Progesterone,microRNA And IRAK1 Participate In Toll-like Receptor Signaling In Monocytes And Macrophages

Toll like receptors are members of a larger superfamily that includes the interleukin-1 receptors (IL-1Rs), and on the basis of considerable homology in the cytoplasmic region.12 TLRs have been identified in mammals. Their expressions are not restricted to monocytes and macrophages but also found in a wide range of nonimmune cells. TLRs are important kinds of pattern recognition receptors(PRRs), the sensing of particular pathogen-associated molecule patterns(PAMPs) by TLRs leads to the activation of downstream signalling cascades, then upregulates the transcription of genes involved in inflammatory responses, which inititates innate immmune response, being the first line of defence against pathogens. However, aberrant activation of this system leads to immunodeficiency, septic shock, or induction of autoimmunity, therefore, represents an important and frequent mechanism of disease. In this study, we investigated the role of progesterone and IRAK1 in Toll like receptor signal pathway in monocytes and macrophages.PartⅠ:Progesterone participates in Toll like receptor signal pathway in macrophagesProgesterone is produced by the granulosa cells and corpus luteum of the ovary. In addition to its essential role in establishment and maintenance of pregnancy, a number of studies have demonstrated its functional role in immune response, mainly at concentrations commensurate with pregnancy. However, the underlying mechanisms remain to be fully understood. Here we present the evidences that progesterone inhibited immune response to lipopolysaccharide (LPS) and polyinosinic polycytidylicacid poly(I:C) through modulating Toll-like receptor (TLR) signaling. Pretreatment with progesterone can significantly inhibit TLR3/4-mediated proinflammatory cytokine (such as IL-6,TNF-α, iNOS) and typeⅠinterferon (such as IFN-β) in macrophages.MicroRNAs (miRNAs), recently identified noncoding small RNAs, are emerging as key regulators in homeostasis of the immune system. Therefore, aberrant expression of miRNAs may be linked to immune dysfunction, such as in chronic inflammation and autoimmunity. In this study, we investigated the potential role of miRNAs in progesterone-mediated regulation of innate immune responses, We found that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was increased in progesterone-pretreated macrophages compared with controls. Inhibition the activity of miR-146a significantly up-regulates its target IRAK1 and LPS-induced IL-6 expression in mouse macrophages. Further, miR-155, which was markedly inhibited by progesterone, upregulated LPS or poly(I:C) induced IL-6 and IFN-βin macrophages, which increased LPS or poly(I:C) induced IFN-βin macrophages. Our data are the first to demonstrate the regulation of miR146 and miR-155 expression in macrophages by progesterone.which are indicative of an important role of miRNAs in progesterone-mediated immune regulation.PartⅡ:IRAK1 regulates in Toll like receptor signal pathway in monocytes and macrophagesInterleukin-1 receptor-associated-1 kinase-1 (IRAK1) is a family member of interleukin-1 receptor-associated-1 kinases, which are a unique family of death domain containing protein kinases that play a key role in the signaling cascades of two receptor families, Toll-like receptors (TLRs) and interleukin-1 receptors(IL-1Rs). As a alternatively spliced variant, IRAK1c contains a death domain, fuction as an adaptor molecule, but lacks a region encoded by exon 11 of the gene, leading to lack kinase activity. IRAK1c broadly expressed in many tissues and is the major form of IRAKI expressed in the brain. In this study, we found that the monocytic human cell line THP-1 predominantly expressed IRAK1 with minimal IRAK1c detected. After LPS stimulation, IRAK1 is activated through its phosphorylation modification, which is reflected by a shift in the apparent molecular mass from about 80 kilodaltons to more than 100 kilodaltons. Transfection of PMA-activated THP-1 cells with plasmid encoding wild-type IRAK1, which can resulted in expression of activated IRAK1. Compare to transfection with empty plasmid, the IRAK1 overexpress upregulated the expression of proinflammatory cytokines (such as IL-6, TNF-α) upon LPS treatment, while typeⅠinterferon(such as IFN-(3)was inhibited after LPS or poly(I:C) stimulation. Using PMA-activated THP-1 cells, we found that IRAK1c overexpression inhibited LPS or poly(I:C)-induced IL-6,TNF-α, but enhanced LPS or poly(I:C)-induced IFN-β. This result was further verified in cells transiently transfected with interfering IRAK1 and IRAKlc, the konckdown of IRAK1 and IRAKlc impaired the regulatory effects of IRAK1 on LPS or poly(I:C)-induced IL-6, TNF-α, IFN-β. We detected the MAPK signal pathway, overexpression of IRAKlc didn't affect the phosphorylation of ERK.In conclusion. IRAK1 increased the production of proinflammatory cytokines but at the same time inhibited the production of typeⅠinterferon, which is perhaps due to the different functions of the death domain and the kinase domain of IRAK1 in Toll like receptor signal pathway. As a alternatively spliced variant, IRAK1c decreases the production of proinflammatory cytokines, such as IL-6, TNF-α, but notably, also promotes the production of typeⅠinterferon (IFN-β), which has been reported to have anti-virus effects, but reduce the damage of inflammation. The results provide insight into the role of IRAK1 and its spliced variants in Toll like receptor signal pathway, especially mechanisms that balance the production of proinflammatory cytokines and typeⅠinterferon in innate responses.