Synergic Effect Of NOD2 Agonists With TLR3 Agonists On Murine LSEC To Trigger HBV Specific T Cell Response

Objective1. To explore whether pretreatment of LSEC with NOD2-TLR3 agonists could reverts their suppressive properties. 2. To investigate the anti-HBV effect mediated liver sinusoidal endothelial cells(LSEC) stimulated simultaneously by NOD2-TLR3 agonists.Methods1. Establish chronic HBV replication mice model by hydrodynamic injection(HI) of p AAV/HBV1.2 into the C57BL/6 mice. 2. Establish acute HBV replication mice model by HI of p SM2/HBV1.3 into the of C57BL/6 mice. CD8+T cell was isolated from the spleen of the mice using Anti-CD8 Micro Beads. 3. T cells were isolated using Anti-CD90.1 Micro Beads from the na?ve BALB/C mice. 4. Livers were perfused using Liberase Blendzymes, and LSEC were isolated using the Anti-CD146 Micro Beads from the na?ve C57BL/6 mice. 5. Flow cytometry was used to detect the proliferation and ability of secreting cytokines(IFN-?,IL2) of na?ve T cells of BALB/C mice and CD8+T cells of acute HBV replication mice induced by LSEC stimulated with the NOD2-TLR3 ligand respectively or costimulatory. 6. LSEC were stimulated with the NOD2-TLR3 ligand respectively or costimulatory, after 20 hours, The peptide of HBs Ag and HBc Ag were used to stimulate LSEC respectively, after 20 hours, washing LSEC, then adding the HBV antigen specific CD8+ T cells to the LSEC, after 5 days, The T cells were collected. And the mice with HBV persistent replication were treated with the T cells by HI. After 1,4,10,20,30,40,50 days, taking blood from the mice, serum was isolated, HBs Ag?HBe Ag in the serum were detected by ELICA, HBV DNA in the serum were detected by Real-time PCR. 7. LSEC were stimulated with the NOD2-TLR3 ligand respectively or costimulatory for 1 hour?6 hours?20 hours. After 1 hour, the LSEC were collected. Phosphorylation levels of the protein(p65 NF-k B?p38 MAPK) were detected by Phosflow. After 6 hours,collecting the LSEC, Total RNA was isolated from the LSEC. Using Real-time RT PCR to detected the m RNA levels of CCL2,CCL3,CCL4, CCL5,CCL7,CCL8,CXCL2,CXCL9,CXCL10,CXCL11,CXCL12,IL-6,IL-2,TNF-?,IL-10,TGF-?,IFN-?,IFN-?,ISG15 in LSEC. After 20 hours, collecting the LSEC and supernatant. surface molecules(MHC?,CD40,CD80,CD86,PD-L1,CD54,CD106) of the LSEC were detected by flow cytometry. Detecte the supernatant cytokines(IL-6,TNF-?) by CBA(cytometric bead array). 8. The figures are made by Grapad software and data are analysed by Statistical Package for Social Science(SPSS).Result1.LSEC that have been costimulated by NOD2 and TLR3 ligand ncan not trigger na?ve T cell proliferation, and can not promote na?ve T cell to secret cytokine(IL-2,IFN-?).2. LSEC that have been costimulated by NOD2 and TLR3 ligand can not trigger HBV-specific CD8+T cell proliferation, but can promote HBV-specific CD8+T cell to secret cytokines(IL-2, IFN-?). 3. LSEC costimulated by NOD2 and TLR3 ligand can trigger HBV-specific CD8+T cell to participate anti-HBV effect in mice. 4.The phosphorylation levels of the protein p65 NF-k B? p38 MAPK in LSEC that have been costimulated by NOD2 and TLR3 ligand were promoted. 5. LSEC that have been costimulated by NOD2 and TLR3 ligand can up-regulate m RNA expression level of the cytokines( ISG15, IL-2, IL-6)and protein expression level of the cytokines(TNF-?, IL-6). 6. LSEC that have been costimulated by NOD2 and TLR3 ligand can up-regulate m RNA expression level of the chemokines(CCL5, CCL8, CXCL 9, CXCL10). 7. LSEC that have been costimulated by NOD2 and TLR3 ligand can up-regulate expression level of the costimulation molecule(CD86).Conclusion1. NOD2-TLR3 can induce LSEC activation. 2. LSEC costimulated by NOD2 and TLR3 ligand can not trigger HBV-specific CD8+T cell and na?ve T cell proliferation, but can promote the ability of secreting cytokines of HBV-specific CD8+T cell. 3. LSEC costimulated by NOD2 and TLR3 ligand can trigger HBV-specific T cell to participate anti-HBV effect in mice.