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Background & Aims: The N6-methyladenosine (m 6 A) reader YTH domain-containing family protein 2 (YTHDF2) is critically involved in a multiplicity of biological processes by mediating the degradation of m 6 A modified mRNAs. Based on our current understanding of this process, we hypothesized that YTHDF2 will play a role in the natural history and function of myeloid-derived suppressor cells (MDSC) and in particular in AIH., Approach & Results: We took advantage of YTHDF2 conditional knock-out mice to first address the phenotype and function of MDSCs by flow cytometry. Importantly, the loss of YTHDF2 resulted in a gradual elevation of MDSCs including PMN-MDSCs both in liver and ultimately in the BM. Notably, YTHDF2 deficiency in myeloid cells attenuated concanavalin (ConA)-induced liver injury, with enhanced expansion and chemotaxis to liver. Furthermore, MDSCs from Ythdf2 CKO mice had a greater suppressive ability to inhibit the proliferation of T cells. Using multi-omic analysis of m 6 A RNA immunoprecipitation (RIP) and mRNA sequencing, we noted RXRα as potential target of YTHDF2. Indeed YTHDF2-RIP-qPCR confirmed that YTHDF2 directly binds RXRα mRNA thus promoting degradation and decreasing gene expression. Finally, by IHC and immunofluorescence, YTHDF2 expression was significantly upregulated in the liver of patients with AIH which correlated with the degree of inflammation., Conclusion: Suppression of YTHDF2 enhances the expansion, chemotaxis and suppressive function of MDSCs and our data reveals a unique therapeutical target in immune mediated hepatitis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In autoimmune hepatitis (AIH), the persisting inflammation contributes to fibrosis progression, for which conventional biochemical markers manifest relatively unsatisfactory prediction. Herein, we assessed the value of serum CD48 (sCD48) as an indicator for inflammation and fibrosis in AIH type 1. The levels of sCD48 were detected first in an exploratory cohort using ELISA. In this cohort, compared with healthy controls (4.90 ng/mL, P < 0.0001), primary biliary cholangitis (7.32 ng/mL, P < 0.0001), and non-alcoholic fatty liver disease (7.76 ng/mL, P < 0.0001), sCD48 levels were elevated in AIH (12.81 ng/mL) and correlated with histological inflammation and fibrosis. Further using multivariate logistic regression analysis, sCD48 was identified as an independent predictor for both significant inflammation (G3-4) and advanced fibrosis (S3-4). Two predictive scores, based on sCD48, were constructed for diagnosing significant inflammation and advanced fibrosis (sCD48-AIH-SI and sCD48-AIH-AF, respectively). Using these data as a premise, predictive abilities were subsequently evaluated and verified in a validation cohort. In the exploratory cohort, the area under the receiver operating characteristic curve of sCD48 and sCD48-AIH-SI, for significant inflammation, were 0.748 and 0.813, respectively. Besides, during treatment follow-up, sCD48 levels gradually decreased from immunosuppression initiation to re-evaluation biopsy, in parallel with aspartate transaminase, total sera IgG, and fibrosis-4 score. For AIH patients in a re-evaluation biopsy cohort, sCD48 could predict significant fibrosis (S2-4). Further using immunohistochemistry, hepatic CD48 expression was elevated in AIH patients and decreased after treatment. In conclusion, sCD48 and sCD48-based predictive scores predict histological inflammation and fibrosis in AIH-1. Detecting sCD48 might help in the clinical management of AIH., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aims: Autoimmune hepatitis (AIH) is a rare and chronic autoimmune liver disease. While genetic factors are believed to play a crucial role in the etiopathogenesis of AIH, our understanding of these genetic risk factors is still limited. In this study, we aimed to identify susceptibility loci to further understand the pathogenesis of this disease., Approach and Results: We conducted a case-control association study of 1,622 Chinese patients with AIH type 1 and 10,466 population controls from two independent cohorts. A meta-analysis was performed to ascertain variants associated with AIH type 1. A single-nucleotide polymorphism within the human leukocyte antigen (HLA) region showed the strongest association with AIH (rs6932730: OR = 2.32; p = 9.21 × 10 -73 ). The meta-analysis also identified two non-HLA loci significantly associated with AIH: CD28/CTLA4/ICOS on 2q33.3 (rs72929257: OR = 1.31; p = 2.92 × 10 -9 ) and SYNPR on 3p14.2 (rs6809477: OR = 1.25; p = 5.48 × 10 -9 ). In silico annotation, reporter gene assays, and CRISPR activation experiments identified a distal enhancer at 2q33.3 that regulated expression of CTLA4. In addition, variants near STAT1/STAT4 (rs11889341: OR = 1.24; p = 1.34 × 10 -7 ), LINC00392 (rs9564997: OR = 0.81; p = 2.53 × 10 -7 ), IRF8 (rs11117432: OR = 0.72; p = 6.10 × 10 -6 ), and LILRA4/LILRA5 (rs11084330: OR = 0.65; p = 5.19 × 10 -6 ) had suggestive association signals with AIH., Conclusions: Our study identifies two novel loci (CD28/CTLA4/ICOS and SYNPR) exceeding genome-wide significance and suggests four loci as potential risk factors. These findings highlight the importance of costimulatory signaling and neuro-immune interaction in the pathogenesis of AIH., (© 2022 American Association for the Study of Liver Diseases.)

Autoimmune hepatitis (AIH) is an inflammatory liver disease driven by the hyperactivation of various intrahepatic antigen-specific T cells due to a breach of immune tolerance. Studies in immunometabolism demonstrate that activated T cells harbor increased levels of reactive oxygen species that cause oxidative DNA damage. In this study, we assessed the potential of DNA damage repair enzyme MutT homolog 1 (MTH1) as a therapeutic target in AIH and karonudib as a novel drug for patients with AIH. We report herein that MTH1 expression was significantly increased in liver samples from patients with AIH compared to patients with chronic hepatitis B and nonalcoholic fatty liver disease and from healthy controls. In addition, the expression of MTH1 was positively correlated with AIH disease severity. We further found abundant T cells that expressed MTH1 in AIH. Next, we found that karonudib significantly altered T-cell receptor signaling in human T cells and robustly inhibited proliferation of human T cells in vitro. Interestingly, our data reflected a preferential inhibition of DNA damage repair in activated T cells by karonudib. Moreover, MTH1 was required to develop liver inflammation and damage because specific deletion of MTH1 in T cells ameliorated liver injury in the concanavalin A (Con A)-induced hepatitis model by inhibiting T-cell activation and proliferation. Lastly, we validated the protective effect of karonudib on the Con A-induced hepatitis model. Conclusion: MTH1 functions as a critical regulator in the development of AIH, and its inhibition in activated T cells reduces liver inflammation and damage., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Autoimmune hepatitis (AIH) is characterized by interface hepatitis, elevated serum alanine aminotransferase and aspartate aminotransferase levels, circulating autoantibodies, and elevated predominantly immunoglobulin G (IgG) levels. The goal in the treatment of autoimmune hepatitis (AIH) is complete disease remission. Here we took advantage of a large cohort of AIH patients to clarify predictors associated with biochemical and histological remission. Of 705 patients with complete follow-up, 569 (80.7%) patients achieved complete biochemical remission. Lower IgG levels (17.8 vs. 25 g/L, p < 0.001) and less liver cirrhosis (19.3% vs. 33.1%, p < 0.001) at diagnosis were observed in these patients. They also had lower serum IgG levels (13 vs. 18.9 g/L, p < 0.001) after 3 months of treatment. Histological remission was achieved in 69.4% of 160 patients with complete biochemical remission after 3 years of treatment. Patients with histological remission had lower IgG levels (16.2 vs. 20.1 g/L, p = 0.006) and Ishak fibrosis scores (3.4 vs. 4.1, p = 0.010) at diagnosis, and they appeared to achieve biochemical remission more rapidly (1 vs. 3 months, p < 0.001). Of note, patients with histological remission had higher frequency of fibrosis regression than those with persisting histological activity (87.5% vs. 60%, p = 0.004). In conclusion, lower serum IgG levels, less fibrosis in liver histology at diagnosis, and rapid response to immunosuppressive therapy are reliable predictors of biochemical and histological remission. Our study underscores the importance of early diagnosis and appropriate treatment., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Myeloid-derived suppressor cells (MDSCs) emerge as a promising candidate for the immunotherapy of autoimmune hepatitis (AIH). However, targets for modulating MDSC in AIH are still being searched. Liver X receptors (LXRs) are important nuclear receptors linking lipid metabolism and immune responses. Despite the extensive studies of LXR in myeloid compartment, its role in MDSCs is currently less understood. Herein, expression of LXRα was found to be upregulated in AIH patients and colocalized with hepatic MDSCs. In ConA-induced hepatitis, deletion of LXRα led to increased expansion of MDSCs in the liver and alleviated the hepatic injury. MDSCs in LXRα -/- mice exhibited enhanced proliferation and survival comparing with WT mice. T-cell proliferation assay and adoptive cell transfer experiment validated the potent immunoregulatory role of MDSCs in vitro and in vivo . Mechanistically, MDSCs from LXRα -/- mice possessed significantly lower expression of interferon regulatory factor 8 (IRF-8), a key negative regulator of MDSC differentiation. Transcriptional activation of IRF-8 by LXRα was further demonstrated., Conclusion: We reported that abrogation of LXRα facilitated the expansion of MDSCs via downregulating IRF-8, and thereby ameliorated hepatic immune injury profoundly. Our work highlights the therapeutic potential of targeting LXRα in AIH., 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 Li, Lian, Li, Qian, Zhang, Liu, Tang and Ma.)

Background and Aims: The diverse inflammatory response found in the liver of patients with autoimmune hepatitis (AIH) is well established, but identification of potentially pathogenic subpopulations has proven enigmatic., Approach and Results: We report herein that CD69 + CD103 + CD8 + tissue-resident memory T cells (T RM ) are significantly increased in the liver of patients with AIH compared to chronic hepatitis B, NAFLD, and healthy control tissues. In addition, there was a significant statistical correlation between elevation of CD8 + T RM cells and AIH disease severity. Indeed, in patients with successful responses to immunosuppression, the frequencies of such hepatic CD8 + T RM cells decreased significantly. CD69 + CD8 + and CD69 + CD103 + CD8 + T cells, also known as CD8 + T RM cells, reflect tissue residency and are well known to provide intense immune antigenic responses. Hence, it was particularly interesting that patients with AIH also manifest an elevated expression of IL-15 and TGF-β on inflammatory cells, and extensive hepatic expression of E-cadherin; these factors likely contribute to the development and localization of CD8 + T RM cells. Based on these data and, in particular, the relationships between disease severity and CD8 + T RM cells, we studied the mechanisms involved with glucocorticoid (GC) modulation of CD8 + T RM cell expansion. Our data reflect that GCs in vitro inhibit the expansion of CD8 + T RM cells induced by IL-15 and TGF-β and with direct down-regulation of the nuclear factor Blimp1 of CD8 + T RM cells., Conclusions: Our data suggest that CD8 + T RM cells play a critical role in the pathogenesis of AIH, and GCs attenuate hepatic inflammation through direct inhibition of CD8 + T RM cell expansion., (© 2021 by the American Association for the Study of Liver Diseases.)

Autoimmune liver diseases are a panel of chronic inflammatory diseases caused by abnormal immune attack against hepatocytes or bile duct epithelial cell, including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), IgG4-related sclerosing cholangitis (IgG4-SC), and overlap syndrome. Currently main drug treatments of autoimmune liver diseases contain corticosteroids, immunosuppressant and other immune-modulation therapy. Using anti-CD20 to deplete B cells has achieved biochemical improvement in patients with IgG4-SC or PBC. There are several clinical trials focus on immune cell transfusion and novel target drug therapy finished or being conducted.

Objective: Emperipolesis is a pathological feature for the diagnosis of autoimmune hepatitis (AIH). We have previously found that CD8 + T cells participated in the emperipolesis in AIH. In this study we aimed to clarify the characteristics and molecular mechanisms of emperipolesis in patients with AIH in vitro and in mice with α-Galactosylceramide (α-GalCer)-induced acute hepatitis., Methods: The peripheral blood mononuclear cells (PBMC) of patients with various chronic liver diseases and healthy controls were co-cultured with hepatic cell lines to induce emperipolesis in vitro. Confocal staining was performed to illustrate the cellular types and potential mechanisms of emperipolesis in AIH. In addition, a murine model of α-GalCer-induced acute hepatitis that mimics human AIH was used to confirm the role of CD44/p-ERM/F-actin in the emperipolesis process in vivo., Results: In the co-cultured system of PBMC and hepatic cell line, emperipolesis was observed most commonly in patients with AIH. The main cells participating in emperipolesis were CD8 + T cells, and they penetrated hepatic cells actively via the CD44/p-ERM/F-actin pathway. As a result, most CD8 + T cells engulfed by hepatic cells underwent apoptosis. In the α-GalCer-induced acute hepatitis model, emperipolesis was observed around the inflammatory foci and was inhibited by the ezrin phosphorylation inhibitor NSC668394. Similarly, activated murine CD8 + T cells penetrated primary hepatocytes via the CD44/p-ERM/F-actin pathway in vitro., Conclusions: CD8 + T cells penetrate hepatic cells actively via the CD44/p-ERM/F-actin signaling pathway and undergo apoptosis. This may be a compensatory mechanism to attenuate the overwhelming immune attack in AIH., (© 2021 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Objective: The significance of the liver-microbiome axis has been increasingly recognised as a major modulator of autoimmunity. The aim of this study was to take advantage of a large well-defined corticosteroids treatment-naïve group of patients with autoimmune hepatitis (AIH) to rigorously characterise gut dysbiosis compared with healthy controls., Design: We performed a cross-sectional study of individuals with AIH (n=91) and matched healthy controls (n=98) by 16S rRNA gene sequencing. An independent cohort of 28 patients and 34 controls was analysed to validate the results. All the patients were collected before corticosteroids therapy., Results: The gut microbiome of steroid treatment-naïve AIH was characterised with lower alpha-diversity (Shannon and observed operational taxonomic units, both p<0.01) and distinct overall microbial composition compared with healthy controls (p=0.002). Depletion of obligate anaerobes and expansion of potential pathobionts including Veillonella were associated with disease status. Of note, Veillonella dispar , the most strongly disease-associated taxa (p=8.85E-8), positively correlated with serum level of aspartate aminotransferase and liver inflammation. Furthermore, the combination of four patients with AIH-associated genera distinguished AIH from controls with an area under curves of approximately 0.8 in both exploration and validation cohorts. In addition, multiple predicted functional modules were altered in the AIH gut microbiome, including lipopolysaccharide biosynthesis as well as metabolism of amino acids that can be processed by bacteria to produce immunomodulatory metabolites., Conclusion: Our study establishes compositional and functional alterations of gut microbiome in AIH and suggests the potential for using gut microbiota as non-invasive biomarkers to assess disease activity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Interleukin-35 (IL-35) is a novel anti-inflammatory cytokine of IL12 cytokine family, however, the role of IL-35 in patients with AIH and its effect on myeloid-derived suppressor cells (MDSCs) has not yet been analyzed. The expression of IL-35 subunits (p35 and EBI3) in liver tissues was quantified by immunochemistry and its correlation with clinical parameters was explored in patients with AIH. The expression of MDSCs and IL-35 receptor (gp130 and IL-12Rβ2) were analyzed using flow cytometry and confocal staining. Besides, we utilized in vitro culture to explore the role of IL-35 on MDSCs expansion and activation. We found that the elevated expression of both IL-35 subunits (EBI3 and p35) in liver tissue was positively associated with degrees of hepatic inflammatory and fibrosis in patients with AIH. Furthermore, the expression of EBI3 in liver was positively correlated with patient age, serum IgG levels and serum AST, and was negatively correlated with hemoglobin and albumin. Moreover, our results showed that ratio of MDSC in peripheral blood increased significantly in AIH patients as compared with healthy controls. Further study showed that CD33, a representative marker of MDSCs, co-localized well with gp130 and IL12Rβ2, suggesting MDSCs as target cell for IL-35. Consistently, MDSCs from AIH displayed a substantial higher abundance of gp130 and IL12Rβ2 and were expanded by IL-35 in vitro . IL-35-induced MDSCs showed a significant increase in Nitric oxide (NO) production but not reactive oxygen species (ROS). Conclusions: IL-35 might play an important role in AIH by regulating MDSCs and it could provide new insights into the therapy of AIH., (Copyright © 2019 Lian, Zhang, Zhao, Chen, Peng, Wang, Chen and Ma.)

There is increasing awareness of the immunologic roles of liver mononuclear populations, including myeloid-derived suppressor cells (MDSCs). We took advantage of a large well-defined cohort of 148 patients with liver inflammation and 45 healthy controls to focus on the qualitative and quantitative characteristics of MDSCs. We investigated the frequency, phenotype, and functional capacities of MDSCs by using peripheral blood MDSCs in a cohort of 55 patients with primary biliary cholangitis (PBC), 40 with autoimmune hepatitis, 39 with chronic hepatitis B, 14 with nonalcoholic fatty liver disease, and 45 healthy controls. This was followed by a liver-targeted determination in 27 patients with PBC, 27 with autoimmune hepatitis, 20 with chronic hepatitis B, 14 with nonalcoholic fatty liver disease, and 6 controls. We then focused on mechanisms of this expansion with PBC as an example, using both ursodeoxycholic acid-naive and treated patients. HLA-DR -/low CD33 + CD11b + CD14 + CD15 - monocytic MDSCs were elevated in diseases characterized by liver inflammation compared to healthy controls. Using PBC as a focus, there was a significant correlation between levels of circulating MDSCs and disease-related biochemical markers (alkaline phosphatase, total bilirubin). We found higher amounts of MDSCs in patients with PBC who were responsive to ursodeoxycholic acid. MDSCs from PBC were found to manifest a potent immunosuppressive function. There was a significant correlation in the accumulation of hepatic MDSCs in the inflamed lesions of PBC with histologic changes, such as fibrosis. We also found that cysteine-rich protein 61 (CCN1), a highly expressed protein in impaired cholangiocytes and hepatocytes, contributes to MDSC expansion and MDSC inducible nitric oxide synthase-associated immune suppression., Conclusion: CCN1 modulates expansion and a suppressive function of MDSCs. Our data highlight the potential functions of CCN1 on MDSCs and suggest therapeutic implications in inflammatory liver diseases. (Hepatology HEPATOLOGY 2018;67:232-246)., (© 2017 by the American Association for the Study of Liver Diseases.)

There is increased interest and recognition of the clinical variants of Sclerosing Cholangitis (SC) namely IgG4-SC, PSC/AIH overlap and PSC. For most Centers, the characteristic of IgG4-SC has not been thoroughly clinically compared with other sclerosing cholangitis variants. Further there are relatively few PSC/AIH overlap patients and the clinical outcome is not well characterized, especially for the PSC/AIH overlap syndrome. Our objective herein is to clarify the differences and similarities of the natural history of IgG4-SC, the PSC/AIH overlap and PSC alone. We also place in perspective the diagnostic value of serum IgG4 for IgG4-SC and investigate biomarkers for predicting the prognosis of sclerosing cholangitis. In this study, we took advantage of our large and well-defined patient cohort to perform a retrospective cohort study including 57 IgG4-SC, 36 PSC/AIH overlap patients, and 55 PSC patients. Firstly, as expected, we noted significant differences among immunoglobulin profiles and all patients exhibited similar cholestatic profiles at presentation. Cirrhotic events were found in 20 of total 57 IgG4-SC, 15 of 36 PSC/AIH overlap, and 18 of 55 PSC patients. Serum IgG4 was elevated in 92.65% of IgG4-SC patients with an 86% sensitivity and 98% specificity for diagnosis. IgG4-SC patients had a better treatment response at 6-month and 1-year than PSC/AIH patients, while the latter responded better with steroids than PSC patients. Importantly the adverse outcome-free survival of IgG4-SC patients was reduced, unlike earlier reports, and therefore similar to the PSC/AIH overlap syndrome. Serum IgG and total bilirubin were useful to predict long-term survival of IgG4-SC and PSC/AIH, respectively. In conclusion, serum IgG4≧1.25 ULN shows an excellent predictability to distinguish IgG4-SC among SC patients. IgG4-SC appears to be immune-mediated inflammatory process, while PSC/AIH overlap more tends to be cholestatic disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)