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P017. The miRNA-29 family regulates NOD2-dependent IL-23 expression in human DCs and limits intestinal pathology in a mouse model of mucosal inflammation

O. Brain1, B. Owens1, M.-Y. Sun2, S. Schonefeldt3, P. Allan1, T. Pichulik2, E. Khatamzas2, D. Jewell1, A. Liston3, A. Simmons1,2, 1University of Oxford, TGU, Nuffield Dept of Medicine, Oxford, United Kingdom, 2Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, Oxford, United Kingdom, 3KU Leuven, VIB Autoimmune Genetics Laboratory, Leuven, Belgium


Crohn's disease (CD) is a polygenic disease, with NOD2 polymorphisms conferring susceptibility to terminal ileal CD. GWAS studies have also identified a number of CD-susceptibility genes within the IL-23 pathway.

NOD2 encodes an intracellular innate immune receptor. Stimulation leads to NFkB activation and release of pro-inflammatory cytokines. CD-associated NOD2 mutations appear to be predominantly loss-of-function, raising the question as to how they predispose to a pro-inflammatory disease. We hypothesized that NOD2 stimulation may affect cellular function through microRNA expression. MiRNAs are short non-coding RNAs that prevent the translation of mRNA into protein, and have been shown to play an important role in the negative regulation of the innate immune response.

We previously presented that NOD2 regulates miR-29 expression in human DCs. This further work describes the functional consequences of reduced miR-29 expression.


Human monocyte-derived DCs expressing either WT or 1007fsinsC NOD2 were simulated with NOD2 and/or TLR ligands, followed by lysis and generation of RNA. MicroRNA microarray analysis was conducted using Illumina microRNA microarrays. MiRNA expression was validated with qPCR. MiRNA mimic and antimir transfection were used to confirm targets by Affymetrix cDNA array, qPCR, western blot, ELISA and 3'UTR luciferase assay. Functional consequence of loss of miR-29 expression in human DCs was determined by intracellular bacterial killing assay, FACS analysis, and T-cell proliferation assay. miR-29a-b1 deficient mice were exposed to DSS and assessed for weight loss and colitis pathology severity. Colonic explant cultures were assessed by qPCR, ELISA and FACS analysis.


NOD2 stimulation is required for expression of miR-29a/b/c. miR-29 up-regulation is dependent on RIPK2 expression. DCs from patients homozygous for NOD2 polymorphisms fail to up-regulate the miR-29 clusters. miR-29 directly targets IL-12p40, and indirectly regulates IL-23p19, in human DCs. miR-29 expression controls the Th17 response in a DC + T cell co-culture. Mice lacking miR-29a/b exhibit more severe inflammation in response to DSS than their WT counterparts, and an enhanced Th17 signature in colonic tissue, including IL-23.


DCs from patients with CD-associated NOD2 mutations fail to up-regulate miR-29. miR-29 controls IL-23 expression and the Th17 response in vitro. miR-29 deficiency in mice confers a greater susceptibility to an enhanced Th17 response and severe colitis.