Function And Mechanism Analysis Of Liver Sinusoidal Endothelial Cells Regulating CD8+ T Cell Immunity During HBV Infection

Objective Liver sinusoidal endothelial cells(LSECs)play a crucial role in maintaining the homeostasis of the hepatic microenvironment through induction of Ag-specific T cell tolerance.However,certain virus infection or TLRs stimulation can induce the maturation of LSECs,which reverts their suppressive properties to induce T cell immunity.So far,little is known about whether and how the function of LSECs in regulating T cell immunity is regulated during HBV infection.Here,we investigated function and mechanism of liver endothelial cells regulating CD8+ T cell immunity during HBV Infection.Methods 1.The male C57BL/6 mice,6-8 weeks old,were purchased from Silaike-Jingda Experimental Animal Co.,Ltd.in Hunan province.2.The HBV acute-resolving and persistent replication mice models were established by hydrodynamically injecting p SM2 and p AAV-HBV1.2 plasmids respectively.Mice were sacrificed at indicated time points and their LSECs,KCs and hepatocytes were freshly isolated and co-cultured with polyclonal TCR-activated T cells.The level of IFN? in the supernatant of 48 h coculture was measured by ELISA.3.Liver sinusoidal antigen presenting cells(LSECs,KCs)freshly isolated from na?ve mice were stimulated by recombinant HBV antigen protein(r HBs Ag,r HBe Ag,r HBc Ag)and co-cultured with polyclonal TCR-activated T cells.The level of IFN? in the supernatant of 48 h coculture was measured by ELISA.4.The experimental group mice were hydrodynamically injected with the HBe Ag short-term expression plasmid pc DNA3.1/HBe Ag and the control group mice were hydrodynamically injected with the pc DNA3.1/null plasmid.LSECs were isolated 2 days post injection(dpi),and cocultured with polyclonal TCR-activated T cells.The level of IFN? in the supernatant of 48 h coculture was measured by ELISA.5.Three different levels of HBe Ag expression mouse models(Normal HBV replication,HBe Ag deficiency expression,and HBe Ag replenishment)were established by hydrodynamically injecting p BS/HBV1.3 plus p CI-neo/null,p BS/HBV1.3-HBe Ag(-)plus p CI-neo/null and p BS/HBV1.3-HBe Ag(-)plus p CI-neo/HBe Ag respectively.The mice were sacrificed at indicated time points and their intrahepatic infiltrating lymphocytes,splenic lymphocytes,hepatic sinusoidal endothelial cells were isolated at indicated time points.The levels of CD25,CD69,CD62 L,PD1,CD44 and T cell function(IFN?,TNF? and IL-2)were detected by flow cytometry.The HBs Ag and HBe Ag were detected by ELISA.LSECs of each group were incubated with polyclonal activated T cells,and the level of IFN? in the supernatant of 48 h incubation was measured by ELISA.6.The expression of MHC ?,MHC ?,CD80,CD86 and PD-L1 on LSECs of HBV intrahepatic replication 14 dpi and HBe Ag stimulation in vitro were detected by flow cytometry.Inflammatory cytokines(TNF?,IL-6,IL-10,MCP 1,IL-27)of supernatants from LSECs cultured 24 h were detected by Cytometric Beads Array and ELISA.7.The TNF? or IL-27 recombinant protein was added into co-incubation system of naive mice LSECs and polyclonal activated T cells,and the IFN? levels in the supernatants of 48 h coculture was measured by ELISA.Results 1.Compared with the control group,acute and chronic intrahepatic HBV replication at 14 dpi could induce LSECs to reverse their tolerant properties and promote T cell immunity.The level of IFN? produced by T cells promoted by LSECs of HBV intrahepatic HBV replication group was higher than that of chronic hepatitis B intrahepatic replication group and control group.HBV intrahepatic replication had no significant impact on the function of Kupffer cells and hepatocytes.2.HBV protein components(HBe Ag and HBs Ag)in vitro stimulation induced functional maturation of LSECs and promoted T cell immunity;HBV protein components(HBe Ag and HBs Ag)in vitro stimulation enhanced the inhibitory properties of KCs and induced T cell tolerance.3.HBe Ag stimulation in vitro and in vivo could induce functional maturation of LSECs and promoted T cell immunity.4.LSECs separated from HBe Ag-deficiency HBV HI mice were less capable of promoting T cell immunity compared to those separated from wild type HBV HI mice.In contrast,replenishing HBe Ag expression in HBe Ag-deficiency HBV HI mice resulted in better LSEC activation and led to accelerated HBV clearance.5.The acute and chronic intrahepatic HBV replication had no significant effect on the expression of co-stimulatory molecules in LSECs.Compared with chronic hepatitis B virus replication group and control group,acute intrahepatic HBV replication induced higher levels of hepatic sinusoidal endothelial cells TNF?,IL-27,IL-10.6.HBe Ag in vitro stimulation induced the production of TNF?,IL-27,IL-10 and MCP1 in LSECs.HBe Ag stimulation up-regulated MHC class ? molecules and down-regulated of PD-L1 in LSECs.7.The addition of TNF? or IL-27 to the coculture system of LSECs and T cells could abrogate the inhibitory effect of LSECs on T cells.In vitro blocking of TNF? or IL-27 partially abrogated the ability of HBe Ag-stimulated LSECs to promote IFN-? production by T cells.Conclusion HBV replication can revert LSECs suppressive properties to promote T cell immunity,which is probably mediated by HBe Ag induced LSECs TNF-? and IL-27 production.Further study is needed to characterize the mechanism of HBV induced LSECs immune functional maturation.