Role of autophagy in gut homeostasis

Genome-wide association studies of inflammatory bowel diseases (IBDs) identified susceptible loci containing an autophagy related-gene. However, the role of autophagy in the colon, a major affected area in IBDs, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7)-conditional knockout (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relm! , intelectin-1 and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against 1 http://www.jbc. org/cgi/doi/10.1074/jbc.M114.632257 The latest version is at JBC Papers in Press. Published on July 6, 2015 as Manuscript M114.632257 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at U niersity of G ergia A tens on July 9, 2015 hp://w w w . jb.org/ D ow nladed from Role of autophagy in gut homeostasis bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and mucus secretion. Inflammatory bowel diseases (IBDs) including Crohn’s disease (CD) and ulcerative colitis (UC) are chronic remittent or progressive inflammatory disorders in the gastrointestinal tract associated with an increased risk of colon cancer (1). UC affects part of the colon or the entire colon in an uninterrupted pattern (1). In contrast, CD generally affects any region of the intestine, including the ileum and colon, often discontinuously (2,3). In CD, the ileum alone was affected in approximately 45% of cases (ileitis); the ileum and colon (ileocolitis) in approximately 19% of cases; and the colon alone (colitis) in approximately 32% of cases (4). Therefore, colonic symptoms are present in the majority of IBD-patients. Although the etiology of IBDs is not well understood, extensive previous studies have suggested the involvement of both environmental and genetic factors that lead to dysfunction of the epithelial barrier, mucosal immune system and responses to gut microflora (5). Genome-wide association studies of CD identified susceptible loci containing autophagy related-gene ATG16L1 (6,7). Autophagy is a conserved process for the bulk degradation of cytoplasmic components. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The ATG5-ATG12/ATG16L1 complex and LC3-II are essential for autophagosome formation (8,9). ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions (ATG12-conjugation and LC3-lipidation). Therefore, ATG7 is also essential for autophagy (10-12). Recent studies have revealed that autophagy is important for the clearance of intracellular microbes, including adherent-invasive Escherichia coli (13), Salmonella typhimurium (7,14) and Mycobacterium tuberculosis (15,16). It was reported that ATG16L1-deficient macrophages exhibited elevated endotoxin-induced IL-1! production (17), indicating that autophagy is also important for the control of endotoxin-induced responses. In agreement with this notion, others have reported that autophagy in the small intestinal epithelium reduced LPS-induced proinflammatory responses by inhibiting NF-"B activation (18). A number of studies have demonstrated the function of autophagy related genes in the gastrointestinal tract. In hypomorphic Atg16L1 mice that were generated with a Gene-trap mediated method, Paneth cells exhibited notable abnormalities in the granule exocytosis pathway (19). Macrophages harboring T300A variants of ATG16L1 showed defective clearance of the ileal pathogen Yersinia enterocolitica and elevated cytokine production (20). Intestinal epithelia-specific Atg16L1 deficient mice exhibited constitutive endoplasmic reticulum stress in Paneth cell and are highly susceptible to experimental colitis (21). Intestinal epithelia-specific autophagy-related gene 7 (Atg7) deficiency in Villin-Cre transgenic mice (22-24) showed enhanced susceptibility against Citrobacter 2 at U niersity of G ergia A tens on July 9, 2015 hp://w w w . jb.org/ D ow nladed from Role of autophagy in gut homeostasis rodentium infection (used as murine models of EHEC and EPEC infection) (25). In these studies, Atg16L1-deleted fetal liver chimeric mice (17), Atg16L1-hypomorphic mice (19), Atg16L1 Villin-Cre mice (21) and Atg7 Villin-Cre mice (25) were used. However, it should be noted that Atg16L1 is more abundantly expressed in the colon than in the small intestine (6). Additionally, colonic Cre recombinase expression in Villin-Cre transgenic mice was much lower than expression in the small intestine (22-24). Therefore, it is unlikely that previous studies using these mutant mice could have clarified the role of autophagy in the colon, which is a major affected area in IBDs. In this study, we took advantage of the specific Cre recombinase expression in colonic epithelial cells in a GlcNAc6ST-2-Cre transgenic mouse model (26) to delete Atg7 in a colonic epithelial cell-specific manner. By using these mutant mice, we analyzed the function of autophagy in maintenance of gut commensal microflora and protection against UC-like colitis. EXPERIMENTAL PROCEDURES Generation of cKO mice—cKO mice were generated by crossing GlcNAc6ST-2-Cre transgenic (26) and Atg7 mice (27). The GlcNAc6ST-2-Cre/Atg7 and GlcNAc6ST-2-Cre/Atg7 mice were used as conditional knockout mice. The Atg7 mice were used as WT controls throughout the study unless otherwise indicated. To detect Cre recombinase expression, GlcNAc6ST-2-Cre were crossed with R26R reporter mice (28). The experimental protocol was approved by the Animal Research Committee of Hoshi University and University of Shizuoka. X-gal staining—X-gal staining was performed as previously described (26). Briefly, frozen sections (7 μm) were fixed in PBS containing 1.5% glutaraldehyde and incubated with X-gal solution and Nuclear Fast Red solution (Sigma-Aldrich). Quantitative RT-PCR for mRNA expression—Tissue RNA was extracted with TRIzol reagent (Life technologies). The cDNA was synthesized with the PrimeScript RT-PCR kit with gDNA Eraser (TaKaRa) and subjected to quantitative RT-PCR using SYBR Premix Ex Taq II (Tli RNase H Plus; TaKaRa). The expression of each mRNA was normalized to the expression of "-actin with the ##Ct method according to the manufacturer’s instructions (TaKaRa Thermal Cycler Dice TP870). The primer sequences are in Table 1. Western blotting—Mouse colons were homogenized in homogenization buffer (0.25 M