Ileal Pouch of Ulcerative Colitis Patients Exhibit Impaired Autophagy

Abstract

BACKGROUND: Total rectocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment and for familial adenomatous polyposis (FAP) with many rectal polyps. The main complication after this procedure is the pouch inflammation (pouchitis) that can affect up to 45 percent of patients who are submitted to IPAA for UC, and only five percent of the FAP patients who undergo the same procedure. Previous studies have shown increased pro-inflammatory cytokines in the ileal pouch (IP) mucosa of UC patients, even in the absence of clinical, endoscopic and histological inflammation. Autophagy is an evolutionarily conserved catabolic pathway that consists of selective degradation of cellular components and a homeostatic mechanism that protects cells exposed to stress situations (toxins, starvation), and defects in this pathway have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. METHODS: Sixteen patients with IP in "J" shape, asymptomatic and with endoscopically normal mucosa were evaluated. The control group consisted of eight patients with normal colonoscopy. Gene expression was analyzed by qPCR and protein levels by immunoblotting and immunofluorescence. This study was approved by the Ethics Committee of the University of Campinas and was performed in accordance with the Declaration of Helsinki. All results were reported as means ± SEM. Data were analyzed by non-parametric Test, comparing all groups. The level of significance was set at P<0.05. RESULTS: There was a significant decrease in the transcriptional levels of ATG5, MAP1LC3A and BAX in the FAP group (P<0.05). There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group (P<0.05). Although the LC3II levels by immunoblot were higher in the UC group, total LC3 and LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group (P<0.05). Corroborating to these results, there was an increase of p62 by immunoblot in the UC group, compared to controls (P<0.05). CONCLUSION(S): These results indicate impaired macroautophagy mechanism in the IP. In FAP, decreased autophagy may be related to impaired apoptosis, otherwise in UC, may be mainly due to increased Toll-Like Receptors (TLR) activation. These findings may explain the inflammation predisposition, mainly in the IP mucosa of UC patients. Acknowledgments: We thank FAPESP (São Paulo Research Foundation) for financial support. Negreiros LMV (co-author) received scholarship from FAPESP. We are grateful to Prof. Tristan Torriani for English grammar revision. We thank Francesca Ramos and José Diego Botezelli for technical assistance and the staff of the Life Sciences Core Facility (LaCTAD) from State University of Campinas (UNICAMP), for the Cell Biology analysis.