Supplementary MaterialsS1 Fig: O1 bacteria clinical isolates VC08 and VC09 secrete both Cholera Toxin (CT) and Cytolysin (VCC) when grown in AKI broth. one from patient VC09 (VC09), that had been grown in AKI broth overnight (upper row) or with culture supernatants from overnight cultures ART1 of the same bacterial strains in AKI (lower row). Levels of miR-146a, miR-155 and miR-375 were determined by real-time qRT-PCR and expressed as relative quantity (RQ) compared to the median ct of tight monolayers incubated in parallel to challenged monolayers with tissue culture medium without added bacteria (Ctr, upper row) or fresh AKI (Ctr, lower row). Bars indicate mean RQ + 1 SD of 4 tight monolayers for each challenge in the upper row and 3 in the lower row. Statistically significant differences from the control monolayers as determined by one-way ANOVA with Dunett’s compensation for multiple comparisons, are shown. * O1 infection. (DOCX) pone.0173817.s003.docx (26K) GUID:?81C33CE4-6599-4D35-96CD-C5052528C0D9 S1 File: Supplementary materials and methods. (DOCX) pone.0173817.s004.docx (107K) GUID:?4E3AA318-40CC-4D1F-83A8-893B9FF3DB1B S2 File: Supplementary data. (XLSX) pone.0173817.s005.xlsx (64K) GUID:?B4B54E18-DCB4-4317-8B83-C42F4D45C014 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. An Excel file denoted “S2 File” with individual data points on which results presented in figures and tables are based and statistic evaluations performed is enclosed. Abstract The potential immunomodulatory role of microRNAs in small intestine of patients with acute watery diarrhea caused by O1 or enterotoxigenic (ETEC) infection was investigated. Duodenal biopsies were obtained from study-participants at the acute (day 2) and convalescent (day 21) stages of disease, and from healthy individuals. Levels of miR-146a, miR-155 and miR-375 and target gene (IRAK1, TRAF6, CARD10) and 11 cytokine mRNAs were determined by qRT-PCR. The cellular source of microRNAs in biopsies was analyzed by hybridization. The ability of bacteria and their secreted products to cause changes in microRNA- and mRNA levels in polarized tight monolayers of GSK690693 inhibitor intestinal epithelial GSK690693 inhibitor cells was investigated. miR-146a and GSK690693 inhibitor miR-155 were expressed at significantly elevated levels at acute stage of infection and declined to normal at convalescent stage (= 0.03 versus convalescent stage, pairwise). Both microRNAs were mainly expressed in the epithelium. Only marginal down-regulation of target genes IRAK1 and CARD10 was seen and a weak cytokine-profile was identified in the acute infected mucosa. No elevation of microRNA levels was seen in ETEC infection. Challenge of tight monolayers with GSK690693 inhibitor the wild type V. O1 strain C6706 and clinical isolates from two study-participants, caused significant increase in miR-155 and miR-146a by the strain C6706 (bacterium. By inducing microRNAs the bacterium can limit the innate immune response of the host, including inflammation evoked by its own secreted factors, thereby decreasing the risk of being eliminated. Introduction O1 and Enterotoxigenic (ETEC) are the predominant causes of acute dehydrating diarrhea in developing countries such as Bangladesh where a large proportion of cases requiring hospitalization are reported [1C4]. Both pathogens are non-invasive and colonize the small intestine. Major virulence factors and inducers of diarrhea are the secreted cholera toxin and heat-labile- and heat-stable enterotoxins in the case of O1 and ETEC, respectively [2, 5]. At the acute stage of disease in infection an innate immune response is GSK690693 inhibitor seen at the epithelial lining and its vicinity in the small intestinal mucosa involving accumulation of neutrophil granulocytes, increased production of inflammatory mediators and anti-microbial peptides [6, 7]. MicroRNAs are non-coding, 19C25 nucleotides long, single stranded RNA molecules that regulate inflammation at the post-transcriptional level [8C13]. Although microRNAs on average have the capacity to repress hundreds of target mRNAs, their action as immunomodulators seems to be highly regulated [8, 14C16]. Two microRNAs shown to be highly involved in the regulation of the acute inflammatory response after pathogen recognition through Toll-like receptors (TLRs) are miR-146a and miR-155 [17]. Identified target genes for miR-146a in immune cells are the cytoplasmic adapter molecules Interleukin(IL)-1 receptor-associated kinase 1 (IRAK1) and Tumor necrosis factor(TNF) receptor-associated factor 6 (TRAF6), key molecules in the signaling pathway of Nuclear factor -light-chain-enhancer of activated B cells (NFB) activation downstream of Myeloid differentiation primary response gene 88 (MyD88) after pathogen recognition through TLRs [8, 11, 18C20]. More recently, Caspase recruitment domain family member 10 (CARD10) was identified as.