Mechanisms involved in the protective effect of short-chain fatty acids against Clostridium difficile-associated colitis

Abstract

Under physiological conditions, commensal intestinal bacteria restrict colonization by Clostridium difficile, a bacillus resistant to various antimicrobial agents. After the use of antibiotics, there is an imbalance of the microbiota and proliferation of the C.difficile, causing diverses pathological disease. It is known that the products of bacterial metabolism, short chain fatty acids (SCFAs), are produced during the fermentation process of dietary fibers and present a protective effect against intestinal infections, such as that caused by C. difficile. However, the molecular mechanisms behind these effects are not known. This work aims to investigate the participation of AGCCs activated pathways in the protection observed in murine model of acute C. difficile colitis. Specifically, we will examine the relevance of the G protein coupled receptor, FFAR2/GPR43, and the hypoxia-induced transcription factor 1 (HIF-1), as well as the possible involvement of miRNAs over the gene expression modifications relevant to the cure. For this, we will use in vivo models (FFAR2 whole-body knockout animals, tissue specific deletion of HIF-1± and/or Dicer, an enzyme involved in the maturation of miRNA) and in vitro (HCT-116 cell line or intestinal organoids). We will perform molecular analyzes including Western blotting, immunofluorescence, RT-qPCR for specific genes and miRNAs and RNA sequencing and histopathological analyzes including analysis of stained colonies of H&E, identification of leukocyte populations in the lamina propria and mesenteric lymph nodes by flow cytometry, as well as analysis of inflammatory mediators by ELISA and epithelial permeability by immunohistochemical labeling of proteins relevant to the process and of bacterial and FITC-dextran translocation after infection. With this study, we intend to advance in the understanding of the interactions between the microbiota and the development of intestinal infections and to identify new molecular targets for therapeutic interventions in the disease caused by C.difficile. (AU)