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P010. Investigation of microRNA regulation by NOD2

O. Brain1, P.J. Allan1, T. Pichulik2, E. Khatamzas2, P. Simpson1, D. Jewell1, A. Simmons1,2

1John Radcliffe Hospital, Oxford, United Kingdom; 2Weatherall Institute of Molecular Medicine, Oxford, United Kingdom

Background: Crohn's Disease (CD) is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th1/Th17 immune response to commensal gut flora. Polymorphisms in NOD2 gene confer susceptibility to Crohn's, and predispose to early-onset, terminal ileal CD.

NOD2 encodes an intracellular receptor that responds to muramyl dipeptide (MDP), a component of bacterial cell wall peptidoglycan. Stimulation leads to NF-κB activation and release of pro-inflammatory cytokines. CD-associated NOD2 mutations appear to be predominantly loss-of-function, which raises the question as to how they predispose to a pro-inflammatory disease. Published data, and previous work in our lab, have revealed NOD2 cross-talks with toll-like receptors, such as TLR2. I am investigating NOD2 signalling and NOD2/TLR cross-talk with reference to microRNAs (miRNAs).

MicroRNAs 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. Loss of these negative regulators might lower the threshold to the development of a pro-inflammatory state in mucosal tissue.

Aims:

  1. Identify microRNAs differentially expressed in human dendritic cells (DCs) on NOD2, and on combined NOD2/TLR, stimulation.
  2. Determine the targets of these microRNAs.
  3. Determine the functional consequence of miRNA expression, and loss-of-expression.

Materials and Methods: Donors were genotyped before generation of monocyte-derived DCs expressing either WT or 1007fsinsC NOD2. Cells were left unstimulated or stimulated with MDP, Pam3CSK4 (TLR2 ligand), or both, before lysis and generation of RNA. MicroRNA microarray analysis was conducted using Illumina microRNA microarrays. MiRNA expression was validated with quantitative PCR. Potential miRNA targets were identified using Targetscan, Pictar and miRBase algorithms, and from published data. MiRNA mimic and antimir transfection was used to confirm targets by western blot, elisa and 3'UTR lucferase assay. Further targets and functional effects were determined by dual-colour Affymetrix cDNA array following miRNA mimic transfection into DCs. Functional consequence of loss of miRNA expression determined by intracellular bacterial killing assay, confocal microscopy, FACS analysis, and T-cell proliferation assay.

Results: NOD2 stimulation is required for expression of two miRNA clusters, in combination with either TLR2 or TLR5 triggering. MiRNA up-regulation is dependent on RIPK2 expression. DCs from patients homozygous for 1007fsinsC NOD2 fail to up-regulate these miRNA clusters. MiRNA cluster over-expression down-regulates the IL12-p40 subunit in human DCs at both mRNA and protein level. This subunit is a component of both IL-12 and IL-23 cytokines. IL-12p40 down-regulation can be ‘rescued’ in FS1007insC DCs by miRNA mimic expression.

Conclusion: DCs from patients with CD-associated mutations fail to up-regulate two miRNA clusters. These clusters regulate a critical component of the Th1/Th17 immune response. These findings may help link NOD2 polymorphisms and the pathogenesis of CD.