Inhibitory And Mechanism Of HBV Infection In Neutrophil Extracelltular Traps Release

Hepatitis B Virus(HBV)is a family of hepadnaviruses and is the main pathogen of human viral hepatitis.HBV infection is distributed worldwide,with different epidemics in different regions.About 2 billion people worldwide have been infected with HBV,and 360 million people are chronically infected with HBV.In China,the rate of HBV infection is about 10%,and about 20 million people with chronic hepatitis B.HBV infection can cause chronic,acute and severe hepatitis,and is closely related to the occurrence of liver fibrosis,cirrhosis and liver cancer.The mechanism of HBV persistent infection formation is still unclear.A large number of studies have shown that the body's immune system cannot effectively eliminate the virus is the main cause of the formation of persistent HBV infection.Neutrophils play an important role in antiviral infection.When pathogens such as bacteria,fungi and viruses are infected,neutrophils are rapidly recruited from the peripheral circulation to the infected site under the action of inflammatory factors and chemokines and participate in the clearance of pathogens.Receptor-mediated endocytosis phagocytosis of pathogens into neutrophils,forming phagosomes,fusion of phagosomes with azurophilic particles,resulting in degranulation of the latter,allowing enzymes to be injected into the phagosomes;Granulocytes trigger NADPH oxidase on the membrane,causing neutrophil respiratory burst,producing a large number of active oxygen metabolites,which are killed and destroyed by enzymes and reactive oxygen species.In addition,neutrophils can release web-like structures called neutrophil extracellular trap(NETs)to capture and eliminate pathogens and participate in anti-infective immunity.NETs are newly discovered neutrophil anti-infective mechanisms and are the main means of neutrophil extracellular anti-infection.NETs are extracellular fibrous structures that are released into the extracellular space by neutrophils stimulated by microorganisms,cytokines and chemicals such as fMLP and PMA.They are composed of depolymerized chromatin and more than 30 neutrophil proteins.The network structure of NETs,which includes various anti-infective factors such as neutrophil elastase(NE),cathepsin G,myeloperoxidase(MPO)cathepsin G,and proteinase 3(PR3).These large extracellular structures trap and kill a variety of microbes by exposing them to high concentrations of NETs-associated microbicidal factors and providing a physical barrier to prevent microbial dissemination.NETs components-antibacterial molecules,histones and even DNA can directly participate in capturing and killing pathogens.Neutrophils release NETs in two ways:Non-cytosolic release.Neutrophils can release untwisted chromatin and antibacterial proteins to the outside of the cell in the form of vesicles,which are assembled extracellularly into NETs.Non-cytosolic release is a rapid release of NETs that takes only 5 to 60 minutes.Cytosolic release is a novel process of activating cell death that takes 120-240 minutes.Activated neutrophil nucleus deformed,its euchromatin and heterochromatin became uniform,followed by cleavage of the nuclear membrane and granule membrane,and finally the cell membrane ruptured by NETs.This process is called NETsosis and is the main pathway for NETs release.The regulation mechanism of NETs release is still unclear.It has been found that reactive oxygen species(ROS)act as a NETs release promoter and play an important role in the release process.Induction factors such as pathogens bind to neutrophil membrane receptors,causing the release of Ca2+ from the endoplasmic reticulum,and intracellularly elevated Ca2+ activates protein kinase C(PKC).PKC phosphorylation of NADPH oxidase(NOX)forms a functional complex(phagocytic oxidase;PHOX)to produce reactive oxygen species(ROS),especially superoxide anion.The superoxide is converted into a ROS product such as hydrogen peroxide and perchloric acid.The ROS product may act as a second messenger to trigger a change in the downstream signal transduction pathway,initiate NETs release:block the apoptosis process by inhibiting the caspase activity necessary for apoptosis;causing destruction of the endoplasmic reticulum and or mitochondria leading to Ca2+ Elevated levels in turn maintain PAD4 activity;mediated activation of elastase(NE)contributes to chromatin decondensation.Recent studies have shown that only ROS is not enough to induce NETs release,and elevated neutrophil autophagy activity is also a necessary condition for NETs release.Inhibition of autophagy activity of neutrophils by wortmannin can lead to abnormal decondensation of chromosomes and inhibition of NETs release;mTOR is a negative regulatory protein of autophagy,inhibits mTOR pathway and increases neutrophil self-Phage activity,which in turn promotes the release of NETs.The study found that the proportion of neutrophils increased in acute and subacute HBV infection;granulocyte colony-stimulating factor treatment can improve the survival rate of patients with acute liver failure associated with HBV.The above findings suggest that neutrophils participate in HBV clearance.In addition,patients with chronic active hepatitis and severe hepatitis have reduced intracellular granules,cytoplasmic loosening,edema and degeneration,and the number of organelles contained in the cytoplasm is significantly reduced,and phagocytic vacuoles,low-density particles,and dense particles are significantly reduced.The proportion of pulp decreased,showing obvious nuclear concentration;recombinant HBe can inhibit the activation of neutrophils and migration to the infected site,reduce the respiratory burst of neutrophils,and then reduce ROS production and reduce phagocytosis;suggesting neutrophils Functional abnormalities may be related to the establishment of persistent HBV infection.ObjectivesTo study the effect of HBV on the release of NETs and the potential mechanism,and to clarify the mechanism of HBV infection inhibiting neutrophil function.Provide experimental basis and theoretical basis for the research of HBV therapeutic targets and new therapeutic drugs.Methods1.Establishment of HBV-carrier mouse mode.HBV-carrier mice were established by hydrodynamic injection of pAAV/HBV 1.2 plasmids via the tail vein into C57BL/6 mice.2.The efficiency of bacterial eradication in vivo.E.coli was injected intraperitoneally into HBV chronically infected mice and control mice.The effect of HBV on neutrophil activity was determined by measuring the number of E.coli in the peritoneal cavity of mice.3.Patients collection.The present study was conducted on 40 patients with chronic hepatitis B(CHB)infection(age 33.8±4.3 years)who met the diagnostic criteria for HBV(WS299-2008)and were recruited from Shandong Provincial Hospital,as well as 40 healthy controls without HBV infection or autoimmune diseases(age 31.4±4.2 years).None of the 40 patients had been treated with antiviral drugs.4.Isolation of primary human neutrophils.Neutrophils were isolated from the peripheral blood of donors via density centrifugation using Polymorph Prep according to the manufacturer's recommendations.Trypan blue staining and flow cytometry were used to detect the activity and purity of neutrophils.5.Detection of NETs release markers.Using Quant-iT?PicoGreen dsDNA Reagent and Kits kit,live-cell imaging and western blot to detect the NETs release from peripheral blood of patients with persistent HBV infection and normal controls.6.Detection of HBV serum markersSerum levels of the HBV markers hepatitis B surface antigen(HBsAg),hepatitis B surface antibody(HBsAb),hepatitis B E antigen(HBeAg),hepatitis B E antibody(HBeAb)and hepatitis B core antibody(HBcAb)were quantified using the Abbot HBV Quantitative Test Kit(Chemiluminescence Microparticle Immuno Assay)using an Architech i4000 particle chemiluminescence detection instrument.7.Detection of HBV-DNA load.HBV-DNA levels were measured using an HBV nucleic acid quantitative detection kit(quantitative PCR-fluorescent probing method)and an Applied Biosystems 7500 fluorescence quantitative PCR instrument.8.Effect of HBV protein on NETs release.Recombinant HBs protein,HBe protein,HBc protein,HBx protein and PBS were incubated with normal control PMNs for 1 hour,after fMLP stimulation,using Quant-iTTM PicoGreen dsDNA Reagent and Kits kit,live-cell imaging and western blot to detect the effect of HBV protein on NETs release.9.Effect of HBV protein on ROS production.Using flow cytometry and cytochrome C reduction assay to modulate the regulation of ROS production by HBV protein.The western blot was used to detect the signaling pathway of HBV protein affecting ROS production.10.Effect of HBV protein on autophagy.Western blot was used to detect autophagy activity markers LC3,SQSTM1/p62 levels and using Autophagy Detection Kit to detect the regulation of HBV protein on autophagy.The effect of intracellular protein levels by HBV protein was detected by Western blot.11.Effect of HBV protein on NETs release after inhibiting mTOR pathway.We used the mTOR inhibitor rapamycin to inhibit intracellular mTOR phosphorylation.The release level of NETs was detected by Quant-iTTM PicoGreen dsDNA Reagent and Kits kit,live-cell imaging and western blot,and the effect of decreased mTOR phosphorylation level on NETs formation release was also observed.Results1.HBV decreases the ability of neutrophils to eradicate bacteria in vivo.Compared with control mice,HBV chronically infected mice were infected with E.coli,and their neutrophil-clearing bacteria decreased in vivo,and the survival time became shorter.This indicates that HBV may inhibit the sterilizing ability of neutrophils.2.HBV can inhibit NETs release.By detecting NETs markers cf-DNA/NETs,citrullinated histone 3(H3Cit)and live-cell imaging,the NETs release ability of patients with chronic HBV infection was significantly lower than that of the normal control group(P<0.05).3.Correlations between serum levels of HBV markers and NETs Correlation.The correlation analysis between NETs release and HBV serum markers showed that NETs release ability was negatively correlated with HBsAg,HBeAg and HBcAb levels at ?=0.05 level,and had no significant correlation with HBsAb and HBeAb levels.4.Correlations between NETs release ability and HBV-DNA load.Correlation analysis between NETs and HBV-DNA load in patients with chronic HBV infection.There was no significant correlation between NETs release ability and HBV-DNA load at a=0.05 level.5.HBc protein and HBe proteins can inhibit NETs release.After co-culture of HBc,HBe,HBs,HBx recombinant protein with normal group peripheral blood neutrophils,and fMLP stimulated NETs release.The results of NETs release markers showed that HBc and HBe proteins can inhibit NETs release,and HBs and HBx proteins have no effect on NETs release.6.HBc and HBe proteins can suppressing ROS production.After co-culture of HBc,HBe,HBs,HBx recombinant protein and PBS with normal group peripheral blood neutrophils,and fMLP stimulated NETs release.Flow cytometry(CM-H2DCFDA)and cytochrome C reduction assay showed that HBc and HBe proteins inhibited ROS production,while HBs and HBx proteins had no effect on ROS.Western Blot results confirmed that HBc and HBe proteins can inhibit the phosphorylation levels of p38(Thrl80)and p-ERK(ser21 7)sites.It is suggested that HBc and HBe proteins can inhibit the production of ROS by inhibiting the phosphorylation of P38 and p-ERK,thereby inhibiting the production and release of NETs.7.HBc and HBe proteins can inhibit autophagy.After co-culture of HBc,HBe,HBs,HBx recombinant protein and PBS with normal group peripheral blood neutrophils,and fMLP stimulated NETs release.The results showed that HBc and HBe protein can inhibit the expression of LC3 protein and increase the level of P62 protein,suggesting that HBc and HBe protein can inhibit autophagy.Autophagy Detection Kit results further confirmed that HBc and HBe proteins can inhibit autophagy.Western Blot results confirmed that HBc and HBe proteins can increase the phosphorylation levels of p-mTOR(Ser2448)and p-ULKl(Ser757)sites.It is suggested that HBc and HBe proteins can inhibit autophagy by regulating mTOR signaling pathway,thereby inhibiting the production and release of NETs.8.HBc and HBe proteins regulate NETs release by regulating the mTOR signaling pathway.The stimulation of NETs release by the mTOR inhibitor rapamycin can be corrected by the action of HBc and HBe proteins.Rapamycin significantly inhibits the phosphorylation level of mTOR(Ser2448)site and enhances autophagy activity,thereby stimulating the release of NETs.However,after HBc and HBe protein treatment,the phosphorylation level of mTOR(Ser2448)was increased by HBc and HBe protein,and the inhibition of mTOR phosphorylation by Rapamycin was no longer significant,and the stimulatory effect on NETs production release disappeared.This indicates that HBc and HBe proteins can regulate the release of NETs by regulating the expression level of mTOR signaling pathway.ConclusionHBV reduces the levels of neutrophil reactive oxygen species and autophagy through HBc and HBe proteins,inhibits the release of NETs,affects the clearance of neutrophils by viruses,and participates in the establishment of HBV immune escape and persistent infection.