Colitis and colon cancer in WASP-deficient mice require helicobacter species

Abstract

Background: Wiskott-Aldrich syndrome protein-deficient patients and mice are immunodeficient and can develop inflammatory bowel disease. The intestinal microbiome is critical to the development of colitis in most animal models, in which Helicobacter spp. have been implicated in disease pathogenesis. We sought to determine the role of Helicobacter spp. in colitis development in Wiskott-Aldrich syndrome protein-deficient (WKO) mice.

Methods: Feces from WKO mice raised under specific pathogen-free conditions were evaluated for the presence of Helicobacter spp., after which a subset of mice were rederived in Helicobacter spp.-free conditions. Helicobacter spp.-free WKO animals were subsequently infected with Helicobacter bilis.

Results: Helicobacter spp. were detected in feces from WKO mice. After rederivation in Helicobacter spp.-free conditions, WKO mice did not develop spontaneous colitis but were susceptible to radiation-induced colitis. Moreover, a T-cell transfer model of colitis dependent on Wiskott-Aldrich syndrome protein-deficient innate immune cells also required Helicobacter spp. colonization. Helicobacter bilis infection of rederived WKO mice led to typhlitis and colitis. Most notably, several H. bilis-infected animals developed dysplasia with 10% demonstrating colon carcinoma, which was not observed in uninfected controls.

Conclusions: Spontaneous and T-cell transfer, but not radiation-induced, colitis in WKO mice is dependent on the presence of Helicobacter spp. Furthermore, H. bilis infection is sufficient to induce typhlocolitis and colon cancer in Helicobacter spp.-free WKO mice. This animal model of a human immunodeficiency with chronic colitis and increased risk of colon cancer parallels what is seen in human colitis and implicates specific microbial constituents in promoting immune dysregulation in the intestinal mucosa.

Figure 1. Helicobacter spp. are required for colitis induction in WKO mice

(a) Representative H&E sections and (b) mean histologic scores ± SEM of WKO mice at 10–15 months of age from Helicobacter spp.-free rederived group (n = 7) and of 6 months-old WKO mice in SPF conditions (n = 30) at MGH. (c) Representative H&E sections and (d) mean histologic scores ± SEM of WKO mice rederived in Helicobacter spp.-free conditions at MIT (n = 13) at 5 months and 14 months old, respectively, compared to age-matched WKO mice housed in SPF conditions (n = 8). (e) Naïve CD4⁺ T cells were transferred into 4–6 month-old WRDKO mice to induce colitis. Percent of initial weight of WRDKO mice from SPF (n = 4) compared to those from Helicobacter spp.-free (n = 4) rooms after WT naïve CD4⁺ T cell transfer. (f) Mean histologic colitis indices of animals ± SEM in (e), p = 0.017. Data are representative of two independent experiments. H&E stained sections taken at 20x objective. *p < 0.05, **p < 0.01, ***p < 0.005 on Mann-Whitney test comparing mice in SPF and Helicobacter-free rooms. H-free = Helicobacter spp.-free; SPF = specific pathogen free; WKO = WASP^−/− mice; WRDKO = WASP^−/−RAG-2^−/− mice.

Figure 2. Sublethal irradiation led to colitis in WKO mice in SPF conditions

(a) Mean histologic colitis indices ± SEM and macroscopic thickening of (b) irradiated WKO mice compared to irradiated WT mice and (c) irradiated WKO mice compared to non-irradiated WKO mice at six months of age two months after irradiation. Representative H&E images of colons from (d) non-irradiated WT mice, (e) irradiated WT mice, (f) non-irradiated WKO mice, and (g) irradiated WKO mice taken at 10x magnification. Kruskal-Wallis analysis with post-hoc Dunn’s test showed * p < 0.05. Irr = irradiated.

Figure 3. Radiation-induced colitis in WKO mice is not dependent on the presence of Helicobacter spp

(a) Mean percent of initial weights ± SEM of sublethally irradiated and non-irradiated WKO mice in SPF and Helicobacter spp.-free conditions over 9 weeks after irradiation starting at 2 months of age. (b) Mean histologic colitis indices ± SEM in sublethally irradiated or non-irradiated Helicobacter spp.-free or SPF WKO mice. Data were pooled from five independent experiments (n = 22 for each irradiated group; n = 15 for non-irr H-free; n = 8 for non-irr SPF). Kruskal-Wallis analysis with post-hoc Dunn’s test showed * p < 0.05. (c) Representative H&E images of non-irradiated (left) and irradiated (middle) Helicobacter spp.-free WKO mice and irradiated SPF WKO (right), 10x objective. H-free = Helicobacter spp.-free; Irr = irradiated.

Figure 4. Rederived Helicobacter spp.-free WKO mice infected with H. bilis develop typhlitis and colitis at 7–9 months post infection

(a) Representative cecum from an uninfected control mouse at 7 mpi with sparse inflammatory cells in the lamina propria and lack of other significant epithelial changes (bar = 80 µM). (b) Representative non-neoplastic cecum of a H. bilis-infected mouse at 7 mpi showing moderate mucosal and submucosal inflammation, edema, surface epithelial tethering, epithelial hyperplasia and minimal dysplasia (bar = 160 µM). (c) Low magnification image of the cecum of a H. bilis-infected male at 7 mpi that developed a broad-based mucosal proliferative lesion (diagnosed as intramucosal carcinoma) showing prominent inflammation, edema, epithelial defects, epithelial hyperplasia, and disorganized glandular architecture (bar = 400 µM). (d) Higher magnification view of (c) showing high-grade dysplastic glands with invasion into the muscularis mucosa and extension/herniation into the submucosa (bar = 80 µM). (e) Mean subscores ± SEM of inflammation (inf), edema, epithelial defect (epi def), crypt atrophy (crypt atr), hyperplasia (hyperpl), dysplasia (dyspl), contribute to the cumulative typhlitis index of all mice. (f) Representative H&E image of the colon from an uninfected control mouse at 7 mpi with no significant mucosal inflammation or other epithelial alterations (bar = 80 µM). (g) Representative image of a non-neoplastic colon from an H. bilis-infected at 7 mpi showing mild mucosal inflammation and minimal hyperplasia (bar = 80µM). (h) Low magnification H&E image of a grossly thickened colon from a H. bilis-infected male at 7 mpi showing severe transmural mixed inflammation, lympho-follicular formation, edema, epithelial defects, fibrosis, crypt loss, marked epithelial hyperplasia, and severely disorganized glandular architecture with high grade dysplasia and multifocal submucosal invasion/herniation (bar = 400 µM). (i) Higher magnification view of (h) showing dysplastic glands with frequent mitotic activity and luminal cellular debris in a background of granulocytic inflammation (bar = 40µM). (j) Higher magnification of (h) showing invasive high grade dysplastic/neoplastic glands in the muscularis mucosa and submucosa surrounded by proliferating fibroblasts and dense lympho-plasmacytic inflammation (bar = 80µM). (k) Higher magnification of (h) showing a focus of invasive glands deep in the submucosa with associated partial loss of epithelial lining, disorganized pools of mucin-like material, and associated dense inflammatory cellular aggregates (bar = 40µM). (l) Subscores contributing to the colitis total index of all mice. *p < 0.05, **p < 0.01, ***p < 0.005 on Mann-Whitney test comparing un-infected and infected mice in that subscore. C = uninfected control mice; Hb = H. bilis-infected mice; mpi = months post infection.

Figure 5. Dysplasia and colon carcinoma found in WKO mice raised under SPF conditions and under Helicobacter spp.-free conditions with H. bilis infection

Dysplasia subscores ± SEM of individual mice in (a) cecum and (b) colon of rederived WKO animals 7–9 months after infection H. bilis with the horizontal bars representing the mean ± SEM shown in Figure 4e&l. (c) Dysplasia subscores ± SEM of colons from 6-month-old WT, 6-month-old WKO in SPF, and 10–15-month-old WKO mice raised in Helicobacter spp.-free conditions. (d) Dysplasia subscores ± SEM of colons from irradiated 4-month-old and 6-month-old WT or WKO mice (two months after irradiation). H-free = Helicobacter spp.-free; irr = irradiated.