Gut Microbiota-Derived Butyrate Induces Epigenetic and Metabolic Reprogramming in Myeloid-Derived Suppressor Cells to Alleviate Primary Biliary Cholangitis.

BACKGROUND AND AIMS: Gut dysbiosis and myeloid-derived suppressor cells (MDSCs) are implicated in primary biliary cholangitis (PBC) pathogenesis. However, it remains unknown whether gut microbiota or their metabolites can modulate MDSCs homeostasis to rectify immune dysregulation in PBC. METHODS: We measured fecal short-chain fatty acids levels using targeted gas chromatography-mass spectrometry and analyzed circulating MDSCs using flow cytometry in 2 independent PBC cohorts. Human and murine MDSCs were differentiated in vitro in the presence of butyrate, followed by transcriptomic, epigenetic (CUT&Tag-seq and chromatin immunoprecipitation-quantitative polymerase chain reaction), and metabolic (untargeted liquid chromatography-mass spectrometry, mitochondrial stress test, and isotope tracing) analyses. The in vivo role of butyrate-MDSCs was evaluated in a 2-octynoic acid-bovine serum albumin-induced cholangitis murine model. RESULTS: Decreased butyrate levels and defective MDSCs function were found in patients with incomplete response to ursodeoxycholic acid, compared with those with adequate response. Butyrate induced expansion and suppressive activity of MDSCs in a manner dependent on PPARD-driven fatty acid ß-oxidation (FAO). Pharmaceutical inhibition or genetic knockdown of the FAO rate-limiting gene CPT1A abolished the effect of butyrate. Furthermore, butyrate inhibited HDAC3 function, leading to enhanced acetylation of lysine 27 on histone 3 modifications at promoter regions of PPARD and FAO genes in MDSCs. Therapeutically, butyrate administration alleviated immune-mediated cholangitis in mice via MDSCs, and adoptive transfer of butyrate-treated MDSCs also displayed protective efficacy. Importantly, reduced expression of FAO genes and impaired mitochondrial physiology were detected in MDSCs from ursodeoxycholic acid nonresponders, and their impaired suppressive function was restored by butyrate. CONCLUSIONS: We identify a critical role for butyrate in modulation of MDSC homeostasis by orchestrating epigenetic and metabolic crosstalk, proposing a novel therapeutic strategy for treating PBC.

Itaconate inhibits CD103 + T RM cells and alleviates hepatobiliary injury in mouse models of primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic progressive liver disease characterized by the infiltration of intrahepatic tissue-resident memory CD8 + T cells (T RM ). Itaconate has demonstrated therapeutic potential in modulating inflammation. An unmet need for PSC is the reduction of biliary inflammation, and we hypothesized that itaconate may directly modulate pathogenic T RM . APPROACH AND RESULTS: The numbers of intrahepatic CD103 + T RM were evaluated by immunofluorescence in PSC (n = 32), and the serum levels of itaconate in PSC (n = 64), primary biliary cholangitis (PBC) (n = 60), autoimmune hepatitis (AIH) (n = 49), and healthy controls (n = 109) were determined by LC-MS/MS. In addition, the frequencies and immunophenotypes of intrahepatic T RM using explants from PSC (n = 5) and healthy donors (n = 6) were quantitated by flow cytometry. The immunomodulatory properties of 4-octyl itaconate (4-OI, a cell-permeable itaconate derivative) on CD103 + T RM were studied in vitro. Finally, the therapeutic potential of itaconate was studied by the administration of 4-OI and deficiency of immune-responsive gene 1 (encodes the aconitate decarboxylase producing itaconate) in murine models of PSC. Intrahepatic CD103 + T RM was significantly expanded in PSC and was positively correlated with disease severity. Serum itaconate levels decreased in PSC. Importantly, 4-OI inhibited the induction and effector functions of CD103 + T RM in vitro. Mechanistically, 4-OI blocked DNA demethylation of RUNX3 in CD8 + T cells. Moreover, 4-OI reduced intrahepatic CD103 + T RM and ameliorated liver injury in murine models of PSC. CONCLUSIONS: Itaconate exerted immunomodulatory activity on CD103 + T RM in both in vitro and murine PSC models. Our study suggests that targeting pathogenic CD103 + T RM with itaconate has therapeutic potential in PSC.

P4HA2 induces hepatic ductular reaction and biliary fibrosis in chronic cholestatic liver diseases.

BACKGROUNDS: Prolyl-4-hydroxylases (P4Hs) are key enzymes in collagen synthesis. The P4HA subunit (P4HA1, P4HA2, and P4HA3) contains a substrate binding and catalyzation domain. We postulated that P4HA2 would play a key role in the cholangiocyte pathology of cholestatic liver diseases. METHODS: We studied humans with primary biliary cholangitis (PBC) and Primary sclerosing cholangitis (PSC), P4HA2 -/- mice injured by DDC, and P4HA2 -/- /MDR2 -/- double knockout mice. A parallel study was performed in patients with PBC, PSC, and controls using immunohistochemistry and immunofluorescence. In the murine model, the level of ductular reaction and biliary fibrosis were monitored by histology, qPCR, immunohistochemistry, and Western blotting. Expression of Yes1 Associated Transcriptional Regulator (YAP) phosphorylation was measured in isolated mouse cholangiocytes. The mechanism of P4HA2 was explored in RBE and 293T cell lines by using qPCR, Western blot, immunofluorescence, and co-immunoprecipitation. RESULTS: The hepatic expression level of P4HA2 was highly elevated in patients with PBC or PSC. Ductular reactive cholangiocytes predominantly expressed P4HA2. Cholestatic patients with more severe liver injury correlated with levels of P4HA2 in the liver. In P4HA2 -/- mice, there was a significantly reduced level of ductular reaction and fibrosis compared with controls in the DDC-induced chronic cholestasis. Decreased liver fibrosis and ductular reaction were observed in P4HA2 -/- /MDR2 -/- mice compared with MDR2 -/- mice. Cholangiocytes isolated from P4HA2 -/- /MDR2 -/- mice displayed a higher level of YAP phosphorylation, resulting in cholangiocytes proliferation inhibition. In vitro studies showed that P4HA2 promotes RBE cell proliferation by inducing SAV1 degradation, eventually resulting in the activation of YAP. CONCLUSIONS: P4HA2 promotes hepatic ductular reaction and biliary fibrosis by regulating the SAV1-mediated Hippo signaling pathway. P4HA2 is a potential therapeutic target for PBC and PSC.

Suppression of YTHDF2 attenuates autoimmune hepatitis by expansion of myeloid-derived suppressor cells.

BACKGROUND & AIMS: The N6-methyladenosine (m6A) reader YTH domain-containing family protein 2 (YTHDF2) is critically involved in a multiplicity of biological processes by mediating the degradation of m6A 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 Ythdf2CKO mice had a greater suppressive ability to inhibit the proliferation of T cells. Using multi-omic analysis of m6A 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.

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.

A regulatory variant at 19p13.3 is associated with primary biliary cholangitis risk and ARID3A expression.

Genome-wide association studies have identified 19p13.3 locus associated with primary biliary cholangitis (PBC). Here we aim to identify causative variant(s) and initiate efforts to define the mechanism by which the 19p13.3 locus variant(s) contributes to the pathogenesis of PBC. A genome-wide meta-analysis of 1931 PBC subjects and 7852 controls in two Han Chinese cohorts confirms the strong association between 19p13.3 locus and PBC. By integrating functional annotations, luciferase reporter assay and allele-specific chromatin immunoprecipitation, we prioritize rs2238574, an AT-Rich Interaction Domain 3A (ARID3A) intronic variant, as a potential causal variant at 19p13.3 locus. The risk allele of rs2238574 shows higher binding affinity of transcription factors, leading to an increased enhancer activity in myeloid cells. Genome-editing demonstrates the regulatory effect of rs2238574 on ARID3A expression through allele-specific enhancer activity. Furthermore, knock-down of ARID3A inhibits myeloid differentiation and activation pathway, and overexpression of the gene has the opposite effect. Finally, we find ARID3A expression and rs2238574 genotypes linked to disease severity in PBC. Our work provides several lines of evidence that a non-coding variant regulates ARID3A expression, presenting a mechanistic basis for association of 19p13.3 locus with the susceptibility to PBC.

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.