IL-17a and IL-22 Induce Expression of Antimicrobials in Gastrointestinal Epithelial Cells and May Contribute to Epithelial Cell Defense against Helicobacter pylori

Abstract

Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some β-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.

Fig 1. IL-17A and IL-22 synergistically stimulated human epithelial cells inducing IL-8.

(A) IL-8 production at 24 hours by AGS cells in response to IL-22 and IL-17A as measured by ELISA. p-value is based on ANOVA test with Dunnett’s correction for multiple comparisons ***p<0.001. (B) 8 hour stimulation of AGS cells or (C) AZ-521 cells with an IL-22/IL-17A cocktail induces upregulation of antimicrobials as measured by real-time rtPCR. Expression is shown as relative units and is relative to RNA from untreated cells. Data shown as ± SEM and are representative of 3 independent experiments.

Fig 2. IL-17A and IL-22 can activate human gastrointestinal epithelial cells to kill H. pylori.

The ability of H. pylori to survive for 6 hours in co-culture with AGS cells pre-stimulated for 16 hours with IL-22, IL-17A or combined IL-22 and IL-17A is presented. (A) SS1 strain or (B) PMSS1 strain percent (%) survival represented on the y-axis is equal to the number of H. pylori CFU from the cytokine-treated cells divided by the number of CFU recovered from untreated AGS cells. Graphs are representative of 3 independent experiments and error bars represented mean ± SEM. Statistical significance is based on one-way ANOVA test with Dunnett’s correction for multiple comparisons to the no cytokine treatment control ***p<0.001,**p<0.01,*p<0.05.

Fig 3. H. pylori infection induces IL-22 expression in murine gastric mucosa.

Expression of IL-22 in H. pylori-infected WT mice at 1, 2 and 3 months post infection was determined by real-time rtPCR. Relative units (relative to uninfected WT mice) is presented as the mean of 4–6 WT mice at each time point. Error bars represent ± SEM.

Fig 4. IL-22 is not required to control bacterial burden or gastric inflammation in the mouse model.

(A) Bacterial burden was measured in WT and IL-22-/- mice that were infected with PMSS1 for 1 month or 3 months. Colony forming units (CFU) per gram of stomach tissues was calculated and is presented in the graph. (B) Levels of acute and chronic inflammation were scored on stomach tissue (in the corpus and antrum) at 1 month and 3 months post infection with strain PMSS1. Total inflammation as presented is the sum of acute and chronic inflammation. Statistical analysis was performed using Mann-Whitney U which resulted in no significant differences between the groups, and error bars represented mean ± SEM. See methods for scoring system (scale is 0–12).

Fig 5. IL-22 deficiency had minimal effect on gene expression in the mouse during H. pylori infection.

At 1 months post infection, no significant difference in expression of Defb3 (p = 0.524), Lcn2 (p = 0.31), S100a8 (p = 0.944), S100a9 (p = 0.31), Cxcl1 (p = 0.413), Cxcl2 (p = 0.944) and Cxcl5 (p = 0.532) as measured between H. pylori–infected WT and infected IL-22-/- mice gastric tissue. Relative units are calculated as described in the methods, relative to GAPDH and calibrated to uninfected WT mice. Statistical analysis was performed using Mann-Whitney U (Error bars represent ± SEM).