Entecavir-induced interferon-λ1 suppresses type 2 innate lymphoid cells in patients with hepatitis B virus-related liver cirrhosis.

The immunomodulatory effects of entecavir (ETV) in anti-hepatitis B virus (HBV) therapy have long been recognized. This study aimed to determine the effects of ETV on non-natural killer innate lymphoid cells (non-NK ILCs) in HBV-related liver disease progression. We enrolled treatment-naïve chronic hepatitis B (CHB) and HBV-related liver cirrhosis (LC) patients treated with ETV for 24 months. Before and after therapy, the frequency and cytokine profiles of ILC2s and non-NK ILCs subset homeostasis and their clinical significance were determined, and serial serum interferon (IFN)-λ levels were analysed. Peripheral blood mononuclear cells (PBMCs) of untreated LC patients were cultured with serum from untreated and ETV-treated LC patients in addition to being subject to IFN-λ1 neutralization and stimulation, and the frequency and cytokine production of ILC2s as well as non-NK ILCs subset ratios were calculated. Furthermore, IFN-λ receptor expression on non-NK ILCs and dendritic cells (DCs) was measured. After 24 months of ETV treatment, the frequency and cytokine production of ILC2s (IL-4, IL-13, IFN-γ, TNF-α) decreased with increased ILC1/ILC2 and decreased ILC2/ILC3 ratios, revealing a close association with disease status in LC patients. Long-term ETV administration-induced serum IFN-λ1 levels were negatively correlated with ILC2s. ETV-treated LC serum culture and IFN-λ1 stimulation yielded similar effects on suppression of ILC2s, and IFN-λ1 neutralization in serum culture partly inhibited this effect. The IFN-λ receptor was detected on DCs but not on non-NK ILCs. In conclusion, ETV suppresses the frequency and cytokine profiles of ILC2s by increasing IFN-λ1 in LC patients.

Peg-interferon alpha add-on Tenofovir disoproxil fumarate achieved more HBsAg loss in HBeAg-positive chronic hepatitis B naïve patients.

Several studies have showed that combining peg-interferon alpha (Peg-IFNα) with nucleotide analogues has complementary effects in chronic hepatitis B (CHB), but the optimal regimen and potential mechanisms remain unclear. This was a prospective, longitudinal and multicentre clinical trial (NCT03013556). HBeAg-positive CHB naïve patients were randomly assigned to three groups: tenofovir disoproxil fumarate (TDF) monotherapy for 96 weeks, TDF alone for 48 weeks and sequentially Peg-IFNα added for 48 weeks, TDF de novo combination with Peg-IFNα for 48 weeks then TDF alone for 48 weeks. The primary endpoint was HBeAg seroconversion at week 96 and HBsAg loss as the secondary endpoint. Furthermore, the levels of 12 cytokines in serum were assessed at different time points. A total of 133 patients were included in the analysis. The rates of HBeAg seroconversion at 96 weeks were not significant different among the three groups (p = 0.157). Interestingly, patients in the Peg-IFNα add-on group showed markedly lower HBsAg level compared with the other two groups at week 96. In addition, only three patients in the Peg-IFNα add-on group achieved HBsAg loss. For the following 24 weeks from week 96, no HBsAg reappearance in the three patients and no new patients with HBsAg loss were observed in the three groups. Serum cytokine analysis showed that the baseline level of interferon-inducible protein-10 (IP-10) was strongly higher in HBeAg conversion patients and HBsAg loss patients. Compared with de novo combination and TDF alone, the addition of Peg-IFNα in TDF-treated group might be an effective strategy for HBsAg loss in HBeAg-positive CHB naïve patients.

Type 3 innate lymphoid cell: a new player in liver fibrosis progression.

Type 3 innate lymphoid cell (ILC3) has recently emerged as a crucial effector in inflammatory and fibrotic diseases. The present study was designed to determine the roles of ILC3 in liver fibrosis. By flow cytometry, we documented increased frequencies of peripheral ILC3 (Lin-CD127+CD117+CD294- lymphocytes) in patients, especially at the advanced stage of hepatitis B virus (HBV)-related chronic liver diseases, and demonstrated their correlations with disease progression. The in vitro fibrogenic effects by ILC3 were determined by co-culture experiments with LX-2 (a human hepatic stellate cell (HSC) line). The data indicate that pathogenic ILC3 can directly promote LX-2 fibrogenesis in non-contact manners by producing interleukin (IL)-17A and IL-22. Additionally, they had indirect fibrogenic effects by producing IL-22 to suppress interferon (IFN)-γ (a well-known anti-fibrotic cytokine) production by other immune cells. In carbon tetrachloride (CCl4)-induced wild-type mouse liver fibrosis models, we also documented significantly increased frequencies of both non-natural killer (NK) ILC (Lin-CD127+ lymphocytes) and ILC3 (Lin-CD127+RORγt+ lymphocytes) in liver and spleen specimens. Furthermore, the ILC3 from fibrotic mice contained more IL-17A+ILC3 and IL-22+ILC3 subsets than those from normal and less-fibrotic mice. The in vivo effects of ILC3 in liver fibrogenesis were further determined using RAG-1-/- mice with ILC depletion and further adoptive transfer of ILC3 from wild-type mice. The immunohistochemical staining of liver specimens showed the beneficial effects by ILC depletion and the detrimental effects by ILC3 transfer in CCl4-induced mouse liver fibrosis models. Collectively, ILC3 plays a pro-fibrotic role in liver fibrosis progression.

Activated hepatic stellate cells directly induce pathogenic Th17 cells in chronic hepatitis B virus infection.

Th17 cells are involved in liver fibrosis by activating hepatic stellate cells (HSCs). We aimed to investigate whether HSCs are able to regulate the function of Th17 cells and to determine the relevant mechanism. Sixty-five patients diagnosed with chronic hepatitis B (CHB) were enrolled in this study. To determine the effect of HSCs on T cells, naïve CD4+T cells and Th17 cells were sorted from CHB patients and cultured with or without activated-HSCs, and cytokine expression and gene transcription were analyzed. In addition, the regulatory mechanism of HSCs was investigated. ELISA and qRT-PCR showed that Th17 cells from CHB patients were more pathogenic, on the basis of the expression of IL-17A, IL-23R, RORC, CCL20 and CCR6, and meanwhile, they could activate the primary HSCs. Co-culture experiments indicated that activated HSCs dramatically promoted proliferation of CD4+T cells in a time- and dose-dependent manner. In addition, they could induce naïve CD4+T cells to become Th17 cells which had a more pathogenic phenotype. Moreover, activated HSCs-mediated induction of Th17 cells might depend on the release of IL-1ß and IL-6 as well as on the COX-PGE2 pathway. Th17 cells cooperated with HSCs in a proinflammatory feedback loop might provide a better understanding of the pathogenic role of Th17 cells in the chronicity of HBV infection.

HMGB1-induced autophagy: a new pathway to maintain Treg function during chronic hepatitis B virus infection.

High-mobility group box-1 (HMGB1) protein, as one of the well-known damage-associated molecular pattern molecules (DAMPs), is enriched in chronic hepatitis B virus (HBV) infection and has a context-dependent role in autophagy, a highly conserved self-digestive process in response to environmental stress. Recent mouse studies indicate that autophagy is highly active in regulatory T (Treg)-cells. In the present study, we evaluated spontaneous and induced autophagy of peripheral Treg cells from 98 patients with chronic hepatitis B (CHB), by measuring levels of lipidated form of microtubule-associated light chain 3 (LC3-II, marker for closed autophagosomes) and observing autophagic vacuoles (AV) with transmission electron microscope. No significant difference was found in spontaneous autophagy of either Treg or CD4+ naive cells when comparing CHB patients with healthy subjects, apart from CHB-Treg showed significantly higher autophagic activity after activation by anti-CD3-CD28 beads. Besides, incubation of CHB-Treg cells with CHB-serum greatly maintained their autophagic behaviour, which could be significantly diminished by blocking HMGB1 with the neutralizing antibody. Further, we characterized time- and dose-dependent effects by recombinant HMGB1 protein on autophagy of CHB-Treg cells. We also documented a significant up-regulation of HMGB1 and its receptors [toll-like receptor (TLR4), receptor for advanced glycation end-product (RAGE)] in both peripheral and intra-hepatic microenvironments of CHB patients. Moreover, the RAGE-extracellular regulated protein kinases (ERK) axis and rapamycin-sensitive components of mammalian target of rapamycin (mTOR) pathways were demonstrated in vitro to be involved in HMGB1-induced autophagy of Treg cells. Additionally, HMGB1-induced autophagy could maintain cell survival and functional stability of CHB-Treg cells. Our findings could open new perspectives in developing therapeutic strategies to activate specific anti-HBV immunity by diminishing Treg autophagy.

IL-10-producing regulatory B-cells suppressed effector T-cells but enhanced regulatory T-cells in chronic HBV infection.

Non-specific immune responses to antigens have been demonstrated as being enhanced during chronic hepatitis B virus (HBV) infection. Here, we evaluated the role of interleukin-10 (IL-10)-producing regulatory B-cells (Bregs) in the pathogenesis of HBV-related liver fibrosis (HBV-LF) and assessed their immunoregulatory effects. Sixty-seven patients diagnosed with chronic hepatitis B (CHB) were enrolled in this study. Numbers and frequencies of peripheral B-cells (memory CD19(+)CD24(hi)CD27(+) cells, immature/transitional CD19(+)CD24(hi)CD38(hi) cells, mature CD19(+)CD24(int)CD38(int) cells) were tested and analysed. Flow cytometry-sorted CD4(+)T cells were cultured with autologous Bregs to elucidate the effects of Bregs on CD4(+)T cells, including effector T and regulatory T-cells (Tregs). The potential immunoregulatory mechanism of Bregs was also investigated. The numbers of total B-cells and Bregs were enriched in CHB patients. The frequency of Bregs was negatively correlated with elevated alanine aminotransferase (ALT) and histological inflammation grades (G), but positively correlated with advanced histological fibrosis stages (S) and enhanced HBV replication. The phenotype of Bregs was predominantly characterized as CD19(+)CD24(hi)CD38(hi) In co-culture with Bregs, CD4(+)CD25(-)T cells from CHB patients produced less interferon-γ (IFN-γ) and IL-17 but more IL-4 than CD4(+)CD25(-)T cells alone, whereas their conversions into Tregs and IL-10(+)T cells were enhanced. In addition, Breg depletion in CHB samples dramatically decreased Treg numbers and expression of cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), IL-10 and transforming growth factor-ß (TGF-ß). Moreover, the observed regulatory effect was partly dependent on IL-10 release and cell-to-cell contact. Elevated Bregs can suppress effector T but enhance Treg functions, which might influence immune tolerance in chronic HBV infection.

Telbivudine therapy may shape CD4(+) T-cell response to prevent liver fibrosis in patients with chronic hepatitis B.

BACKGROUND & AIMS: Nucleos(t)ide analogues (NAs) can indirectly restore host immunity against hepatitis B virus (HBV) by inhibiting virus replication. We aimed to investigate whether telbivudine could prevent HBV-related fibrosis progression by their influence on CD4(+) T-cell response. METHODS: Thirty-six HBeAg-positive patients with chronic hepatitis B (CHB) were enrolled for 52-week telbivudine monotherapy and were followed at treatment week (TW)-0, 4, 12, 24 and 52. By TW-52, the patients were classified into a complete-response group (CR, n = 10) with both negative HBV-DNA and HBeAg, or a part-response group (PR, n = 11) only with negative DNA, or a non-response group (NR, n = 15) still with positive DNA. The peripheral blood mononuclear cells (PBMCs) were prepared for further flow cytometric and real-time PCR analyses, and also for the in vitro experiments with primary hepatic stellate cells (HSCs). RESULTS: Peripherally, all chronic HBV-infected subjects showed the involvement of CD4(+) T-cell responses, among whom the inactive carriers (IC) had Th1 (CD4(+) IFNγ(+) ) dominated, CHB had Th17 (CD4(+) IL-17(+) ) dominated, while the immune tolerant (IT) subjects had Treg (CD4(+) CD25(high) Foxp3(+) ) dominated. Besides, we found the therapeutic responses to telbivudine were especially associated with up-regulation of Th1 and Th17, and down-regulation of Treg. Furthermore, compared to CD4(+) cells from CR, those from NR could in vitro significantly exacerbate cell activation, proliferation and cytokine production of HSCs, which were partly mediated by IL-4 and TGF-ß1. CONCLUSIONS: Telbivudine might slow down HBV-related liver fibrosis progression by restoring CD4(+) T-cell responses against HBV.

Pre-S1 Mutations as Indicated by Serum Pre-S1 Antigen Negative is Associated with an Increased Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients.

Objective: Pre-S1 antigen (pre-S1) is a component of hepatitis B virus large surface antigen (L-HBsAg). This study aimed to investigate the association between clinical pre-S1 antigen (pre-S1) status and adverse prognostic events in chronic hepatitis B (CHB) patients. Methods: This study retrospectively enrolled 840 CHB patients with comprehensive clinical data, including 144 patients with multiple follow-up of pre-S1 status. All patients were tested for serum pre-S1 and divided into pre-S1 positive and negative groups. Single factor and logistic multiple regression analyses were performed to explore the association between pre-S1 and other HBV biomarkers with the risk of hepatocellular carcinoma (HCC) in CHB patients. The pre-S1 region sequences of HBV DNA were obtained from one pre-S1 positive and two pre-S1 negative treatment-naïve patients using polymerase chain reaction (PCR) amplification followed by Sanger sequencing. Results: The quantitative HBsAg level was significantly higher in the pre-S1 positive group than that in the pre-S1 negative group (Z=-15.983, P<0.001). The positive rate of pre-S1 increased significantly with the increase in HBsAg level (χ 2=317.963, P<0.001) and HBV DNA load (χ 2=15.745, P<0.001). The pre-S1 negative group had a higher HCC risk than the pre-S1 positive group (Z=-2.00, P=0.045, OR=1.61). Moreover, patients in the sustained pre-S1 negative group had a higher HCC risk (Z=-2.56, P=0.011, OR=7.12) than those in the sustained pre-S1 positive group. The sequencing results revealed mutations in the pre-S1 region from samples of pre-S1 negative patients, including frameshift and deletion mutations. Conclusion: Pre-S1 is a biomarker that indicates the presence and replication of HBV. Pre-S1 sustained negativity attributed to pre-S1 mutations in CHB patients may be associated with a higher risk of HCC, which has clinical significance and warrant further investigations.

Hepatitis B virus (HBV) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of HBV infection.

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.

Serum amyloid A levels in patients with liver diseases.

BACKGROUND: Serum amyloid A (SAA) is an acute phase protein mainly synthesized by the liver. SAA induces inflammatory phenotype and promotes cell proliferation in activated hepatic stellate cells, the major scar forming cells in the liver. However, few studies have reported on the serum levels of SAA in human liver disease and its clinical significance in various liver diseases. AIM: To investigate the serum levels of SAA in patients with different liver diseases and analyze the factors associated with the alteration of SAA levels in chronic hepatitis B (CHB) patients. METHODS: Two hundred and seventy-eight patients with different liver diseases and 117 healthy controls were included in this study. The patients included 205 with CHB, 22 with active autoimmune liver disease (AILD), 21 with nonalcoholic steatohepatitis (NASH), 14 with drug-induced liver injury (DILI), and 16 with pyogenic liver abscess. Serum levels of SAA and other clinical parameters were collected for the analysis of the factors associated with SAA level. Mann-Whitney U test was used to compare the serum SAA levels of patients with various liver diseases with those of healthy controls. Bonferroni test was applied for post hoc comparisons to control the probability of type 1 error (alpha = 0.05/6 = 0.008). For statistical tests of other variables, P < 0.05 was considered statistically significant. Statistically significant factors determined by single factor analysis were further analyzed by binary multivariate logistic regression analysis. RESULTS: All patients with active liver diseases had higher serum SAA levels than healthy controls and the inactive CHB patients, with the highest SAA level found in patients with pyogenic liver abscess (398.4 ± 246.8 mg/L). Patients with active AILD (19.73 ± 24.81 mg/L) or DILI (8.036 ± 5.685 mg/L) showed higher SAA levels than those with active CHB (6.621 ± 6.776 mg/L) and NASH (6.624 ± 4.891 mg/L). Single (P < 0.001) and multivariate logistic regression analyses (P = 0.039) for the CHB patients suggested that patients with active CHB were associated with an SAA serum level higher than 6.4 mg/L. Serum levels of SAA and CRP (C-reactive protein) were positively correlated in patients with CHB (P < 0.001), pyogenic liver abscess (P = 0.045), and active AILD (P = 0.02). Serum levels of SAA (0.80-871.0 mg/L) had a broader fluctuation range than CRP (0.30-271.3 mg/L). CONCLUSION: Serum level of SAA is a sensitive biomarker for inflammatory activity of pyogenic liver abscess. It may also be a weak marker reflecting milder inflammatory status in the liver of patients with CHB and other active liver diseases.

Oxymatrine therapy for chronic hepatitis B: a randomized double-blind and placebo-controlled multi-center trial.

AIM: To evaluate the efficacy and safety of capsule oxymatrine in the treatment of chronic hepatitis B. METHODS: A randomised double-blind and placebo-controlled multicenter trial was conducted. Injection of oxymatrine was used as positive-control drug. A total of 216 patients with chronic hepatitis B entered the study for 24 weeks, of them 108 received capsule oxymatrine, 36 received injection of oxymatrine, and 72 received placebo. After and before the treatment, clinical symptoms, liver function, serum hepatitis B virus markers, and adverse drug reaction were observed. RESULTS: Among the 216 patients, six were dropped off, and 11 inconsistent with the standard were excluded. Therefore, the efficacy and safety of oxymatrine in patients were analysed. In the capsule treated patients, 76.47% became normal in ALT level, 38.61% and 31.91% became negative both in HBV DNA and in HBeAg. In the injection treated patients, 83.33% became normal in ALT level, 43.33% and 39.29% became negative both in HBV DNA and in HBeAg. In the placebo treated patients, 40.00% became normal in ALT level, 7.46% and 6.45% became negative both in HBV DNA and in HBeAg. The rates of complete response and partial response were 24.51% and 57.84% in the capsule treated patients, and 33.33% and 50.00% in the injection treated patients, and 2.99% and 41.79% in the placebo treated patients, respectively. There was no significance between the two groups of patients, but both were significantly higher than the placebo. The adverse drug reaction rates of the capsule, injection and placebo were 7.77%, 6.67% and 8.82%, respectively. There was no statistically significant difference among them. CONCLUSION: Oxymatrine is an effective and safe agent for the treatment of chronic hepatitis B.

Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis: a randomized, double blind, placebo-controlled, multicenter clinical study.

AIM: To evaluate the efficacy and safety of oxymatrine capsule in treatment of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: It was a randomized, double blind, placebo-controlled, multicenter clinical study. One hundred and forty-four patients were divided into oxymatrine capsule group(group A) and placebo group (group B). The course was 52 wk. Patients were visited once every 12 wk and the last visit was at 12 wk after cessation of the treatment. All patients had liver biopsy before treatment. part of them had a second biopsy at the end of therapy. Clinical symptoms, liver function test, serum markers of hepatic fibrosis were tested. Ultrasound evaluation was performed before, during and at the end of therapy. RESULTS: One hundred and forty-four patients enrolled in the study. Of them 132 patients completed the study according to the protocol,49 patients had liver biopsy twice (25 patients in group A and 24 in group B). At the end of therapy, significant improvements in hepatic fibrosis and inflammatory activity based on Semi-quantitative scoring system (SSS) were achieved in group A. The total effective rate of the treatment was 48.00%, much higher than that of 4.17% in group B (P<0.05). Significant improvement in serum markers of hepatic fibrosis such as hyaluronic acid (HA) and type III procollagenic peptide (P III P) in group A was seen (P<0.05). The total effective rate of serum markers at the end of therapy in group A was 68.19%, much higher than that of 34.85% in group B (P<0.05). The total effective rate of noninvasive markers at the end of therapy in group A was 66.67%, much higher than that of 30.30% in group B (P<0.05). The rate of adverse events was similar in two groups. CONCLUSION: Oxymatrine capsule is effective and safe in treatment of hepatic fibrosis due to chronic viral hepatitis.

[Oxymatrine in the treatment of chronic hepatitis B for one year: a multicenter random double-blind placebo-controlled trial].

OBJECTIVE: To evaluate the efficacy and safety of oxymatrine in the treatment of chronic hepatitis B. METHODS: A multicenter randomized double-blind placebo-controlled trial was conducted. A total of 144 patients with chronic hepatitis B entered the study for 52 weeks; of them 72 received oxymatrine, and 72 received a placebo. Before and after the treatment, clinical symptoms, liver function, serum hepatitis B virus markers, and adverse drug reactions were observed. RESULTS: In 144 patients, 14 were dropped and excluded due to inconsistencies in the included standard. Therefore, the efficacy and safety of 130 patients were analyzed. After being treated for 52 weeks, 70.77% of the patients in the study group had a normal ALT level, and in 43.08% and 33.33% their HBV DNA and HBeAg became negative. In the placebo group, 39.68% had normal ALT level, and 12.31% and 3.33% had their HBV DNA and HBeAg become negative. The rates of complete response and partial response in the oxymatrine group were 23.08% and 58.46%, and in the placebo group they were 3.08% and 44.62%. They were significantly higher in the oxymatrine group than in the placebo group. In the oxymatrine treated patients, 12 weeks after its withdrawal, 60.00% had a normal ALT level, 41.54% and 23.33% had both HBV DNA and HBeAg negative. In the placebo group, 31.75% had a normal ALT level, 3.08% and 1.67% had both HBV DNA and HBeAg negative. The rates of complete response and partial response in the oxymatrine group were 21.54% and 47.69%, and in the placebo group they were 0 and 41.54%. They were significantly higher in the study group than in the placebo group. The adverse reaction rates of oxymatrine in the study and the placebo group were 7.69% and 6.15%, respectively, but there was no statistical significant difference between them. CONCLUSION: Oxymatrine is an effective and safe agent for the treatment of chronic hepatitis B.

3, 3'-Diindolylmethane alleviates steatosis and the progression of NASH partly through shifting the imbalance of Treg/Th17 cells to Treg dominance.

This study was designed to discuss the effects of 3, 3'-diindolylmethane (DIM) on methionine-choline-deficient (MCD)-diet induced mouse nonalcoholic steatohepatitis (NASH) and the potential mechanisms. NASH mice were administrated with or without DIM at different concentrations for 8 weeks. Both the in-vivo and in-vitro effects of DIM on Treg/Th17 imbalance during NASH progression were analyzed. The in-vivo blocking of CD25 or IL-17 was performed to respectively deplete respective function of Treg or Th17 subset. Besides, with the assistance of AhR antagonist CH223191 and anti-TLR4 neutralizing antibody, we designed the in-vitro DIM-incubation experiments to discuss the roles of aryl hydrocarbon receptor (AhR) (CYP1A1, CYP1B1) and toll-like receptor 4 (TLR4) on DIM's effects when shifting Treg/Th17 imbalance. Notably, in NASH mouse models, DIM alleviated hepatic steatosis and inflammation, and shifted the Treg/Th17 imbalance from MCD diet-induced Th17 dominance to Treg dominance. In-vitro, DIM not only significantly up-regulated the mRNAs of Foxp3 (Treg-specific) in purified spleen CD4(+) T cells, but also enhanced the immunosuppressive function of these Treg cells. Besides, DIM significantly up-regulated the proteins of CYP1A1 and CYP1B1 whereas down-regulated those of TLR4 on CD4(+) T cells from MCD-diet mice. Moreover, blocking AhR attenuated while blocking TLR4 enhanced the effects of DIM when regulating Treg/Th17 imbalance. Conclusively, DIM could be used as a potential therapeutic candidate to treat NASH based on its dramatic induction of Treg dominance to alleviate intra-hepatic inflammation, suggesting us a clue that the dietary cruciferous vegetables (containing abundant DIM) might exist as a protective factor for patients with NASH-related liver diseases.

Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis.

BACKGROUND: Hepatitis B virus-related liver fibrosis (HBV-LF) always progresses from inflammation to fibrosis. However, the relationship between these two pathological conditions is not fully understood. Here, it is postulated that the balance between regulatory T (Treg) cells and T helper 17 (Th17) cells as an indicator of inflammation may predict fibrosis progression of HBV-LF. METHODOLOGY/PRINCIPAL FINDINGS: The frequencies and phenotypes of peripheral Treg and Th17 cells of seventy-seven HBeAg-positive chronic hepatitis B (CHB) patients who underwent liver biopsies and thirty healthy controls were determined by flow cytometry. In the periphery of CHB patients, both Treg and Th17 frequencies were significantly increased and correlated, and a lower Treg/Th17 ratio always indicated more liver injury and fibrosis progression. To investigate exact effects of Treg and Th17 cells during HBV-LF, a series of in vitro experiments were performed using purified CD4(+), CD4(+)CD25(+), or CD4(+)CD25(-) cells from the periphery, primary human hepatic stellate cells (HSCs) isolated from healthy liver specimens, human recombinant interleukin (IL)-17 cytokine, anti-IL-17 antibody and HBcAg. In response to HBcAg, CD4(+)CD25(+) cells significantly inhibited cell proliferation and cytokine production (especially IL-17 and IL-22) by CD4(+)CD25(-) cells in cell-contact and dose-dependent manners. In addition, CD4(+) cells from CHB patients, compared to those from HC subjects, dramatically promoted proliferation and activation of human HSCs. Moreover, in a dramatically dose-dependent manner, CD4(+)CD25(+) cells from CHB patients inhibited, whereas recombinant IL-17 response promoted the proliferation and activation of HSCs. Finally, in vivo evidence about effects of Treg/Th17 balance during liver fibrosis was obtained in concanavalin A-induced mouse fibrosis models via depletion of CD25(+) or IL-17(+) cells, and it's observed that CD25 depletion promoted, whereas IL-17 depletion, alleviated liver injury and fibrosis progression. CONCLUSIONS/SIGNIFICANCE: The Treg/Th17 balance might influence fibrosis progression in HBV-LF via increase of liver injury and promotion of HSCs activation.