MHC class II
+ cells comparable to treatment
with LPS (Figure3-2C).(Figure 3-2B). Infection with HK bacteria also increased the proportion
of positive cells albeit to a lesser degree than WT bacteria. Similar results were observed when
assessing the markers CD80+ and MHC class II
+ (Figure 3-2D). Infection with WT bacteria
increased the proportion of these cells compared to control cells. However, incubation with HK
bacteria did not increase the expression of these maturation markers above control cells (Figure
3-2E). Incubation with IL-6, which activated the STAT3 pathway comparable to H. pylori
infection, did not result in increased expression of maturation markers on DCs compared to
control cells. These studies demonstrate that H. pylori infection induces DC maturation.
FSC
103
102
101
100
0 200 400 600 800 1000
SSC
103
102
101
100
103102101100
CD
11b
A
49.7688.93
C
CD11c
B
103
102
101
100
CD86
MH
CII
Control LPS
103102101100
CD86
MH
CII
103
102
101
100
103102101100
CD86
MH
CII
IL-6
WT 7.13
103
102
101
100
103102101100
CD86
MH
CII
103
102
101
100
CD80
MH
CII
CD80
MH
CII
CD80
MH
CII
Control LPS IL-6
WT 7.13
CD80
103
102
101
100
MH
CII
103
102
101
100103102101100
103102101100103102101100 103102101100
103
102
101
100
103
102
101
100
103102101100
17.60 5.69
72.06 4.66
15.56 16.81
59.15 8.48
12.69 11.83
68.49 7.00
18.62 3.06
76.05 2.26
HK 7.13
103
102
101
100
103102101100
CD86
MH
CII
16.78 9.38
70.27 3.57
10.30 6.87
76.11 6.72
9.94 12.53
70.69 6.48
7.41 10.15
79.57 2.86
10.45 4.17
79.28 6.11
HK 7.13
103
102
101
100
CD80
MH
CII
103102101100
9.16 8.86
77.97 4.01
DE
*
*
45
46
Figure 3-2: H. pylori infection upregulates surface expression of DC maturation markers.
A) BMDC purity was determined using staining for CD11c+ and CD11b
+ and analyzed using
FACS analysis. B) DCs were incubated with either E. coli LPS (1μg/ml), IL-6 (100ng/ml), WT
bacteria (MOI 25:1) or HK bacteria for 24h. FACS analysis was used to determine the proportion
of cells positive for maturation markers MHC class II and CD86. Cells in upper right quadrant of
each scatter plot demonstrate the percentage of cells positive for both maturation markers. C)
Quantification of CD86+ and MHC class II
+ cells normalized to control. D) Scatter plots
represent cells stained with MHC class II and CD80. Upper right quadrants represent cells
positive for both surface markers. E) Quantification of CD80+ and MHC class II
+ cells. Columns,
mean; bars, SE;*, P < 0.05 using one way ANOVA, n=5.
47
H. pylori stimulated DCs induce an unusual cytokine profile
To determine the functional effects of H. pylori induced DC maturation, the cytokine expression
profile was assessed using the corresponding culture supernatants of BMDCs. Using a Luminex
multiplex panel, we examined a Th1/Th2 panel of cytokines. Of all the cytokines profiled in this
kit (IL-2, IL-4, IL-5, IL-10, IL-12, IL-17 and IFN-γ) only IL-12 (p40/p70) and IL-10 had levels
within detectable limits (Figure 3-3A). In comparison with supernatants obtained from either
control or IL-6 treated cells, WT infection increased the levels of both IL-12 and IL-10.
Supernatants from DCs incubated with HK bacteria showed a slight increase in IL-10 secretion
but not to the same levels as cells incubated with WT bacteria as results were not statistically
significant. When we compared the ratio of IL-10:IL-12 (Carrieri et al., 2008;Hwang et al.,
2007;Stax et al., 2008) infection with WT H. pylori increased the IL-10:IL-12. In contrast, the
proinflammatory E. coli LPS exerted an increased IL-12:IL-10 ratio (Figure 3-3B).
To further assess the cytokine profiles we employed ELISA-based assays. We first confirmed the
increase in IL-10 and IL-12 p40 with WT bacteria stimulation with ELISA analysis and an
increased immunosuppressive ratio of IL-10:IL-12 (Figure 3-4A, B). We could not detect an
increase in IL-12 p70 using the ELISA based assay. Additionally, we also saw an increase in IL-
6 and TNF-α stimulation with WT infection. (Figure 3-4A).
**
****
A
B
48
49
Figure 3-3: Luminex assay of H. pylori induced DC cytokine secretion.
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) WT or HK bacteria (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.
**
**
*
**AA
B
50
51
Figure 3-4: Determination of H. pylori induced DC cytokine secretion by ELISA based
assay.
A) ELISA was used to detect cytokine secretion for IL-10, IL-12, IL-6 and TNF-a in cell
supernatants obtained from BMDCs incubated with either E. coli LPS (1μg/ml), IL-6 (100ng/ml)
WT or HK bacteria (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). B)
Graph depicts ratios of IL-10:IL-12 and IL-12:IL-10.
52
H. pylori induced DC maturation and cytokine release is dependent on activation of the
STAT3 pathway.
To determine if the activation of the STAT3 pathway was responsible for the DC phenotype
observed, we employed the STAT3 inhibitor S3I-201. S3I-201 specifically inhibits the SH2
domains on STAT3 monomers, preventing dimerization and phosphorylation of the STAT3
pathway. S3I-201 inhibits constitutive Stat3 DNA-binding and transcriptional activities but has
no effect on Src activation or the EGFR-mediated activation of the Erk1/2(MAPK) pathway,
indicating that this inhibitor is specific for the STAT3 pathway (Zhang et al., 2010).
Cells were pretreated with S3I-201 for 30 min followed by incubation with WT bacteria or IL-6
treatment and the DC phenotype determined. Phosphorylation of STAT3 was inhibited in cells
pretreated with the STAT3 inhibitor and infected with WT H. pylori as assessed by
immunoblotting (Figure 3-5A). In addition, a decrease in expression of the DC maturation
markers was observed in comparison to cells infected with WT bacteria alone (Figure 3-5B,
n=2). Furthermore, S3I-201 pre-treatment prevented H. pylori-mediated secretion of IL-12p40,
IL-10 and TNF-α (Figure 3-5C). S3I-201 pre-treatment also resulted in a decrease in levels of
IL-6, although this did not reach significance. In addition, the IL-10:IL-12 ratio observed with
WT bacteria alone was reduced with S3I-201 treatment, (Figure 3-5D).
A
B
C
D
*** ***
** **
IL-6
WT 7
.13S3I-
201 +
WT 7
.13
PSTAT3
STAT3Con
trol
53
54
Figure 3-5: H. pylori induced DC maturation and cytokine secretion is dependent on
activation of the STAT3 pathway.
A) Western blot image of DC cell lysates obtained from cells pretreatment with S3I- 201 for
30min followed by incubation with WT bacteria for 24h. Corresponding blot below demonstrates
native STAT3 levels. B) Cells were incubated with WT bacteria (MOI 25:1) or S3I-201 (25μM)
(30 min pretreatment) plus WT bacteria for 24 h. Cells were assessed via flow cytometry for
expression of surface markers MHC class II, CD86 and CD80. Graphs represent cells positive
for either MHC class II and CD86 (left) or MHC class II and CD80 (right) (n=2). C) ELISA
analysis of DC culture supernatants. Columns, means; bars, SE; **, P < 0.01; ***, P < 0.001
using one-way ANOVA (n=3). D) Ratios of IL-10:IL-12 from mean ELISA
55
S3I-201 does not affect DC viability.
To ensure that treatment with S3I-201 does not affect DC viability, an MTT assay was utilized.
BMDCs were grown and infected for 24h as described. Viable cells contain mitochondrial
dehydrogenase, which cleaves the tetrazolium substrate ring, yielding purple formazan crystals.
Analysis demonstrated that cell viability is not affected with S3I-201 infection (Figure 3-6).
56
57
Figure 3-6: S3I-201 does not affect BMDC viability.
MTT analysis of DCs. DCs were incubated with IL-6 (100ng/ml), WT bacteria (MOI 25:1) or
S3I-201 (25μM) plus WT bacteria for 24 h. Spectrophotometric measurement of cell viability by
mitochodrial dehydrogenase activity which converts MTT to a water-insoluble colored formazan
derivative which is then solubilized in acidic isopropanol.
58
DISCUSSION
An increasing body of work is emerging studying the effects of H. pylori on DCs, linking
alterations in DC response to immune evasion. Despite the increasing interest, the exact
mechanisms by which H. pylori affects DC function is not known. In this study, we demonstrate
for the first time that H. pylori activates the STAT3 pathway in DCs. Additionally, we show that
H. pylori induces DC maturation as assessed by upregulation of the maturation markers MHC
class II, CD80 and CD86. Finally we show that H. pylori infected DCs cytokine profile shows an
enhanced IL-10: IL-12 ratio indicating a immunoregulatory phenotype. Both the DC maturation
and phenotype was dependent on STAT3 activation suggesting that STAT3 activation in DCs
may be a mechanism by which the bacterium subverts the host immune response.
We have shown that H. pylori mediated STAT3 activation in DCs occurs independently of the
CagA virulence factor and that live bacteria are required for maximal activation. However, our
studies also implicate a role for heat stable factors in this activation (e.g. LPS). In support of this
finding, Samavati et al. demonstrated that E. coli LPS can activate the STAT3 pathway in RAW
246.7 cells and BMDCs, leading to increased IL-1β, IL-6 and TNF-α production (Samavati et al.,
2009).
Use of the new inhibitor S3I-201, which blocks the phosphotyrosine binding region of the SH2
domain on STAT3, demonstrates the necessary involvement of STAT3 activation in H. pylori
induced DC maturation and cytokine release. Near-complete reversal of DC maturation and
cytokine release was seen when cells were pretreated with S3I-201 before WT incubation,
demonstrating for the first time the importance of STAT3 activation in these processes. Although
this inhibitor is thought to be specific it remains possible that other nonspecific effects may
occur. The use of additional mechanisms to inhibit STAT3, are now required to confirm the
STAT3 dependance on the observed DC phenotype.
STAT3 activation in immune cells is thought to have immunosuppressive effects. Lin and
collegues have demonstrated that Salmonella enteric serovar Typhimurium activates STAT3 in
macrophages, limiting excessive inflammatory responses and providing a hypothesized
mechanism of Salmonella’s immune evasion (Lin and Bost, 2004). Previous studies have
demonstrated that increased STAT3 activation in DCs can limit the inflammatory responses of
bacterial products. In liver DCs, STAT3 activation is upregulated. When challenged with
59
bacterial LPS, IL-6 release is noted, however due to elevated STAT3, DC activation is limited
(Lunz, III et al., 2007). STAT3 activation inhibits the production of DC IL-12, reducing
proinflammatory effects and downregulating the Th1 response (Wang et al., 2004). STAT3
inhibits IL-12p35 gene expression in tumour-associated DCs but the paralysis of tumour DCs
into an immature state has been shown to be reversed by blocking IL-10 activity, allowing de
novo IL-12 production (Vicari et al., 2002).
Additionally, STAT3 signalling has been linked to decreased tumour surveillance, decreasing the
ability of DCs to mature in a tumour environment (Lin et al., 2010c). STAT3 has also been
shown to inhibit DC maturation through the secretion of inhibitory IL-10, promoting the
development of inhibitory DCs (Corinti et al., 2001). Due to the role of STAT3 in directing DC
tumour immunosurveillance, our studies may demonstrate a mechanism by which H. pylori alters
DC signalling to induce tumourigenesis.
In our studies, we see a paradoxical increase in both IL -10 and IL-12 with H. pylori stimulated
STAT3 activation. However increases were only seen in IL-12p40, and not IL-12p70 (data not
shown) in our studies. IL-12p70 is the biologically active form of IL-12 composed of two
covalently linked chains of 40 and 35 kDa (IL-12p40 and IL-12p35) and is associated with
increased proinflammatory responses. Since in our studies, only IL-12p40 was induced, we
hypothesize that the increase in IL-12 with H. pylori infection does not induce a pro-
inflammatory response due to the lack of IL-12p70 formation, instead producing only IL-12p40.
Instead, IL-10 secretion is of greater importance. We report an increased IL-10:IL-12p40 ratio
observed with H. pylori infected DCs when compared to proinflammatory E.coli LPS. These
observations indicate a shift in importance from a Th1-IL-12 phenotype to an IL-10 response,
especially when noting the lack of IL-12p70. TNF-α elevation in response to H. pylori infection
seen in our studies may appear contradictory as TNF-α is known to be a proinflammatory
cytokine and associated with a Th1 response. However, evidence shows that TNF-α has also
been proven to be critical for the generation of IL-10 producing CD4+ T cells during the antigen
presentation by immature DCs, therefore providing an immunosuppressive role for TNF-α in our
DCs (Hirata et al., 2010).
Recently, activation of the STAT3 pathway has been linked to the development of a new class of
inflammation, the IL-23/Th17 response. The presence of only IL-12p40 can have increased
60
significance when considering that IL-23 shares the p40 chain and some properties with IL-12
and enhances Th17 responses. Reports have indicated that shifts from a anticarcinogenic IL-12
response to a pro-carcinogenic IL-23 is regulated through STAT3 signalling, leading to the
development of T-regulatory (Treg) cells, which would also induce IL-10 expression
(Kortylewski et al., 2009). We thus hypothesize that the increase in IL-10, IL-12p40 and IL-
10:IL-12 preferentially induces the development of Treg cells and possibly activation of the
Th17 pathway.
In conclusion, this study indentifies that H. pylori induces activation of the STAT3 signalling
pathway in DCs which is responsible for an induction of DC maturation, as well as an altered
cytokine profile, shifting the balance toward a potential Treg/Th17 polarization. These studies
indicate a novel mechanism by which H. pylori alters the innate immune response to prevent
elimination by the host and also potentially subvert tumour surveillance. Thus, the findings from
this study set the stage for investigating the effect of STAT3 inhibition on disease outcome
during H. pylori infection.
61
Chapter 4 Helicobacter pylori’s VacA Toxin Induces Reactive Oxygen Species
Production and Alters Parkin Distribution
INTRODUCTION
Oxidative stress has been noted in many tumours and is caused by an increase in the generation
of reactive oxygen species (ROS) alongside a decreased ability of the cell to clear these ROS.
High levels of ROS are associated with damage of proteins, lipids and DNA and all major
cellular constituents, whereas low ROS levels has been linked to the activation of intracellular
pathways leading to proliferation, promoting angiogenesis and metastasis (Droge,
2002;Weinberg and Chandel, 2009). ROS generation occurs with oxygen reduction, resulting in
the formation of the hydroxyl radical (•OH) and superoxide (•O2), which are easily integrated
into the activation of signalling pathways and react with iron to generate hydroxyl radicals
(Imlay et al., 1988).
Several sources of ROS have been identified. NADPH oxidase is one of the best studied sources
of •O2, catalyzing the production of •O2 from oxygen and NADPH (Lambeth, 2004). The
mitochondrial electron transport chain is also a major site of non-enzymatic •O2 formation, with
•O2 being created at complexes I, II and III. Additionally, the H2O2 created in these steps can be
further converted to •OH after contact with iron. Mitochondrial production of ROS is also an
important factor for the overall ROS level within a cell because it underlies oxidative damage in
many pathologic conditions, contributing to retrograde redox signalling from organelle to the
cytosol and the nucleus. Accumulation of mitochondrial derived ROS can lead to lipid
peroxidation, mitochondrial dysfunction, apoptosis and necrosis (Murphy, 2009). Additionally,
the ROS produced by the mitochondria have been reported to regulate autophagy and autophagic
cell death, as autophagy is induced by rotenone, H2O2, and superoxide (Chen and Gibson, 2008).
Recently, Parkin a gene first implicated in juvenile Parkinsonism has been shown to be recruited
to damaged, depolarized mitochondria selectively targeting these mitochondria for degradation
by autophagy (Narendra et al., 2008). Parkin is an E3-ligase, responsible for conjugating
ubiquitin to proteins, targeting them for degradation via the ubiquitin-proteasome degradation
pathway, where misfolded proteins are transported to aggresomes whose clearance is facilitated
by autophagy (Farrer, 2006;Olzmann and Chin, 2008). Mutations in the Parkin gene result in the
62
development of hepatocellular carcinoma, and the loss of Parkin contributes to the
overproliferation of hepatocytes (Ikeuchi et al., 2009). These combined observations indicate
that the Parkin gene may be important in tumourigenesis and cell cycle regulation in epithelial
cells (Farrer, 2006). Studies using Parkin knock-out mice demonstrated deficiencies in proteins
involved in mitochondrial function and oxidative stress, including reductions in proteins
involved in complexes I and IV (Palacino et al., 2004). Parkin deficient mice also exhibited
decreased levels of proteins involved in protection from oxidative stress, as well as decreased
serum antioxidant capacity and increased protein and lipid peroxidation, indicating that Parkin
mutations are associated with mitochondrial dysfunction (Palacino et al., 2004).
We have shown that H. pylori’s VacA activates the autophagy pathway. However the exact
mechanisms involved remain unknown. VacA localizes to mitochondria where it forms channels
in the membrane, reducing mitochondrial transmembrane potential, causing mitochondrial
dysfunction and cytochrome c release (Galmiche et al., 2000;Willhite et al., 2003;Willhite and
Blanke, 2004a). Therefore, we hypothesized that VacA may induce increased mitochondrial
derived ROS and Parkin redistribution to trigger autophagy. Therefore we determined if VacA
altered ROS production and Parkin distribution in gastric epithelial cells in vitro.
RESULTS
H. pylori’s VacA toxin induces ROS production
To determine if VacA increased ROS production, AGS cells were treated for 4 hours with
inactive or activated pure toxin and ROS measured using CM-H2DCFDA, a compound that
fluoresces when in contact with ROS, and assessed by FACS. As a positive control cells were
incubated with H2O2 (200µM, 40min). Activated VacA induced increased ROS production
comparable to levels seen in cells treated with H2O2 (Figure4 -1B). In marked contrast, ROS
levels were not increased in cells treated with inactive VacA toxin and resembled control
untreated cells (Figure 4-1A, B). We next determined if antioxidants could reduce VacA-
mediated ROS production. The antioxidants DPI, rotenone and α-tocopherol all failed to prevent
ROS induction (data not shown). However, in cells pre-treated with 5mM N-acetyl-cysteine
(NAC) VacA-mediated ROS was prevented (Figure 4-1A, B). Taken together, these results
demonstrate that VacA induces the production of ROS, which can be inhibited using NAC
treatment.
A
NAC
103102101100
100
60
20
80
0
40% o
f max
FL-1
12.2824.33
NAC + pure toxin
103102101100
100
60
20
80
0
40% o
f max
FL-1
12.2829.53
0 200 400 600 800 1000
FSC
SSC
0
200
400
600
800
1000
H2O2
% o
f max
FL-1
12.2874.81
103102101100
100
60
20
80
0
40
Pure VacA Toxin%
of m
ax
12.2859.25
FL-1103102101100
100
60
20
80
0
40
Inactive VacA Toxin
103102101100
100
60
20
80
0
40% o
f max
FL-1
12.2825.44
FL-1
# o
f cel
ls
Control
200
100
0
300
103102101100
12.28
B**
* *
63
64
Figure 4-1: VacA pure toxin induces ROS production in AGS cells.
AGS cells were incubated with activated VacA pure toxin or inactive toxin for 4h. Where
indicated, NAC (5mM) was utilized alone, or with pure toxin. Cells incubated with H2O2
(200μM, 30min) served as a positive ROS control. Cells A) ROS production was measured by
using a redox-sensitive dye (CM-H2DCFDA) on live cells, followed by flow cytometry analysis.
Overlaid curves indicate fluorescence curve for condition stated (green) versus control curve
(red) with corresponding numbers representing the percentage of ROS + cells assessed within the
gated region. B) Graph shows quantification of ROS experiments. Fold increase is expressed as
ratio of the %ROS+ cells in each condition/control. Columns, means; bars, SE; *, P < 0.05, **, P
< 0.01, using one-way ANOVA (n=3).
65
Inhibition of VacA induced ROS does not prevent VacA induced autophagy.
To determine if VacA induced ROS is responsible for VacA-mediated autophagy, we used
immunofluorescence to detect the redistribution of cytosolic LC3I to autophagic, membrane-
bound LC3II puncta. AGS cells were transfected with LC3-GFP plasmid and treated with EBSS
starvation conditions (a potent inducer of autophagy), or inactive or activated VacA pure toxin
for 4 hours in the presence or absence of NAC. As shown previously, starvation was sufficient to
induce the formation of autophagosomes indicated by green puncta formation (Figure 4-2A,
arrowheads). Similarly active but not inactive VacA induced the formation of LC3 puncta
(Figure 4-2A). However, treatment with NAC prior to incubation with active VacA did not
reduce the percent of autophagic cells (Figure 4-2A, B). These results suggest that inhibition of
VacA induced ROS does not alter VacA induced autophagy, indicating that ROS production and
autophagy induction by VacA occur independently.
Control Starvation
Pure VacA Toxin Inactive VacA Toxin
NAC NAC + Pure VacA
LC3-GFP
LC3-GFP
LC3-GFP
LC3-GFP
LC3-GFP
LC3-GFPContro
l
Starva
tion
Pure Toxin
Inactiv
e Toxin NAC
NAC + pure
toxin0
10
20
30
40
50
60
70
% A
utop
hagi
c ce
lls
*
*** ***
***
A
B
66
67
Figure 4-2: VacA induced ROS production is not responsible for VacA induced autophagy.
A) Confocal mircographs of AGS cells transfected with LC3-GFP (green) incubated with
activated VacA pure toxin, inactive toxin, NAC alone and NAC pretreatment followed by pure
toxin incubation. Starvation was used as a positive control for autophagy. Autophagy induction
is denoted by the formation of LC3 puncta (white arrowheads). Scale bars equals 15 μM. B)
Quantification of the percent autophagic cells for each treatment. Columns, means; bars, SE; *, P
< 0.05, ***, P < 0.001, using one-way ANOVA (n=4).
68
VacA induced autophagosomes do not colocalize with the mitochondria
VacA can localize to mitochondria and induce a variety of effects including increasing
mitochondrial permeability and the release of cytochrome c (Galmiche et al., 2000;Kimura et al.,
1999). Mitochondrial damage can induce mitochondrial specific autophagy (termed mitophagy),
particularly if the mitochondrial membrane potential is altered (Blanke, 2005;Kim et al., 2007b).
Therefore, due to VacA’s ability to alter the mitochondria, we next determined if VacA induced
mitophagy.
AGS cells transfected with LC3-GFP and the mitochondria marker Mito-RFP which fluoresces
in viable mitchondria were incubated with VacA pure toxin for 4h. Cells were then stained with
HSP60 (a mitochondrial marker staining the mitochondrial matrix) antibody with Cy5
conjugation to ensure all mitochondria were labelled. Immunofluorescence was used to
determine colocalization of the different markers.
Under starvation conditions, LC3-GFP puncta did not colocalize with mitochondrial markers as
seen in the merged image, indicating that the autophagosomes were not associated with the
mitochondria (Figure 4-3). Similarly, in cells treated with active purified VacA the majority of
autophagosomes did not colocalize with mitochondrial markers (Figure 4-3). Furthermore, NAC
pretreatment did not affect the degree of mitochondrial and LC3 colocalization (Figure 4-3).
Therefore, VacA induced autophagy does not selectively target mitochondria, to induce
mitophagy.
Control
Starvation
Pure VacA Toxin
Inactive VacA Toxin
NAC
NAC + Pure VacA Toxin
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
LC3-GFP Mito-RFP Hsp60-Cy5 LC3-GFPMito-RFPHsp60-Cy5
69
70
Figure 4-3: VacA induced LC3 aggregates do not colocalize with mitochondrial antibodies.
Confocal micrographs of AGS cells expressing LC3-GFP (green) and Mito-RFP (red) treated for
4h with either starvation media, pure activated VacA toxin, inactive toxin, NAC alone or NAC
pretreatment for 30min followed by incubation with pure toxin. HSP60 was immunolabelled
utilizing Cy5 (blue) conjugated secondary antibody. Arrows point to autophagosome formation.
Bars equal 15μM. Insets represent magnification of boxed area. Experiment was performed 3
times with representative images from one experiment shown.
71
H. pylori’s VacA toxin causes Parkin redistribution that is not dependent on VacA induced
autophagy
Although we did not demonstrate that VacA induced mitophagy, this process may be cell type
specific. Therefore we next determined if VacA mediated effects on mitochondria may alter
Parkin distribution. AGS cells were transfected with LC3-RFP and Parkin-YFP plasmids and
infected for 4h. Rotenone (50μM), which causes inhibition of the electron transport chain at
complex II and thus mitochondrial instability, was used as a positive control for Parkin
aggregation. Cells were assessed via spinning disc microscopy to determine if Parkin and LC3
aggregates colocalize. As expected, a diffuse cytosolic distribution of LC3 and Parkin was
detected in control cells (Figure 4-4A). Rotenone induced Parkin redistribution but no change in
LC3 compared to control cells (Figure 4-4A). Cells treated with inactive toxin resembled control
cells, (Figure 4-4A). In contrast, in cells incubated with pure toxin LC3 and Parkin were
redistributed in aggregates(Figure 4-4A, B). However significant colocalization of LC3 and
Parkin aggregates was not detected. Furthermore, LC3 and Parkin aggregation was not always
seen in tandem, as some cells contain only LC3 aggregation, while others have only Parkin
redistribution (Figure 4-4C). In addition, NAC treatment which reduced ROS failed to prevent
pure toxin induced Parkin redistribution. These results indicate that VacA treatment can cause
Parkin redistribution. However VacA’s ability to induce autophagy appears to be independent of
Parkin aggregation and VacA induced ROS.
Control
Starvation
NAC
NAC + Pt
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
Inactive Toxin
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
Pure Toxin
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
Contro
l
Starva
tion
Roteno
ne
Pure V
acA Tox
in
Inacti
ve Tox
inNAC
NAC + Pure
Toxin
05
101520253035404550556065
% Autophagic% (+)Parkin aggregation
% o
f cou
nted
cel
ls
AB
Control
Starva
tion
Rotenone
Pure Toxin
Inactiv
e Toxin
0
10
20
30
40
% c
ells
with
par
kin
aggr
egat
ion
C
**
Rotenone
LC3-RFP Parkin-YFP LC3-RFPParkin-YFP
72
73
Figure 4-4: VacA pure toxin induces redistribution of Parkin that is independent of its
induction of autophagy.
A) Confocal micrographs of AGS cells expressing Parkin-YFP (green) and LC3-RFP (red)
treated with pure activated VacA toxin, inactive toxin, NAC alone or NAC pretreatment for
30min followed by incubation with pure toxin for 4h. Arrows represent autophagosome
formation in LC3-RFP panels or Parkin aggregates in Parkin-YFP panels. Insets show
magnifications of Parkin redistribution and autophagy formation. B) Graph shows the percent of
cells with Parkin aggregation. Columns, means; bars, SE; **, P < 0.01, using one-way ANOVA
(n=4). C) Graph demonstrates percent of autophagic cells (light grey) or Parkin redistributed
cells (dark grey). Columns, means; bars, SE (n=4).
74
VacA induced Parkin aggregation does not colocalize with the mitochondria.
Since Parkin aggregation has been shown to selectively target the mitochondria for autophagic
degradation, we next examined if VacA toxin induces Parkin recruitment to the mitochondria.
AGS cells were transfected with LC3-RFP and Parkin-YFP plasmids and treated for 4h with
VacA pure toxin or starvation conditions (Figure 4-5). Cells were subsequently stained with
HSP60 Cy5 and examined using spinning disc microscopy. Colocalization between Parkin
aggregates and mitochondrial antibodies was not detected. Therefore, despite Parkin’s ability to
induce mitophagy, VacA induced Parkin aggregation does not lead to mitochondrial autophagy.
LC3-RFP Parkin-YFP HSP 60 Cy5
LC3-RFP Parkin-YFP HSP 60 Cy5
LC3-RFP Parkin-YFP HSP 60 Cy5
Control
Starvation
Pure Toxin
75
76
Figure 4-5: VacA induced Parkin aggregation does not target to the mitochondria.
Confocal micrographs of AGS cells expressing Parkin-YFP (green) and LC3-RFP (red) treated
for 4h with either starvation conditions or pure activated VacA toxin. Cells were stained for
HSP60 conjugated to Cy5 and colocalization between HSP60 and Park-YFP was not noted.
Insets show magnifications of Parkin redistribution and autophagy formation.
77
DISCUSSION
Autophagy is a mechanism used by host cells to eliminate intracellular pathogens, having a role
in antigen processing and influencing the host immune response. We have shown previously that
H. pylori’s VacA toxin is capable of inducing autophagy in gastric epithelial cells. VacA can
affect mitochondria. The toxin localizes to the mitochondria where it forms channels in its
membrane, disrupting mitochondrial membrane potential and inducing the release of cytochrome
c. Since alterations in mitochondria can induce autophagy we determined if VacA’s
mitochondrial effects induce the production of ROS, thereby inducing VacA induced autophagy.
In our studies, autophagosomes induced by VacA toxin did not colocalize with mitochondria
suggesting that VacA induced autophagy was not specifically targeting the mitochondria for
degradation. However, due to VacA’s ability to interact with the mitochondria, we further
investigated the role of ROS.
The production of ROS has been previously linked with H. pylori infection. Increased reactive
oxygen metabolites have been found in H. pylori positive patients, but not in H. pylori negative
patients (Davies et al., 1994). Additionally, H. pylori induces enhanced production of ROS in
gastric cells, and enhances membrane damage (Bagchi et al., 1996). H. pylori can also induce the
recruitment and activation of monocytes and neutrophils that create ROS (Mashimo et al., 2006).
H. pylori induces ROS with increasing levels of DCF fluorescence as well showing increasing
levels of MitoSOX Red fluorescence, an indicator of mitochondrial superoxide production
(Calvino-Fernandez et al., 2008). As well, an increase in ROS-mediated DNA oxidation is seen
in the gastric mucosa upon H. pylori infection, and these levels are reduced upon H. pylori
eradication (Katsurahara et al., 2009). Therefore, ROS production in association with H. pylori
infection is well established.
Our studies are the first to demonstrate that VacA alone can induce ROS production. The origin
of these ROS remains unknown. Lack of ROS inhibition by DPI, which specifically blocks
NADPH oxidase, indicates that the NOX-2 pathway is not responsible for ROS production in
VacA treatment despite being implicated in other antibacterial autophagic processes (Huang and
Brumell, 2009). We speculate that VacA interaction with mitochondria may be the source of
ROS induction. However, further studies are required to define the exact source of ROS.
78
ROS production can induce the autophagic process, with ROS being specifically required to
regulate Atg4 activity (Scherz-Shouval et al., 2007), and superoxide being responsible for
regulating autophagy (Chen et al., 2009). However the ROS production in our studies does not
seem to play a role in VacA induction of autophagy, as NAC (a whole-cell antioxidant)
treatment, which reduces ROS did not inhibit VacA induced autophagy. Host cells may induce
ROS to limit bacterial replication (Mastroeni et al., 2000). VacA increases intracellular bacterial
survival, (Terebiznik et al., 2006) therefore host cells may induce ROS production to limit VacA
induced intracellular survival and reduce microbial burden.
Since ROS production did not appear to be the cause of VacA induced autophagy, we next
looked at the E3-ligase Parkin to examine if VacA can induce Parkin aggregation. Parkin has
recently been linked to autophagy in a mitochondrial specific manner (Narendra et al., 2008).
Our studies demonstrate that VacA can cause re-distribution of Parkin. These Parkin aggregates
did not appear to colocalize with VacA (data not shown) or mitochondrial antibodies. The
significance of VacA induced Parkin aggregation is not yet known.
In summary, we demonstrate that VacA toxin induces ROS production in AGS cells. However
these ROS do not mediate autophagy. Parkin aggregation is also seen with VacA incubation, and
the implications of this are not yet known. Although the specific mechanisms by which H. pylori
induces autophagy are still unknown, knowledge that VacA induces ROS could potentially
provide an avenue by which host cells limit VacA bacterial survival. These studies provide the
platform for future studies that may elucidate the specific mechanisms by which autophagy is
induced, providing more insight into H. pylori’s effects on host immune response.
79
Chapter 5 Discussion and Future Directions
Affecting or altering the host immune response is crucial in the long term infection and survival
of H. pylori. Alterations in innate immune signalling ultimately determine the outcome of the
bacterial infection, orchestrating the response of secondary T-cells, and coordinating the adaptive
immune response.
Two key components of the innate immune response are the activation of DCs and the induction
of autophagy. Both play crucial roles in detecting and responding to pathogenic bacteria
infection and the manipulation of these responses by bacteria are crucial in their survival. In our
studies, we determined that H. pylori alters the responses of both the autophagic pathway and the
activation of DCs, providing possible mechanisms for immune subversion by the bacterium.
In DCs, H. pylori activates STAT3 and induces maturation and activation of a unique phenotype
of DCs, differing from that seen with IL-6 treatment or induced by E. coli LPS. The cytokine
profiles induced by H. pylori are skewed to IL-10 induced immunoregulatory responses, likely
preventing robust pro-inflammatory responses and allowing H. pylori to avoid elimination by the
host innate immune response. STAT3 regulates the activity of IL-10, as STAT3 knock-out DCs
are resistant to IL-10 suppression of DC activation, allowing enhanced inflammatory cytokine
secretion (Melillo et al., 2010b). Thus the increased IL-10 and STAT3 signalling we detected
may be crucial to the regulation of DCs, These developments may assist in producing STAT3
based therapies to prevent H. pylori long term colonization.
Our studies also indicate the potential involvement of enhanced IL-23 secretion in H. pylori
infected DCs as discussed in chapter 3. STAT3 signalling has been linked to the development of
a Th17 response, inducing IL-17 and IL-23 expression (Harris et al., 2007;Yang et al., 2007). IL-
23 has been implicated in other chronic disorders such as inflammatory bowel disease (IBD),
Crohn’s disease and colitis as IL-23 deficiency or blockade protects from Crohn’s disease as well
as human IBD (Abraham and Cho, 2009). Similarly, the proposed initiation of an IL-23/Treg
response may be a mechanism by which H. pylori promotes long term and chronic infection.
Furthermore, it has been shown that STAT3 signalling within the tumour microenvironment
induces IL-23, which was shown to be a procarcinogenic cytokine, while inhibiting
80
anticarcinogenic, IL-12, thereby shifting the balance of tumour immunity toward
carcinogenesis(Kortylewski et al., 2009). IL-23 also has been linked to tumourigenesis and the
development of cancer. It has also been demonstrated that IL-23 has an important role in the
production of IL-17 producing CD8+ T-cells leading to the development of tumourigenesis and
disease (Ciric et al., 2009). As well, antibody-mediated blockade of the receptor for IL-23
inhibits Bacteroides fragilis, a commensal colonic bacterium, STAT3 induced colitis, colonic
hyperplasia and tumour formation (Wu et al., 2009).
The STAT3 pathway has been linked to oncogenesis, as STAT3 is persistently activated in many
human cancers and transformed cell lines (Bromberg et al., 1999). Indeed, STAT3 expression
correlates with a poor prognosis in gastric cancer (Kim et al., 2009). H. pylori’s activation of
STAT3 in DCs could promote oncogenesis. Recent evidence indicates that dendritic cells can
integrate signals from the tumour microenvironment to modulate tumour immunity (Lin et al.,
2010b). STAT3 hyperactivation has been observed in DCs infiltrating tumours and decreases
their immunostimulatory capacity thereby facilitating tumour immune evasion (Cheng et al.,
2003). Indeed, specific targeted deletion of STAT3 in DCs in mice confirms the role for STAT3
as a negative regulator of DC function (Melillo et al., 2010a). Hyperactivation of STAT3 in DCs
increases IL-10 production, common in the tumour microenvironment and similar to the results
we observed in H. pylori treated DCs (Lin et al., 2010a). Our findings suggest that in addition to
epithelial STAT3 activation, STAT3 activation in DCs could also be involved in promotion of
carcinogenesis during chronic H. pylori infection.
Using a specific STAT3 inhibitor, S3I-201 we observed a reversal of STAT3 induced DC effects
in vitro. Previous studies demonstrate that inhibition of STAT3 with JSI-124 can inhibit the
growth of STAT3 hyperactivated cell lines in mice as well as reduce the tumour burden of
melanoma cells (Blaskovich et al., 2003). Therefore our studies set the stage for investigating the
role of STAT3 as a promoter of a pro-tumour microenvironment due to DC alteration in vivo.
Autophagy induction can function as an innate defence mechanism by killing intracellular
microorganisms attempting to establish a replicative niche in the host cytoplasm (Levine and
Deretic, 2007). However, previous studies have shown several pathogens such as Listeria
monocytogenes and Shigella flexneri are able to evade autophagy to promote intracellular
survival (Hussey et al., 2009;Orvedahl and Levine, 2009). Results from our lab suggest that
81
during acute exposure, VacA-induced autophagy reduces the effects of the toxin in host cells.
The exact mechanisms by which VacA induces autophagy remain unknown and were the focus
of chapter 4. We did not identify VacA-induced mitophagy but did determine that VacA could
also cause redistribution of Parkin, a protein which localizes to damaged mitochondria. Our
studies also demonstrated that VacA induces ROS production, however VacA induced
autophagy is not dependent on ROS production. The significance of VacA induced ROS is not
yet known. Increased ROS levels are known to cause oxidative DNA damage, potentially leading
to deregulation of tumour suppressing elements such as p53, and enhancing tumourigenesis.
Thus VacA induced ROS may contribute to H. pylori induced carcinogenesis.
Manipulations of the innate immune response have been implicated in shifting the balance to a
protumour type of immunity (Diacovich and Gorvel, 2010). Although H. pylori evades
elimination by the host immune response, chronic inflammation is associated with infection.
Chronic inflammation has been associated with increased tumour risk, is involved in polarizing
immunity toward those effectors that facilitate tumour growth (Ostrand-Rosenberg, 2008). Thus
alteration and manipulation of the innate immune response by H. pylori can not only result in
successful immune evasion thereby promoting chronic infection and also promoting
tumourigenesis.
Overall, our results demonstrate new and novel mechanisms by which H. pylori may alter the
host immune response to promote long term survival. By unravelling the various mechanisms by
which H. pylori alters the innate immune response there will be a greater understanding into its
colonization, chronic infection and disease outcomes, leading to the development of new
preventative and treatment strategies .
FUTURE DIRECTIONS
The results of our studies investigating the effects of H. pylori infection on DCs have uncovered
several future directions that need to be addressed. To further define the mechanism of STAT3
mediated DC activation, a more specific STAT3 inhibitor, SFI-001, has been developed, which
binds the SH2 domains on STAT3 monomers. This new inhibitor can more definitively
demonstrate if STAT3 is responsible for the DC phenotype we observe.
82
Due to the postulated involvement of IL-23, it would also be of interest to examine DC secretion
for presence of IL-23. At the time of our studies, a mouse IL-23 kit was not available to us,
however examination of this cytokine would be a crucial next step.
Another future consideration would look into the functional effect of H. pylori infected DCs on
T-cells to definitively determine if these DCs would lead to the development of T-regulatory
cells. H. pylori stimulated DCs could be incubated with T-cells to determine the effects on T-cell
proliferation, cytokine secretion, and finally determining the overall T-cell polarization. It would
also be of interest to determine if the DC cytokine secretion we observed would be sufficient to
polarize T-cells to Treg cells. T-cells could be stimulated with DC supernatant secretion and
assessed for functional changes.
Mellilo and colleagues have recently developed a DC targeted STAT3 conditional knock-out
(CKO) mouse to determine specific effects of STAT3 in DCs (Melillo et al., 2010c). Using these
DCs, we could further define the role of STAT3 activation by H. pylori. To extend these studies
to human subjects STAT3 activation in DCs could be confirmed via immunofluorescence in
tissue sections of H. pylori infected stomachs.
The purpose and importance of VacA induced ROS needs additional study as well. The role of
ROS as a mechanism to limit intracellular bacterial survival could be assessed, using intracellular
survival assays to determine if inhibiting ROS leads to increased bacterial survival. Other aspects
of VacA induced ROS should also be examined, as the mechanism of ROS production is not yet
known. The lack of ROS inhibition by DPI, suggests that ROS production is not dependant on
the NOX/NADPH pathway. Use of other antioxidants could be used to characterize the ROS, as
well as examination via RTPCR for upregulation of genes related to oxidative stress.
The effects of VacA on endogenous Parkin needs further study. Little is known about the
potential effects of Parkin in epithelial cells as most studies focus on neuronal cells.
Additionally, although we did not see colocalization of VacA with Parkin at 4h, later timepoints
should be assessed to confirm these findings. Additionally, to determine the relevance of Parkin
aggregation siRNA to inhibit Parkin or essential autophagy genes would allow us to determine
definitively if Parkin redistribution is linked to the autophagy pathway, or if autophagy is
essential for Parkin redistribution. Finally, it would also be important to confirm these findings in
tissue sections to determine if Parkin aggregation plays a role in H. pylori infected tissues.
83
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