The Regulation Of TLR4 Signaling Pathway By OCILRP2 In Murine Macrophage

Background Osteoclast inhibitory lectin related protein 2,OCILRP2,also called clr-g,is a kind of Type-II transmembrane protein which is belonged to C-type lectin-related protein family.OCILRP2 is a gene that originally cloned from osteoblasts for its ability to inhibit osteoclast formation.It is selectively expressed in osteoblasts,B cells,dendritic cells(DCs),and activated T cells.Interacted with NKRP1 f,OCILRP2 provides an additional positive signal to enhance the proliferation of T cells as well as the production of IL-2.OCILRP2-silenced T cells are incapable of proliferation and the production of IL-2,upon antigen priming.Moreover,the activiation of NF-?B is also impaired as the result of OCILRP2 silencing.Macrophages,which help maintain homeostasis during embryonic development and throughout life.Toll-like receptor(TLR)is an important type of pattern recognition receptor on the surface of macrophages.Its main member Toll-like receptor 4(TLR4)is mainly distributed in macrophages and dendritic cells and T lymphocytes,which can recognize the lipopolysaccharide(LPS)of gram-negative bacteria and be activated by it,LPS can initiating TLR4 signaling pathway,leading to the production of pro-inflammatory cytokines and interferon,thereby causing related inflammatory reactions.In this study,we investigate OCILRP2 regulating TLR4 signaling pathway affects the function of macrophage.Objective To Eestablish Osteoclast inhibitory lectin related protein 2 gene silenced in RAW264.7 macrophage.To investigate the effect of OCILRP2 regulating TLR4 signaling pathway on the function of RAW264.7 macrophages.Methods The specific sh RNA sequence of OCILRP2 was synthesized and the sh RNA lentiviral vector targeting OCILRP2 was specifically designed to silence the expression of OCILRP2 in RAW264.7 cells.Western blot and RT-qPCR were used to detect the expression of OCILRP2 after infecting the RAW264.7 cells by lentiviruses.Control cells and silenced cells were cultured and observed with microscope.Adhesion assay was used to detect the adhesion ability,scratch test to detect the mi gration ability,transwell assay was used to detect cell migration and chemotactic ability;flow cytometry to detect cell phagocytry,the phagocytosis ability of silenced OCILRP2 RAW264.7 was detected by neutral red staining assay,and ELISA kits as well as RT-qPCR to detect the influence of the cell secrete inflammatory ability of IL-1β,IL-6,TNF-α and NO;Western blot was used to detect the level of i NOS,C-Raf,AKT,p44/42,p38,TAK1,IκB-α,p65,IRF3,IRAK4,TBK1.Nuclear factor analysis was used to detect the activation of p65 and the distribution of p65 by confocal analysis.Results Western blot and RT-qPCR results showed that OCILRP2 in RAW264.7 macrophages was significantly decreased than the wild group and control group(P <0.05).After culturing for 24 h,the pseudopodium of the silenced group were significantly reduced,and become round.Adhesion assay showed that the amount of adhesion cells in the knockdown group was significantly less than the control group(P <0.05).Meanwhile,wound-healing assay showed that the number of cells in the empty area of silenced group was significantly less than the control group(P <0.05).Transwell results showe d that the number of cells through the up chamber was significantly decreased(P <0.05).Chemotaxis assay showed that the silenced group had less cells through the up chamber.And phagocytosis of OCILRP2-silenced RAW264.7 cells induced by LPS was decreased.The results of ELISA or RT-qPCR showed that the silenced group secreted less IL-1β,IL-6,TNF-α and NO;Western blot results showed that silencing OCILRP2 inhibited the ability of macrophages expressing i NOS and the protein of My D88-independent signaling.The results of nucleoprotein isolates showed that the silenced OCILRP2 inhibited the activation of the transcription factor NF-κB in the nucleus.Confocal results showed that silencing of OCILRP2 inhibited the activation of transcription factor NF-κB into the nucleus.Conclusion OCILRP2 regulates My D88-independent road of TLR4 signaling pathway to impact the function of macrophage.