T cell responses in helicobacter pylori associated gastroduodenal diseases 3

3.1.5 IL-22 in the gastric mucosa Th17 cells also secrete IL-22, a cytokine belonging to the IL-10 family, that enhances epithelial integrity and regeneration in the gut, but promotes inflammation under certain experimental settings (reviewed in Introduction, Chapter 1.6). Gastric mucosal IL-22 was quantified using real time PCR, immunofluorescence microscopy, and ELISA. IL-22 mRNA levels were significantly higher in group A, as compared to group P (figure 3.6A). However, for all other protein data (ex vivo cytokine concentration and IF staining cell counts), such a suppression of IL-22 protein levels was not seen, in fact, there was no significant difference across the three groups (figure 3.6B and D). Additionally, there was no difference in the total amounts of IL22 in group P individuals, whether subjects had PC lesions in the gastric mucosa or not (figure 3.6C). Nevertheless, when CD4+IL-22+ cells counts were analysed from IF-stained frozen sections, an association with HP infection was seen (figure 3.6E), which is in line with the observation that there is a heightened Th17 response during an active HP infection. There was no significant difference between the CD4+IL-22+ cell counts in groups P and N, suggesting that there might be a fairly low level of Th17-derived IL-22 in group P individuals. However, within group P, a higher median number of CD4+IL-22+ cells associated with PC lesions (figure 3.6G). The median and IQR values of the above-mentioned IL-22 protein data is summarized in table 3.5. Taken together, these data demonstrate that CD4+ T cells are not the only source of IL-22 in the gastric mucosa. Although higher amounts of CD4+ T celldervied IL-22 associated with an ongoing HP infection, total IL-22 levels and cell counts showed that there were other sources contributing to what is observed in the gastric mucosa, and this did not correlate with HP infection. 65 IL-22 levels were fairly similar across all three groups, and since it is reported to play homeostatic and protective roles in the gut (Zenewicz, Yancopoulos et al. 2008). IL-22 in the absence of IL-17A has tissue-protective effects; however IL-22 and IL-17 synergistically provoke inflammation when co-expressed (Sonnenberg, Nair et al., 2010). Intra-individual ratios of the pro-inflammatory IL-17A or IFNγ to IL-22 were calculated as a measure of cytokine co-expression in all individuals. IL-17A/IL-22 ratios were significantly elevated in samples from groups A and P compared to group N (figure 3.6H), and IFNγ/IL-22 ratios were elevated in group P compared to N (figure 3.6I). This suggests that the gastric cytokine milieu in group P subjects continues to be biased against the baseline state of mucosal maintenance found in HP-naïve patients, despite the absence of ongoing HP infection. Active IL-22 ex vivo concentrations (pg/gram tissue, IQR) Past Naïve 173, 77-261 149, 87-335 171, 119-397 PC+ PC169, 69-261 212, 105-301 16, 11-22 Total IL-22+ 31, 17-43 17, 9.0-28 PC+ PCIF cell counts 16, 11-25 18, 14-21 4.0, 3.0-5.1 CD4+IL-22+ 11, 5.0-15 3.0, 3.0-6.5 PC+ PCIF cell counts 4.2, 3.2-6.6 3.3, 1.8-4.0 Table 3.6 Summary of median and interquartile range (IQR) values for IL-22 protein data reported in figure 3.6. 3.1.6 Strong intra-individual correlation of pro-inflammatory cytokines in group P subjects Having established that the gastric microenvironment in group P patients had increased Th17 infiltrate and a pro-inflammatory profile, it was of interest whether 66 Figure 3.6 IL-22 levels in human gastric biopsies. (A) Semi-quantitative SYBR-Green real time PCR for IL-22 mRNA. Gene expression was normalized to the housekeeping gene β-actin and arbitrary values were further normalized to group N. (B) ELISA for ex vivo IL-22 concentrations. (C) Subclassification of group P individuals into PC+ or PC- groups for analysis of ex vivo concentrations of IL-22. (D and E) Total number of IL-22+ and CD4+IL-22+ cells determined by the average number of cells counted per HPF of IF-labelled frozen sections. (F and G) Frequencies of total IL-22+ or CD4+IL22+ cells in PC+ and PC- groups. (H and I) Ratio of IL-17A to IL-22 and IFNγ to IL-22 ex vivo cytokine concentrations. 67 there was any correlation across the pro-inflammatory cytokine levels within individuals, that may increase the significance of the data. The associations between several HP-associated cytokines and IL-17A were analysed. The high levels of IL-1β in group P patients were striking (figure 3.4G), as IL-1β is a well recognized risk factor for gastric cancer and is also important in the maintenance of human Th17 cells. In line with this, there was a significant correlation between IL-1β and IL-17A in group P individuals (figure 3.7A). There was no statistically significant correlation in group A, although this might be due to the small sample size compared to group P. the concurrently high levels of both IL-1β and IL-17A indicates that there may be an association between these two cytokines, and that during chronic inflammation, IL-1β and IL-17A are present at elevated levels at the same time. There was also a correlation between IL-17A and IFNγ, IL-8 and IL-10 levels in group P subjects (figures 3.7B, C and E respectively). However, there was no correlation between IL17A and IL-22 nor IL-1β and IL-22 levels within individuals, in line with the finding that total gastric IL-22 levels were not affected by history of HP infection (figure 3.7D). These correlations provide the evidence that the persistent inflammation observed in individuals with a previous HP infection occurs as a collective presence of several pro-inflammatory cytokines, which may work together to worsen or prolong inflammation. 3.1.7 Increased expression of IL-1R1 on IL-17A-expressing cells in group P individuals 68 Figure 3.7 Ex vivo IL-17A levels in biopsies correlates with several other pro-inflammatory cytokines. (A-E) Intra-individual cytokine concentrations correlated with IL-17A. Spearmans’ test for correlation was used. (F) Intra-individual cytokine correlation between IL-1β and IL-22. Given the elevated levels of IL-1β and IL-17A observed in group A and P individuals, and since IL-1β is a cytokine responsible for maintenance and proliferation of Th17 cells, the expression of the receptor for this cytokine, IL-1 receptor-1 (IL-1R1) was investigated alongside IL-17A expression by immunostaining in frozen biopsy sections in three subject groups. Frozen biopsy sections were co-stained with antibodies for the two antigens IL-1R1 and IL-17A and double positive cells were counted in biopsies sections. A number of individuals from group P had a strikingly strong expression of IL-1R1 in their gastric mucosa, and 69 some of this was co-expressed with IL-17A. Not only was the intensity of the staining much brighter than in other samples, the frequency of positive stains were higher. out of of these patients that had a bright staining and high frequency of IL-1R1 staining had severe IM in their gastric mucosa according to histopathology reports. ? out of of group A individuals also had severe IM in their gastric mucosa. Figure 3.8A shows a section showing bright and frequent IL-1R1 staining from a group P individual having severe IM in the gastric mucosa, and this is contrasted with figure 3.8B, a group P individual with IM (not severe), having dimmer and much less IL1R1 staining. These staining patterns were not observed for any of the group N subjects. Figure 3.8C and D shows all the data points from subjects from the three groups. Although there was no statistical significance between the three groups, it was clear that the bright and frequent IL-1R1 staining were only seen for group P subjects, less in group A and not in group N at all. It is however, of note that histological diagnoses of IM in patient biopsies were made on separate biopsies from those used in frozen section staining, and sampling variation within each patient may partially confound these results. These results suggest that the expression of IL-1R1 in the gastric mucosa of some individuals may increase as PC lesions advance. On top of the already elevated presence of IL-1β in the gastric mucosa of group P subjects, the increase in sensitivity to IL-1R1 may fuel precancerous changes. IL-1R1 polymorphisms have been reported to be associated with increased HP infection incidences, but not with gastric cancer. (Hartland, Newton et al. 2004) 70 A B 71 Figure 3.8 High frequencies of IL-1R1 bright cells in group P individuals with severe IM. (A) 40X magnification image showing staining of IL-17A and IL-1R1 on frozen sections from a group P individual with severe IM. (B) Same staining performed on a group P individual with IM (not severe) (C) Frequencies of IL-1R1+ cells determined by the average number of positively-stained cells counted per HPF of IF-labelled frozen sections. (D) Frequencies of IL-1R1+IL-17A+ cells in the same sections. 3.1.8 Pro-inflammatory cytokines induced hBD-2 mRNA expression from human GECs In Chapter 3.1.4, group P subjects were described to have an elevated level of numerous pro-inflammatory cytokines, especially IL-17A and IL-1β. These two cytokines have been reported to induce hBD-2 expression from epithelial cells (Jang, 72 Lim et al. 2004; Kao, Chen et al. 2004). Furthermore, ex vivo hBD-2 levels in group P subjects were also slightly elevated. Since IL-22 is also known to induce the expression of anti-microbial peptides from epithelial cells in host innate immune responses (Wolk, Kunz et al. 2004), the effects of IL-22 on gastric epithelial cells (GECs) were investigated alongside the pro-inflammatory cytokines. GECs were isolated from fresh biopsy samples from several group P and N subjects and these were stimulated with IL-1β. IL-1β significantly up-regulated hBD-2 expression in the gastric mucosa by a median of 50-fold (IQR 24-79) across all samples (figure 3.9A). There were no significant differences between the responses of GECs isolated from group P and group N samples towards IL-1β stimulation. Due to the lack of clinical samples and scarce numbers of GECs isolated from each individual sample each time, stimulation of GECs with IL-22 and IL-17A in combination with IL-1β were not repeated as many times as for IL-1β alone. For two individuals, IL-22 alone or in combination with IL-1β was used to stimulate GECs. IL-22 alone did not result in any significant increase in hBD-2 gene expression. However, IL-22 in combination with IL-1β resulted in a synergistic increase in hBD-2 gene expression level as compared to either one of the cytokines alone (figure 3.9B). IL-17A resulted in a median of 76fold increase in hBD-2 gene expression, slightly higher than the IL-1β-induced increase (55-fold). Together, IL-17A and IL-1β co-stimulation resulted in an additive effect, with a 126-fold increase in hBD-2 gene expression (figure 3.9C). Earlier data reported a presence of IL-22 across the three patient groups. The effects of IL-22 together with pro-inflammatory IL-17A and IL-1β on GECs include the increase in hBD-2 expression. In a single group P patient where there were enough GECs isolated to perform an extra condition, IL-22 together with both IL-17A and IL-1β 73 resulted in the highest increase in hBD-2 gene expression (151-fold) (figure 3.9D). This observation is partly in line with previous reports that IL-22 can synergize IL17A in the induction of human β-defensin production (Liang, Tan et al. 2006), and shows for the first time, the cooperation between IL-22 and IL-1β in hBD- induction. This increase in hBD-2 levels (also seen in ex vivo biopsy analyses) may enhance chemotaxis and retention of CCR6+ Th17 cells in the gastric mucosa of group P individuals. To establish a biological relevance of this finding, the ex vivo concentrations of hBD2 measured in biopsies were correlated with the cytokines used in stimulation, to determine if there was any biological association between the cytokines and the induced gene. For IL-17A and IL-1β, there was no statistically significant correlation in all three groups of individuals, but there was a trend towards an increased correlation in group P subjects as compared to group N (figure 3.9E and F). 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Immunobiology 216(1-2): 200-7. 141 [...]... housekeeping gene GAPDH and arbitrary values were further normalized to the unstimulated cells effects of cytokines on gastric epithelial cell proliferation were investigated in the cell lines Using an optimized range of concentrations of cytokines, AGS and KatoIII cell proliferation was assessed using tritiated thymidine (3H) incorporation Concentration of cytokines used were determined by starting with the... reports implicating a vital role for it in gut homeostasis and innate immunity Total IL-22 levels across three subject groups were not different, indicating that this cytokine was perhaps not involved in chronic inflammation in the gastric mucosa However, in line with its role in innate immunity, CD4+IL-22+ cells were significantly elevated in group A subjects, but not group P and N 99 Intra-individual... count per minute, indicating that there may have been apoptosis induced in those concentrations 89 Figure 3. 14 Increase in epithelial cell proliferation in the presence of gastric cytokines (A-D) Tritiated thymidine (3H) incorporation assay for AGS and KatoIII cells Concentrations of cytokines are shown on a log10 scale (E) Cell lysates of AGS and Kato III, immuno-blot for p-Tyr705 STAT3 Cells were... starved for 4 hours prior to a 10-minute stimulation with IL-22 (200ng/ml) in serumfree media, after which cells were lysed Since IL-22 had an effect on the gastric epithelial cells, it was of interest to determine which cellular signaling pathway it utilized Gastric cancer is typically associated with an increase in STAT3 phosphorylation and IL-22 is known to activate downstream STAT signaling STAT3... result in IL-22 having a pro-inflammatory effect on the gastric 97 environment, while on the other hand, this pro-inflammatory effect may be less apparent or non-existent in normal healthy individuals despite the presence of IL-22 in their gastric environment 3. 3 Summary The results presented in this thesis have demonstrated that there are persistent Th17 responses accompanied by other inflammatory responses. .. elevated level of IL-1β, important for maintaining Th17 cells, and several other cytokines which could contribute to the inflammatory environment that is conducive for the persistence of Th17 cells The definite reason or mechanism for the observed increase in Th17 infiltrate is however, not clear from the results presented IL-22, another Th17 derived cytokine was also studied at depth due to many recent... suggesting 82 that proliferation may contribute to the increase in cellular infiltrate seen over time Finally, chemotaxis and retention of lymphocytes was proved to be another possible explanation for the large cellular infiltrate Lastly, by demonstrating the presence of HP-specific responses in this group of individuals, the hypothesis that ‘HP-specific Th17 responses, which were primed during initial infection,... stimulation with inflammatory stimuli semi-quantitative real time PCR (figure 3. 13) IL-1R1 was not significantly affected by the inflammatory stimuli being investigated The largest effects were seen at the 24 hour time point – a down-regulation in IL-22R1 expression Hence the effects of a few of the inflammatory stimuli were tested in freshly isolated GECs As in cell lines, IL-1R1 was not significantly... hypothesis stated in Chapter 1.7 that ‘Th17 cells contribute to immunopathology associating with precancerous lesions, and perhaps indirectly, cancer risk’ Firstly, it was demonstrated in a proportion of this cohort of subjects (made up mostly of Chinese individuals above the age of 50) that were followed up for 2 -3 time points, that after treatment for HP, there was little or no regression of PC lesions In. .. (20ng/ml) where indicated (E-G) Correlation of ex vivo hBD-2 concentrations with IL-1β, IL17A and IL-22 respectively 3. 1.9 IL-1β-stimulated GEC supernatant is chemoattractive to autologous CCR6+Th17 cellls In order to determine if the induced hBD-2 gene expression in GECs translated into increased chemotaxis of infiltrating Th17 cells, a chemotaxis assay was conducted Supernatant from the GEC stimulation assay . cytokines in group P subjects Having established that the gastric microenvironment in group P patients had increased Th17 infiltrate and a pro-inflammatory profile, it was of interest whether. elevated in group P compared to N (figure 3. 6I). This suggests that the gastric cytokine milieu in group P subjects continues to be biased against the baseline state of mucosal maintenance found in. 3. 1.5 IL-22 in the gastric mucosa Th17 cells also secrete IL-22, a cytokine belonging to the IL-10 family, that enhances epithelial integrity and regeneration in the gut, but promotes inflammation