HBV Inhibits LPS-induced NLRP3 Inflammasome Activation And IL-1? Production Via Suppressing NF-?B Pathway And ROS Production

ObjectiveHepatitis B virus(HBV),an enveloped DNA virus that belongs to the Hepadnaviridae family,chronically infects more than 400 million people worldwide.Its persistence is a major cause of liver cirrhosis and hepatocellular carcinoma(HCC).HBV has been considered to be a "stealth virus" that induces negligible innate immune responses during the early phase of infection.Viral proteins can inhibit distinct innate immune pathways,leading to immunosuppression and viral persistence.NLRP3 inflammasome is a cytosolic multiprotein complex detecting pathogen infections,tissue damage,or metabolic imbalances,leading to maturation and release of several proinflammatory cytokines,including IL-1? and IL-18.Full activation of NLRP3 inflammasome usually requires two signals.The first signal(priming signal)is triggered by various pathogen-associated molecular patterns(PAMPs),and induces synthesis of NLRP3 and pro-IL-1? via activation of nuclear factor-kappa B(NF-?B);the second stimulus(activation signal),provided by danger-associated molecular patterns(DAMP),leading to caspase-1 auto-activation,cleavage of pro-IL-1?,and then release of the biologically active,mature IL-1?.As a pleiotropic inflammatory factor,IL-1? activates the expression of other immune genes and facilitates lymphocyte recruitment to the site of primary infection,thereby controlling invading pathogens.It has been reported that a variety of pathogens develope multifaceted strategies to evade the surveillance of immune responses,while,no clear report exists concerning the relationship between HBV infection and inflammasome activation.On one hand,as the most important organ of metabolism and detoxification,liver usually receives an increased level of bacterial LPS in the portal and/or systemic circulation due to gut-liver axis;on the other hand,liver is the most affected organ by HBV.The low levels of NLRP3 and related proteins make us to investigate whetherHBV infection suppresses LPS-induced NLRP3 inflammasome activation,thus to promote immune escape.In order to discuss the question,we firstly confirmed that HBV was not able to directly activate NLRP3 inflammasome in in vitro and in vivo models for HBV infection.Then,LPS was used to activate NLRP3 inflammasome in the liver,and we identifed that Kupffer cells were the major producers of NLRP3 and IL-1?,while HBV infection could significantly inhibit NLRP3 inflammasome activation.Morover,using HBV infected cell line and muring model as well as CHB and HCC patients,we proved that this inhibitory activity was primarily mediated by HBeAg.Lastly,the inhibitory activity of HBV was further confirmed in mice infected with S'.Typhimurium.Methods1.NLRP3 inflammasome related components in HBV infected cells,mice and patients were detected by Real-time PCR and Western blotting.2.HBV-carrier mice and HBV-e-null mice were established by hydrodynamic injection of pAAV/HBV1.2 plasmids or pAAV/HBV 1.2-e-null plasmids via the tail vein into C57BL/6 mice.3.The HBsAg and HBeAg serum levels were assayed by ELISA;HBcAg was explored by immunohistochemistry;the serum HBV DNA level was detected by Real-time PCR.4.The IL-1? expression in Kupffer cells was assayed by FACS and immunofluorescence.5.The NF-?B phosphorylation in THP-1 was detected by FACS and Western blotting.6.Protein docking was performed utilizing ZDOCK 3.0.2.with the crystal structures of HBeAg,TRAM and MAL.7.The levels of IL-1?,IL-18 and IL-6 in supernatants from cell cultures,liver homogenates,or serum were assayed by ELISA.8.The ROS production was measured from dichlorofluorescein(DCF)fluorscence intensity by FACS and laser confocal microscope.9.The p47-phox and Na+/K+ ATPase in cytomembrane was detected by Western blotting.10.The Ca2+ concentration and lysosomal integrity were measured from Fluo-3/AM fluorescence and LysoTracker Green DND-26 uptaken by laser confocal microscope.11.HBV-carrier mice and control mice were orally infected with S.typhimurium,bacteria load in liver and survival were detected to evaluate the susceptibility of these mice to S.typhimurium.Results1.HBV could not activate NLRP3 inflammasomeThe expression of NLRP3,pro-caspase-1 and pro-IL-1? were significantly lower in HepG2.2.15 cells compared with HepG2 cells;no significant differences in NLRP3 inflammasome related components in the liver and IL-1? level in the serum were observed between the HBV-carrier mice and control mice.Besides,concentrated HBV virions could not activate NLRP3 inflammasome in differentiated THP-1 cells.These results suggested that HBV and related proteins could not activate NLRP3 inflammasome.2.HBV suppresses LPS-induced NLRP3 inflammasome activationChronically HBV infection significantly inhibited LPS-induced NLRP3 and pro-IL-1? expression,caspase-1 activation and IL-1? maturation in mice;Kupffer cells,rather than hepatocytes,as the major producers of NLRP3 and IL-1?,and HBV mainly inhibited the IL-1? produced by Kupffer cells;pre-incubation of differentiated THP-1 cells with supernatants containing HBV virions could obviously repress LPS-induced NLRP3 and pro-IL-1? expression,caspase-1 activation and IL-1?maturation.These results suggested that HBV could suppresse LPS-induced NLRP3 inflammasome activation3.HBeAg,but not HBsAg,inhibits LPS-induced NLRP3 inflammasome activationHBeAg pretreatment significantly down-regulated LPS-induced intracellular NLRP3 and pro-IL-1? expression,caspase-1 activation and IL-1? maturation in THP-1 cells.Moreover,in vivo experiments have shown that LPS-induced NLRP3 inflammasome activation was significantly downregulated in HBV-persistent mice,while slightly downregulated in HBV-persistent mice without HBeAg.Besides LPS,HBeAg impairs other NLRP3 agonists-induced NLRP3 inflammasome activity.However,HBsAg had no such inhibitory effect on NLRP3 inflammasome activation.4.HBeAg inhibits LPS-induced NLRP3 and pro-IL-1? expression via repressing the NF-?B signal pathwayHBeAg pretreatment significantly down-regulated LPS-induced intracellular NF-?B phosphorylation and downstream cytokines such as IL-6 and TNF-a in THP-1 cells.Protein docking was performed by ZDOCK 3.0.2.to analyze the possible interactions between HBeAg and two downstream molecules of NF-?B,TRAM and MAL.As expected,protein docking performed with the crystal structure of HBeAg dimer revealed the potential protein binding pockets and predicted the possible interactions between HBeAg and TRAM or MAL.5.HBeAg inhibits the caspase-1 activation and IL-1? production by decreasing ROS productionHBeAg pretreatment obviously inhibited LPS-induced intracellular ROS production through suppressing NADPH oxidase activation.While,H202 treatment effectively reversed the suppressive effect of HBeAg on LPS-induced activation of NLRP3 inflammasome.6.Decreased levels of IL-1? and NLRP3 activation are detected in HBeAg-positive HCC and CHB patientsAnalysis of liver tissues in HCC patients revealed that the protein levels of NLRP3,activated caspase-1 p20,pro-IL-1?,and mature IL-1? p17 in the liver para-carcinoma tissues and the IL-1? expression in Kupffer cells were significantly lower in HBeAg-positive HCC patients than in HBeAg-negative ones.In CHB patients,the serum IL-1? level in IT patients(HBeAg-positive)was lower than in small part of IN patients(HBeAg-negative).It is worth noting that the levels of serum IL-1? in most of IN patients are not different from IT patients,and that although the IA group consists of almost the same number of HBeAg+ and HBeAg-patients,no obvious differences of IL-1? levels were observed due to different levels of HBeAg.We proposed that the high level of inhibitory factors such as IL-10 and PEG-2 probably play a major role in these CHB patients,leading to the low level of IL-1?.7.HBV increases the susceptibility of mice to Salmonella Typhimurium infectionHBV infection dramatically reduced the expression of NLRP3,pro-IL-1?,cleaved IL-1?,and activated caspase-1 in liver,increased the bacterial load in liver and reduced the survival time of mice infected with S.Typhimurium.These results indicated that HBV infection increased the susceptibility of mice to S.Typhimurium infection,possibly via inhibiting the activation of NLRP3 inflammasome and the production of IL-1?.Conclusions1.Kupffer cells,rather than hepatocytes,as the major producers of NLRP3 and IL-1?.2.HBV could not activate NLRP3 inflammasome,while it was able to inhibit LPS-induced NLRP3 inflammasome activation and IL-1? production,leading to immune suppression and HBV persistence.3.HBeAg suppresses LPS-induced NLRP3 inflammasome activation and IL-1?production in two ways,one is to repress NLRP3 and pro-IL-1? expression via inhibiting NF-?B phosphorylation,and the other is to repress caspase-1 activation and IL-1? maturation via inhibiting ROS production.4.HBV inhibites inflammation by targeting NLRP3 inflammasome,which provides a more enhanced understanding of the interplay between HBV and innate immune responses,and reveal a novel mechanism of HBV-mediated suppression of intracellular innate immune responses,which contributes to HBV-induced immunotolerance.The research may also provide new therapeutic targets for chronic HBV infection and related diseases.