Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.