ROLE OF MICRORNA IN THE REGULATION OF TLR SIGNALING PATHWAY

Toll-like receptors (TLRs) play key roles in detecting pathogens and initiating inflammatory responses via the activation of specific signaling pathways. The TLRs activity must be tightly regulated to avoid excessive inflammation and consequent immunopathology, ranging from autoimmunity to cancer. MicroRNAs (miRNAs) are a new class of negative regulators involved in setting the balance of the immune response to inflammatory triggers. In this study, we identified miR-125a~99b~let-7e cluster and miR-146b as miRNAs that, after LPS engagement on human monocytes, are induced by the anti-inflammatory IL-10 and TGFβ, but are inhibited by the pro-inflammatory IFNγ. Bioinformatic analysis predicted and experimental evidence demonstrated that miR-125a-5p, let-7e-5p and miR-146b directly target the TLR pathway at multiple levels, including receptors (TLR4, CD14), signaling molecules (IRAK1, MyD88, TRAF6), and effectors (TNFα, IL-6, CCL3, CCL7, CXCL8). We showed that over-expression or inhibition of miR-125a, let-7e and miR-146b expression with lentiviral vector in human monocytes had a significant impact on the production of pro-inflammatory cytokines in response to LPS. In particular, we identified a role for miR-125a-5p and miR-146b in mediating the LPS hyporensponsiveness observed after IL-10 or TGFβ priming or during the endotoxin tolerance, the phenomenon of reduced sensitivity to subsequent challenge of LPS. The up-regulation of miR-125a-5p and miR-146b into THP-1 cells mimicked the LPS, IL-10 or TGFβ priming, whereas the inhibition of them by lentiviral vector or a pre-treatment with IFNγ reverted, partially, the tolerant phenotype. In an in vivo model of acute inflammatory response, we obtained that miR-125a-5p, miR-99b-5p and miR-146b were induced in macrophages recruited at the site of inflammation during the resolution process, and this was impaired in macrophages of IL-10 KO mice. Our studies indicated that miRNA cluster and miR-146b represent a new negative feedback mechanism of the TLR signaling pathway.