| Invariant NKT(iNKT)cells are unique cell subsets with the characteristics of both NK cells and T cells.They are CD1d-restricted and can secrete a large number of cytokines after activation.They have important immunomodulatory effects and are a bridge between innate immunity and adaptive immunity.Existing studies have shown that iNKT cells are abundantly enriched in the human liver and can regulate the intrahepatic immune microenvironment after HBV infection,which can affect the outcome of diseases after HBV infection.However,there have been conflicting reports on the changes of number and cytokine secretion function of iNKT cells in HBV infection.The effects of human iNKT cells in the process of HBV infection is still unclear.In this study,the meta-analysis of the number and cytokine secretion function of iNKT cells in patients with chronic HBV infection in different disease statuses was perpromed.To further verify the effect of HBV on iNKT cell cytokine producing capapcity,iNKT cell clone hiNKT38 was stimulated with CD1d-bearding HBV-infected(HepG2.2.15-tmCD1d)and non-infected(HepG2-tmCD1d)cells,followed by cytokine detection.In addition,stimulator-based mechanisms was explored by RNA-seq analysis and public dataset analysis.The paper is divided into two parts,the main contents and results are as follows.1.Meta-analysis of the number and cytokine secretion function of iNKT in patientswith chronic HBV infection in different clinical typesAfter systematically searching for studies about the changes in the number and cytokine secretion function of iNKT in peripheral blood or liver tissues of patients with chronic HBV infection on PubMed,Embase,Cochrane,Cnki and WanfangData,the results of meta-analysis showed that the ratio of iNKT to CD3~+T in peripheral blood changes dynamically in different clinical types of the disease.Compared to healthy controls,it increased in chronic HBV carrier,whose statistical indicator comprise that MD(mean difference)is 0.16%,95%CI is[0.10%,0.23%],I~2 is 0%,and p value is less than 0.00001.It has varying degrees of decline in patients with CHB(chronic hepatitis B),CHB patients in immune clearance phase,inactive HBsAg carriers(also known as inactive carriers),and patients with HBV-LC(HBV-liver cirrhosis).The statistical indicator of CHB patients comprise that MD is-0.10%,95%CI is[-0.15%,-0.06%],I~2is 42%,and p value is less than 0.00001.The statistical indicator of CHB patients in immune clearance phase comprise that MD is-0.14%,95%CI is[-0.20%,-0.09%],I~2is 0%,and p value is less than 0.00001.The statistical indicator of inactive HBsAg carriers comprise that MD is-0.05%,95%CI is[-0.09%,-0.02%],I~2 is 0%,and p value is 0.004.The statistical indicator of patients with HBV-LC comprise that MD is-0.07%,95%CI is[-0.09%,-0.04%],I~2 is 0%,and p value is less than 0.00001.Analysis of cytokine secretion ability in patients with different clinical types showed that the ratio of IFN-γ~+iNKT increased in CHB patients in immune clearance phase,whose statistical indicator comprise that MD is 9.40%,95%CI is[3.33%,15.46%],I~2 is 0%,and p value is 0.002.The ratio of IL-4~+iNKT increased in patients with HBV-LC,whose statistical indicator comprise that MD is 8.88%,95%CI is[3.94%,13.82%],I~2 is 0%,and p value is 0.0004.The results suggested that IFN-γ+iNKT cells promoted antiviral immune response and IL-4~+iNKT cells promoted the development of cirrhosis.2.Differential cytokine secretion by iNKT cells stimulated by two kinds of humanhepatoma cell lines positive or negative for HBV expression and transcriptomicanalysis of hepatoma cells and public data miningThe iNKT cell line derived from Jurkat CD8~+cells transfected with TCR of iNKT(hiNKT38)and two hepatoma cell lines expressing CD1d(HepG2-tmCD1d cells,HepG2.2.15-tmCD1d cells expressing HBV)were selected as effector cells and stimulating cells,respectively.The concentration of cytokines in the culture supernatant was determined through CBA after 24 hours of co-culture.The results showed that the IFN-γsecretion of hiNKT38 cells was significantly decreased in the HepG2.2.15-tmCD1d stimulation group imitating HBV infection compared with the HepG2-tmCD1d stimulation group.The stimulating cell samples were sent for the transcriptome sequencing.The results of RNA-seq analysis indicated that the expression of IL-18 and IL-12A was declined in HepG2.2.15-tmCD1d cells.In addition,the results of two datasets from the GEO database(high-throughput gene expression database)also showed down-regulated expression of the IL-18-encoding gene in human primary hepatocytes infected with HBV.These results suggested that HBV can reduce the IFN-γsecretion of iNKT cells by inhibiting IL-18 and IL-12A expression in hepatocytes.The conclusions and significance of this study1.The number and cytokine secretion function of iNKT cells in peripheral blood ofpatients with chronic HBV infection is related to clinical types.The proportion ofIFN-γ+iNKT cells and IL-4~+iNKT cells were related to antiviral immune responseand the occurrence of cirrhosis respectively.2.The human iNKT cell line hiNKT38 showed a decrease in IFN-γsecretion underthe stimulation of HepG2.2.15-tmCD1d cells imitating HBV infection.Transcriptome analysis indicated that HBV inhibited IL-18 and IL-12A geneexpression in HepG2.2.15-tmCD1d cells.Results of two datasets from the GEOdatabase(high-throughput gene expression database)also showed down-regulatedexpression of the IL-18-encoding gene in human primary hepatocytes infected withHBV.The innovation of this researchA meta-analysis of quantity and function of iNKT in chronic HBV infection was performed.The differential cytokine secretion of hiNKT38 in the presence of two hepatoma cell lines positive or negative for HBV expression was analyzed through cell experiment.The differentially expressed genes of HepG2.2.15-tmCD1d under HBV stimulation were studied.The results suggested that HBV may evade immune attack of iNKT cells by inhibiting IL-18 and IL-12A expression in HepG2.2.15-tmCD1d cells. |
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