436g M-CSF Deficient Op/Op Mice Are Protected Against the Development of Diabetic Gastroparesis in Streptozotocin-Induced Diabetes

Abstract

Background and Aims: The dysregulation of microRNAs (miRNAs, miR) in Crohn's disease (CD) was observed, suggesting a role for miRNAs as contributors to CD susceptibility. We previously showed that the adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of CD patients, are able to adhere to and invade intestinal epithelial cells (IECs). Here, we elucidated the mechanism by which AIEC infection modulates expression of miRNAs to suppress the host defense response. Methods: Mature miRNA expression was quantified by quantitative RT-PCR. Direct binding of miRNAs to the 3'-untranslated regions of their mRNA targets was determined by luciferase assay. Binding of NFκB to miRNA gene promoter was assessed by Chromatin immunoprecipitation assay. Autophagic activity was assessed by Western blot analysis and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by invasion assay and confocal microscopy. In vivo transfection of miRNAs or anti-miRNAs was performed using a mouse ileal loop model. Results: AIEC infection activated NF-kB pathway, driving the transcription of miR-30c and miR130a genes. The AIEC-induced miR-30c and miR-130a upregulation in turn resulted in a decrease in ATG5 and ATG16L1 expression and inhibited autophagic activity, leading to abnormal intracellular persistence of AIEC and enhanced pro-inflammatory response. A reverse correlation between expression of miR-30c and miR-130a and that of ATG5 and ATG16L1 in CD ileal biopsies was observed, supporting the pathological relevance of the in vitro data. Inhibition of miR-30c and miR-130a in vitro and in vivo effectively blocked the AIEC-induced defect in autophagy, suppressing AIEC intracellular replication and AIECinduced inflammation. Conclusions: We show here that AIEC infection suppresses autophagy response to replicate within host cells by regulating miRNA expression. Our study strongly reinforces the increasing evidence showing autophagy deficiency as a molecular pathway underlying the pathogenesis of CD. This raises autophagy-targeting miRNAs as promising therapeutic targets to inhibit the intracellular replication of CD-associated bacteria and the consequent bacterial-induced inflammation.