Cell atlas of CCl 4-induced progressive liver fibrosis reveals stage-specific responses.

Chronic liver injury leads to progressive liver fibrosis and ultimately cirrhosis, a major cause of morbidity and mortality worldwide. However, there are currently no effective anti-fibrotic therapies available, especially for late-stage patients, which is partly attributed to the major knowledge gap regarding liver cell heterogeneity and cell-specific responses in different fibrosis stages. To reveal the multicellular networks regulating mammalian liver fibrosis from mild to severe phenotypes, we generated a single-nucleus transcriptomic atlas encompassing 49 919 nuclei corresponding to all main liver cell types at different stages of murine carbon tetrachloride (CCl 4)-induced progressive liver fibrosis. Integrative analysis distinguished the sequential responses to injury of hepatocytes, hepatic stellate cells and endothelial cells. Moreover, we reconstructed cell-cell interactions and gene regulatory networks implicated in these processes. These integrative analyses uncovered previously overlooked aspects of hepatocyte proliferation exhaustion and disrupted pericentral metabolic functions, dysfunction for clearance by apoptosis of activated hepatic stellate cells, accumulation of pro-fibrotic signals, and the switch from an anti-angiogenic to a pro-angiogenic program during CCl 4-induced progressive liver fibrosis. Our dataset thus constitutes a useful resource for understanding the molecular basis of progressive liver fibrosis using a relevant animal model.

[A recombinant adenovirus vector carrying murine interleukin-21 gene controls chronic HBV infection in mice].

OBJECTIVE: To investigate the effect of an adenovirus vector containing murine interleukin-21 gene (Ad-GFP-mIL-21) in virus clearance and on the production of HBV-specific antibodies in mice with persistent HBV infection. METHODS: ELISA and Western blot analysis were used to detect the expression of mIL-21 in the supernatant and cytoplasm of cultured HepG2.2.15 cells after infection by Ad-GFP-mIL-21. Mouse models of chronic HBV infection established by in vivo transduction with rAAV8-1.3HBV were divided into 3 groups for treatment 12 weeks later with injection of Ad-GFP-mIL-21, GFP recombinant adenovirus or PBS via the tail vein. Serum levels of HBsAg, HBsAb, HBcAb, and mIL-21 in the mice were detected using ELISA, and the expression of Ad-GFP-mIL-21 in the organs was observed by fluorescent microscopy at different time points after the injection. RESULTS: Ad-GFP-mIL-21 was capable of infecting HepG2.2.15 cells in vitro, and the levels of mIL-21 in the supernatant were correlated with the titers of adenovirus administered and the infection time. In the mice with persistent HBV infection, green fluorescence expression was observed almost exclusively in the liver on day 4 after injection of Ad-GFP-mIL21, and serum levels of IL-21 increased significantly compared with the level before treatment (P<0.05). Although HBsAb was undetectable in both Ad-GFP-mIL21-injected and control mice on day 13, a significantly higher serum level of HBcAb was detected in the mice with Ad-GFP-mIL21 injection (P<0.05). CONCLUSION: Ad-GFP-mIL-21 can efficiently express mIL-21 in mice with chronic HBV infection to downregulate serum levels of HBsAg and promote HBcAb production, suggesting its efficacy in controlling chronic HBV infection.

[Characteristics of lymphocyte phenotypes in HBV transgenic mice and the effect of interferon-α: a preliminary study].

OBJECTIVE: To analyze the characteristics of lymphocyte phenotypes in hepatitis B virus (HBV) transgenic mice and the effect of exogenous interferon-α on virological profiles and lymphocytes phenotypes of the mice. METHODS: HBV transgenic mice and wild-type (WT) mice were examined for serum levels of HBsAg, HBcAb, IL-21, and IL-6 using ELISA. The frequencies of CD4(+)T and CD19(+)B cells separated from the liver, spleen, and peripheral blood were detected by flow cytometry. Nine HBV transgenic mice were injected subcutaneously with recombinant mouse interferon alpha (rmIFN-α) and another 9 transgenic mice were injected with PBS, and their HBsAg, HBV DNA, IL-6, and IL-21 levels and frequencies of peripheral blood CD4(+)T and CD19(+)B cells were detected. RESULTS: HBV transgenic mice showed a high level of HBsAg with a detectable level of HBcAb and significantly increased serum levels of IL-21 and IL-6 as compared with WT mice (P<0.05). The transgenic mice had a significantly lower frequency of CD4(+) T cells in the peripheral blood, liver and spleen (P<0.05) but a significantly higher frequency of CD19(+) B cells in the liver (P<0.05). An inverse correlation between intrahepatic CD4(+) T cell frequency and serum HBsAg level while a positive correlation between intrahepatic CD19(+) B cell frequency and HBcAb level were found in HBV transgenic mice. Administration of rmIFN-α significantly increased the frequencies of CD4(+) T and CD19(+) B cells in the peripheral blood and the serum level of IL-6 in HBV transgenic mice (P<0.05). CONCLUSION: HBV transgenic mice have lymphocyte subset dysregulation and exogenous interferon-α can modulate the immune function of the mice by regulating the frequencies of lymphocyte subsets.

High serum IL-21 levels after 12 weeks of antiviral therapy predict HBeAg seroconversion in chronic hepatitis B.

BACKGROUND & AIMS: Interleukin-21 (IL-21) stimulates T cell and B cell responses and plays a role in control of chronic viral infections. The role of IL-21 in chronic hepatitis B virus (HBV) infection is not understood. METHODS: Serum IL-21 levels were measured by enzyme immunoassay in 75 HBeAg-positive chronic hepatitis B (CHB) patients undergoing telbivudine treatment. The findings were validated in 103 patients from a separate clinical trial of telbivudine. A complete response to telbivudine was defined as having both HBeAg seroconversion and serum HBV-DNA level <300 copies/ml by treatment week 52. The proportions of T-cells producing IL-21 and/or expressing programmed death 1 (PD-1) in peripheral blood mononuclear cells were assessed longitudinally during treatment by intracellular cytokine staining and flow cytometry. RESULTS: Median serum IL-21 levels at treatment week 12 were significantly higher in patients who did achieve vs. patients who did not achieve a complete response in both the initial (128.4 vs. 69.2 pg/ml, p=0.003) and the validation (142.2 vs. 89.9 pg/ml, p=0.004) trials. Serum levels of IL-21 (p=0.005) or HBV-DNA (p=0.003) levels at treatment week 12 independently predicted HBeAg seroconversion in the first year of treatment. The decrease in PD-1 expression on CD4(+) and CD8(+) T cells during the first 12 weeks on telbivudine treatment was not correlated with changes in IL-21 concentrations. CONCLUSIONS: Serum IL-21 levels may be a biomarker for HBeAg seroconversion, and may contribute to individualization of antiviral therapy in HBeAg-positive CHB. IL-21 may also have a role in immunotherapy for CHB.

Complementarity-determining region 3 size spectratypes of T cell receptor beta chains in CD8+ T cells following antiviral treatment of chronic hepatitis B.

An increased CD8(+) T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8(+) T cells at these time points predict the virological response to therapy. Peripheral blood CD8(+) T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) ß chain variable region (Vß) gene family was analyzed, and the changes in the numbers of Vß families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8(+) TCR Vß families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus -1 [-3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8(+) T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8(+) T cells are a potential target for a therapeutic vaccine for chronic hepatitis B.

[Characteristics of CDR3 of TCR beta on CD8+ T cells in chronic hepatitis B patients].

OBJECTIVE: To analyze the characteristics of CDR3 of TCRbeta on CD8+ T cells in chronic hepatitis B patients. METHODS: Eight patients with chronic hepatitis B (ALT more than 2 ULN) were enrolled in this study. CD8+ T cells were isolated from peripheral blood. RT-PCR was proformed to amplify the CDR3 of TCRbeta, and the PCR products were sequenced and analyzed. RESULTS: The chronic hepatitis B patients showed obvious clonal expansion of T cell, and three perturbation patterns of T cell expansion were showed in the CDR3 of TCRbeta, including monoclonicity, oligoclonicity and skewed peak patterns. The number of perturbation families of CD8+ subpopulation was significantly higher than that of CD8- subpopulation (10.6+/-4.7 vs. 4.1+/-3.1, t = 6.619, P less than 0.01). In 3 out of 8 patients, the number of perturbation families of CD8+ subpopulation was also higher than that of PBMCs without depleting CD8+ subpopulation. CONCLUSIONS: The characteristics of CDR3 of TCRbeta may help to understand the inflammatory response in CHB patients.