1111 Innate Immune Sensing of a Bacterial Protein in the Endoplasmic Reticulum by Inositol-Requiring Enzyme 1 (Ire1α)

Abstract

Background & Aims: Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated distinct miRNA profiles in a wide range of human disorders including inflammatory bowel disease and colon cancer. Surprisingly little is known about miRNA responses in immune cells that are challenged with relevant proinflammatory stimuli, i.e. cytokines or pathogen-associated microbial patterns. With this study we thus aim to generate a prototypic picture of inflammatory miRNA networks in monocytes as a relevant immune cell population and to understand functional consequences of selected regulated miRNAs on inflammatory signalling pathways. Methods: Levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-alpha as pro-inflammatory control in primary human monocytes were qunatified using real time PCR. Selected miRNAs were verified in parallel in LPMNCs and biopsies from IBD patients. Transfection of premiRs and antago-miRs in THP1 monocytic cells were employed in order to understand functional consequences of miRNA regulation. Results: We identified distinct miRNA response clusters pointing to unique and shared mechanisms for different proinflammatory stimuli even in a single cell type. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding preor antimiRNAs . The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK-gamma (Nemo). Interestingly, regulation of several overlap miRNAs shared between stimuli could also be detected in samples from IBD patients. Conclusions: Our findings on co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Investigation in the field of miRNA responses in intestinal inflammation will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases.