Search

The present work aims to evaluate the efficacy of Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules (LCBLECs), a probiotic drug containing Bifidobacterium , in the treatment of autoimmune hepatitis (AIH). In this study, a mouse model of experimental autoimmune hepatitis (EAH) was established to investigate the effects of LCBLECs on AIH. The results showed that LCBLECs improved dysbiosis of gut microbiota, reduced liver injury, restored liver function, and maintained Treg/Th17 balance in EAH mice. In addition, LCBLECs restored Treg/Th17 balance in EAH mice by downregulating IL-33 production. Besides, LCBLECs also suppress IL-33 upregulation in EAH mice by inhibiting the TLR2/4 signaling pathway. Furthermore, LCBLECs also mitigated dysbiosis of gut microbiota and enhanced the efficacy of conventional treatment for AIH patients. To sum up, our findings revealed that LCBLECs exerted therapeutic effects on EAH mice by improving Treg/Th17 imbalance in an IL-33-dependent manner via the TLR2/4 signaling pathway and relieved the clinical symptoms of AIH patients, indicating Bifidobacterium supplementation with LCBLECs might be a potential adjuvant therapy for AIH treatment., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Intestinal microbiota (IM) dysbiosis contributes to the development of autoimmune hepatitis (AIH). This study aimed to investigate the potential effect of fecal microbiota transplantation (FMT) in a murine model of experimental AIH (EAH), a condition more similar to that of AIH patients. Changes in the enteric microbiome were determined in AIH patients and EAH mice. Moreover, we established an experimental model of secondary EAH mice harboring dysbiosis (ABx) to analyze the effects of therapeutic FMT administration on follicular regulatory T (TFR) and helper T (TFH) cell imbalances and IM composition in vivo . Alterations of the IM composition and bacterial translocation occurred in AIH patients compared to nonalcoholic fatty liver disease patients and healthy controls (HCs). Therapeutic FMT significantly attenuated liver injury and bacterial translocation and improved the imbalance between splenic TFR cells and TFH cells in ABx EAH mice. Furthermore, therapeutic FMT also partially reversed the increasing trend in serum liver enzymes (ALT and AST) of CXCR5-/-EAH mice on the 28th day. Finally, therapeutic FMT could effectively restore antibiotic-induced IM dysbiosis in EAH mice. Taken together, our findings demonstrated that FMT was capable of controlling hepatitis progression in EAH mice, and the associated mechanism might be involved in the regulation of the TFR/TFH immune imbalance and the restoration of IM composition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liang, Liwen, Jianguo, Juan, Fei, Yin, Changping and Jianping.)

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) are widely used to treat advanced metastatic cancers. Neutralisation of PD-1 or CTLA-4 by ICIs results in immune-related adverse events (irAEs). The clinicopathological features of twelve patients with hepatic irAEs were evaluated and compared to those of ten patients with autoimmune hepatitis (AIH) or graft-versus-host disease (GVHD). No significant difference was seen in serum levels of transaminases, whereas serum levels of IgG and anti-nuclear antibody were higher in patients with AIH than in those with GVHD or hepatic irAEs. Inflammation was limited to the liver lobes in patients with GVHD or hepatic irAEs, whereas patients with AIH exhibited both portal and lobular inflammation. Immunohistochemical analyses revealed a predominant infiltration of CD8 + T cells and defective accumulation of regulatory T cells (Tregs) expressing forkhead box p3 (FOXP3) in the lobular areas of patients with hepatic irAEs and GVHD. In contrast, periportal lesions of patients with AIH were characterised by an infiltration of CD4 + T cells, CD8 + T cells, CD20 + B cells, and FOXP3 + Tregs. Overall, the activation of CD8 + T cells in the absence of activation of Tregs potentially underlies the immunopathogenesis of hepatic irAEs.

Background & Aims: In autoimmune hepatitis (AIH), the imbalance between regulatory T cells (Tregs) and T-helper type 17 (Th17) cells has been linked to low levels of CD39, an ectoenzyme that hydrolyses ATP, ultimately generating immunosuppressive adenosine. Upregulation of CD39 results from activation of aryl hydrocarbon receptor (AHR), which mediates toxin responses to modulate T-cell immunity. In this study, we investigated whether altered AHR signalling underlies defective CD39 expression and function in AIH Tregs and Th17 cells, therefore contributing to regulatory/effector cell imbalance., Methods: Tregs and Th17 cells, obtained from the peripheral blood of 49 patients with AIH and 21 healthy individuals (HI), were tested for response to endogenous and exogenous AHR ligands., Results: When compared to those of HI, AIH-derived Tregs and Th17 cells displayed impaired responses to AHR activation, reflected by impaired upregulation of CD39, delayed increase in ectoenzymatic activity, and defective Treg suppressive function. These impairments resulted, at least in part, from heightened levels of AHRR and Erα in Tregs and high HIF-1α in Th17 cells, and were reverted upon molecular blockade. Importantly, in AIH-derived Tregs, the binding affinity of AHR was higher for Erα than ARNT., Conclusions: In AIH, high levels of AHRR and HIF-1α inhibit AHR signalling in Tregs and Th17 cells. AHR non-canonical binding to Erα further amplifies the lack of effective CD39 upregulation. Blockade of these inhibitory and/or non-canonical activation pathways represents a potential therapeutic approach to restore CD39 and immunohomeostasis in AIH., Lay Summary: In patients with autoimmune hepatitis, the imbalance between regulatory T cells and T helper type-17 cells is linked to dysfunction of the aryl hydrocarbon receptor pathway, resulting from aberrant inhibition or non-canonical activation. These alterations impair Treg- and Th17 cell-induced upregulation of CD39, an ectoenzyme key to immunoregulation. Blockade of excessive inhibition or non-canonical activation of the aryl hydrocarbon receptor pathway might represent a novel therapeutic strategy to control inflammation while restoring immune balance in autoimmune hepatitis., Competing Interests: Conflicts of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Nanoparticles (NPs) displaying autoimmune disease-relevant peptide-major histocompatibility complex class II molecules (pMHCII-NPs) trigger cognate T-regulatory type 1 (Tr1)-cell formation and expansion, capable of reversing organ-specific autoimmune responses. These pMHCII-NPs that display epitopes from mitochondrial protein can blunt the progression of both autoimmune hepatitis (AIH) and experimental autoimmune encephalomyelitis (EAE) in mice carrying either disease. However, with co-morbid mice having both diseases, these pMHCII-NPs selectively treat AIH. In contrast, pMHCII-NPs displaying central nervous system (CNS)-specific epitopes can efficiently treat CNS autoimmunity, both in the absence and presence of AIH, without having any effects on the progression of the latter. Here, we develop a compartmentalized population model of T-cells in co-morbid mice to identify the mechanisms by which Tr1 cells mediate organ-specific immunoregulation. We perform time-series simulations and bifurcation analyses to study how varying physiological parameters, including local cognate antigenic load and rates of Tr1-cell recruitment and retention, affect T-cell allocation and Tr1-mediated immunoregulation. Various regimes of behaviour, including 'competitive autoimmunity' where pMHCII-NP-treatment fails against both diseases, are identified and compared with experimental observations. Our results reveal that a transient delay in Tr1-cell recruitment to the CNS, resulting from inflammation-dependent Tr1-cell allocation, accounts for the liver-centric effects of AIH-specific pMHCII-NPs in co-morbid mice as compared with mice exclusively having EAE. They also suggest that cognate autoantigen expression and local Tr1-cell retention are key determinants of effective regulatory-cell function. These results thus provide new insights into the rules that govern Tr1-cell recruitment and their autoregulatory function., (© 2020 John Wiley & Sons Ltd.)

Autoimmune hepatitis (AIH) is a severe and chronic liver disease, and its incidence has increased worldwide in recent years. Research into the pathogenesis of AIH remains limited largely owing to the lack of suitable mouse models. The concanavalin A (ConA) mouse model is a typical and well-established model used to investigate T cell-dependent liver injury. However, ConA-induced hepatitis is acute and usually disappears after 48 h; thus, it does not mimic the pathogenesis of AIH in the human body. Several studies have explored various AIH mouse models, but as yet there is no widely accepted and valid mouse model for AIH. Immunosuppression is the standard clinical therapy for AIH, but patient side effects and recurrence limit its use. Regulatory T cells (Tregs) play critical roles in the maintenance of immune homeostasis and in the prevention of autoimmune diseases, which may provide a potential therapeutic target for AIH therapy. However, the role of Tregs in AIH has not yet been clarified, partly because of difficulties in diagnosing AIH and in collecting patient samples. In this review, we discuss the studies related to Treg in various AIH mouse models and patients with AIH and provide some novel insights for this research area., (Copyright © 2020 Wang, Feng, Yan and Tian.)

Peptide MHC class II-based (pMHCII-based) nanomedicines trigger the formation of multicellular regulatory networks by reprogramming autoantigen-experienced CD4+ T cells into autoimmune disease-suppressing T regulatory type 1 (TR1) cells. We have shown that pMHCII-based nanomedicines displaying liver autoimmune disease-relevant yet ubiquitously expressed antigens can blunt various liver autoimmune disorders in a non-disease-specific manner without suppressing local or systemic immunity against infectious agents or cancer. Here, we show that such ubiquitous autoantigen-specific T cells are also awakened by extrahepatic tissue damage and that the corresponding TR1 progeny can suppress experimental autoimmune encephalomyelitis (EAE) and pancreatic β cell autoreactivity. In mice having EAE, nanomedicines displaying either ubiquitous or CNS-specific epitopes triggered the formation and expansion of cognate TR1 cells and their recruitment to the CNS-draining lymph nodes, sparing their liver-draining counterparts. Surprisingly, in mice having both liver autoimmunity and EAE, liver inflammation sequestered these ubiquitous or even CNS-specific TR1 cells away from the CNS, abrogating their antiencephalitogenic activity. In these mice, only the ubiquitous antigen-specific TR1 cells suppressed liver autoimmunity. Thus, the scope of antigen spreading in autoimmune disorders is larger than previously anticipated, involving specificities expected to be silenced by mechanisms of tolerance; the regulatory activity, but not the retention of autoreactive TR1 cells, requires local autoantigen expression.

Follicular helper T (TFH) cell provides germinal centre (GC) B cell with critical signals for autoantibody production in the immunopathogenesis and progression of autoimmune hepatitis (AIH). However, the immunoregulatory functions of follicular regulatory T (TFR) cell in AIH are still unclear. The numbers of circulating TFR/TFH cells were measured in AIH patients. Moreover, we established experimental autoimmune hepatitis (EAH) model to examine the function of TFR cells on B-cell differentiation and autoantibody production in vivo and vitro. AIH patients had significantly increased numbers of TFH cells and decreased numbers of TFR cells as well as imbalanced TFR/TFH-type cytokines (IL-10, TGF-β1 and IL-21) compared with healthy controls (HCs). In addition, TFR cell numbers negatively correlated with TFH cell numbers. Also, serum hypergammaglobulinaemia (IgG and IgM) concentration negatively correlated the levels of serum IL-21, but positively correlated with the levels of serum IL-10 in AIH patients. Furthermore, in comparison with control group, significantly higher frequencies of spleen TFR cells but lower frequencies of spleen TFH cells were detected in the EAH group. Further analysis found that TFR cells simultaneously express the phenotypic characteristics of Treg and TFH cells, but exercise as negative regulators of autoantibody production in vitro culture. Our findings demonstrated that dysregulated between TFR and TFH cells might cause excessive production of autoantibodies and destruction of the immune homeostasis, leading to the immunopathological process in AIH., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

To evaluate the pathophysiology of autoimmune hepatitis (AIH) following acute hepatitis A (AHA) in immunologic aspects, we performed multi-color flow cytometry with peripheral blood mononuclear cells of a patient who underwent liver transplantation due to AIH-induced liver failure. Unlike general AHA patients, the proportion of tumor necrosis factor-α-producing Treg cells remained high for 6 months after diagnosis of AHA until she underwent a liver transplantation. The conversion of Treg cells into mediators of inflammation may have played a role in the autoimmune pathogenesis following AHA., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2020.)

Objective: Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to its immunoregulatory and hepatoprotective activities. This study aimed to evaluate the anti-inflammatory effects and underlying mechanisms of total flavonoids from T. hemsleyanum(TFT)on Con A-induced hepatitis in mice., Methods: TFT (1, 2 and 4 g/kg) and a positive control drug bifendate (200 mg/kg) were administered intragastrically to mice once daily for 10 consecutive days. On the 10th day, the model autoimmune of hepatitis was established by intravenous injection of Con A (20 mg/kg) 1 h after drug administration. Liver injury was assessed by serum levels of alanine amino transferase (ALT and AST) and histopathology 8 h after Con A injection. The levels of pro-inflammatory Th17 cytokines (IL-17, IL-6) and anti-inflammatory Treg cytokines (IL-10, TGF-β1) in serum were evaluated by ELISA, the levels of Th17 and Treg cells infiltrated into spleen were investigated by flow cytometry methods, and hepatic tissue transcription factor Foxp3 and RORγt mRNA were determined using quantitative real-time PCR., Results: Pretreatment with TFT and bifendate significantly reduced the serum levels of ALT and AST, and attenuated histopathological alterations in Con A-induced liver injury. With respect to samples treated with Con A alone, TFT and bifendate pretreatments differentially attenuated the increase of serum inflammatory factors interleukin (IL)-17 and IL-6 levels, the proportions of Th17 cells in spleen and the expression of RORγt in hepatic tissues. Meanwhile, TFT and bifendate pretreatments could enhance the percentage of Treg cells in spleen and the expression of Foxp3 in hepatic tissues, as well as the levels of transforming growth factor (TGF)-β1, IL-10 in serum., Conclusion: The anti-inflammatory effects of TFT was mediated by regulating Treg/Th17 immune homeostasis, which, therefore, suppressed the inflammatory immune response. This study provided scientific basis for the further researches and clinical applications of Tetrastigma hemsleyanum.

BACKGROUND Autoimmune hepatitis (AIH) is a chronic hepatic disorder. This study investigated role of Foxp3⁺ regulatory T cells (Treg) and methylation-regulated Tregs in AIH pathological processes. MATERIAL AND METHODS Forty consecutive patients diagnosed with hepatitis were enrolled and divided into a virus hepatitis (n=20) group and an AIH group (n=20). Twenty healthy individuals were assigned to the healthy control group (HC, n=20), Liver function biomarkers were detected on an automatic biochemical analyzer. Serum auto-antibodies were evaluated using immunofluorescence method. Histopathological evaluation was conducted with liver tissues. Treg cells were counted using FACS flow cytometry. Peripheral lymphocytes surface/intracellular biomarkers, CD4⁺CD25⁺, CD127, and Foxp3, were examined. Serum cytokines were evaluated using cytometric bead array. Methylation-specific PCR (MS-PCR) was conducted to identify the status of Foxp3 gene methylation. RESULTS Levels of liver function biomarkers were significantly increased in the AIH group compared to the HC group (p<0.05). Levels of ANA and ASMA were significantly enhanced in the AIH group compared to the HC group (p<0.05). Other auto-antibodies, including anti-AHA, anti-ribosome P protein, and anti-RO-52, were also discovered in the AIH group. Severe lymphocytic infiltration and inflammatory cells clustering were discovered in AIH patients. There were significantly fewer CD4⁺CD25⁺ T cells in the AIH group, and interleukin 6 (IL-6) and IL-10 levels were significantly decreased compared to the HC group (p<0.05). CD127⁺ Treg and Foxp3⁺ Treg expressions were decreased in the AIH group compared to the HC group (p<0.05). Foxp3 in Treg cells of AIH patients exhibited higher methylation frequency compared to that of HC patients (p<0.05). CONCLUSIONS Foxp3⁺ regulatory T cells were involved in pathological processes by activating methylation modification in autoimmune hepatitis patients.

Regulatory T cell (Treg)-based therapy can effectively control autoimmune hepatitis (AIH). Hepatic stellate cells (HSC) can selectively stimulate allogeneic Treg proliferation following liver transplantation. This study tested the therapeutic effect and potential mechanisms underlying the action of HSC-stimulated Tregs on AIH in a mouse model of Concanavalin A (ConA)-induced AIH. HSC were isolated from BALB/c mice and characterized. Splenic CD4 + CD25 + Tregs were isolated from C57BL/6 mice by immunomagnetic beads. The cells were co-cultured with primary (HSC-0), the second generation of HSC (HSC-2) for 72 h. The proliferation of Tregs was determined by flow cytometry. Similarly, the Tregs were co-cultured with HSC in transwell plates to determine the potential cell-cell contact dependent. The CD4 + CD25 - effector T cells (CFSE-Teffs) were co-cultured with Teff or Tregs in the presence or absence of HSC to determine the suppressive capacity of Tregs. The effects of Tregs or HSC-stimulated Tregs on AIH severity and the frequency of splenic Tregs and Th17 cells were examined in mice. Co-culture with HSC-2 significantly promoted Treg proliferation in a dose- and cell-cell contact-dependent manner, and allogeneic HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff in vitro. Adoptive transfer of Tregs, particularly of HSC-stimulated Tregs, significantly reduced liver injury, inflammation and Ishak modified histology activity index in AIH mice, which were associated with improving the balance of Treg and Th17 cell responses. Our data indicated that mature HSC stimulated allogeneic Treg proliferation in a dose and cell-cell contact-dependent manner, and HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff. Adoptive transfer of HSC-stimulated Tregs significantly reduced liver injury in AIH mice by modulating the balance of Treg and Th17 cell responses., (Copyright © 2019. Published by Elsevier Inc.)

Autoimmune hepatitis (AIH) is an orphan disease characterized by an autoimmune attack against hepatocytes. The exact sequence of events that leads to a breach of tolerance is incompletely understood. Current hypotheses suggest that environmental agents such as toxins or infectious agents like viruses cause a tissue damage that initiates autoimmunity in genetically susceptible individuals. The growing knowledge of the multi-facetted immune dysregulation, which involves Th1/Th17 polarization and the suspected inability of regulatory T cells to revert autoimmunity in the otherwise tolerogenic milieu of the liver, offers multiple new therapeutic approaches and targets. Standard of care (SOC) is treatment with corticosteroids with or without azathioprine, which is sufficient to induce remission in the majority of patients. However, it rarely cures AIH or restores intrahepatic immune tolerance. Hence, life-long therapy is required in the majority of patients. In addition, several studies suggest a weakening of immune regulation mediated by intrahepatic T cells under current therapies. Furthermore, second line therapies for non-responders, intolerant or otherwise difficult to treat patients are urgently needed as this is relevant for at least one fifth of all patients with inefficacy or intolerance to SOC. Current second line therapies are mainly based on single center retrospective experiences and none of them have been approved by regulatory authorities for AIH, yet. This article highlights new therapeutic approaches based on our growing knowledge on the pathophysiology of AIH. It focuses on cell-based therapies that strengthen or restore immune tolerance. An additional focus lies on new therapeutic agents showing promising results in non-hepatic autoimmune diseases that have a potential for treating AIH. The dynamics in the whole field of innovative therapies for non-hepatic autoimmune diseases will hopefully improve the therapeutic armamentarium for AIH patients in the future., (Copyright © 2018. Published by Elsevier Ltd.)

A dysbalance between effector T cells (Tconv) and regulatory T cells (Tregs) and impaired Treg function can cause autoimmune liver disease. Therefore, it is important to identify molecular mechanisms that control Treg homeostasis. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a) is an immune coreceptor with dichotomous roles in T-cell regulation: its short isoform (CEACAM1S) can activate T cells and induce Tregs, whereas its long isoform (CEACAM1L), containing two intracellular immune receptor tyrosine-based inhibitory motifs, can inhibit activated T-cell function. In the liver, CEACAM1 has antifibrotic effects in models of nonalcoholic steatohepatitis. However, its role in immune-mediated hepatitis is unknown. In the mouse model of concanavalin A-induced CD4 + T-cell-dependent liver injury, liver damage was aggravated and persisted in Ceacam1 -/- mice. Concomitantly, we observed hyperexpansion of Tconv, but reduction of interleukin (IL)-2 production and hepatic forkhead box protein P3 + (Foxp3 + )CD4 + Treg numbers. CEACAM1 -/- CD4 + T cells showed impaired IL-2-mediated signal transducer and activator of transcription 5 (STAT5) phosphorylation, which correlated with a failure of naïve CEACAM1 -/- CD4 + T cells to convert into Tregs in vitro. Furthermore, CEACAM1 -/- Tregs expressed reduced levels of Foxp3, CD25, and B-cell lymphoma 2. Adoptive transfer experiments demonstrated that hepatic Treg expansion and suppressive activity required CEACAM1 expression on both CD4 + T cells and Tregs. We identified predominant CEACAM1S expression on hepatic CD4 + T cells and Tregs from mice with acute liver injury and expression of both isoforms in liver-derived CD4 + T-cell clones from patients with liver injury., Conclusion: Our data suggest that CEACAM1S expression in CD4 + T cells augments IL-2 production and STAT5 phosphorylation leading to enhanced Treg induction and stability, which, ultimately, confers protection from T-cell-mediated liver injury. (Hepatology 2018;68:200-214)., (© 2018 by the American Association for the Study of Liver Diseases.)

This study is aimed at examining the potential role of regulatory T- (Treg-) Th1-Th17-Th22 cells in the pathogenic process of autoimmune hepatitis (AIH). The numbers of Foxp3 + Tregs and Th1, Th17, and Th22 cells were measured in 32 AIH patients using flow cytometry. Moreover, a murine model of experimental autoimmune hepatitis (EAH) was also established and used to investigate the function of Treg-Th1-Th17-Th22 cells in disease progression. AIH patients undergoing an active state had significantly decreased numbers of CD3 + CD4 + CD25 + Foxp3 + Tregs and increased numbers of CD3 + CD4 + CD25 - Foxp3 + T, CD3 + CD4 + IFN- γ + Th1, CD3 + CD4 + IL-17 + Th17, and CD3 + CD4 + IL-2 + Th22 cells as well as higher levels of Th1/Th17/Th22-type cytokines compared to AIH patients in remission and HC. Additionally, the numbers of CD3 + CD4 + CD25 + Foxp3 + Tregs were negatively correlated with the numbers of Th1-Th17-Th22 cells. Also, the serum levels of IL-17A and IL-22 were correlated positively with liver injury (ALT/AST), whereas the serum levels of IL-10 were correlated negatively with hypergammaglobulinaemia (IgG, IgM) in AIH patients. Interestingly, the percentages of spleen Tregs, expression of Foxp3 mRNA, and liver IL-10 levels decreased, whereas the percentages of spleen Th1-Th17-Th22 cells, expression of T-bet/AHR/ROR γ t mRNA, and liver IFN- γ , IL-17, and IL-22 levels increased in the murine model of EAH. Our findings demonstrated that an imbalance between Tregs and Th1-Th17-Th22 cells might contribute to the pathogenic process of AIH.

Imbalance between T regulatory (Treg) and T effector (Teff) cells is likely to contribute to the induction and perpetuation of liver damage in autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (AISC) either through inability of Tregs to restrain proliferation and effector cytokine production by responders or through conversion of Tregs into T helper type 1 (Th1) or type 17 (Th17) effector lymphocytes. We investigated the effect of Treg skewing on the phenotypic and functional properties of CD4 + CD127 + CD25 high cells, an activated subset of Teff, in 32 patients with AIH and 20 with AISC and in 36 healthy subjects. In AIH/AISC we noted a substantial increase in peripheral blood-derived CD4 + CD127 + CD25 high cells that display a Th1/Th17 phenotypic profile, as reflected by heightened interferon gamma and interleukin 17 (IL-17) production as well as by high levels of T-bet and related orphan receptor 3 expression, which is strongly correlated with disease activity. CD4 + CD127 + CD25 high cells are unresponsive to low-dose IL-2 and in patients have marked proliferative ability, further enhanced by stimulation with IL-7. CD4 + CD127 + CD25 high cells obtained from CD4 + cells exposed to Treg polarizing conditions display enhanced IL-10 production; up-regulate CD49b and LAG-3, markers of T regulatory 1 cells; and effectively suppress responder cell proliferation in both healthy subjects and AIH/AISC patients through a mechanism which is dependent on interferon gamma and IL-17. The suppressive function of CD4 + CD127 + CD25 high cells is maintained upon proinflammatory challenge in healthy subjects but not in AIH/AISC., Conclusion: Treg skewing confers activated Teff phenotypic and functional properties of T regulatory 1 cells in health and in AIH/AISC, though suppressive function is lost in patients upon proinflammatory challenge; protracted modulation of the inflammatory environment is required to attenuate the effector potential while boosting immunoregulatory properties in Teff. (Hepatology 2017;66:1570-1584)., (© 2017 by the American Association for the Study of Liver Diseases.)

Background & Aims: The autoimmune hepatitis (AIH) is a chronic hepatitis driven by the adaptive immunity that affects all age groups. A functional and numerical regulatory T cell (Treg) defect has been reported in pediatric AIH (pAIH), while an intrahepatic increase in adult AIH (aAIH) patients has been detected in current research findings., Methods: Therefore, we quantified the intrahepatic numbers of Treg, T and B cells, as well as serum cytokine levels before and during therapy in pAIH., Results: We found a disproportional intrahepatic enrichment of Tregs in untreated pAIH compared to pediatric non-alcoholic fatty liver disease. The increase of Treg/total T cells was even more pronounced than in aAIH due to fewer infiltrating T and B cells. Portal densities of Treg, as well as total T and B cells, declined significantly during therapy. However, portal Treg densities decreased disproportionately, leading to even decreasing ratios of Treg to T and B cells during therapy. Out of 28 serum cytokines IL-2 showed the strongest (10fold) decrease under therapy. This decline of IL-2 was associated with decreasing intrahepatic Treg numbers under therapy. None of the baseline T and B cell infiltration parameters were associated with the subsequent treatment response in pAIH., Conclusions: Intrahepatic Tregs are rather enriched in untreated pAIH. The disproportional decrease of Tregs during therapy may be caused by a decrease of IL-2 levels. New therapies should, therefore, aim in strengthening intrahepatic immune regulation.

Autoimmune hepatitis (AIH) is characterized by overwhelming effector immune responses associated with defective regulatory T cells (T regs ). Several lines of evidence indicate CD4 as the main effectors involved in autoimmune liver damage. Herein we investigate the in-vitro effects of prednisolone, 6-mercaptopurine, cyclosporin, tacrolimus, mycophenolic acid (MPA) and rapamycin, immunosuppressive drugs (ISDs) used in AIH treatment, on the expression of proinflammatory cytokines, co-inhibitory molecules and ability to proliferate of CD4 + CD25 - cells, isolated from the peripheral blood of treatment-naive patients with AIH. We note that in healthy subjects (HS) following polyclonal stimulation and in the absence of ISDs, the expression of interferon (IFN)-γ, interleukin (IL)-17 and tumour necrosis factor (TNF)-α by CD4 effectors peaks at 48 h and decreases at 96 h to reach baseline levels. In contrast, in AIH the expression of all these proinflammatory cytokines continue rising between 48 and 96 h. Levels of programmed cell death-1 (PD-1), T cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) and cytotoxic T lymphocyte antigen-4 (CTLA-4) increase over 96-h culture both in HS and AIH, although with faster kinetics in the latter. Exposure to ISDs contains IFN-γ and PD-1 expression in AIH, where control over CD4 + CD25 - cell proliferation is also noted upon exposure to MPA. Treatment with tacrolimus and cyclosporin render CD4 + CD25 - cells more susceptible to T reg control. Collectively, our data indicate that in treatment-naive patients with AIH, all ISDs restrain T helper type 1 (Th1) cells and modulate PD-1 expression. Furthermore, they suggest that tacrolimus and cyclosporin may ameliorate effector cell responsiveness to T regs ., (© 2017 British Society for Immunology.)

Unlabelled:  Background. T-cell populations regulate the balance of immune responses. The CD (Cluster of differentiation) 4+CD25+ regulatory T cells (Tregs) are crucial for maintaining negative control of various immune responses. There are different T-cell subpopulations with regulatory functions, as natural killer T cells, CD8+ and CD28. The forkhead box P3 (FOXP3) regulates Treg development and is required for its suppressive function., Aim: To evaluate the hepatic expression of the intrahepatic Tregs, Ig (immunoglobulin) G and IgM plasma cells in autoimmune hepatitis (AIH) and other chronic liver diseases (CLDs)., Material and Methods: This study included 100 pediatric patients; 50 AIH and 50 CLDs other than AIH. All patients were subjected to routine investigations of CLDs plus immune-staining of liver tissue for FOXp3, IgG and IgM plasma cells, CD4 and CD8 T-cells., Results: The FOXP3+ T cells in patients with AIH (6.3 ± 5) were significantly higher than that in the non-AIH (2.1 ± 2.6). FOXP3+ T cells were abundant in liver tissue with marked inflammatory cellular infiltrate. CD4+ and CD8+ infiltrating the liver tissue and IgG positive cells were significantly higher in AIH group, while the expression of IgM positive cells showed no significant difference. The IgG/IgM was significantly higher in the AIH treatment responders (3 ± 3) than non-responders (1.6 ± 0.5), while there was no significant difference regarding the intrahepatic expression of FOXP3+, CD4+, CD8+ cells, T-cells, IgG and IgM plasma cells., Conclusion: Intrahepatic Tregs were increased in number in patients with AIH in the initial presentation, and their presence is associated with increased activity and inflammation in liver biopsy.

A subset of human regulatory T cells (Tregs) can secrete IFN-γ or IL-17, and thus share features of TH1 or TH17 effector cells and lose suppressive function. The main factors driving this differentiation of Tregs toward a proinflammatory phenotype include IL-12 for TH1-like and IL-6 for TH17-type Tregs. In this study we show that Tregs of patients with de novo autoimmune hepatitis (dAIH) display increased frequencies of proinflammatory IFN-γ and IL-17 cytokines. Irrespective of a fully demethylated FOXP3 locus, Tregs of subjects with dAIH are functionally impaired. In line with the observed Treg phenotype, we detected the presence of two dominant cytokines (IL-12 and IL-6) clustering with CD68(+) monocyte/macrophage cells in livers of subjects with dAIH, and isolated monocytes of subjects with dAIH secrete high levels of proinflammatory IL-12 and IL-6, suggesting that this inflammatory milieu is key for functional impairment of Tregs. Importantly, the blockade of IFN-γ partially restores suppressive function of Tregs of subjects with dAIH, indicating that monocyte/macrophage-derived triggers might play a central role in Treg dysfunction and pathogenesis of dAIH., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background & Aims Autoimmune hepatitis (AIH), an immune-mediated liver disease, originates as a consequence of interacting genetic and environmental risk factors. Treatment remains non-specific and prone to side effects. Deficiencies in regulatory T cell (Treg) function are hypothesized to contribute to the pathogenesis of AIH. Methods We describe an adult patient who presented with AIH in the context of monocytopenia. The patient was characterized by GATA2 gene sequencing, flow cytometry of peripheral blood for leucocyte subsets, ELISA for serum Flt-3 ligand, and immunohistochemistry of liver biopsy tissue. Results Sequencing confirmed a GATA2 mutation. Peripheral Treg were absent in the context of a preserved total T cell count. Immunostaining for the Treg transcription factor FOXP3 was reduced in liver tissue as compared to a control AIH specimen. There were marked deficiencies in multiple antigen-presenting cell subsets and Flt-3 ligand was elevated. These findings are consistent with previous reports of GATA2 dysfunction. Conclusions The association of a GATA2 mutation with AIH is previously unrecognized. GATA2 encodes a hematopoietic cell transcription factor, and mutations may manifest as monocytopenia, dendritic and B cell deficiencies, myelodysplasia, and immunodeficiency. Tregs may be depleted as in this case. Our findings provide support for the role of Tregs in AIH, complement reports of other deficiencies in T cell regulation causing AIH-like syndromes, and support the rationale of attempting to modulate the Treg axis for the therapeutic benefit of AIH patients., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3(+) regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by "omics" and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients.

Unlabelled: Defective immune regulation plays a permissive role enabling effector cells to initiate and perpetuate tissue damage, eventually resulting in autoimmune disease. Numerical and functional regulatory T-cell (Treg) impairment has been previously reported in autoimmune liver disease (AILD; including autoimmune hepatitis and autoimmune sclerosing cholangitis ASC). However, in these early reports, Tregs were phenotypically defined as CD4(+) CD25(+) or CD4(+) CD25(high) cells. In the current study, we reexamined phenotypic and functional properties of Tregs by adopting a more refined definition of these cells that also includes negativity or low level of expression of CD127. We studied 43 AILD patients and 22 healthy subjects (HSs) and found that CD4(+) CD25(+) CD127(-) Tregs were decreased in the former. This decrease was more marked in patients with active disease than in those in remission. In AILD, Treg frequencies correlated inversely with parameters of disease activity and were not affected by immunosuppressive treatment. We also document, for the first time, that, in AILD, bona-fide Tregs produce less interleukin (IL)-10 and are impaired in their ability to suppress CD4(+) CD25(-) target cell proliferation, a feature that in HSs, but not in AILDs, is dependent, at least in part, on IL-10 secretion. Decreased IL-10 production by Tregs in AILD is linked to poor responsiveness to IL-2 and phospho signal transducer and activator of transcription 5 up-regulation., Conclusion: Tregs are numerically impaired in AILD, this impairment being more prominent during active disease. Notably, defective IL-10 production, resulting from low Treg responsiveness to IL-2, contributes to Treg functional impairment., (© 2015 by the American Association for the Study of Liver Diseases.)

Adoptive cell transfer is an intervention in which autologous immune cells that have been expanded ex vivo are re-introduced to mitigate a pathological process. Tregs, mesenchymal stromal cells, dendritic cells, macrophages and myeloid-derived suppressor cells have been transferred in diverse immune-mediated diseases, and Tregs have been the focus of investigations in autoimmune hepatitis. Transferred Tregs have improved histological findings in animal models of autoimmune hepatitis and autoimmune cholangitis. Key challenges relate to discrepant findings among studies, phenotypic instability of the transferred population, uncertain side effects and possible need for staged therapy involving anti-inflammatory drugs. Future investigations must resolve issues about the purification, durability and safety of these cells and consider alternative populations if necessary.

There are three classic liver diseases with probable autoimmune etiology: primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. The occurrence of these autoimmune conditions is determined by the breakdown of immune-regulatory mechanisms that in health are responsible for maintaining immunological tolerance against self-antigens. Among the multiple T cell subsets with suppressive function, the regulatory T cells (Tregs), defined by the expression of CD4, the IL-2 receptor α chain (CD25), and the transcription factor FOXP3, have emerged as having a central role in maintaining immune-tolerance to autoantigens. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways, and the production of anti-inflammatory cytokines. In all the three autoimmune liver diseases mentioned above, there is evidence pointing for either a reduced frequency and/or function of Tregs. Here, we review the definition, phenotypic characteristics, and mechanisms of suppression employed by Tregs and then we discuss the evidence available pointing to their impairment in patients with autoimmune liver disease., (© 2015 International Union of Biochemistry and Molecular Biology.)

Regulatory T cells (Tregs) have a profound ability to control immune responses. A majority of Tregs are derived from the thymus; yet a substantial Treg fraction is derived from the periphery. The liver seems to be an important source of peripherally derived Tregs. Indeed, the liver's well-known ability to induce immune tolerance is at least partly based on hepatic Treg generation. With recently developed tools to deliver antigens to tolerance-inducing liver cells, it is now possible to harness liver-derived Tregs for specific control of unwanted immune responses. Indeed, the selective delivery of autoantigens to liver sinusoidal endothelial cells could induce autoantigen-specific Tregs in vivo, providing effective treatment of autoimmune disease. Owing to the fundamental role Tregs play in controlling immune responses, an impairment of Tregs seems to be a plausible explanation for the development of autoimmune diseases, for example, in the liver. However, the actual role of Treg impairment in autoimmune liver diseases, such as autoimmune hepatitis (AIH), remains controversial. Major obstacles for clarifying the role of Tregs in autoimmune liver diseases are related to the difficulty to identify human Tregs unambiguously and to the difficulty to identify those Tregs and effector T cells that specifically recognize disease-driving autoantigens. However, even if AIH turned out to be a disease that is not driven by Treg impairment, Treg-based therapies for autoimmune liver diseases might still be effective, provided the Tregs for therapeutic use recognize the relevant antigens., (© 2015 S. Karger AG, Basel.)

In both autoimmune liver disease and chronic viral hepatitis, the injury results from an immune-mediated cytotoxic T cell response to liver cells. As such, it is not surprising that CD4(+) regulatory T cells, a key regulatory population of T cells able to curb immune responses, could be involved in both autoimmune hepatitis and chronic viral hepatitis. The liver can induce the conversion of naïve CD4(+) T cells to CD4(+) regulatory T cells and induce tolerance to locally expressed antigens. This tolerance mechanism is carefully regulated in physiological conditions but any imbalance could be pathological. An overly tolerant immune response can lead to chronic infections while an overreactive and unbridled immune response can lead to autoimmune hepatitis. With the recent advent of monoclonal antibodies able to target regulatory T cells (daclizumab) and improve immune responses and several ongoing clinical trials analysing the impact of regulatory T cell infusion on autoimmune liver disease or liver transplant tolerance, modulation of immunological tolerance through CD4(+) regulatory T cells could be a key element of future immunotherapies for several liver diseases allowing restoring the balance between proper immune responses and tolerance.  .

Background & Aims: Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH., Methods: We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients., Results: Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production., Conclusions: Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease associated with interface hepatitis, raised plasma liver enzymes, the presence of autoantibodies and regulatory T-cell (Tregs) dysfunction. The clinical course is heterogeneous, manifested by a fulminant or indolent course. Although genetic predisposition is well accepted, the combination with currently undefined environmental factors is crucial for the development of the disease. Progress in the development of reliable animal models provides added understanding of the pathophysiology of AIH, and these will be very useful in evaluating potential therapeutics. It appears that artificially breaking tolerance in the liver is easy. However, maintaining this state of tolerance breakdown, to get chronic hepatitis, is difficult because liver immune homeostasis is strongly regulated by several immune response inhibitory mechanisms. For example, Tregs are crucial regulators in acute and chronic hepatitis, and C57BL/6 mice are most prone to experimental AIH. Immunization of C57BL/6 mice with liver (AIH) autoantigens (CYP2D6/FTCD or IL-4R) and the disturbance of liver regulatory mechanism(s), leading to experimental AIH, are likely to be most representative of human AIH pathology., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Imbalance between effector and regulatory T-cells (Treg) underlies the loss of immune-tolerance to self-antigens in autoimmune disease. In autoimmune hepatitis type 2 (AIH-2), effector CD4 T-cell immune responses to cytochrome P450IID6 (CYP2D6) are permitted by numerically and functionally impaired Treg. Restoration of CYP2D6-specific Treg in AIH-2 would enable control over effectors sharing the same antigen specificity, leading to re-establishment of immune-tolerance. We have previously developed a protocol for generating antigen-specific Treg through co-culture with semi-mature dendritic cells presenting CYP2D6 peptides. In this study, we aimed to explore phenotypic and functional features of patient Treg compared to health, to test Treg stability under pro-inflammatory conditions, and to investigate the potential benefit of supplementation with all-trans-retinoic acid (RA) or rapamycin (RP), agents proven to enhance Treg function. We show that antigen-specific Treg from patients have comparable phenotypic and functional features to those from healthy controls, suppressing both proliferation and pro-inflammatory cytokine production by effector cells. Treg exposure to inflammatory challenge results in decreased suppressive function and up-regulation of Th1/Th2/Th17 transcription factors both in health and AIH-2. The increase of Th1 and Th17 transcription factors is limited by addition of RA in controls and Th1 expression is decreased by RP in patients. Importantly, inflammation-induced decrease in Treg function is also abrogated by RA/RP in health and RA in patients. Our data provide important information for the optimization of protocols aiming at generating antigen-specific Treg for treatment of autoimmune disease and for understanding their biology upon pro-inflammatory challenge and RP/RA supplementation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Unlabelled: Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T-cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39(pos) Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39(pos) Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4(pos) CD25(high) , CD4(pos) CD25(high) CD39(pos) , and CD4(pos) CD25(high) CD39(neg) subsets to suppress both proliferation of effector T cells and interleukin (IL)-17 production was evaluated. In AIH, CD39(pos) Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL-17 production by effector CD4 T cells. Moreover, these CD39(pos) Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon-gamma or IL-17 upon challenge with proinflammatory stimuli., Conclusions: In AIH, CD39(pos) Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL-17 production by effector CD4 T cells. CD39(pos) Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells., (© 2014 by the American Association for the Study of Liver Diseases.)

Autoimmune hepatitis is an immuno-mediated inflammatory liver disorder of unknown etiology and is characterized by hypergammaglobulinaemia, circulating autoantibodies and interface hepatitis. The disease may often present as an acute icteric hepatitis, or run an insidious and progressive course, and in most of the cases it is expected to evolve toward liver cirrhosis and end-stage liver failure, without prompt and appropriate treatment with steroids and other immunosuppressive drugs. Nonetheless, several patients are non-responsive or become non-tolerant to conventional therapy with prednisone/prednisolone with or without azathioprine. Recent findings highlight the role of the interplay between CD4+CD25+ regulatory T cells and Th17 cells in the pathogenesis of autoimmune hepatitis. A numerical and functional imbalance between regulatory and effector cells in favor of the latter appears to be pivotal in the progression of the disease. In addition, the intra-hepatic microenvironment of autoimmune hepatitis is particularly rich in pro-inflammatory cytokines such as IL-6, IL-17, IL-23, IL-1β which play a crucial role in perpetuating and expanding effector cells and subsequent liver damage, whereas regulatory T cells are greatly disadvantaged and inhibited in such polarized habitat. Novel therapeutic interventions should aim at modulating the intra-hepatic pro-inflammatory milieu while favoring the expansion of regulatory T cells. Liver autoantigen-specific regulatory T cells generated and expanded in vitro from patients' own cells might offer a potentially curative approach to autoimmune hepatitis by inhibiting effector cells of the same specificity without inducing pan-immunosuppression., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinaemia, autoantibodies and interface hepatitis, is occasionally associated with systemic autoimmune manifestations [systemic lupus erythematosus (SLE); mixed connective tissue disease (MCTD)]. In both AIH and SLE/MCTD numerical and/or functional impairment of regulatory T-cells (T-regs) is believed to favour autoimmunity. To investigate whether immune-tolerance breakdown profiles differ in patients with AIH and SLE/MCTD, isolated AIH or systemic autoimmunity, we studied phenotypic and functional features of T-regs in 10 patients with AIH-SLE/MCTD, 22 with AIH, 12 with SLE and 20 healthy subjects. Compared to health, CD4(pos)CD25(pos) cells were decreased in number and expressed high levels of the CD127 activation marker in all three disease groups; in AIH-SLE/MCTD and in SLE they displayed low levels of FOXP3. In AIH-SLE/MCTD, they also contained a high proportion of IFNγ positive cells, indicating a Th1 profile. Similarly, in AIH-SLE/MCTD, CD4(pos)CD25(pos)CD25(high) T-regs were reduced in number and contained an increased proportion of activated CD127(pos) and IFNγ(pos) cells. Such skewing towards a Th1 profile was also present at effector level, as a high frequency of IFNγ-producing cells was observed within AIH-SLE/MCTD CD4(pos)CD25(neg) responder cells. Impairment in suppressor function both of CD4(pos)CD25(pos) cells and CD4(pos)CD25(pos)CD127(neg) T-regs was observed in all three autoimmune conditions, but while addition of CD4(pos)CD25(pos)CD127(neg) T-regs decreased CD4(pos)CD25(neg) responder cell proliferation in healthy subjects and partially in AIH patients, it had no effect in AIH-SLE/MCTD and SLE patients. In conclusion, in AIH-SLE/MCTD T-regs display a distinctive phenotypic and functional signature, characterized by marked activation, elevated IFNγ production and by a profound impairment of suppressive function, suggesting that multiple autoimmune manifestations may derive from a complex defect of immune-regulation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To determine the metabolic and immunological effects of the oral administration of DT56a, an enzymatic isolate of soybeans., Methods: DT56a was orally administered to mice in three animal models: leptin deficiency, high-fat diet (HFD) supplementation and immune-mediated hepatitis. Liver damage and immunological status were assessed., Results: Oral administration of DT56a to leptin-deficient (ob/ob) and HFD mice led to a significant reduction in serum triglyceride (TG) and total cholesterol (TC) levels. DT56a-treated mice in both models exhibited a significant reduction in hepatic levels of TG and marked alleviation of glycemic control as indicated by significant decreases in fasting blood glucose levels and glucose tolerance tests. The levels of liver enzymes were reduced. These metabolic effects were associated with altered distributions of regulatory T (Tregs) and natural killer T (NKT) cells. DT56a suppressed the immune-mediated liver damage induced by concanavalin A indicated by decreased liver enzymes and serum interferon-γ levels and by improved histology and decreased hepatic apoptosis. Oral administration of DT56a also alleviated immune-mediated hepatitis and affected Tregs and NKT cells., Conclusions: Oral administration of DT56a promotes a hepatoprotective effect associated with an alteration in the distribution of Tregs and NKT cells., (© 2012 The Authors. Journal of Digestive Diseases © 2012 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Unlabelled: Autoimmune hepatitis (AIH) is characterized by a loss of immunological tolerance to hepatocytes. Patients respond well to immunosuppression but progression to endstage liver disease occurs in 10%-20% of cases, leading to liver transplantation. Using a murine model of type 2 AIH, we identified susceptibility factors for autoimmune hepatitis and attempted to restore immunological tolerance to liver autoantigens. An increased ectopic expression of a liver autoantigen (FTCD) in the thymus leading to reduced numbers of circulating autoreactive T cells was sufficient to prevent development of AIH in mice. However, in the presence of a reduced central tolerance to FTCD, a strong regulatory T-cell response was able to inhibit proliferation of liver-specific autoreactive T cells and prevent AIH. Development of a severe AIH stemmed from reduced numbers of functional regulatory T cell (Tregs) leading to an increased proliferation of FTCD-specific autoreactive T and B cells. Adoptive transfer of ex vivo expanded CXCR3(+) Tregs in mice with AIH efficiently targeted the inflamed liver, restored peripheral tolerance to FTCD, and induced remission of AIH., Conclusion: Peripheral tolerance to liver autoantigens in AIH is paramount. Autologous infusion of ex vivo expanded CXCR3(+) Tregs in AIH patients could be an effective therapeutic approach to restore peripheral tolerance and induce remission of AIH., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background & Aims: The pathogenesis of autoimmune hepatitis (AIH) is not understood, but it was suggested that AIH may be related to a numerical or functional impairment of CD4+CD25+FOXP3+ regulatory T cells (Treg), which are important mediators of immune tolerance to self-antigens. However, the role of Treg in AIH is not clear, since earlier studies reporting Treg impairment had used only CD25 as marker that cannot unambiguously distinguish Treg from activated effector T cells., Methods: We assessed the frequency and suppressor function of Treg using current staining protocols that can distinguish Treg from activated effector T cells., Results: The frequency of CD4+CD25(high)CD127(low)FOXP3+ Treg cells in blood of AIH patients was not reduced compared to healthy subjects, as shown by flow cytometry and confirmed by quantifying Treg-specific demethylation of the FOXP3 gene. Moreover, the suppressor function of Treg isolated from AIH patients was similar to that of Treg isolated from healthy subjects, indicating that Treg function was not impaired in AIH patients. However, we observed that the Treg frequency was significantly higher in those AIH patients with active disease than in those who were in a state of remission, suggesting that the Treg frequency may increase with the degree of inflammation. Indeed, analysis of FOXP3+ Treg in liver histology revealed that the intrahepatic Treg frequency was higher in AIH patients than in NASH patients and correlated with the inflammatory activity of the liver., Conclusions: The frequency and function of circulating Treg cells is not impaired in AIH., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4+CD25(high)FOXP3+ T regulatory cells (Tregs). Tregs can be generated from CD25⁻ (ngTreg) cells, which suppress the immune response less efficiently than Tregs. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4+ progenitors as Tregs, accounts for the reduced suppressive functions of ngTregs. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Tregs., Methods: ngTregs were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor β or neutralizing antibodies against IL-6 and IL-1β (to promote differentiation of ngTregs vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches., Results: ngTregs from patients with AIH contained greater proportions of IL-17+ and RORC+ cells than Tregs from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTregs and their suppressive functions. Inhibition of IL-17 led to development of ngTregs that were phenotypically stable and did not acquire proinflammatory properties after exposure to IL-6 and IL-1β., Conclusions: Blocking Th17 allows ngTregs to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Myeloid-derived suppressor cells (MDSCs) are getting increased attention as one of the main regulatory cells of the immune system. They are induced at sites of inflammation and can potently suppress T cell functions. In the current study, we demonstrate how activation of TRPV1 vanilloid receptors can trigger MDSCs, which in turn, can inhibit inflammation and hepatitis., Methodology/principal Findings: Polyclonal activation of T cells, following injection of concanavalin A (ConA), in C57BL/6 mice caused acute hepatitis, characterized by significant increase in aspartate transaminase (AST), induction of inflammatory cytokines, and infiltration of mononuclear cells in the liver, leading to severe liver injury. Administration of cannabidiol (CBD), a natural non-psychoactive cannabinoid, after ConA challenge, inhibited hepatitis in a dose-dependent manner, along with all of the associated inflammation markers. Phenotypic analysis of liver infiltrating cells showed that CBD-mediated suppression of hepatitis was associated with increased induction of arginase-expressing CD11b(+)Gr-1(+) MDSCs. Purified CBD-induced MDSCs could effectively suppress T cell proliferation in vitro in arginase-dependent manner. Furthermore, adoptive transfer of purified MDSCs into naïve mice conferred significant protection from ConA-induced hepatitis. CBD failed to induce MDSCs and suppress hepatitis in the livers of vanilloid receptor-deficient mice (TRPV1(-/-)) thereby suggesting that CBD primarily acted via this receptor to induce MDSCs and suppress hepatitis. While MDSCs induced by CBD in liver consisted of granulocytic and monocytic subsets at a ratio of ∼2∶1, the monocytic MDSCs were more immunosuppressive compared to granulocytic MDSCs. The ability of CBD to induce MDSCs and suppress hepatitis was also demonstrable in Staphylococcal enterotoxin B-induced liver injury., Conclusions/significance: This study demonstrates for the first time that MDSCs play a critical role in attenuating acute inflammation in the liver, and that agents such as CBD, which trigger MDSCs through activation of TRPV1 vanilloid receptors may constitute a novel therapeutic modality to treat inflammatory diseases.

Unlabelled: Effector CD4 and CD8 T cell immune responses to cytochrome P450IID6 (CYP2D6), the autoantigen of autoimmune hepatitis type 2 (AIH-2), are permitted by a numerical and functional impairment of CD4(pos) CD25(high) regulatory T cells (T-regs). We aimed to investigate whether T-regs specific for CYP2D6 immunodominant regions and restricted by the appropriate human leukocyte antigen (HLA)-DR molecule can be generated in patients with AIH-2 and can control CD4 and CD8 T cell effectors targeting identical or overlapping CYP2D6 regions. CYP2D6-specific regulatory T cells (CYP2D6 T-regs) were obtained from peptide-pulsed monocyte-depleted peripheral blood mononuclear cells of 17 patients with AIH-2, who were positive for the predisposing HLA-DR7 and/or HLA-DR3 alleles. Their antigen specificity was assessed by cytofluorimetry using HLA class II tetramers and their cytokine profile by intracellular staining. T-reg ability to suppress was ascertained by measuring reduction of CD4(pos) CD25(neg) cell proliferation/effector cytokine secretion and of CD8 T cell cytotoxicity. The most efficient suppression of effector T cell proliferation, inflammatory cytokine release, and cytotoxicity was obtained by coculturing T-regs with CYP2D6-peptide-loaded semimature dendritic cells (smDCs), and smDC-CYP2D6 T-regs also expressed high levels of FOXP3 (forkhead box P3). Possession of the appropriate HLA-DR molecule and recognition of the CYP2D6 autoantigenic sequence were critical to the synergistic smDC-CYP2D6 T-reg immunoregulatory functions, and lack of either element led to poor control of responder cell proliferation and cytokine secretion. Moreover, interferon-γ neutralization significantly boosted the suppressive ability of CYP2D6 T-regs., Conclusion: T-regs generated under CYP2D6-specific conditions and cocultured with smDCs are highly effective at controlling autoreactive T cells, thus providing the basis for a powerful and tailored form of immunotherapy for AIH-2., (Copyright © 2010 American Association for the Study of Liver Diseases.)