Modulation Of TLR3 Signal And Polarization In Macrophage

Macrophages, a type of phagocyte, are widely distributed in almost all tissues. Macrophages can be produced by differentiation of monocytes which moved to the organization from circulating blood. There are many Toll-like receptors (TLRs) expressed by macrophages, such as TLR1, TLR2, TLR3, TLR4 and so on. These TLRs can recognize pathogen-associated molecular patterns (PAMPs) from different pathogens, and this process makes macrophage activate and eliminate pathogens. Therefore, macrophages are the first line of immune system to defence against pathogens. Inevitably, inflammation will happen followed by the course of eliminating pathogens. So in that sense, macrophages are inflammatory cell for it is one of the first cells to contac with pathogens. Excessive activation of macrophage can cause the dysregulation of inflammation and pathological damage, even the inflammatory diseases such as sepsis.During the TLRs expressed by macrophage, activation mediated by TLR4 was the most studied TLR in inflammation. many TLR4 related compounds have been put into clinical researchs. Unfortunately, they were withdrawn for the serious side dffects or they became ineffective in clinical research. It is anticipated that the less-studied TLR3, which located in endosome, might be the therapeutic target. TLR3 is an sensor of double-strained RNA (dsRNA) from virus or host cells and its analogy poly(I:C). TLR3 signals were transmitted through the only adaptor TIR-domain-containing adaptor inducing interferon-β (TRIF). In the early studies, TLR3 was identified as an participator in antiviral reactivity. Recently, studies showed that TLR3 was an endogenous sensor independence of virus infection and acted in the positive feedback loop. TLR3 and its signals are related to the pathogenesis of sepsis, rheumatoid arthritis, chronic neurodegenerative diseases and so on. Inhibition of TLR3 signal excessive activation might be a new strategy for treatment of inflammation.Besides inflammatory activation, macrophages have other effects taken through different polarization. There are two subtypes of macrophage:one is M1, participated in infection clearance and tissue damage; another is M2, which is termed as anti-inflammatory macrophages. The balance between M1 and M2 is important to maintain physiologic stability. Studies showed that the increase of M2 number was contributed to the suppression of inflammation.FC-99 is a small molecular compound with simple structure and stable property. During preliminary studies FC-99 showed effect on dendritic cells (DCs) mature induced by poly(I:C) without studies on the effect of FC-99 on macrophages activity. In this studies, we aimed to regulate activity and polarization of macrophage throuth this exogenous compound FC-99, which could help to alleviate inflammatory diseases.1. FC-99 inhibited inflammatory response modulated by TLR3 in macrophage.Firstly, we identified the effect of FC-99 on macrophage activity mediated by TLR3 using analogy of dsRNA poly(I:C). After recognition with ligand, TLR3 induces phosphorylations of IRF3, NF-κB and MAPK through the only adaptor TRIF. These signals are involved in anti-virus infection, cell lesion or death and inflammatory disease. Inflammation is a pathological process, and dysregulation of inflammatory response leads to amplification of inflammatory signals and inflammatory disease. Previous studies showed that TLR3 signal took part in sepsis, rheumatoid arthritis and chronic neurodegenerative diseases. It is suggested that TLR3 was a potential target in acute or chronic inflammation. Our results showed that a novel 1,2-benzenediamine derivate FC-99 suppressed poly(I:C)-induced inflammatory response in mouse peritoneal macrophage, including 1) inhibiting expression of inflammatory factors such as TNF-α、IL-6、CCL-5 and IP-10 induced by poly(I:C); 2) inhibiting phosphorylation levels of ERK、JNK、P38 and IκB. All these were established on the basis that FC-99 performed no effect on cytotoxicity and uptake of poly(I:C). These results suggested for the first time that novel small molecular compound FC-99 inhibited inflammatory response induced by poly(I:C).2. FC-99 suppressed TLR3 expression through IRF3/IFN-a/JAK/STATl signal.Next, we aimed to clarify the effect of FC-99 on TLR3, the key factor in the positive feedback loop. As a sensor of dsRNA, TLR3 could recognize nucleic acid and mRNA during virus replication or degraded bacteria, damaged tissue necrotic cells to induce type I IFN synthesis, which contribute to suppress virus infection. Studies showed that TLR3 served as an endogenous sensor of danger-associated molecular patterns (DAMPs) independent of viral activation, and overexpression of TLR3 was involved in aggravation of inflammatory damage, autoimmunity disease and systemic inflammatory response. So expression and activation of TLR3 keep the balance between physiology and inflammation. TLR3, one of IFN-stimulated genes (ISGs), can be induced by type I IFN. During TLR3-involved inflammation, dsRNA from exogenous pathogen or nucleic acid from endogenous tissue can induce expression of inflammatory factors and type I IFN via binding with TLR3. Accordingly, interaction of type I IFN and IFNAR promotes the activation of JAK/STAT pathway, which further enhances the expression of TLR3. Up-regulation of TLR3 forms a key in the positive feedback loop and amplifies inflammation. Thus we focused on the analysis of TLR3 expression influenced by FC-99. Results showed that IFN-a induced by poly(I:C) enhanced TLR3 expression via interacting with IFNR. On the one hand, FC-99 inhibited phosphorylations of IRF3, and this reduced the expression of type I IFN, the one that served as a source of resulting in TLR3 expression. On the other hand, FC-99 suppressed phosphorylations of JAK/STAT1 induced by IFN-a and further inhibited TLR3 expression. The combination of such two effects helped FC-99 suppressed TLR3-modulated positive feedback loop.3. FC-99 induced M2 macrophage polarization through PPAR-y.Macrophage is the key participator in inflammatory response, which performed different function through different cell types. Classic activated macrophage (Ml) is pro-inflammatory macrophage involved in sterilization and expresses high level of inflammatory factors such as iNOS, TNF-a, IL-12 and IL-6. Alternative activated macrophage (M2) is anti-inflammatory macrophage involved in wound repair and clearance of infection. The key factors released from M2 are anti-inflammatory cytokines such as IL-10 and TGF-β. Studies showed that modulation of balance between Ml and M2 was pivotal for metabolism and homeostasis. Mechanism involved in macrophage polarization is complex and some researchers indicated that IL-4 stimulated the activation of STAT6 and PPAR-y to promote M2 polarization. Our results showed that FC-99-treated bone marrow-derived macrophage exhibited a phenotypic switch from M1 to M2. Similar results were observed in FC-99 treated macrophage line RAW264.7. Given that FC-99 did not induce phosphorylations of STAT6, and it was not siRNA for STAT6 but inhibitor of PPAR-y suppressed FC-99-induced Mrc (a marker of M2) expression, it is indicated that FC-99 induced M2 macrophage polarization through PPAR-y rather than STAT6.4. Regulation of TLR3 expression and M2 polarization were involved in the improvement of mouse septic symptoms.Sepsis is an overwhelming systemic inflammation which was associated with a high mortality rate despite appropriate therapy and advances in supportive care. Death frequently follows the development of severe complications such as acute lung injury, acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Many strategies were attempted to treat sepsis for clinical trials, including anti-PAMP antibodies, neutralizing antibody directed against cytokines and TLR4 inhibitors. Disappointingly, all these were withdrawn for their lack of clinical efficacy. Hence effective agents are desiderated for the treatment of sepsis. TLR3 is an endogenous sensor of DAMPs and amplifies of immune response during sepsis. TLR3 deficient mice showed an increased survival rate in cecal ligation and puncture (CLP)-septic model. We thus presume that the synthetic small molecule compound FC-99, which exhibited inhibitory effect on poly(I:C)-induced inflammatory response and TLR3 expression, could be used in the treatment of sepsis. To elucidate this hypothesis, a septic mice model has been introduced by CLP surgery. Data showed that FC-99 administration significantly reduced the serum levels of inflammatory factors including IL-6, TNF-a, CCL-5 and KC, remitted acute lung injury and organ function of liver and kidney, reduced cell death and bacterial burden in blood and peritoneal and improved survival. Besides, FC-99 suppressed expression of TLR3 in splenic CD11b+ cells, lung tissue, liver and kidney in vivo, and promoted expression of M2 macrophage-related genes in liver. Our findings suggested that FC-99 might be a potential therapeutic candidate for the treatment of sepsis.Above all, FC-99 suppressed inflammatory response induced by poly(I:C) and inhibited TLR3 expression through IRF3/IFN-a/JAK/STAT1 pathway, which repressed TLR3-modulated positive feedback loop. Besides, FC-99 promoted M2 macrophage polarization through PPAR-y. All these may help FC-99 improve septic symptoms induced by CLP.