Effects Of IL-33 And Its Receptor ST2 On HBV Replication And The Mechanism Study

Background and aims:Chronic hepatitis B is a global public health problem,especially when our country has a large number of people infected with hepatitis B virus nowadays.Although remarkable progress has been made in the antiviral treatment of chronic hepatitis B,up to now there is still no drug that can completely remove the hepatitis B virus.Once the infection becomes chronic,it is necessary to take antiviral drugs such as nucleoside analogs for life.Due to the continuous progress of the disease,the corresponding medical expenses also increase with the severity of the disease,which eventually leads to a serious physical and economic burden.Therefore,finding new targets and developing drugs with strong antiviral effect,few side effects,that can cure hepatitis B without damaging the liver are the hot spots and difficulties of anti-HBV research.Interleukin-33(IL-33),a member of the interleukin-1 family,is mainly derived from endothelial and epithelial cells,but fibroblasts,monocytes,macrophages,dendritic cells and hepatocytes can also express IL-33.Its receptor oncogenic inhibitor 2(ST2)can be expressed by a variety of immune cells such as CD4~+T cells,CD8~+T cells,mast cells,ILC2,macrophages and NK cells.The biologically active precursor IL-33 is released in the form of"alarmin"when cells and tissues are destroyed.IL-33 is believed to play a key role in infectious and inflammatory diseases by binding to its receptor ST2 to induce a variety of downstream immune responses after being released from cells.As part of innate immunity,natural killer(NK)cells constitute the first and most important line of defense against pathogenic microorganisms due to their cytotoxic and cytokine-secreting functions.However,the relationship between IL-33 and NK cells in HBV infection is not fully understood.This study is based on the background of chronic hepatitis B infection,through the study of the expression characteristics of IL-33 receptor ST2 in the plasma of patients with different stages of chronic hepatitis B patients and healthy people,so as to clarify its clinical significance.Subsequently,the effect of IL-33 on HBV replication was studied through in vivo animal experiments and in vitro cell experiments,using HBV high-pressure hydrodynamic mouse model and Hep G2 cell model.Finally,the m RNA level of IL-33 in the process of inhibiting HBV replication was studied and enrichment analysis was carried out.Trying to find new therapeutic targets,to provide more theoretical basis for the therapeutic strategy of IL-33 treating chronic hepatitis B,this study aims on providing new drug candidates for anti-HBV therapy.Materials and methods:This study firstly enrolled HBV patients of different stages and healthy controls,detecting the level of IL-33 soluble receptor ST2 in their plasma.Human peripheral blood samples were obtained from 71 chronic hepatitis B patients of different stages and 12 healthy volunteers who received outpatient and inpatient treatment at the Bethune First Hospital of Jilin University from January 2019 to December 2020.Among them,63 chronic hepatitis B patients who did not receive antiviral therapy and8 patients who received nucleoside analog antiviral therapy were included.Then ELISA method was used to detect the level of IL-33 soluble receptor ST2(s ST2)in plasma,and Luminex method was used to detect the level of cytokines in plasma.Finally,the correlation analysis between the levels of s ST2 and cytokines in plasma and the laboratory indexes of patients with different stage s of chronic hepatitis B was performed to explore their clinical significance in chronic hepatitis B infection.Subsequently,wild-type,ST2 knockout,CD8~+T knockout,NK knockout C57BL/6 mice,C.B-17 SCID and nod SCID mice were transinfected with HBV and then treated with different doses of IL-33,respectively.The levels of HBV DNA,HBs Ag,HBe Ag and liver function-related indexes were detected in serum collected from model mice at different time points;the expression of HBc Ag in liver tissue was observed by immunohistochemical staining.In vitro,Hep G2 cells were transfected with p AAV-HBV 1.2 and then treated with different concentrations of IL-33 to detect HBV DNA,HBs Ag and HBe Ag in the cell supernatant.Finally,in order to further study the changes in gene expression of IL-33 in the process of inhibiting HBV replication from the m RNA level,we isolated the livers of HBV mice treated with IL-33 and performed m RNA transcriptome sequencing(m RNA sequencing,m RNA-seq).The differentially expressed m RNAs(DE m RNAs)obtained by sequencing were included in the volcano map and cluster heat map respectively,and the functions of these DE m RNAs were enriched by Go function enrichment and KEGG and Reactome pathway enrichment methods respectively.and preliminary exploration of pathways.Results:1.The level of soluble receptor ST2 of IL-33 in the plasma of chronic hepatitis B patients was significantly higher than that of healthy adults,and the level of s ST2 was significantly decreased after nucleoside analog treatment.In addition,the level of s ST2 in patients with immune activation was higher than that in othergroups;2.Plasma s ST2 in patients with chronic hepatitis B was positively correlated with liver function indexes ALT,AST,?GT,ALP and TBIL,cytokines IP-10 and TNF- ?,and negatively correlated with TP and ALB;3.IL-33 decreased the levels of serum HBs Ag,HBe Ag and HBV quantification in a dose-dependent manner in a mouse model of HBV and also decreased the expression of HBc Ag in the liver;compared with the control group,ALT in mice treated with IL-33,The decrease of AST proves that after IL-33 exerts antiviral effect,it is accompanied by the reduction of liver inflammation;4.After the high-pressure hydrodynamic HBV mouse model was established in ST2 gene knockout mice and treated with IL-33,the levels of HBs Ag,HBe Ag and hepatitis B virus in the serum of the mouse model were not different from those in the control group,proving that IL-33 33 exerts its antiviral effect mainly through its receptor ST2;5.There were no differences in HBs Ag,HBe Ag and HBV DNA levels between wild-type HBV mice and CD8~+T cell-depleted HBV mice after IL-33 treatment. However,the levels of HBs Ag and HBV DNA were higher in NK cell knockout HBV mice compared with wild-type HBV mice,suggesting that IL-33 may exert antiviral effects through NK cells;6.IL-33 reduced HBs Ag levels in C.B-17 SCID HBV mice,but had no effect on HBe Ag and HBV DNA levels.In addition to this,for nod SCID HBV mice,IL-33 could reduce HBs Ag and HBV DNA concentrations at day 3,but until day 6, there was no difference between the two groups.Consistent with the results in C.B-17 SCID and NK cells knockout HBV mice,IL-33 had no effect on HBe Ag, suggesting that IL-33 could partially inhibit HBV replication in immunodeficient mice;7.In vitro,IL-33 can inhibit HBV replication in Hep G2 cells in a dose-dependent manner;8.High-throughput sequencing technology and transcriptomics were used to compare and analyze the livers of high-pressure hydrodynamic HBV mice treated with different doses of IL-33.Various enrichment analysis results showed that the enrichment analysis mainly focused on adaptive immune responses.,cell activation regulation,positive regulation of immune response,migration of leukocytes,positive regulation of cytokines and other immune responses.Conclusion:Our study showed that the plasma level of ST2,a soluble receptor for IL-33,was significantly increased in patients with chronic hepatitis B and decreased after nucleoside analog treatment.In addition,the level of s ST2 in patients with immune activation was higher than that in other groups,suggesting that it may have a predictive effect on the inflammatory state of the liver.Animal experiments confirmed that IL-33 inhibits hepatitis B virus through its receptor ST2 in a hydrodynamic HBV mouse model,and this effect may be attenuated in the absence of NK cells.In addition,in vitro cell experiments confirmed that IL-33 has a direct anti-HBV effect,all of the above results suggest that IL-33 may be an effective inducer of HBV clearance and a promising drug candidate.Transcriptomic comparative analysis of the livers of high-pressure hydrodynamic HBV mice treated with different doses of IL-33,multiple enrichment analysis results showed that the main focus was on adaptive immune response,cell activation regulation,positive regulation of immune response,Immune responses such as migration of leukocytes and positive regulation of cytokines provide more targets and new drug candidates for chronic hepatitis B treatment strategies targeting IL-33.