OCILRP2Signaling Synergies LPS Inducing Maturation And Functional Differentiation Of Mouse Bone Marrow-derived Dendritic Cells

Background and Objective Osteoclast Inhibitory Lectin Related Protein2(OCILRP2) which selectively and dominantly expresses in immune tissues, activated lymphocytes and dendritic cells (DCs) is a member of C-type lectin-related (Clr) protein family. NKRP1f as its ligand also expresses in activated lymphocytes and DCs and the expression in DCs are hundreds fold higher than that in lymphocytes. OCILRP2signaling coordinates with B7/CD28-mediated cositimulation enhances T cell proliferation as well as IL-2secretion both in vitro and in vivo. Blockage of OCILRP2signaling leads to intrinsic impairment in T cell response to antigenic stimulation. So far, there is no report about the roles of OCILRP2plays in DCs maturation and differentiation even though it dominately expresses in DCs.DCs are the unique cells possess the ability to stimulate naive T cells, act as a bridge between the innate and adaptive immunity systems. Pathogen-associated molecular patterns (PAMP) or danger signals of cellular stress are recognized by pattern recongnition receptors (PRR) induces DCs maturation and triggers immnune response, Toll like receptors (TLR)are important PRR of DCs for inducing DCs maturation and differentiation, and TLRs are modulated by other PRRs in these process. However, insights into OCILRP2signaling pathway that affects TLR signaling have not been described.Therefore, this study will focus on the roles of OCILRP2signaling in DCs maturation and differentiation and try to disclose how OCILRP2signaling regulates TLR signaling pathway in DCs. It is helpful for developing DCs vaccine to apply in cancer and autoimmune diseases therapy.MethodsCHO cells were transfected with Expression vector pIg-CD5-OCILRP2constructed with extracellular domain of OCILRP2and human IgG1Fc fragement by using Lipofectamine2000, and adding G418into selective medium for stable cell line. Amplified stable cell culture and OCILRP2-Fc in serum-free supnantants was purified by using Protein A column. The purity and the nature of the protein were confirmed by SDS-PAGE and western blot, respectively. The male New Zealand rabbits were immuned with OCILRP2-Fc and anti-OCILRP2polyclonal antibodies were purified from anti-OCILRP2serum by using zinc sulfate sedimentation and Protein G column.Bone marrow cells were collected from tibias and femurs of6-8weeks old BALB/c mice, recombinant murine granulocytemacrophage colony stimulating factor (rmGM-CSF) and murine interleukin-4(rmIL-4) were added into medium for DCs produce, and LPS treatment for other24hrs induces DCs maturation. Medium supplemented with OCILRP2or anti-OCILRP2was used in overall cell culture time in this research. LPS binding ELISA was performed for detecting interaction between LPS and OCILRP2-Fc. OCILRP2expression in DCs was measured through Real time RT-PCR and Flow cytometry analysis. Cell surface markers expression in DCs, and the capacity of DCs take up FITC-dextran were analysed by Flow too. Allostimulatory activity of DCs measured through [3H]thymidine incorporation mixed lymphocytes reaction. Proinflammatory cytokines concentration in cell culture supernatants and gene levels in cells were tested by sandwich ELISA and Real time RT-PCR, respectively. Electophoretic mobility shift assy (EMSA) was performed to monitor the activation of NF-κB and Western blots were done for testing degradation of I-κB.Balb/c mice were given the dose of20mg/kg body weight LPS by intraperitoneal injection. And2hrs prior to inject5mg/kg body weight anti-OCILRP2. Inspected and recorded the mice status and survival every6hours, and weighed spleen weight after mice were dissected when they were found death or were euthanized, organs of lung and liver were prepared for HE staining.ResultsCHO stable cell line was gotten after3times of subclone. OCILRP2-Fc fusion protein secreted by CHO stable cells was purified and more than10mg OCILRP2-Fc were produced in1L cell culture supernatants. And this cell line remained effective expression of OCILRP2-Fc protein after passaged for several times. One protein band with molecular weight about55kDa was found in the Polyacrylamide Gel after coomassie blue staning and can be detected by goat anti-human IgG after transformed into PVDF membrane. Anti-OCILRP2polyclonal antibody was purified in this study also can bind to OCILRP2-Fc antigen and OCILRP2that in OCILRP2overexpressing cell lysates.Both relative real-time PCR and flow cytometry analysis indicated that OCILRP2expression on LPS inducing mature DCs is higher than immature DCs. LPS binding ELISA showed LPS cannot bind to OCILRP2-Fc. The expression of CD11c, MHC class Ⅱ molecule, CD80and CD86on DCs were downregulated by OCILRP2-Fc treatment. Flow analysis showed that OCILRP2-Fc not only prevented the endocytosis of immature DCs effectively, but also prevented the decrease in endocytosis of mature DCs. DCs treated with OCILRP2-Fc were less effective in stimulating naive allogeneic T cells than that without treatment. OCILRP2-Fc or anti-OCILRP2treated DC reduced pro inflammatory cytokines including IL-12p70, IL-6, TNFa produce in contrast to untreated DC, but the anti-inflammatory cytokine IL-10expression had no significantly difference in different group. In addition, OCILRP2-Fc inhibited the release of cytokine in a dose-dependent manner. EMSA demonstrated that OCILRP2-Fc suppressed NF-κB activation, and Western blots confirmed that OCILRP2-Fc treated DC contained higher I-κB compare to control human IgG.After injected LPS for72h,71.4%mice treated with anti-OCILRP2were survival, but mice without anti-OCILRP2treatment all died. The mice spleen weight in anti-OCILRP2treatment group is significantly lower than that in PBS control group. And Inflammatory cell infiltration and vasodilatation in the lung of anti-OCILRP2treated mice are slighter than control.Conclusion1.OCILRP2Signaling Synergies LPS Inducing maturation and Functional Differentiation of Mouse Bone Marrow-derived Dendritic Cells by enhancing NF-κB activation.2. OCILRP2can be a potential target of immune diseases therapy.