A+T rich interaction domain protein 3a (Arid3a) impairs Mertk-mediated efferocytosis in cholestasis.

BACKGROUND & AIMS: Macrophages are key elements in the pathogenesis of cholestatic liver diseases. Arid3a plays a prominent role in the biologic properties of hematopoietic stem cells, B lymphocytes and tumor cells, but its ability to modulate macrophage function during cholestasis remains unknown. METHODS: Gene and protein expression and cellular localization were assessed by q-PCR, immunohistochemistry, immunofluorescence staining and flow cytometry. We generated myeloid-specific Arid3a knockout mice and established three cholestatic murine models. The transcriptome was analyzed by RNA-seq. A specific inhibitor of the Mertk receptor was used in vitro and in vivo. Promoter activity was determined by chromatin immunoprecipitation-seq against Arid3a and a luciferase reporter assay. RESULTS: In cholestatic murine models, myeloid-specific deletion of Arid3a alleviated cholestatic liver injury (accompanied by decreased accumulation of macrophages). Arid3a-deficient macrophages manifested a more reparative phenotype, which was eliminated by in vitro treatment with UNC2025, a specific inhibitor of the efferocytosis receptor Mertk. Efferocytosis of apoptotic cholangiocytes was enhanced in Arid3a-deficient macrophages via upregulation of Mertk. Arid3a negatively regulated Mertk transcription by directly binding to its promoter. Targeting Mertk in vivo effectively reversed the protective phenotype of Arid3a deficiency in macrophages. Arid3a was upregulated in hepatic macrophages and circulating monocytes in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Mertk was correspondingly upregulated and negatively correlated with Arid3a expression in PBC and PSC. Mertk+ cells were located in close proximity to cholangiocytes, while Arid3a+ cells were scattered among immune cells with greater spatial distances to hyperplastic cholangiocytes in PBC and PSC. CONCLUSIONS: Arid3a promotes cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes by macrophages during cholestasis. The Arid3a-Mertk axis is a promising novel therapeutic target for cholestatic liver diseases. IMPACT AND IMPLICATIONS: Macrophages play an important role in the pathogenesis of cholestatic liver diseases. This study reveals that macrophages with Arid3a upregulation manifest a pro-inflammatory phenotype and promote cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes during cholestasis. Although we now offer a new paradigm to explain how efferocytosis is regulated in a myeloid cell autonomous manner, the regulatory effects of Arid3a on chronic liver diseases remain to be further elucidated.

Altered faecal microbiome and metabolome in IgG4-related sclerosing cholangitis and primary sclerosing cholangitis.

OBJECTIVE: Multiple clinical similarities exist between IgG4-related sclerosing cholangitis (IgG4-SC) and primary sclerosing cholangitis (PSC), and while gut dysbiosis has been extensively studied in PSC, the role of the gut microbiota in IgG4-SC remains unknown. Herein, we aimed to evaluate alterations of the gut microbiome and metabolome in IgG4-SC and PSC. DESIGN: We performed 16S rRNA gene amplicon sequencing of faecal samples from 135 subjects with IgG4-SC (n=34), PSC (n=37) and healthy controls (n=64). A subset of the samples (31 IgG4-SC, 37 PSC and 45 controls) also underwent untargeted metabolomic profiling. RESULTS: Compared with controls, reduced alpha-diversity and shifted microbial community were observed in IgG4-SC and PSC. These changes were accompanied by differences in stool metabolomes. Importantly, despite some common variations in the microbiota composition and metabolic activity, integrative analyses identified distinct host-microbe associations in IgG4-SC and PSC. The disease-associated genera and metabolites tended to associate with the transaminases in IgG4-SC. Notable depletion of Blautia and elevated succinic acid may underlie hepatic inflammation in IgG4-SC. In comparison, potential links between the microbial or metabolic signatures and cholestatic parameters were detected in PSC. Particularly, concordant decrease of Eubacterium and microbiota-derived metabolites, including secondary bile acids, implicated novel host-microbial metabolic pathways involving cholestasis of PSC. Interestingly, the predictive models based on metabolites were more effective in discriminating disease status than those based on microbes. CONCLUSIONS: Our data reveal that IgG4-SC and PSC possess divergent host-microbe interplays that may be involved in disease pathogenesis. These data emphasise the uniqueness of IgG4-SC.

NUDT1 promotes the accumulation and longevity of CD103+ TRM cells in primary biliary cholangitis.

BACKGROUND & AIMS: Pyruvate dehydrogenase (PDC)-E2 specific CD8+ T cells play a leading role in biliary destruction in PBC. However, there are limited data on the characterization of these autoantigen-specific CD8+ T cells, particularly in the liver. Herein, we aimed to identify pathogenic intrahepatic CD8+ T-cell subpopulations and investigate their immunobiology in PBC. METHODS: Phenotypic and functional analysis of intrahepatic T-cell subsets were performed by flow cytometry. CD103+ TRM cell frequency was evaluated by histological staining. The transcriptome and metabolome were analyzed by RNA-seq and liquid chromatography-mass spectrometry, respectively. Cytotoxicity of TRM cells against cholangiocytes was assayed in a 3D organoid co-culture system. Moreover, the longevity (long-term survival) of TRM cells in vivo was studied by 2-octynoic acid-BSA (2OA-BSA) immunization, Nudt1 conditional knock-out and adoptive co-transfer in a murine model. RESULTS: Intrahepatic CD103+ TRM (CD69+CD103+CD8+) cells were significantly expanded, hyperactivated, and potentially specifically reactive to PDC-E2 in patients with PBC. CD103+ TRM cell frequencies correlated with clinical and histological indices of PBC and predicted poor ursodeoxycholic acid response. NUDT1 blockade suppressed the cytotoxic effector functions of CD103+ TRM cells upon PDC-E2 re-stimulation. NUDT1 overexpression in CD8+ T cells promoted tissue-residence programming in vitro; inhibition or knockdown of NUDT1 had the opposite effect. Pharmacological blockade or genetic deletion of NUDT1 eliminated CD103+ TRM cells and alleviated cholangitis in mice immunized with 2OA-BSA. Significantly, NUDT1-dependent DNA damage resistance potentiates CD8+ T-cell tissue-residency via the PARP1-TGFßR axis in vitro. Consistently, PARP1 inhibition restored NUDT1-deficient CD103+ TRM cell durable survival and TGFß-Smad signaling. CONCLUSIONS: CD103+ TRM cells are the dominant population of PDC-E2-specific CD8+ T lymphocytes in the livers of patients with PBC. The role of NUDT1 in promoting pathogenic CD103+ TRM cell accumulation and longevity represents a novel therapeutic target in PBC. LAY SUMMARY: Primary biliary cholangitis (PBC) is a rare inflammatory condition of the bile ducts. It can be treated with ursodeoxycholic acid, but a large percentage of patients respond poorly to this treatment. Liver-infiltrating memory CD8+ T cells recognizing the PDC-E2 immunodominant epitope are critical in the pathogenesis of PBC. We identifed the key pathogenic CD8+ T cell subset, and worked out the mechanisms of its hyperactivation and longevity, which could be exploited therapeutically.

The Clinical Significance of Hepatic CD69+ CD103+ CD8+ Resident-Memory T Cells in Autoimmune Hepatitis.

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 (TRM ) 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+ TRM cells and AIH disease severity. Indeed, in patients with successful responses to immunosuppression, the frequencies of such hepatic CD8+ TRM cells decreased significantly. CD69+ CD8+ and CD69+ CD103+ CD8+ T cells, also known as CD8+ TRM 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-ß on inflammatory cells, and extensive hepatic expression of E-cadherin; these factors likely contribute to the development and localization of CD8+ TRM cells. Based on these data and, in particular, the relationships between disease severity and CD8+ TRM cells, we studied the mechanisms involved with glucocorticoid (GC) modulation of CD8+ TRM cell expansion. Our data reflect that GCs in vitro inhibit the expansion of CD8+ TRM cells induced by IL-15 and TGF-ß and with direct down-regulation of the nuclear factor Blimp1 of CD8+ TRM cells. CONCLUSIONS: Our data suggest that CD8+ TRM cells play a critical role in the pathogenesis of AIH, and GCs attenuate hepatic inflammation through direct inhibition of CD8+ TRM cell expansion.

Serum Biomarkers for Autoimmune Hepatitis Type 1: the Case for CD48 and a Review of the Literature.

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.

MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells.

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.