Studies On Regulation Mechanism Of Pattern Recognition Receotor(PRR)-triggered Innate Immune Response In Macrophages

The innate immune system is the first line of defense, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner, and innate immune systems will provide immediate defense against infection. Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play significant roles in the recognition of and responses to microbial pathogens. TLRs trigger a signaling cascade through the MyD88-dependent and TRIF-dependent signaling pathways, which in turn transmit a series of signaling cascades that leads to the activation of MAPK, NF-κB and IRF3, resulting in the production of pro-inflammatory cytokines and type I interferon. MicroRNAs (miRNAs) are highly conserved, small noncoding RNAs with about18-25nucleotides in lenth. By binding to partially complementary sites in the3’untranslated region of mRNA transcripts, miRNAs reduce translation of these transcripts and/or lead to their degradation. Currently, miRNAs have been found to play key roles in many biological processes, such as cellular development and tumorigenesis. Now, more and more miRNAs have been shown to participate in both innate and adaptive immune responses,such as miRNA-146、miRNA-101.miR-92a has been reported to paly a role in cancer and angiogenic process,but whether miR-92a or its family takes part in TLR-induced innate immune response is still unclear. In this study, we found that the expression profile of miR-92a is down-regulated quickly in macrophages after activation through multiple TLR ligations, such as LPS, PGN and Polyl:C. We next investigated the underlying mechanism by which miR-92a was reduced, and found it was dependent on both Myd88and TRIF signaling.Then we transfected RAW264.7cells with miR-92a mimics, miR-9a inhibitors or their controls and analyzed the production of pro-inflammatory cytokines. It was showed that miR-92a overexpression significantly inhibited LPS-stimulated TNF-aand IL-6production, also JNK/C-JUN pathway, while inhibition of miR-92a increased the production of these cytokines and the JNK/C-JUN pathway. We next investigated possible targets by which miR-92a may act through to modulate the LPS-triggered JNK/JUN pathway in regulating the inflammatory response. The results of luciferase reporter assay and western blot showed that MKK4was a direct target of miR-92a and endogenous MKK4can be directly regulated by miR-92a in macrophages. Knockdown of MKK4also markedly inhibited the JNK/C-JUN pathway, and then reduced LPS-triggered TNF-. and IL-6production in macrophages.Thus, miR-92a negatively regulates TLR-triggered inflammatory cytokine production through targeting MKK4involved in the JNK/C-JUN pathway in TLR signaling. Our findings provide a new explanation characterizing the molecular mechanism responsible for the tight regulation of TLR-triggered macrophage activation. Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play significant roles in the recognition of and responses to microbial pathogens. Most innate cells such as macrophages, dendrtic cells express TLRs. TLRs trigger a signaling cascade through the MyD88-dependent and TRIF-dependent signaling pathways, which in turn transmit a series of signaling cascades that leads to the activation of MAPK, NF-kB and IRF3, resulting in the production of pro-inflammatory cytokines and type I interferon. MicroRNAs (miRNAs) are highly conserved, small noncoding RNAs with about18-25nucleotides in lenth. By binding to partially complementary sites in the3’untranslated region of mRNA transcripts, miRNAs reduce translation of these transcripts and/or lead to their degradation. Currently, miRNAs have been found to play key roles in many biological processes, including immune response.miR-33interested many researchers as it was reported to regulate several important genes in homeostatic control of cholesterol. But its function in innate immunity is not very clear. In this study, we found that the expression level of miR-33is down-regulated in the late time point in macrophages after activation through multiple TLR ligations, such as LPS, HKLM and PolyI:C. We next investigated the underlying mechanism by which miR-33was reduced, and found it was dependent on TRIF signaling. Then we transfected peritoneal macrophages with miR-33mimics, inhibitors or their controls and analyzed the production of pro-inflammatory cytokines. We found that miR-33overexpression significantly increased LPS-stimulated TNF-a and IL-6production, while inhibition of miR-33reduced the production of these cytokines. It was reported that ABCA1and ABCG1were the target of miR33, and they could inhibit the inflammation response. So our hypothesis is that miR33could regulate TLR-induced inflammatory response in macrophages by targeting ABCA1and ABCG1and thereby controlling raft cholesterol. At first, we confirmed that miR33could directly inhibit the expression of ABCA1and ABCG1in peritoneal macrophages. Absence or overexpression of ABCA1and ABCG1both attenuated the effect of miR-33. ABCA1and ABCG1were known regulators of lipid raft abundance, so we detected the lipid raft after miR-33overexpression. It showed miR-33increased lipid rafts of macrophages, and lipid raft modulators attenuated the effect of miR-33on LPS-induced cytokines.In addition, miR-33also decreased the IL-10production, coordinately inhibiting the inflammation response.Thus, in the peritoneal macrophages, TLRs ligands stimulation reduced the expression of miR-33through a TRIF-dependent mechanism. And miR-33increased the production of pro-inflammatory cytokines. This function may be through targeting both ABCA1and ABCG1, thereby increasing lipid rafts and inhibiting IL-10secretion in macrophages. Our findings provide new and useful insights to understand the molecular mechanism responsible for the tight regulation of TLR-triggered macrophage activation. Interleukin9(IL-9) was first purified and characterized as a T cell and mast cell growth factor. In recent years, more and more functional activities and production mechanism of IL-9have been revealed, arousing the interest of many researchers. In vitro, naive CD4+T cells primed in the combination of TGF-β and IL-4produced high levels of IL-9which was termed Th9cells, and showed diminished expression of other lineage-specific cytokines and transcription factors.IL-9serves its multiple biologic functions via IL-9R, a cytokine receptor that consists of a ligand-specific a-subunit and a common y-chain. Beyond the first description as T cell or mast cell growth factor, IL-9may affect other immune cells, such as B cells, innate lymphoid cells, monocytes and so on. IL-9strongly up-regulated the production of TGF-β by LPS-stimulated human monocytes, while down-regulated the production of TNF-α, consequently inhibited the oxidative burst. IL-9also inhibited APC induced Thl immune response.IL-9plays different roles in various diseases. In allergic inflammation and autoimmune diseases, IL-9can promote the development of disease as a proinflammatory cytokine.While; IL-9provides a protective role in immunity to parasites infection by increasing clearance of pathogen. IL-9also can greatly reduce allograft rejection in tolerant mice. Macrophages play a key role in the pathogen infection and inflammation, as the main cells of innate immune response. Pathogen-associated molecular patterns (PAMPs) are molecules associated with groups of pathogens, that are recognized by pattern recognition receptors (PRRs) expressing on innate immune cells. Once activated by PAMPs, PRRs trigger a series of signaling cascades that leads to the the production of pro-inflammatory cytokines and type I interferon. So far, the functional activities of IL-9in PAMPs induced immune response in macrophages remain far from clear. Therefore, the purpose of this study is to understand whether IL-9participate in this process and the role of IL-9in inflammatory diseases.In this study, we found that IL-9-/-bone marrow derived macrophages showed impaired proinflammatory responses after various PAMPs stimulation. We next treated bone marrow derived macrophages and peritoneal macrophages cells with IL-9. And we found that IL-9in high concentration could promote proinflammatory cytokines production in primary macrophages. In additon, IL-9in low concentration promoted proinflammatory cytokines induced by PAMPs in peritoneal macrophages. Whether in these responses IL-9modulates cytokine production through direct or indirect mechanisms remains to be determined, as well the role of IL-9in different inflammation diseases. We hope our further findings can provide new and useful direction in the diagnosis and treatment of related inflammation diseases.