IFN-γ inhibits gastric carcinogenesis by inducing epithelial cell autophagy and T-cell apoptosis - PubMed (original) (raw)

IFN-γ inhibits gastric carcinogenesis by inducing epithelial cell autophagy and T-cell apoptosis

Shui Ping Tu et al. Cancer Res. 2011.

Abstract

IFN-γ mediates responses to bacterial infection and autoimmune disease, but it is also an important tumor suppressor. It is upregulated in the gastric mucosa by chronic Helicobacter infection; however, whether it plays a positive or negative role in inflammation-associated gastric carcinogenesis is unexplored. To study this question, we generated an H(+)/K(+)-ATPase-IFN-γ transgenic mouse that overexpresses murine IFN-γ in the stomach mucosa. In contrast to the expected proinflammatory role during infection, we found that IFN-γ overexpression failed to induce gastritis and instead inhibited gastric carcinogenesis induced by interleukin-1beta (IL-1β) and/or Helicobacter infection. Helper T cell (Th) 1 and Th17 immune responses were inhibited by IFN-γ through Fas induction and apoptosis in CD4 T cells. IFN-γ also induced autophagy in gastric epithelial cells through increased expression of Beclin-1. Finally, in the gastric epithelium, IFN-γ also inhibited IL-1β- and Helicobacter-induced epithelial apoptosis, proliferation, and Dckl1(+) cell expansion. Taken together, our results suggest that IFN-γ coordinately inhibits bacterial infection and carcinogenesis in the gastric mucosa by suppressing putative gastric progenitor cell expansion and reducing epithelial cell apoptosis via induction of an autophagic program.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1. H/K-ATPase IFN-γ mice fail to develop spontaneous gastric metaplasia

(A) The _IFN_-γ construct contains the mouse _H+/K+-ATPase_-s subunit gene and murine IFN-γ cDNA. (B) ELISA determined murine IFN-γ in stomachs of 4-month-old H+/K+-ATPase-IFN-γ mice and control mice. Data are mean ± SD of 10 mice. (C) Gene expression levels of mouse IP-10 and Mig mRNA in the stomach. (D) Representative macroscopic and histologic appearance of IFN-γ transgenic mouse stomach at 24-month

Figure 2

Figure 2. Overexpression of IFN-γ alleviates development of _H. felis_-induced gastric inflammation and neoplasia

Representative photomicrographs of H. felis infected H+/K+-ATPase-IFN-γ mouse stomach at (A) 12 months and (B) 24 months. (C) Representative macroscopic pictures of H. felis infected H+/K+-ATPase-IFN-γ and WT mice at 12 months (D) Histopathologic scores for stomachs of indicated mice (data represent the mean ± SD of 20 mice (*p< 0.05, WT vs. IFN-γ mice (both lines)). (E) Overexpression of IFN-γ does not affect the colonization of H. felis in the stomach. Stomach sections from H+/K+-ATPase-IFN-γ and WT mice infected for 2 months were stained with H&E. Arrow and insert indicate H. felis localization in the lumen of gastric glands. (F) Quantification of H. felis DNA in the stomach from indicated mice by PCR. The data represent the mean ± SD of 5 mice.

Figure 3

Figure 3. Overexpression of IFN-γ inhibits IL-1β-induced gastric inflammation, cell proliferation and progenitor cell expansion

12-month-old uninfected mice (A) and _H. felis_-infected mice (B) Proliferation index was calculated using the number of positive epithelial cells per total epithelial cell number per gland in 10 different high power fields (×400). The presented data represent the mean ± SD of 5 mice (*p < 0.05). (C) Representative macroscopic pictures of _IL-1β mice and IL-1β/_H+/K+-ATPase-IFN-γ stomachs at 12 months

Figure 4

Figure 4. Overexpression of IFN-γ induces apoptosis of T cells and inhibits the production of pro-inflammatory cytokines

(A) TUNEL staining of stomach sections from 12-month old mice showing percentage of TUNEL+ cells in different locations from each of the groups (n = 5). All data represent the mean ± SD. *P < 0.05. (B) Apoptotic epithelial cells were assessed in H&E stained sections and Caspase-3 stained cells were counted in 10 high power fields and numbers are presented as mean ± SD. *P < 0.05. (C) Immune cells isolated from the indicated mice were stained with PerCP-CD4, APC-CD8, TITC-Annexin V and PI and analyzed by FACS (n=5) to analyze apoptosis. (D-E) mRNA expression levels of cytokines in 12 month-old mice, as indicated.

Figure 5

Figure 5. Overexpression of IFN-γ induces autophagy in gastric epithelial cells in vivo

(A) Immunohistochemical staining for LC3 in stomach sections from 12-month-old mice, as indicated. Representative results from 8 different groups are shown (original magnification, × 200). (B and C) Western blot showing expression of LC3 in the stomach tissue of indicated mice. (D) Accumulation of LC3-II due to increased autophagosome formation after chloroquine treatment (30mg/kg for 10 days intraperitoneal). (E) Gastric cancer MKN-28 cell line was transfected with GFP–LC3 and then treated with 40μg IFN-γ for 24 hours and 48 hours. (original magnification, × 400). (F) IFN-γ increased LC3-II expression in MKN-28 cells after treatment with 40μg IFN-γ for 48 hours. GFP–LC3 was detected by Western blot. (G) GFP–LC3-positive punctate dots per cell. Data are from 100–200 cells from three independent experiments. Data are mean ± SEM, n = 100–200 cells from three independent experiments; * P < 0.01.

Figure 6

Figure 6. Overexpression of IFN-γ induces autophagy via Beclin 1 in gastric cancer cells in vitro

(A) Immunohistochemical staining for Beclin 1 in stomach sections from 12-month-old mice. Representative photomicrographs are shown (original magnification, × 200). (B) mRNA expression of Beclin-1 in the stomach from indicated mice, normalized to GAPDH expression. The data represent the mean ± SD of 5 mice. (*p < 0.001, vs. indicated group of mice). (C) MKN-28 cells were transfected with control and Beclin 1 siRNA for 6 hours and cultured with or without IFN-r 20ng/ml for another 48 hours. (E) Downregulation of Beclin-1 expression by siRNA decreased IFN-γ-induced autophagy in MKN-28 cells. *p<0.05. (F) IFN-γ activated the promoter activity of Beclin 1 in MKN-28 cotransfected with the indicated Beclin 1-promoter plasmids or empty pGL3 vector and Renilla luciferase vector for 6 h before IFN-γ treatment. Data are shown as means ± SD of 3 independent experiments. *p < 0.05.

Figure 7

Figure 7. IFN-γ infusion inhibits Dclk-1+ cell expansion and induces autophagy

(A) Representative stomach sections from indicated mice infused with IFN-γ for 4 weeks were stained with H&E (first lane), Ki-67 (second lane), DCAMKL-1 (Dclk-1) (third lane) and LC3 (Fourth lane) (original magnification, × 400). (B-D) Quantification of Ki-67-, DCAMKL-1-and LC3-positive cells in indicated mice with and without IFN-γ infusion. The percentage of positive epithelial cells was calculated using the number of positive cells to total cell number per field at 10 different locations in each stomach under light microscopy (× 400). The presented data represent the mean ± SD of 5 mice (*p < 0.05, vs. infused mice). (E) mRNA expression levels of Beclin1 from indicated mice was measured by qPCR and normalized to GAPDH expression. The data represent the mean ± SD of 5 mice (*p < 0. 01, vs. control group mice).

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