Impacts Of Innate Immune Response Mediated By NOD1/NOD2on HBV Persistent Replication And Its Mechanism

Objective1. To explore impacts of innate immune response mediated by NOD1/NOD2on theHBV replication in the HBV persistent replication mice model and its possiblemechanism.2. To investigate anti-HBV effect and mechanism mediated by NOD1/NOD2receptorin liver sinusoidal endothelial cells (LSEC).Methods1. HBV persistent replication mice model was established by hydrodynamic injectionof pAAV/HBV1.2into the tail veins of C57BL/6mice. And the mice with HBVpersistent replication were treated with the ligands of NOD1or NOD2byhydrodynamic injection.2. After injection, the mice were regularly bled to monitor the serum levels of HBsAg,HBeAg, HBV DNA. The serum levels of HBsAg and HBeAg were detected byquantitative ELISA or General ELISA. And the serum samples fromhydrodynamically injected C57BL/6mice were also assayed for the presence ofencapsidated HBV DNA by real-time PCR. Liver tissues were collected from mice killed at the indicated time points. Intrahepatic HBcAg was visualized byimmunohistochemical staining. At the10day after hydrodynamic injection, micewere killed. Then the separated splenocytes were cultured and assayed for thefrequencies of antigen-specific IFN-γ secreting cells by using ELISPOT.3. Livers were perfused through the portal vein with Liberase Blendzymes, and LSECswere isolated using Anti-LSEC MicroBeads.4. The NOD1and NOD2mRNA express levels at baseline from LSEC were detectedby Real-time RT-PCR.5. LSECs were stimulated with the C12-ieDAP, MDP and ssRNA, respectively, for6hours or20hours. Then the LSECs and cells supernatant were collected. Total RNAwas isolated from LSECs and was used to detect the mRNA of NOD1, NOD2, IL-6,RIP2, TNF-α, CXCL-1, CXCL-2, CXCL-9, CXCL-10, CCL-2, CCL-3and CCL-5by real time RT-PCR. The cytokines (IFNα/β/γ, TNF-α, IL-1β, IL-6and IL-18) inthe cells supernatant were detected by quantitative ELISA. LSEC surface molecules(PDL-1, CD40, CD86, MHCII, CD106, CD54and CD80) were detected by flowcytometry.6. Data are analysed by Statistical Package for Social Sciences (SPSS) version18.0and the figures are made by Graphpad software.Results1. In vivo, the treatment of NOD1ligands (C12-ieDAP), but not NOD2ligands (MDP)by hydrodynamic injection into the tail veins of C57BL/6mice with HBV persistentreplication obviously inhibit the serum HBsAg and HBeAg expression and serumHBVDNA replication. The expression of the HBcAg in the liver is alsodown-regulated. However, NOD1/NOD2agonists by imtramuscular andsubcutaneous injection have no anti-HBV effects.2. The frequency of HBcAg-specific IFN-γ-producing cells in the splenoncytes ofC57BL/6mice with the treatment of NOD1ligand (C12-ieDAP) is higher than the mice with the treatment of NS at10day post injection by using the IFNγenzyme-linked immunospot (ELISPOT) assay.3. The NOD1mRNA expresses highly in the LSECs, but NOD2mRNA doesrelatively lowly.4. After stimulated by NOD1ligands (DAP or C12-ieDAP) for6or20hours, LSECsare induced to up-regulate the NOD1, RIP2, IL-6, CXCL-1, CXCL-2, CXCL-9,CCL-2and CCl-5mRNA expression. And NOD2ligands (MDP) activate the NOD2in the LSECs, then increases RIP2and IL-6mRNA expression levels. AndssRNA40induce the up-regulation of expression of the RIP2, CXCL-2, CXCL-9,CXCL-10, CCL-2and CCl-5mRNA by perhaps stimulated NOD2in the LSECs.5. Stimulation of LSECs with the NOD1ligands (C12-ieDAP) and NOD2ligands(MDP) induce the production of IL-6proteins. The quantity of IL-6proteins ishighest at the stimulation with the10μg/ml C12-ieDAP,10μg/ml MDP and0.2μg/ml ssRNA40, respectively. In addition, Stimulation of LSECs with the NOD1ligands (C12-ieDAP) induces the production IFNγ proteins. However, the proteinsof other cytokines (IL-1β, IL-18, TNF-α, IFN-α and IFN-β) couldn’t be detectedafter the stimulation.6. It was found that up-regulation of CD40, CD86and PD-L1in the LSECs detectedby flow cytometry is in response to the activation of NOD1ligands (C12-ieDAP),but not found the up-regulation of MHCII, CD106, CD54and CD80. And theup-regulation of CD40and CD86is obvious at the stimulation with10μg/mlC12-ieDAP, the up-regulation of PD-L1at2μg/ml C12-ieDAP. Similarly,stimulation of LSECs with NOD2ligands (MDP) enhances the expression of CD40,CD86and PD-L1, but not up-regulate MHCII, CD106, CD54and CD80. Theup-regulation of the expression of CD40, CD86and PD-L1is relatively obvious atthe stimulation of10μg/ml MDP. The ssRNA40stimulation induces theup-regulation of PDL-1, but not CD40, CD86, MHCII, CD106, CD54and CD80. Conclusion1. NOD1could be activated by its ligands with hydrodynamic injection in the HBVpersistent replication mice model and mediate anti-HBV immune response effect,but not NOD2. However, NOD1/NOD2agonists by imtramuscular andsubcutaneous injection have no anti-HBV effects.2. It is clear from our data that LSECs highly express functional NOD1and NOD2andcould be activated by both NOD1and NOD2specific ligands. Thus NOD1andNOD2mediate NF-κB signaling pathways and induce the productions of IL-6andsome chemokines. And NOD1still induces the productions of IFN-γ,but notNOD2.3. Impacts of innate immune response mediated by NOD1on HBV replicationpossibly is due to the inflammatory response, neutrophil recruitment andproductions of IFN-γand theregulation of the HBcAg specific adaptive immuneresponse.