Inducible MicroRNA-223Down-regulation Promotes TLR-triggered IL-6and IL-1βProduction In Macrophages By Targeting STAT3

Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific microbial components (pathogen-associated molecular patterns, or PAMPs) derived from pathogens including bacteria, viruses and fungi. Stimulation of TLRs by microbial components triggers the induction of inflammatory cytokines such as interleukin-6(IL-6), IL-1β and TNF-a. IL-6has diverse roles in the regulation of immune response, and the detailed mechanism that regulates IL-6production has attracted much attention. Although multiple regulators, including signaling molecules, transcription factors have been extensively investigated in the regulation of IL-6production, there are limited reports about the regulation of IL-6production and its functions by microRNA(miRNAs). miRNAs, the new-found small non-coding RNAs, have been demonstrated to be important regulators in various biological process, including TLR-triggered inflammatory response. miR-223was in the first cadre of miRNAs discovered to be highly expressed in myeloid cells of the bone marrow, where it is mainly expressed in the myeloid, granulocytic and monocytic compartments. And it negatively regulates progenitor proliferation, granulocyte differentiation and activation. However, whether miR-223is regulated by innate signals and involved in the subsequent innate response need to be further determined. In the present study, miR-223in mouse macrophages treated with LPS or polyI:C showed significant down-regulation. In order to investigate its biological significance and the underlying mechanism in TLR-activated innate immune reponse, we used miR-223mimics and the data showed that miR-223could decrease IL-6and IL-1β production in LPS-treated macrophages but not TNF-a. We assessed the effect of miR-223mimics on the expression levels of several components of the NF-κB and MAPK pathway in RAW264.7cells. Over-expression of miR-223did not influence the phosphorylation of IκBα, ERK1/2or P38upon LPS stimulation. Surprisingly, a decrease in the protein levels of phosphorylated and total STAT3was seen. As miRNAs function mainly through posttranscriptional inhibition of their target genes, miR-223was suggested to exert its immune modulatory function through directly targeting STAT3expression, a transcription factor which promotes IL-6and IL-1β expression, but not TNF-α. MiR-223mimics markedly decreased the luciferase activity and protein expression of STAT3. These results suggested that miR-223can regulate STAT3protein expression. More interestingly,, the IL-6produced after TLRs activation could down-regulates miR-223expression, consequently, relieves miR-223-mediated translational suppression of STAT3resulting in STAT3protein expression. Taken together, our findings provide a new explanation characterizing the molecular mechanism responsible for the regulation of IL-6production after TLR-triggered macrophage activation. It may provide useful insights to the understanding of molecular mechanisms by which the IL-6/miR-223/STAT3pathway controls the inflammatory response and promotes the pathogenesis of inflammatory diseases.