Targeted deletion of Atg5 in intestinal epithelial cells promotes dextran sodium sulfate-induced colitis

Abstract

Autophagy-associated genes have been identified as susceptible loci for inflammatory bowel disease. We investigated the role of a core autophagy factor, Atg5, in the development of dextran sodium sulfate (DSS)-induced colitis. Intestinal epithelial cell (IEC)-specific Atg5 gene deficient mice (Atg5 ^ΔIEC mice) were generated by cross of Atg5-floxed mice (Atg5 ^fl/fl ) with transgenic mice expressing Cre-recombinase driven by the villin promotor. Mice were given three cycles of 1.5% DSS in drinking water for 5 days and regular water for 14 days over a 60-day period. The dysfunction of autophagy characterized by a marked accumulation of p62 protein, a substrate for autophagy degradation, was detected in epithelial cells in the non-inflamed and inflamed mucosa of inflammatory bowel disease patients. DSS-colitis was exacerbated in Atg5 ^ΔIEC mice compared to control Atg5 ^fl/fl mice. Phosphorylation of inositol-requiring transmembrane kinase/endonuclease1α (IRE1α), a sensor for endoplasmic reticulum stress, and c-Jun N-terminal kinase, a downstream target of IRE1α, were significantly enhanced in IECs in DSS-treated Atg5 ^ΔIEC mice. Accumulation of phosphorylated IRE1α was enhanced by the treatment with chloroquine, an autophagy inhibitor. Apoptotic IECs were more abundant in DSS-treated Atg5 ^ΔIEC mice. These findings suggest that Atg5 suppresses endoplasmic reticulum stress-induced apoptosis of IECs via the degradation of excess p-IRE1α.

Keywords: IBD; IRE1α; autophagy.

Fig. 1

The expression of p62 protein in the epithelium of patients with IBD. Immunostaining for p62 was performed in normal mucosa and in the inactive and active mucosa of UC and CD patients. Pictures are shown from one of six independent samples with similar results. Original magnification: ×100.

Fig. 2

The deficiency of Atg5 increased susceptibility to DSS-induced colitis. IEC specific autophagy-deficient mice (Atg5^ΔIEC) were generated by crossbreeding Atg5 floxed (Atg5^fl/fl) mice with Villin-Cre mice. The experimental chronic colitis was induced by 3 five-day cycles of 1.5% w/v DSS treatment followed by 14 days of water. The mice were sacrificed on day 60. (A) Body weight. A statistically significant difference between the Atg5^fl/fl DSS group and the Atg5^ΔIEC DSS group was shown (*p<0.05). (B) Disease activity index. (C) Representative photographs of the colon. (D) Colonic weight per length on day 60. The data are expressed as means ± SEM (n = 10 mice/group). Values not sharing a letter are significantly different (p<0.05).

Fig. 3

Histological evaluation of chronic DSS-induced colitis. (A) Histological pictures of the colonic tissue on day 60. Original magnification; ×200. (B) Histological score. The data are expressed as means ± SEM (n = 10 mice/group). Values not sharing a letter are significantly different (p<0.05).

Fig. 4

The deficiency of Atg5 enhanced an activation of IRE1α in IECs. (A) Immunofluorescence staining was used to evaluate expression of p-IRE1α (red fluorescence), EP-CAM (green fluorescence) and DAPI (blue fluorescence). Representative pictures are shown from one of four independent experiments with similar results. (B) Effects of chloroquine, an inhibitor of autophagy, in DSS-colitis developed in Atg5 normal mice. Chloroquine (50 µg/g body weight) (Sigma-Aldrich) or physiological saline was intraperitoneally administered 6 h before euthanasia on day 60.

Fig. 5

Immunoblot for phosphorylated (p)-IRE1α, IRE1α, p-JNK and JNK in the isolated colonic epithelial cells. (A) Total protein was isolated from colonic epithelial cells. GAPDH was used as a loading control. The pictures are representative of four independent experiments. Relative expression of p-IRE1α (B) and p-JNK (C). The data are expressed as means ± SEM (n = 4 mice/group). Values not sharing a letter are significantly different (p<0.05).

Fig. 6

The deficiency of Atg5 increased the apoptosis of colonic epithelial cells. (A) TUNEL staining in the colon sections. (B) Number of TUNEL positive epithelial cells per 10 crypts. The data are expressed as means ± SEM (n = 10 mice/group). Values not sharing a letter are significantly different (p<0.05). (C) Correlation between number of TUNEL positive epithelial cells and histological score. (D) Number of TUNEL positive epithelial cells per 10 crypts in the comparison using samples with a similar degree of histological score (from 3 to 6 points). The data are expressed as means ± SEM. **p<0.01.