Effect Of Programmed Cell Death-1 In Chronic HBV Infection

Background:The mechanism hepatitis B is a complex, hepatitis B virus(HBV), host and their interaction affected the disease. The effect from virus including viral protein and genotype, and which from host mainly including host genetic background and immunity. The balance between virus and host is the mainly factor affect progression and outcome of the disease. Nucleoside/Nucleotide analogues(NAs) is a class of oral anti-HBV drug. The main mechanism of NAs is that they become triphosphate compound after their access to the cells, inhibit viral polymerase or reverse transcriptase by competition with substrate, and terminate HBV DNA chain extension. Recent studies showed that NAs may also have immuno-modulatory effect. In CHB patients who undertaking NAs anti-HBV therapy, study showed that NAs therapy can regulate Th1/Th2 balance, improve proliferation of Treg cells, and enhance or recover immune function. Efficacy of anti-HBV infection with NAs therapy in clinic have individual differences, and its influencing factors not only viral genotype, but also the genetic background of the host. Single nucleotide polymorphisms(SNP) plays a mainly role in the genetic background of the host. SNP refers to changes in DNA sequences caused by changing of a single nucleotide A, T, C or G, which lead the diversity of genome chromosomes in the human individuals or different species. SNP constitute more than 90% of the human genome variation, which is widely used in linkage analysis and gene mapping research, disease susceptibility, population genetics and evolutionary biology, pharmacogenomics, environmental genomics and so on. Current literatures reported SNPs associated with prognosis of HBV infection included: HLA DRBA*1302, MHC-I, MHC-II in human leukocyte antigen region, tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), estrogen receptor-α, mannose binding protein, vitamin D receptor, interleukin-10(IL-10), interferon-γ inducible protein-10,programmed cell death-1 in non-human leukocyte antigen region and other genes. PD-1 is a member of the CD28 family and Ig superfamily, which is a type I transmembrane protein with 55,000 relative molecular mass. It is expressed on activated CD4+ T cells and CD8+ T cells, B cells and bone marrow cells, not expressed in the non-activated lymphocytes. PD-L1 overexpression on hepatic cell surface when chronic HBV infection, which inhibit the expression of CD8+ T cells through the PD-1/PD-L1 signaling pathway, T cell exhaustion leads to reduce expression of anti-viral cytokines such as IFN-γ, TNF-α, etc. thus viral persistence. Different alleles of PD-1 expressed different in patients with HBV infection, cirrhosis and healthy people, and its SNPs in intron were associated with peripheral blood viral load. Recent study showed that peripheral expansion of Treg cells levels correlated with HBV viral load and decreased HBV-specific CD8+ T cells. Adefovir dipivoxil therapy can block the expression of PD-1 receptor, inhibit HBV replication and reduce the number and function of Treg cells, significantly increased HBV-specific CD8+ T effector cells, thereby enhance the immune response. Current researches on the role of PD-1 in chronic HBV infection is not yet very clear, PD-1 SNPs in spontaneous clearance of HBV infection and efficacy of HBV antiviral therapy were less, and the results were inconsistent. The correlation between NAs antiviral therapy and PD-1 as well as its related factors, and effect of NAs antiviral therapy on host immune function are still controversial.Objective: To explore the role of PD-1 in the development and outcome of HBV infection in individuals of spontaneous clearance of HBV infection, CHB patients(including undertake NAs treatment and not undertake any anti-HBV therapy) and HBV-related acute on chronic liver failure(ACLF) patients by measuring PD-1 expression in serum and liver tissue, assess effect of NAs on PD-1 expression, analyze the correlation between serum PD-1 with chronic HBV infection in viral load, clinical and biochemical indicators. To evaluate the association between frequency of genotype distribution of SNPs in PD-1 and different individuals infected HBV and response in NAs therapy.To assess the effect of NAs on PD-1/PD-L1 signaling pathway and host immunity by measuring serum levels of PD-1, IL-10, IFN-γ, HBV DNA load, as well as the peripheral blood of HBV-specific CD8+ T cell frequency in individuals of spontaneous clearance of HBV infection, CHB patients(including undertake NAs treatment and not undertake any anti-HBV therapy) and HBV-related ACLF patients.Methods: 1 Clinical significance of programmed cell death-1 in chronic HBV infection patients.The cases with chronic HBV infection and spontaneous clearance of HBV in The Third Hospital of Hebei Medical University from October 2013 to January 2015 were included. All of the CHB and HBV-related ACLF patients were diagnosed according to the Guidelines on Prevention and Treatment for Chronic Hepatitis B in China(2010 edition) or the Diagnostic and Treatment Guidelines for Liver Failure(2012 edition) developed by the Chinese Society of Hepatology and the Chinese Society of Infectious Disease respectively. The antiviral therapy was single or combined NAs treatment, without any NAs or IFN treatment was non-antiviral therapy. Spontaneous clearance of HBV was defined as two or three positive in anti-HBs、anti-HBe and anti-HBc, negative in HBV DNA, normal in liver function indicators, ultrasound examination of liver, gallbladder and spleen, without any antiviral therapy. The exclusion criteria:(1) pregnancy, the patients with HAV, HCV, HDV, HEV, HIV, EBV, CMV and other hepatitis virus infection;(2) the patients who have taken NAs combined with IFN or traditional Chinese medicine;(3) the patients with malignant tumors, severe immune system diseases, blood diseases and history of mental illness. Demographic and characteristic data of the cases were collected, including gender, age, diagnosis, treatment, medication time. All the patients treated with NAs monitored liver function, HBV viral markers(HBs Ag, anti-HBs, HBe Ag, anti-HBe, anti-HBc) and HBV DNA load. Serum albumin(ALB),alanine arninotransferase(ALT),and total bilirubin(TB) were measured by a fully automatic biochemicalanalyzer. Serum PD-1 concentrations were measured with sandwich enzyme-linked immunosorbent assay(ELISA).HBV DNA was quantified by real-time quantitative polymerase chain reaction(PCR) assay. PD-1 expression in liver tissue was measured with western-blot. Use IBM SPSS Statistics 19 statistical software for statistical analysis, measurement data expressed as(mean±SD). Normality and homogeneity of variance were tested before data analysis. Use t-test to analyze the data if the data is normally distributed and the homogeneity of variance(P > 0.05), use t’-test to analyze the data if the heterogeneity of variance(P < 0.05). Comparisons in multiple groups using single factor analysis of variance, comparisons in two groups were using LSD test. Use Kruskal Wallis H rank sum test if the data were not normally distributed or heterogeneity of variance.Count data use Chi-square test. Correlation analysis using Spearman rank correlation analysis. All the tests are two-side test, P < 0.05 was considered as statistical difference.2 Correlation in single nucleotide polymorphisms of programmed cell death-1 and efficacy of anti-HBV infection.The cases with chronic HBV infection and spontaneous clearance of HBV in The Third Hospital of Hebei Medical University from October 2013 to January 2015 were included. All of the CHB and HBV-related ACLF patients were diagnosed according to the Guidelines on Prevention and Treatment for Chronic Hepatitis B in China(2010 edition) or the Diagnostic and Treatment Guidelines for Liver Failure(2012 edition) developed by the Chinese Society of Hepatology and the Chinese Society of Infectious Disease respectively. The antiviral therapy was single or combined NAs treatment, without any NAs or IFN treatment was non-antiviral therapy. Spontaneous clearance of HBV was defined as two or three positive in anti-HBs、anti-HBe and anti-HBc, negative in HBV DNA, normal in liver function indicators, ultrasound examination of liver, gallbladder and spleen, without any antiviral therapy. The exclusion criteria:(1) pregnancy, the patients with HAV, HCV, HDV, HEV, HIV, EBV, CMV and other hepatitis virus infection;(2) the patients who have taken NAs combined with IFN or traditional Chinese medicine;(3) the patients with malignant tumors, severe immune system diseases, blood diseases and history of mental illness. After 12 weeks treatment with NAs, serum HBV DNA by PCR undetectable or below the detection limit(500copies/ml), or a decrease from baseline ≥2log10 is early responders; decrease from baseline <2log10 is non-early responders. After 4 weeks treatment with NAs, serum HBV DNA by PCR undetectable or below the detection limit(500copies/ml), or a decrease from baseline ≥2log10 is rapid responders; decrease from baseline <2log10 is non- rapid responders. 3 ml of venous blood from cases was collected, rs10204525, rs2227982, rs7421861 and rs6710479 were genotyped by polymerase chain reaction-restrictive fragment length polymorphism(PCR-RFLP) analysis. Use IBM SPSS Statistics 19 statistical software for statistical analysis. Ratio was compared by Pearson Chi-Square test or Fisher? s exact test in the case of P ≈ a or n < 40. All the tests are two-side test, P < 0.05 was considered as statistical difference. SHEsis software was used to Hardy-Weinberg equilibrium analysis, P > 0.05 in the control group is consistent with the genetic equilibrium.3 Effect of HBV-mediated PD-1/PD-L1 signaling pathway in chronic hepatitis B disease progression.The cases with chronic HBV infection and spontaneous clearance of HBV in The Third Hospital of Hebei Medical University from October 2013 to January 2015 were included. All of the CHB and HBV-related ACLF patients were diagnosed according to the Guidelines on Prevention and Treatment for Chronic Hepatitis B in China(2010 edition) or the Diagnostic and Treatment Guidelines for Liver Failure(2012 edition) developed by the Chinese Society of Hepatology and the Chinese Society of Infectious Disease respectively. The antiviral therapy was single or combined NAs treatment, without any NAs or IFN treatment was non-antiviral therapy. Spontaneous clearance of HBV was defined as two or three positive in anti-HBs、anti-HBe and anti-HBc, negative in HBV DNA, normal in liver function indicators, ultrasound examination of liver, gallbladder and spleen, without any antiviral therapy. The exclusion criteria:(1) pregnancy, the patients with HAV, HCV, HDV, HEV, HIV, EBV, CMV and other hepatitis virus infection;(2) the patients who have taken NAs combined with IFN or traditional Chinese medicine;(3) the patients with malignant tumors, severe immune system diseases, blood diseases and history of mental illness. Demographic and characteristic data of the cases were collected, including gender, age, diagnosis, treatment, medication time. All the patients treated with NAs monitored liver function, HBV viral markers(HBs Ag, anti-HBs, HBe Ag, anti-HBe, anti-HBc) and HBV DNA load. Serum ALB, ALT, and TB were measured by a fully automatic biochemical analyzer. Serum PD-1, IL-10, and IFN-γ concentrations were measured with ELISA.HBV DNA was quantified by PCR assay. Frequency of HBV-specific CD8+ T cells were analyzed using flow cytometer. Use IBM SPSS Statistics 19 statistical software for statistical analysis, measurement data expressed as(mean ± SD). Normality and homogeneity of variance were tested before data analysis. Use t-test to analyze the data if the data is normally distributed and the homogeneity of variance(P > 0.05), use t’-test to analyze the data if the heterogeneity of variance(P < 0.05). Comparisons in multiple groups using single factor analysis of variance, comparisons in two groups were using LSD test. Use Kruskal Wallis H rank sum test if the data were not normally distributed or heterogeneity of variance.Count data use Chi-square test. Correlation analysis using Spearman rank correlation analysis. All the tests are two-side test, P < 0.05 was considered as statistical difference.Results: 1 Clinical significance of programmed cell death-1 in chronic HBV infection patients.1.1 Demographic and clinical features of the enrolled cases.There were 86 cases enrolled in this study, 46 of CHB patients group(including 23 of undertake NAs treatment and 23 of not undertake any anti-HBV therapy), 20 of HBV-related ACLF patients group, and 20 of spontaneous clearance of HBV infection group. Gender and age of cases in groups have no significant difference(P > 0.05). HBV DNA load and ALT level in CHB un-treated group were higher than in CHB treated group(P < 0.05), no significant difference was found in TB and ALB level(P > 0.05). ALB level was significantly higher in spontaneous clearance of HBV infection group than in CHB group and HBV-related ACLF group(P < 0.05), while ALT and TB level were significantly lower than them(P < 0.05). ALB level was significantly higher in CHB group than in HBV-related ACLF group(P < 0.05), while HBV DNA load, ALT and TB level were significantly lower than it(P < 0.05).1.2 Serum PD-1 level in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. Serum PD-1 level in HBV-ACLF group(17.542 ± 2.265 ng/ml) was significantly higher than CHB un-treated group(15.533 ± 2.194 ng/ml), CHB NAs treated group(12.849 ± 2.193 ng/ml) and spontaneous clearance of HBV infection group(10.641 ± 4.192 ng/ml). All the differences were significant(P < 0.001). Serum PD-1 level was significantly higher in CHB un-treated group than in CHB NAs treated group and spontaneous clearance of HBV infection group, in CHB NAs treated group than in spontaneous clearance of HBV infection group(P < 0.05).1.3 Expression of PD-1 in liver tissue in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. A total of 40 cases took westernblot, 10 of each group. PD-1 expression in HBV-ACLF group(1.274 ± 0.362) was significantly higher than CHB un-treated group(1.074 ± 0.088), CHB NAs treated group(0.813 ± 0.156) and spontaneous clearance of HBV infection group(0.593 ± 0.116). All the differences were significant(P < 0.001). PD-1 expression was significantly higher in CHB un-treated group than in CHB NAs treated group and spontaneous clearance of HBV infection group, in CHB NAs treated group than in spontaneous clearance of HBV infection group(P < 0.05).1.4 Correlation analysis in serum PD-1 level and HBV DNA load, ALT, TB and ALB. Serum PD-1 level was positive correlated with HBV DNA load in CHB group and HBV-ACLF group(r = 0.568, P < 0.001;r = 0.729, P < 0.001). Serum PD-1 level was positive correlated with ALT(r = 0.373, P = 0.011), but negative correlated with ALB(r =-0.457, P = 0.001) in CHB group. Serum PD-1 level was negative correlated with ALB(r =-0.756, P < 0.001), but not correlated with ALT and TB in HBV-ACLF group.2 Correlation in single nucleotide polymorphisms of programmed cell death-1 and efficacy of anti-HBV infection.2.1 Demographic and clinical features of the enrolled cases. There were 195 cases enrolled in this study, 90 of CHB patients group(including 65 of early response and 25 of non-early response patients), 45 of HBV-ACLF patients group(30 of rapidly response and 15 of non-rapidly response patients), and 60 of spontaneous clearance of HBV infection group. Gender and age of patients in groups have no significant difference(P > 0.05). Gender, age, HBV DNA load, serum level of ALT, TB and ALB before NAs treatment all have no significant difference between patients early response and non-early response patients, rapidly response and 15 of non-rapidly response patients(P > 0.05).2.2 Allele frequency.2.2.1 Allele A frequency of rs10204525 have no significant difference not only in CHB group(76.7%), HBV-ACLF group(67.8%) and spontaneous clearance of HBV infection group(75.8%)(χ2 = 2.684, P = 0.261), but also in spontaneous clearance of HBV infection group(75.8%) and chronic HBV infection group(CHB group plus HBV-ACLF group, 73.7%)(χ2 = 0.955, P = 0.328), CHB group and HBV-ACLF group(χ2 = 2.446, P = 0.118)(P > 0.05). Allele A frequency of rs10204525 in CHB early response group(80.8%) was significantly higher than in non-early response group(66.0%)(χ2 = 4.403, P = 0.036), in HBV-ACLF rapidly response group(75.0%) was significantly higher than in non-rapidly response group(53.3%)(χ2 = 4.299, P = 0.038).2.2.2 Allele A frequency of rs6710479 significantly lower in spontaneous clearance of HBV infection group(59.2%) than in CHB group(73.3%) and HBV ACLF group(68.9%)(χ2 = 54.052, P < 0.001), and in spontaneous clearance of HBV infection group(59.2%) than in chronic HBV infection group(CHB group plus HBV-ACLF group, 71.9%)(χ2 = 6.138, P = 0.013). There were no significant differences of allele A frequency of rs6710479 in CHB group and HBV-ACLF group(χ2 = 0.586, P = 0.444), in CHB early response group and non-early response group(χ2 = 3.084, P = 0.079), in HBV-ACLF rapidly response group and in non-rapidly response group(χ2 = 1.435, P = 0.231)(P > 0.05).2.2.3 There were no significant differences of allele frequency of rs2227982 and rs7421861 in spontaneous clearance of HBV infection group, CHB group and HBV-ACLF group, in CHB group and HBV-ACLF group, in CHB early response group and non-early response group, in HBV-ACLF rapidly response group and in non-rapidly response group(P > 0.05).2.3 Genetic equilibrium test. SHEsis software was used to Hardy-Weinberg equilibrium analysis. Genetic equilibrium was found in rs10204525 CHB early response group(P <0.001) and non-early response group(P = 0.922), ACLF group rapidly response group(P = 0.039) and non-rapidly response group(P = 0.447), and in rs6710479 chronic HBV infection group(P = 0.011) and pontaneous clearance of HBV infection group(P = 0.286).3 Effect of HBV-mediated PD-1/PD-L1 signaling pathway in chronic hepatitis B disease progression.3.1 Demographic and clinical features of the enrolled cases.There were 86 cases enrolled in this study, 46 of CHB patients group(including 23 of undertake NAs treatment and 23 of not undertake any anti-HBV therapy), 20 of HBV-related ACLF patients group, and 20 of spontaneous clearance of HBV infection group. Gender and age of cases in groups have no significant difference(P > 0.05). HBV DNA load and ALT level in CHB un-treated group were higher than in CHB treated group(P < 0.05), no significant difference was found in TB and ALB level(P > 0.05). ALB level was significantly higher in spontaneous clearance of HBV infection group than in CHB group and HBV-related ACLF group(P < 0.05), while ALT and TB level were significantly lower than them(P < 0.05). ALB level was significantly higher in CHB group than in HBV-related ACLF group(P <0.05), while HBV DNA load, ALT and TB level were significantly lower than it(P < 0.05).3.2 Serum indicators. 3.2.1 Serum PD-1 level in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. Results were in the first part.3.2.2 Serum IL-10 level in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. Serum IL-10 level in HBV-ACLF group(62.43 ± 21.93 pg/ml) was significantly higher than CHB un-treated group(41.59 ± 16.46 pg/ml), CHB NAs treated group(25.10 ± 11.94 pg/ml) and spontaneous clearance of HBV infection group(20.83 ± 9.71 pg/ml). All the differences were significant(P < 0.001). Serum IL-10 level was significantly higher in CHB un-treated group than in CHB NAs treated group and spontaneous clearance of HBV infection group(P < 0.01), but not significantly different in CHB NAs treated group and in spontaneous clearance of HBV infection group(P > 0.05).3.2.3 Serum IFN-γ level in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. Serum IFN-γ level in HBV-ACLF group(79.08 ± 31.79 pg/ml) was significantly higher than CHB un-treated group(32.66 ± 20.47 pg/ml), CHB NAs treated group(51.95 ± 25.58 pg/ml) and spontaneous clearance of HBV infection group(58.63 ± 17.7 pg/ml). All the differences were significant(P < 0.001). Serum IFN-γ level was significantly lower in CHB un-treated group than in CHB NAs treated group and spontaneous clearance of HBV infection group(P < 0.01), but not significantly different in CHB NAs treated group and in spontaneous clearance of HBV infection group(P > 0.05).3.3 Peripheral blood frequency of HBV-specific CD8+ T cells in CHB un-treated group, CHB NAs treated group, HBV-ACLF group and spontaneous clearance of HBV infection group. Frequency of HBV-specific CD8+ T cells in HBV-ACLF group(0.85 ± 0.39 %) was significantly higher than CHB un-treated group(0.21 ± 0.25 %), CHB NAs treated group(0.41 ±0.47 %) and spontaneous clearance of HBV infection group(0.00 ± 0.00 %). All the differences were significant(P < 0.001). Frequency of HBV-specific CD8+ T cells was significantly higher in CHB un-treated group than in CHB NAs treated group and spontaneous clearance of HBV infection group(P < 0.05), and in CHB un-treated group than in spontaneous clearance of HBV infection group(P < 0.05).3.4 Correlation analysis3.4.1 Correlation analysis in HBV DNA load and level of serum PD-1, IL-10 and IFN-γ. HBV DNA load was positive correlated with serum PD-1 level in CHB group and HBV-ACLF group(r = 0.568, P < 0.001; r = 0.729, P < 0.001), was positive correlated with serum IL-10 level in CHB group and HBV-ACLF group(r = 0.541, P < 0.001; r = 0.560, P = 0.01), but negative correlated with serum IFN-γ level(r =-0.319, P < 0.05; r =-0.832, P < 0.001).3.4.2 Correlation analysis in peripheral blood frequency of HBV-specific CD8+ T cells and level of serum PD-1, HBV DNA load. Peripheral blood frequency of HBV-specific CD8+ T cells was positive correlated with serum PD-1 level(r = 0.438, P = 0.002), not correlated with HBV DNA load in CHB group(r = 0.072, P = 0.634). Peripheral blood frequency of HBV-specific CD8+ T cells was positive correlated with serum PD-1 level and HBV DNA load in HBV-ACLF group(r = 0.838, P < 0.001; r = 0.595, P = 0.006).Conclusion: 1 PD-1 expression was correlated with HBV infection disease progression.2 NAs antiviral therapy can inhibit HBV replication, promote HBV-specific CD8+ T cells, thus inhibit the PD-1 expression of PD-1/PD-L1 signaling pathway, regulate Th1/Th2 balance, and then improve immune function of the host.3 PD-1 allele A in polymorphism of rs6710479 was not conducive to spontaneous clearance of HBV infection, lead to chronic HBV infection; allele A in polymorphism of rs10204525 was conducive to NAs antiviral therapy response.