| BackgroundHepatitis B virus infection is a global public health problem. An estimated400million persons worldwide are chronically infected with HBV, who are at increased risk of developing liver cirrhosis, hepatic decompensation, and hepatocellular carcinoma (HCC).In the nature history of chronic HBV infection, there is2to15percent people emerging spontaneous HBeAg seroconversion, and most of them become the inactive carriers (IC), who show the well control of HBV replication and inflammation. Compared with HBeAg positive patients, they have lower risk of cirrhosis and HCC.The importance and goal of the antiviral therapy in the Clinical Practice Guidelines for chronic hepatitis B is to improve quality of life and survival by preventing progression of the disease to cirrhosis, decompensated cirrhosis, end-stage liver disease, HCC and death. This goal can be achieved if HBV replication can be suppressed in a sustained manner.For HBeAg positive patients, HBeAg seroconversion is a satisfactory endpoint, which defined as the loss of HBeAg from the patient’s serum, together with the appearance of T cell-dependent, neutralizing antibodies that recognize the HBeAg (anti-HBe); and patients who have successfully undergone this process usually become the inactive HBsAg carrier (IC).The viral genetic and host immune mechanisms that drive HBeAg seroconversion are not fully understood. This is an important issue, because current antiviral drugs that potently suppress HBV replication to very low levels in patients with CHB do not always induce HBeAg seroconversion, even though the liver inflammation is controlled. As a result, reactivation with high levels of HBV replication usually occurs when the antiviral drugs are stopped, and the liver inflammation resumes. The consequence of this is that CHB patients who do not undergo HBeAg seroconversion are committed to the expense and inconvenience of lifelong drug therapy.Interleukin-21(IL-21) is a type I cytokine that shares the common cytokine receptor c chain with IL-2, IL-4, IL-7, IL-9, and IL-15. It is produced by multiple CD4+T cell subsets, including T helper17(Th17) cells, follicular helper T (TFH) cells, and natural killer T (NKT) cells. IL-21is thought of as a bridge between innate and adaptive immunity with immune-enhancing and immune-regulatory effects on B, T, and NK cell responses.In particular, IL-21has been shown to stimulate exhausted CD8+T cells to control a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, and has been associated with the development of CD8+T cell immunity to HIV infections in humans, Moreover, in the mouse model of human acute hepatitis B, The immune response to HBV antigen is age dependent, viral clearance occurs in mouse adults, whose IL-21-producing cells in liver were greatly increased. Thus, it is possible that IL-21has a role in promoting HBeAg seroconversion and in the control of HBV replication by stimulating both T cell and B cell responses.Roles of IL-21in the chronic HBV infection are not fully understood. Our hypothesis is that IL-21could promote HBeAg seroconversion and viral clearance. AimsHere, two clinical trials provide a structured framework for standardized longitudinal evaluation of serum IL-21levels during treatment with an oral anti-HBV agent, and provide an opportunity to analyze the relationship between IL-21and treatment-induced HBeAg seroconversion. This reflects, in a condensed time-frame, the changes that occur during the natural history of chronic HBV infection.Materials and Methods1. SubjectsNinety-nine patients with a chronic HBV infection were recruited from the Hepatology Clinics of Nanfang Hospital, Guangzhou, China. Fifty-eight subjects had been referred for management of mild to moderate chronic hepatitis B. Twenty IT and21IC were recruited from a cohort of inactive disease patients who have routine screening for active hepatitis and hepatocellular carcinoma. IT was defined as being HBsAg-positive on at least three occasions in one year, HBeAg-positive and having normal ALT levels. IC was defined as being HBsAg-positive on two occasions at least six months apart, HBeAg-negative, anti-HBe-positive with persistently normal ALT levels and HBV DNA<10,000copies/mL. CHB was defined as being HBsAg-positive, HBeAg-positive and anti-HBe-positive, with HBV DNA higher than100,000copies/mL, and having a flare ALT levels (>1ULN). Twenty laboratory staff and students with normal ALT levels who were negative for HBsAg were enrolled as healthy controls (HC). Ten to twenty ml of heparinized blood and5mL of serum were collected from the CHB patients at baseline and from the IT and IC subjects at the time of a routine blood test. Subjects with previous antiviral therapy and subjects with HCV, HDV or HIV infections, hepatocellular carcinoma, autoimmune liver disease, bacterial infection, any malignant tumor or any severe metabolic disease were excluded.Forty-eight patients with HBeAg-positive CHB from Nanfang Hospital (Guangzhou, China) and27patients with HBeAg-positive CHB from The First Affiliated Hospital, Xinjiang Medical University (Urumqi, China) participated in a phase IV, multi-center, open-label clinical trial on telbivudine (600mg/d, trial CLDT600ACN07T). The criteria for inclusion in the trial were an alanine aminotransferase (ALT) greater than the upper limit of normal (ULN) and an HBV DNA level greater than100,000copies/mL. Twenty milliliter of heparinized blood was taken at Nanfang Hospital for immune studies and1ml of serum was obtained from subjects at both hospitals for measurement of cytokine concentrations. These samples were taken at baseline and at12,24, and52weeks after starting telbivudine treatment. The serum samples were transported on dry ice and stored at-80℃in the Hepatology Unit of Nanfang Hospital. The subjects were classified into either a complete response (CR, n=14) group, if they have undergone HBeAg seroconversion and achieved serum HBV DNA level less than300copies/ml by week52, or a non-complete response (NCR, n=61) group, if serum HBV DNA was reduced but HBeAg remained positive. The same subjects were classified into either a virological response (VR, n=32) or no virological response (NVR, n=43) groups based on having a serum HBV DNA of less than300copies/ml by week52, independent of HBeAg status. To validate the relationship between serum IL-21concentration and treatment response, the study was duplicated in a second set of103patients (CR, n=42; NCR, n=61) from another clinical trial with telbivudine treatment of CHB.The study was conducted according to the Declaration of Helsinki guidelines, and was approved by the Ethical Committee of Nanfang Hospital. Written, informed consent was obtained from all subjects.2. Serum IL-21Levels detectionThe serum concentrations of IL-21were measured in duplicate using a commercial human IL-21ELISA kit.3. Detection of IL-21-producing cells and co-expression of other cytokines.Intracellular staining analysis was performed to detect the distribution of IL-21-producing cells. A minimum of200,000lymphocytes were collected, and analyzed with BD FACSDiva(BD Bioscience, San Jose, CA) and FlowJo (Tree Star Inc., Ashland, OR) software.The percentage of PBMC that were IL-21+CD4+was calculated as the mean of the two values obtained when assessing co-staining for IFN-γ, IL-4, IL-17A, and CXCR5in the two separate staining reactions. One million fresh PBMC from IC and CHB patients were incubated with or without HBcAg stimulation for detection of HBV-specific IL-21-producing cells.The expression of PD-1on PBMC were also analyzed in patients under treatment.4. The expression of IL-21R and other transcription factors in mRNA levelsTotal RNA were extracted from PBMC freshly isolated, then reversed into cDNA. The expression levels of IL-21R mRNAs was measured in PBMC from76subjects involved by quantitative real-time RT-PCR. The expression levels of Bcl-6and ROR-γT,which were IL-21-related transcriptional factors, were determined between17inactive carriers and21CHB patients.5. The effect of IL-21on T and B cells responses in vitroAfter PBMC incubating with HBcAg and IL-21for10days, the supernatant were collected for the detection of anti-HBc and anti-HBe levels. The expression of IL-21R and perforin on CD8+T cells were determined, after incubating CD8+T cells from CHB patients with CD8-T cells from IC in transwell plate for3days under HBcAg stimulation.Results1. IL-21expression in different status of chronic HBV infection.Serum IL-21concentrations in different status of chronic HBV infection. Disease status had a significant effect on serum IL-21concentrations (p<0.0001). Although the IT group had higher levels of IL-21than the HC group, the difference did not reach statistical significance (p=0.26). The serum IL-21concentrations were higher in both the CHB (p<0.0001) and the IC (p=0.011) groups than in the IT group. The serum IL-21concentrations in the CHB and IC groups were similar (p=0.26), There were no correlations between the serum IL-21concentration and either ALT (p=0.180) or HBV DNA (p=0.23) levels in the CHB group.IL-21R and other transcriptional facters expression in different status of chronic HBV infection. Disease status had a significant effect on the levels of IL-21R mRNA in PBMC (p<0.0001). The IT group had lower levels of IL-21R mRNA (p=0.001) in PBMC than the HC group. The CHB group had higher IL-21R mRNA (p=0.006) than IT groups respectively, but similar levels with IC group (p=0.15).The IL-21related transcriptional factors of Bcl-6and ROR-γ T were determined in CHB and IC groups, which represent the differentiation of TFH and Th17cells. The CHB group has significantly higher ROR-γT level and lower Bcl-6level than IC group (p<0.0001, p<0.0001)The frequency of IL-21+CD4+T cells in different status of chronic HBV infection. CD4+T cells were the only lymphocyte subset in PBMC that contained significant levels of IL-21by ICS. Disease status had a significant effect on the frequency of IL-21+CD4+T cells in PBMC (p<0.0001). The IT group had higher levels of IL-21+CD4+T cells than the HC group (p=0.004). The IC group had higher levels of IL-21+CD4+T cells than the IT group (p=0.020).2. Dynamics of IL-21expression during52weeks of telbivudine treatmentHBV DNA, ALT and IL-21concentrations over52weeks of telbivudine treatment Changes in serum HBV DNA levels and ALT levels over52weeks. Serum HBV DNA levels differed significantly between the CR (n=14) and NCR (n=61) groups were significant at week12(p<0.0002), week24(p=0.002) and week52(p=0.002). Serum ALT levels returned to normal range by week24, and were similar in the two groups at all timepoints.Repeated measures analysis of the IL-21data showed that the serum IL-21levels decreased with time (p=0.009and p=0.001for multivariate and univariate analyses respectively), and that the IL-21levels were higher in the CR than the NCR group (p=0.004), whereas the IL-21levels were not higher in the VR than the NVR group (p=0.23). In particular, the median IL-21concentrations were higher in the CR than in the NCR group at week12(p=0.005) but not significantly higher at other time points. Although the serum ALT level of CHB patients at treatment week52was normal, the serum IL-21concentration at week52was higher than in the IT group(p=0.035).Predictors of a complete response to telbivudine treatment. Variables that were significant in the univariate analysis were HBV DNA at baseline,12and24weeks and serum IL-21levels at12weeks. All variables with a p<0.10were included in multivariate analy-sis. The strongest multivariate general linear model predicting treatment response included the12-week IL-21concentration and the12-week HBV DNA level (r2=0.31). None of the interaction terms were statistically significant, and there were independent effects of12-week IL-21(p=0.002) and12-week HBV DNA (p=0.003). Logistic regression analysis also found independent effects of12-week IL-21(p=0.050) and12-week HBV DNA (p=0.007). Receiver operating characteristic curves were generated to assess the use of12-week HBV DNA, IL-21concentrations and the ratio of IL-21to HBV DNA to predict a CR at52weeks. The optimal cut-off value for12-week HBV DNA concentration was3.83log10copies/mL, which gave a sensitivity for detection of a complete response of85.7%and a specificity of70.4%. The optimal cut-off value for12-week IL-21concentration was112.3pg/ml, which gave a sensitivity for detection of a CR of73.3%and a specificity of81.8%. A12-week IL-21concentration of less than51.4pg/mL gave a negative predictive value for HBeAg seroconversion of95%.Serum IL-21concentrations were also measured in103non-consecutive patients from another clinical trial. The median serum IL-21concentration at week12(p=0.004) in the CR group n=42) was higher than that in NCR group (n=61), and there was also a significant difference in baseline serum IL-21concentrations (p=0.003) between the CR and NCR groups.Percentage of CD4+T cells in PBMC secreting IL-21over52weeks of telbivudine treatment. The mean frequencies of IL-21+CD4+T cells were higher in the CR patients compared with NCR, although the overall difference was not significant (p=0.08). When comparing individual time-points during treatment between these two groups, the frequency at24weeks was significantly higher (p=0.003) in the CR than in the NCR group. There was no correlation between the percentage of IL-21+CD4+T cells and serum IL-21concentration at any time point. The changes in the frequencies of the subsets of IL-21+CD4+T cells that co-expressed IL-4, IL-17A, CXCR5, IFN-γ or of the subset that did not co-express any of these markers were generally similar to the changes in the total IL-21+CD4+subset.Serum IL-21concentrations and PD-1expression on PBMC. Repeated measures showed a decrease in frequency of CD8+and CD4+T cells expressing PD-1over52weeks (p=0.030, p=0.210). The higher frequency of CD8+and CD4+T cells expressing PD-1in the CR group (p=0.110, p=0.040). There was a statistically significant decrease in the frequency of CD8+(p=0.050) and CD4+(p=0.030) expressing PD-1between the baseline and12-week samples in the whole group of20subjects. There was a positive correlation between serum IL-21concentrations and the frequency of CD8+T cells (r=0.222, p=0.048) but not CD4+T cells (r=0.111, p=0.328) that expressed PD-1.3. IL-21promote T and B cells immune responses in vitroBoth mitogen-stimulated as well as virus-specific CD4+T cells producing IL-21were significantly higher in IC than CHB patients (p<0.0001, p=0.0005)The expression of IL-21R on CD8+T cells of CHB patients increased after incubation with CD4+Tcells from IC for3days under HBcAg stimulation (p=0.006). Then after IL-21/IL-21R antibody blockade, the expression of IL-21R on CD8+T cells significantly decreased (p=0.013).IL-21could promote the HBcAb and HBeAb production in5of20HBeAg negative patients under HBeAg/HBcAg stimulation.ConclusionsIn the cross-sectional and longitudinal study, all the subjects achieved HBeAg seroconversion either spontaneous or telbivudine-induced have significantly higher serum IL-21levels. Moreover, HBeAg seroconversion during telbivudine therapy for HBeAg-positive CHB was associated with high serum IL-21concentrations at week 12time point, which was a strong predictor, independent of HBV DNA and ALT levels on a multivar-iate analysis. Serum IL-21concentration is a plausible biomarker that might contribute to individualization of antiviral therapy for HBeAg-positive chronic hepatitis B.There is higher frequency of HBV-specific IL-21+CD4+T cells in IC subjects, which could enhance IL-21R expression on CD8+T cells. IL-21could promote HBV-specific antibody production from B cells. Therefore, IL-21may also have a role in the development of immunotherapies for chronic hepatitis B. The mechanism of IL-21involved in HbeAg seroconversion will be further investigated. |
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