T cell responses in helicobacter pylori associated gastroduodenal diseases 1

T CELL RESPONSES IN HELICOBACTER PYLORI-ASSOCIATED GASTRODUODENAL DISEASES VICTORIA SERELLI LEE SHAN (B.SC. HONS, NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE 2012 Dedicated to My Ah Ma, the late Madam Kum Ah Peng (1930-2011), & Jess Lam AnF. I ACKNOWLEDGEMENTS I express my deepest gratitude to my two Ph.D. mentors, Assistant Professor, Dr Justin Wong Soon Boon, and Adjunct Professor, Dr Ling Khoon Lin, for their supervision and invaluable input into every step of my research. You have time and time again gone the extra mile for me. I especially thank the research nurses from the Department of Gastroenterology in the Singapore General Hospital, Celine Wong, Cathy Li, Yan Ping and Yvonne Lim, for faithfully helping me with patient sample collection every week for the past years, and Madam Lim Gek Keow for her assistance in performing HP serology tests. Thank you to the undergraduate students who have taken up parts of my research project - Christine Lee, Cassandra Ho, and Tan Hui Qing. Your help in the lab has been immense and I have learnt much from you during the short course of your research career. My friends in the programme – Hazel, thank you for your help in every way, everyday, I’m lost without you. Kar Wai, thank you for always being one step ahead of me. Fei Chuin, thank you for your advice in both lab and non-lab stuff. Veronique, thank you for your advice and help in picking up IF techniques. All other lab members, Daniel, Chenyu, Karen and Amos, thank you for the intellectual and not-so intellectual exchanges. II Finally, I thank both my parents and aunties Rosie and Betty. For letting me hog their car to travel between SGH and NUS. I would not have achieved as much in this years without this help. Thank you all for your un-ending and unconditional support during the course of my (very long) education. III TABLE OF CONTENTS Acknowledgements II Table of contents IV Summary VII List of Tables IX List of Figures X List of Abbreviations XII 1. Introduction 1.1. Immune evasion of pathogens – failure of the human immune system. 1.2. Helicobacter pylori-associated diseases. 1.3. Helicobacter pylori and gastric cancer risk. 13 1.3.1. Epidemiological studies 13 1.3.2. The COX-2 pathway 15 1.3.3. Genetic polymorphisms in immune genes 16 1.4. Adaptive immune responses to Helicobacter pylori. 18 1.4.1. Sensing HP bacterium 18 1.4.2. Protective T cell responses 19 1.4.3. Mouse models of HP infection 20 1.4.4. T regulatory responses 22 1.5. T helper-17 responses and cytokines associated with Helicobacter pylori infection. 24 1.6. Th17-Associated immunopathology 29 1.7. Rationale and hypotheses 34 2. Materials and Methods 37 2.1. Patient samples and classification. IV 37 2.2. Isolation and culture of peripheral blood mononuclear cells. 37 2.3. Isolation and expansion of primary human gastric epithelial cells and lamina propria mononuclear cells from gastric biopsies 38 2.4. GEC stimulation experiments 39 2.5. Culture of cell lines. 39 2.6. Intracellular antibody labelling and flow cytometry. 40 2.7. Immunofluorescent antibody labelling of frozen sections. 41 2.8. HP lysate preparation co-culture experimental setup 41 2.9. mRNA isolation and real time PCR. 42 2.10. Cytokine quantification. 43 2.11. SDS-PAGE and western blot 44 2.12. Thymidine incorporation assay 45 2.13. Chemotaxis assay 45 2.14. Statistical analyses 46 3. Results 49 3.1. FUEL 49 3.1.1. Patient demographics and histopathology 3.1.2. Th17 frequencies remained elevated in the blood of individuals with previous HP infection up to 10 years post-HP eradication 3.1.3. 49 51 Th17 and IL-17A levels remained high in the gastric mucosa of individuals with previous HP infection and this associates with PC lesions 3.1.4. 54 Gene expression and cytokine environment in the gastric mucosa is highly pro-inflammatory in group A subjects and low levels of inflammation exists in group P subjects V 57 3.1.5. IL-22 in the gastric mucosa 65 3.1.6. Strong intra-individual correlation of pro-inflammatory cytokines in group P subjects 66 3.1.7. Increased expression of IL-1R1 on IL-17A-expressing cells in group P individuals 68 3.1.8. Pro-inflammatory cytokines induced hBD-2 mRNA expression from human GECs 72 3.1.9. IL-1β-stimulated GEC supernatant is chemoattractive to autologous CCR6+Th17 cellls. 76 3.1.10. HP-specific Th17 responses persist in subjects with evidence of past HP infection. 77 3.2. FLAMES 85 3.2.1. Expression of cytokine receptors IL-22R1, IL-1R1 and IL-17R1 in the human gastric epithelium and their regulation by inflammation 85 3.2.2. IL-22, IL-1β and IL-17A enhances gastric epithelial cell proliferation 87 3.2.3. IL-22-induced genes in gastric epithelium 3.3. SUMMARY 91 98 4. Discussion 103 4.1. Active IL-17A versus chronic IL-17A 103 4.2. Other sources of gastric IL-17A 109 4.3. The mixed CD4+ T cell response and T helper cell plasticity 112 4.4. Tight regulation of IL-1β production 119 4.5. Th17 cells in the development of gastric cancer – Role of STAT3 121 VI 4.6. Concluding remarks and future directions 5. Bibliography 126 126 SUMMARY This project begins with investigating the adaptive T cell immunology of Helicobacter pylori (HP) infection of the gastric mucosa in human patients, and concludes in the study of the effects of chronic inflammation on gastric epithelial cells. HP causes a chronic on active gastritis and is associated with an increased risk of developing the intestinal type of gastric cancer. A large body of evidence suggests that the cancer develops via a chronic progression of precancerous changes in the gastric mucosa. In many instances, risk of cancer progression is still evident after the bacteria is eradicated. Concurrent with the cancer risk that precancerous changes associate with, the chronic immune cell infiltrate is apparent for years subsequent to antibiotic treatment. Therefore, it is the main hypothesis of this project that the immune response triggered by HP infection persists even after eradication of the bacteria, and that this chronic inflammation causes immunopathology to the gastric epithelium, which may subsequently predispose to the persistence of precancerous lesions and contribute to cancer risk. Patient subjects were classified into three main groups for most of the analyses – group A: subjects with an active HP infection, group P: subjects with no active HP infection, but evidence of a past infection and group N: naïve individuals who have never had a HP infection. Focus was given to the comparison of antigen-specific and non-antigen-specific Th17 responses and its associated cytokines in these subjects. VII Presence and persistence of HP-specific Th17 cells were demonstrated in the peripheral blood and gastric mucosa of subjects in group P, and in some individuals, the persistence lasted up to 10 years post HP eradication therapy. Ex vivo cytokine concentrations of a panel of human pro-inflammatory and T cell cytokines in gastric biopsies were quantified. IL-1β, IFNγ and IL-17A were the cytokines found to be significantly elevated in group P subjects compared to group N. Several other proinflammatory cytokines were also elevated in group P subjects compared to group N, and an association with precancerous lesions was found. Expression of IL-22, another Th17 cytokine was demonstrated in the gastric mucosa of all subjects and no difference was detected across the three groups. In vitro experiments on gastric epithelial cells demonstrated that IL-22 acted in synergy with IL-1β and IL-17A to elicit pro-inflammatory downstream effects like the induction of S100 proteins and human β-defensin 2. This thesis takes an old model of infection- and inflammation-associated cancer and studies the related immune responses in light of new advances in T cell immunology. The clinical data presented here further contributes to the field of knowledge on T cell responses in HP-associated gastritis and precancerous lesions. VIII Antigenspecificity Conjugate Application Final concentration Company No. 1. 2. CD3 CD4 FC IF 1μl/test 1:100 eBioscience eBioscience 3. CD8 FC 1μl/test eBioscience 4. FC 2μl/test 5. 6. CCR6 (CD196) IL-17A IFNγ PE-Cy7 Alexa Fluor 488 APCeFluor780 PE FC FC 0.5μl/test 1μl/test 7. IL-22 FC 1μl/test eBioscience 8. 9. 10. 11. 12. 13. 14. 15. 16. IL-1β IL-17A IL-1R1 IL-22 CD4 EpCAM IL-22R1 p-STAT3 Total STAT3 FITC Alexa Fluor 647 Alexa Fluor 647 Biotin Biotin unconjugated unconjugated unconjugated unconjugated unconjugated unconjugated unconjugated BD Pharmingen eBioscience eBioscience IF IF IF IF IF IF IF, WB, FC WB WB 1:400 R&D eBioscience R&D R&D Novocastra R&D R&D Cell Signaling Cell Signaling Table 2.1 List of Antibodies. FC: Flow Cytometry; IF: Immunofluorescence; WB: Western blot. No . 1. Gene Chemistry Primer Sequence IL-17A SYBR-Green (ABI) FWD: 5'ACTACAACCGATCCACCTCAC-3’ REV: 5'ACTTTGCCTCCCAGATCACAG-3' 2. IL-23p19 SYBR-Green (ABI) FWD: 5'GGGACACATGGATCTAAGAG-3' REV: 5'GCAAGCAGAACTGACTGTTG-3' 3. IL-22 SYBR-Green (ABI) Fwd: 5'GCAGGCTTGACAAGTCCAACT-3' Rev: 5'GCCTCCTTAGCCAGCATGAA-3' 4. CCL20 SYBR-Green (ABI) Fwd: 5'CTGGCTGCTTTGATGTCAGTG-3' Rev: 5'GCAGTCAAAGTTGCTTGCTGC-3' 5. IL-1β TaqMan (TaqMan Gene Expression Assay ID Hs01555413_m1) 6. hBD-2 SYBR-Green (Biorad) Table 2.2 List of primers and their PCR cycling conditions. 47 PCR Cycling conditions 95°C 5mins, 40 x(95°C 30s, 60°C 30s, 72°C 45s), 72°C 5mins. 95°C 5mins, 40 x(95°C 30s, 58°C 30s, 72°C 45s), 72°C 5mins. 95°C 5mins, 40 x(95°C 30s, 58°C 30s, 72°C 45s), 72°C 5mins. 95°C 5mins, 40 x(95°C 30s, 58°C 30s, 72°C 45s), 72°C 5mins. (Fast PCR reaction) PART I THE FUEL This part forms the bulk of the thesis. It describes the presence of increased Th17 cell frequencies and associated pro-inflammatory cytokines levels in individuals with a past history, but no ongoing HP infection. The persistent Th17 infiltrate is possibly triggered by the initial HP infection. 48 3. RESULTS 3.1.1 Patient demographics and histopathology Most clinical samples in this study came from individuals recruited by the Singapore Gastric Cancer Consortium’s Gastric Cancer Epidemiology, Clinical and Genetic Programme (GCEP). According to the design of the clinical study, subjects who had an active HP infection at their first scope (year 0) were required to undergo follow up gastroscopy at year (vist 1; v1) and (visit 3; v3). Over and a half years, time points were captured for some individuals, but not all. A smaller proportion of the clinical samples came from the HP Study conducted by the Department of Gastroenterology, SGH. All subjects were primarily grouped into three groups, depending on whether they had an active HP infection (Group A), a past HP infection (Group P) or were never infected (Group N). Classification criterion was described in Chapter 2.1. Table 3.1 shows the demographic data of all subjects’ data points, excluding repeat visit data. n Mean Age Male (%) All 153 54.8 ± 11.5 70 (45.6) Active (Group A) 55 55.6 ± 10.5 34 (62) Past (Group P) 39 59.4 ± 9.1 17 (44) Naïve (Group N) 59 51.2 ± 12.6 19 (32) Histology MCG1 97 30 11 56 CAG2 IM3 47 20 24 Seropositive 52 25 27 Table 3.1. Clinical sample demographic data 1Mild Chronic Gastritis 2Chronic atrophic gastritis; Intestinal metaplasia. 49 Figure 3.1 Progression/regression of precancerous (PC) lesions years later in subjects who underwent follow-up endoscopy. Baseline n Mean age at baseline Male (%) MCG 61.0 ± 6.4 CAG 63.7 ± 8.5 IM 62.6 ± 6.4 (50) (66.7) (57.1) Table 3.2. Demographic data of subjects who were followed-up after HP eradication therapy. Figure 3.1 shows the histopathology data from subjects who were HP positive, given treatment and subsequently followed up for more time points (v1 and v3) during the course of this study. There were a total of 18 of these individuals, of which had no precancerous (PC) lesions at baseline (mild chronic gastritis; MCG), had CAG, and had IM. These individuals were assessed based on histology reports provided by the hospital, on whether they had no change, regression or progression of PC lesions. Results of histology were reported as either MCG, CAG or IM (ascending order of progression; see Introduction, Chapter 1.2, figure 1). Of the MCG at baseline, 50% progressed (developed more advance PC lesions in the gastric mucosa), while the other 50% remained the same after years. In those with CAG at baseline, regressed to MCG, another had no change and the last progressed to IM. In individuals with IM at baseline, only regressed to MCG and the rest retained the IM 50 phenotype years later, despite eradication of HP infection. These findings are in line with previous clinical studies reporting that despite the absence of bacteria in the gastric mucosa, PC lesions may persist in some individuals (especially those above the age of 45), or even develop towards more advanced lesions, leaving the individuals at the same or even higher risk of gastric cancer even after antibiotic treatment. Table 3.2 shows the demographic data of these 18 individuals. 3.1.2 Th17 frequencies remained elevated in the blood of individuals with previous HP infection up to 10 years post-eradication Since it was described that HP infection resulted in an increase in IL-17A and Th17 cells in the gastric mucosa, (Luzza, Parrello et al. 2000; Caruso, Fina et al. 2008) PBMCs from the blood of all three groups of individuals were analysed for frequencies of cytokine-secreting T cells. Th17 cells were gated as CD3+CD8- cells that were also IL-17A+ and CCR6+ (figure 3.2A). Individuals from group P exhibited elevated Th17 frequencies in the blood (median 1.4%, interquartile range (IQR) 0.81.8%) that was statistically different from individuals from group N (median 0.8%, IQR 0.5-1.4%) (figure 3.2B). Frequencies of peripheral blood Th17 cells in individuals from group A were not statistically different from the other two groups (median 1.1%, IQR 0.6-2.0%). Th1 responses are the classic CD4+ T-helper response raised against HP infection. CCR6+IFNγ+ events in the CD3+CD8- gate were analyzed in the three groups (figure 3.2C), but unlike for Th17 cells, no differences were found (figure 3.2D). Since it has been previously reported that chronic inflammation persisted in individuals for up to 12 years after HP eradication therapy (Mera, Fontham et al. 2005), subjects from group P were further subdivided into subgroups 51 according to the number of years post-HP eradication the samples were analysed (1 year, 1-3 years, 4-9 years and ≥10 years). It is apparent that many recently eradicated individuals had higher Th17 frequencies. However, the median percentages of Th17 cells across the groups were not statistically different, suggesting that the high frequencies of Th17 cells were not all from individuals from recently cleared infections, but also from individuals whom had infections cleared more than 10 years ago (figure 3.2E). Individuals in the 2-3 year group had the least variation in Th17 frequencies. This result further confirms that despite the clearance of bacteria from the gastric mucosa, Th17 responses persist in the blood, and this associates with the high rate of non-regression of precancerous lesions in this cohort of individuals. Lastly, subjects were classified according to whether they had PC lesions in the gastric mucosa, and Th17 cell frequencies were analyzed in these groups. Group P individuals who did not have PC lesions (PC-) had the highest median number of circulating Th17 cells (1.5%; IQR 1.0-3.3%), and this was significantly higher group P individuals with PC lesions (PC+) (1.0%; IQR 0.7-1.8%) compared with group N individuals (0.8%, IQR 0.5-1.4%). This group of individuals corresponds (but not entirely) to the group with the most recently cleared infections – figure 3.2E, ‘1year’ group. In group A subjects, there was no difference between PC+ or PC- individuals (1.0%; range 0.2-3.2% and 1.1; range 0.3-2.7% respectively) (figure 3.2F). PBMC data show that PC lesions associate with a lower Th17 frequency in group P individuals. 52 Figure 3.2 Th17 frequencies in PBMCs. (A and C) Zebra-plot (showing outliers) of PMA and ionomycin-stimulated PBMCs from a group A individual. Subgate of CD3+CD8- cells is shown. (B) Dot plot showing median with interquartile ranges (IQR) of CCR6+IL-17A+ (Th17) events as a percentage of CD4+ T cells in three groups of subjects. (D) Dot plot showing median with IQR of CCR6+IFNγ+ events as a percentage of CD4+ T cells in the same three groups. (E) Th17 percentages from group P subjects, further sub-grouped according to the number of years since HP eradication. (F) Th17 percentages with group A and P further sub-grouped according to whether subject harbored precancerous (PC) lesions in their gastric mucosa. 53 3.1.3 Th17 and IL-17A levels remained high in the gastric mucosa of individuals with previous HP infection and this associates with PC lesions Next, the gene and protein expression of IL-17A was examined in gastric mucosal biopsies. In agreement with previous studies, the mRNA expression levels of IL-17A were significantly the highest of the three groups in subjects with an ongoing HP infection (group A) (figure 3.3A). Consistent with the PBMC data, there was also a trend towards elevated IL-17A expression in gastric mucosal biopsies from group P individuals, although these levels were not significantly different from group N individuals. Ex vivo protein levels of IL-17A and IFNγ in gastric biopsies were measured by multiplex cytokine bead array. Subjects from group A exhibited the highest median amounts of IL-17A and IFNγ in gastric biopsies, followed by those in group P, which were also significantly higher than that detected in group N individuals (figure 3.3B and D). Table 3.3 summarises the median and interquartile ranges for the concentrations of IL-17A and IFNγ. Looking at associations with PC lesions within the group P subjects, higher amounts of both IFNγ and IL-17A were found in PC+ as compared to PC- subjects (figure 3.3C and E, table 3.3). This difference was significant for IFNγ levels, but not IL-17A levels, although it is noteworthy that the median levels of IL-17A in the PC+ group was almost twice more than the PC- group, and the sample size was small. Together, this data shows that the IL-17A and IFNγ responses persist in group P individuals, and associates with PC lesions. Inflammation in PC lesions consists of elevated IL-17A and IFNγ levels. 54 Active IL-17A (pg/gram tissue, IQR) Past Naïve 36, 24-62 89, 67-111 15, 0-20 PC+ PC42, 26-119 25, 15-29 158, 96-251 IFNγ 173, 73-367 49, 12-105 (pg/gram tissue, PC+ PCIQR) 176, 149-276 95, 41-96 9.3, 5.0-16 + + CD4 IL-17A 8.0, 6.2-13 PC+ PC4.9, 2.4-6.8 IF cell counts 9.3, 6.2-15 7.6, 4.9-16 Table 3.3. Summary of median and interquartile range (IQR) values for IL-17A and IFNγ values in the three subject groups. ‘Past’ group is further divided into Precancerous lesion positive (PC+) or negative (PC-). Frozen sections from fresh biopsies were next examined by immunofluorescent (IF) microscopy to identify CD4+IL-17A+ cells infiltrating the gastric mucosa (figure 3.3G). Numbers of double positive cells were counted in high-powered fields (HPFs). Gastric mucosal CD4+IL-17A+ cell numbers were significantly higher in group P individuals compared to group N (figure 3.3F). The median numbers of infiltrating CD4+IL-17A+ cells is summarized in table 3.3. This demonstrates that at least part of the elevated IL-17A responses detected in the gastric mucosa of group P subjects were derived from CD4+ cells. Stratification of these individuals into groups based on the number of years since HP eradication did not reveal any large differences in the numbers of infiltrating Th17 cells across all groups although sample size was small (figure 3.3H). Finally, PC+ group P individuals had a higher number of infiltrating Th17 cells (median 9.3; IQR 6.2-14.8) compared to group N individuals (median 4.9; IQR 2.4-6.8) (figure 3.3I), demonstrating a link between PC lesions in group P subjects and more Th17 infiltrate. Due to the small sample size, it was difficult to correlate PBMC Th17 data with biopsy Th17 data. Altogether, these results together with the PBMC data provide several independent lines of evidence to suggest that gastric mucosal inflammation persists in group P 55 Figure 3.3 IL-17A levels and Th17 frequencies in patient gastric biopsies. (A) Semi-quantitative SYBR Green real time PCR for IL-17A mRNA. Gene expression was normalized to the housekeeping gene βactin and arbitrary values were further normalized to group N. (B and D) Milliplex xMAP assay for ex vivo IL-17A and IFNγ concentrations respectively (C and E) Ex vivo IL-17A and IFNγ concentrations respectively of Group P individuals divided into PC+ and PC-. (F) Frequencies of Th17 cells determined by the average number of CD4+IL-17A+ cells counted per high-powered field (HPF) of immunofluorescent (IF)-labelled frozen sections. (G) 40X magnification image of IF-labelled frozen sections from a group A subject. (H) IF Th17 cell counts of group P subjects divided into groups according to the number of years since their HP eradication. (I) IF Th17 cell counts with group A and P further sub-grouped according to whether subject harbored precancerous (PC) lesions in their gastric mucosa. 56 patients despite the absence of ongoing HP infection and identify CD4+IL-17A+ cells as a component of this persistent inflammation. This inflammation may even persist for up to 10 years after eradication of the bacterium. Furthermore, although this Th17related inflammation from PBMC data did not associate with PC lesions, data from patient biopsies reflected that inflammation associated with PC lesions, suggesting that inflammation may drive the progression or non-regression of PC lesions seen in the study subjects. 3.1.4 Gene expression and cytokine environment in gastric mucosa is highly proinflammatory in group A subjects and low levels of inflammation exists in group P subjects In order to further characterize the persistent inflammation and to investigate why there is such an accumulation of CD4+IL-17A+ cells in the gastric mucosa of individuals with a cleared HP infection, gene and protein expression of several cytokines and chemokines were assessed by real time PCR and a xMAP multiplex cytometric bead array assay respectively. There were three hypotheses as to why there was such an accumulation of Th17 cells observed in group P individuals : 1) Prolonged inflammation in the presence of Th17-maintaining cytokines like IL-1β, IL-6 and TNFα, 2) proliferation of cellular infiltrate and 3) increased retention or chemotactic homing of Th17 cells via CCR6. Chemotactic factors for Th17 cells acting via the CCR6 axis was investigated. CCL20 is a ligand for the chemokine receptor CCR6, which is expressed on Th17 cells (Jang, 57 Lim et al. 2004; Acosta-Rodriguez, Rivino et al. 2007). Human β-defensin-2 (hBD-2) is an alternative ligand for CCR6 (Yang, Chertov et al. 1999; Ghannam, Dejou et al. 2011). Hence, gene expression levels of these genes were investigated to determine if increased homing of Th17 cells to the gastric mucosa was the reason for the chronic lymphocytic infiltrate seen, especially in patients with PC lesions. As expected, the levels of CCL20 and hBD-2 were significantly elevated in group A subjects (figure 3.4A and B). However, there was no significant differences between group P and group N, hence, increased homing and retention of Th17 cells via the CCR6 receptor may not be a significant mechanism contributing to the infiltrate and persisting inflammation. Gene expression of several other cytokines associated with the priming and maintenance of Th17 cells were investigated. These include IL-6, IL-8, TGF-β, TNFα, IL-1β and IL-23p19. Gene expression data for IL-6, IL-8, TGF-β and TNF-α were not significantly different across three groups and are not shown. IL-23p19 is produced by APCs during infection and is required for the priming of human Th17 cells and IL-1β is essential in the maintenance of Th17 cells. IL-23p19 expression is elevated in group A subjects, in line with the hypothesis that it is produced by APCs during infection (figure 3.4C). Levels of IL-23p19 went down in group P and N subjects as expected, and hence was probably not the reason for more Th17 cells and IL-17A observed in the gastric mucosa. Gene expression of IL-1β was the highest amongst group P patients and this was significantly higher than group A patients (figure 3.4D). 58 Ex vivo protein concentrations of IL-6, TNF-α and IL-1β were measured using the xMAP Milliplex kit. Unlike for the gene expression data, the protein concentrations of IL-6 and TNF-α were significantly higher in individuals with an ongoing HP infection as expected (figure 3.4E and F). Although not significant, the median levels of both cytokines were higher in group P than in group N, suggesting a low level of inflammation in these individuals. IL-1β levels were significantly higher in group A compared to group N, and the median value of group P was 2.9-fold higher than in group N (figure 3.4G), demonstrating that IL-1β also contributes to the inflammation. Due to the high amounts of IL-17A and IL-1β seen in group P patients, and since these cytokines have been reported to induce the production of hBD-2 from epithelial cells, the ex vivo proteins levels of hBD-2 were also measured by ELISA. Group A individuals demonstrated the highest median concentration of hBD-2, followed by group P then the lowest in group N. Significant differences were detected in between groups A and P, and groups A and N (figure 3.4H). Although there was no significant difference between group P and N, the median from group P is 1.5 fold higher than that of group N, and the mean is more than 2-fold higher. Table 3.4 summarises the median values of concentration for the protein concentrations described in figure 3.4. Group P was also further divided into PC+ and PC- groups but no significant differences were detected, but this was mainly due to the lack of sample size for the PC- group. Generally, the PC+ group had a higher median concentration of all the inflammatory cytokines and hBD-2 as compared to PC- group. These data suggest that in group P individuals, despite the lack of bacterial load, the gastric mucosa still harbors a low level of inflammation, characterized not only by IL-17A but other proinflammatory cytokines like IL-6, TNFα and IL-1β, and also hBD-2, which may 59 function in recruiting more Th17 cells via the CCR6 receptor. For all these factors, an association with PC lesions is found, indicating that there might be more inflammation in gastric mucosa harboring PC lesions. The increased inflammation in these individuals may contribute to the increased risk of developing gastric cancer. Other cytokine and chemokine concentrations were also assayed for as part of the xMAP multiplex panel (figure 3.5). Several other cytokines were significantly upregulated in group A individuals – these include IL-1α, IL-8, IL-10, IL-12p40, IL-15 and MCP-1. The significant lack of IL-8 levels (figure 3.5D) indicates the absence of an active infection, as presence of HP is known to trigger an IL-8 response (Roth, Vogl et al. 2003). Gamma (γ)-chain cytokines like IL-2 and IL-15 were also elevated in group P as compared to group N (see table 3.5 for median values), suggesting that proliferation (in the theoretical absence of antigen) may play a role in either maintaining or increasing the T cell infiltrate in the inflamed mucosa during chronic inflammation (figure 3.5C and G). For these cytokines within group P, there was an association between higher cytokine concentration and PC lesions. In line with some previous studies, IL-10 levels were significantly higher in group A compared to the other groups. In group P, the median concentration was slightly more than twice compared to group N. The same trend was seen for IL-12p40, and for both cytokines, there was a higher median concentration in individuals who were PC+. The high levels of IL-10 in group A subjects may aid in providing a balance to the strong Th1 and Th17 inflammatory responses during HP infection. This has been reported to occur via the induction of Treg responses during HP infection (Kandulski, Wex et al. 2008). The lack of this strong IL-10 or Treg response in group P subjects may mark a 60 significant difference between the inflammatory environments in group A versus group P individuals, and also possibly enhance the extent of inflammation-induced pathology, especially since there is a significantly persistent Th17 response detected in these individuals. Although there is theoretically no more presence of antigen, the elevated levels of APC-derived IL-12p40 is not surprising, given that IL-1β, also APC-derived, is elevated in group P individuals. Nevertheless, the reason for this is unknown. Generally, albeit statistically insignificant, there was a higher level of the investigated cytokines and chemokines in group P as compared to normal healthy individuals from group N. These results re-iterate the fact that low levels of inflammation occur in the gastric mucosa of individuals with a past history of HP infection, and this may contribute to immunopathological damage and hence, cancer risk. Active IL-6 (pg/gram tissue, IQR) Past Naïve 87, 57-130 130, 99-186 50, 20-92 PC+ PC108, 58-148 65, 31-87 19, 8.9-57 TNFα 48, 28-75 11, 2.1-27 PC+ PC(pg/gram tissue, IQR) 26, 11-59 5.9, 5.4-19 71, 53-177 IL-1β 132, 108-242 28, 11-59 PC+ PC(pg/gram tissue, IQR) 94, 12-220 58, 53-71 207, 113-352 hBD-2 1390, 936-2072 110, 48-134 PC+ PC(pg/gram tissue, IQR) 213, 120-337 114, 89-347 Table 3.4. Summary of median and interquartile range (IQR) values for ex vivo cytokine concentrations reported in figure 3.4 61 Active IL-1α (pg/gram tissue, IQR) Past Naïve 20, 4.0-47 36, 29-65 0, 0-2.8 PC+ PC24, 12-59 0, 0-0 18313, 8082-33925 IL-1Ra 33649, 2110012271, 6403PC+ PC(pg/gram tissue, IQR) 69157 23437 17358, 787431782, 1061648662 33527 82, 42-143 IL-2 115, 98-231 50, 22-111 PC+ PC(pg/gram tissue, IQR) 92, 51-161 65, 40-91 65, 51-146 IL-8 1250, 747-2323 27, 9.1-156 PC+ PC(pg/gram tissue, IQR) 71, 51-157 55, 45-64 16, 11-36 IL-10 81, 56-99 6.8, 2.1-15 PC+ PC(pg/gram tissue, IQR) 27, 12-48 13, 11-13 149, 84-295 IL-12p40 295, 191-505 PC+ PC61, 23-228 (pg/gram tissue, IQR) 175, 79-328 100, 84-177 83, 53-193 IL-15 164, 120-226 PC+ PC41, 11-92 (pg/gram tissue, IQR) 100, 59-243 59, 30-98 527, 297-780 MCP-1 827, 662-1835 PC+ PC440, 245-584 (pg/gram tissue, IQR) 542, 316-875 527, 297-613 4242, 2763-5506 RANTES PC+ PC5439, 4876-5572 3750, 1842-4994 (pg/gram tissue, IQR) 4297, 27833907, 27635385 5967 Table 3.5. Summary of median and interquartile range (IQR) values for ex vivo cytokine concentrations reported in figure 3.5 62 [...]... microenvironment, fuelling cancer progression Hence the immune system has to perform a balancing act to protect itself from disease Studies on inflammation and T cell responses associated with HP infection supporting either one of the two concepts above is reviewed below 1. 4.2 Protective T cell responses Literature on T cell responses to HP covers a few subsets of T cells, the T- helper 1 (Th1), Th17 as well as T. .. complement system and inflammatory mediators in the blood The innate immune system recognizes pathogen -associated molecular patterns and reacts to these foreign bodies The adaptive immune system, in contrast to the innate immune system, is not poised to act immediately upon infection Infections trigger adaptive immune responses that are specific for a particular protein antigen from the pathogen, and this... group reported a correlation between the T- T haplotype of IL1B- 511 and IL1RN*2 allele with increased protein levels of IL -1 (Hwang, Kodama et al 2002) Sugimoto et al have conducted several studies in Japan and reported polymorphisms in TNFA-85 7T, TNFA-863A, TNFA -10 31C and IL10-592C and IL10 819 C but not IL1B- 511 /- 31 being significantly associated with gastric cancer development (Sugimoto, Furuta et al 2007;... reporting the ability of HP to control and skew the T cell response to favor a regulatory rather than pro-inflammatory response to itself The T cell responses to HP will be reviewed separately in another section 7 Considering the multitude of mechanisms that HP possesses to ensure in its survival in the host, it is not surprising that infections can last for prolonged periods of time even up to decades... protecting itself from infections, and that there are short-comings in the mechanisms of our self-defense that leads to outcomes which may not be directly related to the initial infection (eg cancer) The next few chapters of this introduction will focus on the central theme of this thesis – the association between chronic T cell responses, inflammation and precancerous lesions The evidence in the literature... cancer (El-Omar, Carrington et al 2000) They also demonstrated by an electrophoretic mobility shift assay that there was a five-fold increase in transcription factor binding to the IL1B-3 1T oligonucleotide A later study conducted on a Chinese population validated the association between IL1B- 51 1T and gastric cancer risk (Li, Xia et al 2007) Complementing the data on genetic associations with cancer risk,... proliferation (Sundrud, Torres et al 2004) VacA is recently reported to enter T cells via binding to an integrin subunit CD18 (β2) and exploiting the cellular shuttling of integrins (LFA -1) and their receptors from the cellular surface to intracellular lipid rafts (Sewald, Gebert-Vogl et al 2008) Outer membrane proteins (OMPs) expressed by HP have specific receptors on the gastric epithelium in which they... killing the cell (Albina, Cui et al., 19 93) HP modulates the NO levels in the cell by inducing the activity of inducible nitric oxide synthase (iNOS) in macrophages (Wilson, Ramanujam et al 19 96) and hence the production of NO Increase in NO concentrations in cells induces cellular stress and an increase in p53 levels within the cell Following this, p53-dependent apoptosis follows In addition to this,... Multi-analyte profiling XIII 1 INTRODUCTION 1. 1 Immune Evasion By Pathogens – Failure of the Human Immune System Since the beginning of life, pathogen-host interactions have shaped the co-evolution of the host immune system and pathogen survival mechanisms, resulting in the constant evolutionary development of the pathogens’ invasive or evasive mechanisms to either protect itself from, or attack the host in. .. mature into antibody secreting plasma cells, to secrete antibodies against the specific pathogen These cells are retained in the hosts’ circulation as ‘memory cells’ and are ready to respond to re-infections Even with the elaborate structure and multitude of functional abilities our immune system has, humans are still prone to diseases The reason for this is that pathogens, in the face of pressures exerted . lymphocytes. The T cells are able to secrete cytokines and mount cytotoxic responses on infected cells, whilst B cells differentiate and mature into antibody secreting plasma cells, to secrete antibodies. genes 16 1. 4. Adaptive immune responses to Helicobacter pylori. 18 1. 4 .1. Sensing HP bacterium 18 1. 4.2. Protective T cell responses 19 1. 4.3. Mouse models of HP infection 20 1. 4.4. T regulatory. 72 3 .1. 9. IL -1 -stimulated GEC supernatant is chemoattractive to autologous CCR6 + Th17 cellls. 76 3 .1. 10. HP-specific Th17 responses persist in subjects with evidence of past HP infection.