Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer

Abstract

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Figure 1. Hh-infection induces acute inflammation in the lower bowel

Uninfected 129RAG2^−/− mice, or 129RAG2^−/− mice infected with Hh by oral gavage were euthanized at 2 weeks post-infection (WPI) and 6 WPI. Sections of the cecum, proximal colon, and distal colon were processed for histological analysis and RNA isolation. (A) Representative hematoxylin and eosin staining of cecum, proximal colon, and distal colon. (B) Histological Activity Index (HAI) from individual mice (n=10-11). (C) RT-PCR results were obtained from RNA isolated from indicated tissue at 2 WPI. n=17-20.

Figure 2. Hh-induces expression of iNOS and production of NO within the lower bowel epithelium

(A) RT-PCR results were obtained from RNA isolated from the cecum at 2 WPI, n=8-11. (B) Representative sections from uninfected 129RAG2^−/− mice or mice infected with Hh for 2 weeks evaluated for expression of iNOS within the epithelium (EpCAM) or macrophages (F4/80). Boxed areas in center images are shown at higher magnification on right. (C) Representative images from uninfected mice, or infected mice treated either with NMA or acetate (control for NMA), evaluated for the presence of nitrotyrosine (NT).

Figure 3. Hh-induces iNOS-dependent DNA damage

Uninfected 129RAG2^−/− mice or mice infected with Hh for 2 weeks were treated either with NMA or acetate in the drinking water for the final week of infection. (A) Representative histological sections from the cecum were evaluated by immunofluorescence with indicated stains. (B) Bar graphs demonstrating the percent of epithelial cells that were Ki67⁺ (top), the percent of epithelial cells that were γH2AX⁺ (middle), and the percent of Ki67⁺ epithelial cells were also γH2AX⁺ (bottom). n = 7-10. (C) Higher magnification image of cecum an Hh-infected mouse demonstrating punctate nuclear staining with γH2AX.

Figure 4. IL-22 is necessary for induction of iNOS

Uninfected 129RAG2^−/− mice or mice infected with Hh for 2 weeks were treated either with 100 µg control Ab or anti-IL22 Ab every other day during the second week of infection. (A) RNA was isolated from the cecum and expression of indicated gene was analyzed by RT-PCR. n=9-11. (B) Western blot of total colonic extracts from 3 individual mice per group with indicated antibodies. (C) Representative histological sections from the cecum were analyzed with indicated stains. (D) The fraction of crypts that exhibited iNOS staining was compared (n=3-4). (E) RNA isolated from mouse intestinal organoids treated with IL-22 at indicated dosages was analyzed by RT-PCR. n=6.

Figure 5. Hh-induced DNA damage depends on IL-22

Uninfected 129RAG2^−/− mice or mice infected with Hh for 2 weeks were treated either with depleting IL-22 Ab or a control Ab. (A) Representative histological sections from the cecum were analyzed with indicated stains. (B) Graphs representing the percent of epithelial cells that were γH2AX⁺ (left), the percent of epithelial cells that were Ki67⁺ (center), and the percent of the percent of Ki67⁺ epithelial cells that were also γH2AX⁺ (right) in indicated groups. n=8-14. (C) Histological Activity Index (HAI) of individual mice from each group.

Figure 6. IL-22 depletion inhibits dysplasia in mice chronically infected with Hh

(A) Representative hematoxylin and eosin staining of cecum from uninfected 129RAG2^−/− mice or mice infected with Hh for 10 weeks treated either with control or IL-22 depleting Ab for the final week of infection, as indicated. (B) Inflammation, hyperplasia, and dysplasia scores for uninfected mice (n=7) or mice infected with Hh for 10 weeks and treated with control Ab (n=8) or anti-IL-22 depleting antibody (n=8) as above. (C) Expression of RegIIIβ, RegIIIγ, and iNOS in total RNA isolated from cecum or colon of mice treated as above (n=7-8).

Figure 7. IL-22 depletion inhibits DNA damage in mice chronically infected with Hh

Uninfected 129RAG2^−/− mice or mice infected with Hh for 10 weeks were treated either with depleting IL-22 Ab or a control Ab for the final week of the experiment. (A) Representative histological sections from the cecum were analyzed with indicated stains. (B) Graphs representing the percent of epithelial cells that were Ki67⁺ (top) or γH2AX⁺ (bottom), n=9. (C) Representative histological sections with indicated stains comparing localization of iNOS within epithelial cells (EPCAM) and macrophages (F4/80). Single channel stains of iNOS alone are provided to more clearly demonstrate the differential effects of IL-22 depletion on epithelial cells and macrophages. (D) The fraction of crypts that exhibited iNOS staining was compared (n=3-4).

Figure 8. IL-22 depletion does not inhibit neutrophil recruitment to the cecum following Hh-infection

(A) Representative histological sections from the cecum of Uninfected 129RAG2^−/− mice or mice infected with Hh for 2 and 10 weeks were treated either with depleting IL-22 Ab or a control Ab for the final week of the experiment. (A) Micrographs show representative sections stained with antibody to MPO. (B) The number of MPO⁺ cells per 1000 cells within the cecum was determined (n=4-8 mice/group).

Figure 9. Hh infection induces IL-22 dependent dysbiosis

(A) Quantification of Hh colonization density in the cecal content. n=9-11. (B) Chao and Shannon diversity indices from 16S rRNA sequencing data obtained from fecal pellets of uninfected mice and Hh infected mice treated with control or anti-IL-22 antibody as indicated. (C) Relative abundance of indicated taxa from groups described in B. (D) Relative abundance of indicated taxa as determined by taxa specific 16s rRNA qPCR from groups described in B.