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.

Immunosuppression induces regression of fibrosis in primary biliary cholangitis with moderate-to-severe interface hepatitis.

BACKGROUND: In patients with primary biliary cholangitis (PBC) treated with ursodeoxycholic acid (UDCA), the presence of moderate-to-severe interface hepatitis is associated with a higher risk of liver transplantation and death. This highlights the need for novel treatment approaches. In this study, we aimed to investigate whether combination therapy of UDCA and immunosuppressant (IS) was more effective than UDCA monotherapy. METHODS: We conducted a multicenter study involving PBC patients with moderate-to-severe interface hepatitis who underwent paired liver biopsies. Firstly, we compared the efficacy of the combination therapy with UDCA monotherapy on improving biochemistry, histology, survival rates, and prognosis. Subsequently we investigated the predictors of a beneficial response. RESULTS: This retrospective cohort study with prospectively collected data was conducted in China from January 2009 to April 2023. Of the 198 enrolled patients, 32 underwent UDCA monotherapy, while 166 received combination therapy, consisting of UDCA combined with prednisolone, prednisolone plus mycophenolate mofetil (MMF), or prednisolone plus azathioprine (AZA). The monotherapy group was treated for a median duration of 37.6 months (IQR 27.5-58.1), and the combination therapy group had a median treatment duration of 39.3 months (IQR 34.5-48.8). The combination therapy showed a significantly greater efficacy in reducing fibrosis compared to UDCA monotherapy, with an 8.3-fold increase in the regression rate (from 6.3% to 52.4%, P < 0.001). Other parameters, including biochemistry, survival rates, and prognosis, supported its effectiveness. Baseline IgG >1.3 × ULN and ALP <2.4 × ULN were identified as predictors of regression following the combination therapy. A predictive score named FRS, combining these variables, accurately identified individuals achieving fibrosis regression with a cut-off point of ≥ -0.163. The predictive value was validated internally and externally. CONCLUSION: Combination therapy with IS improves outcomes in PBC patients with moderate-to-severe interface hepatitis compared to UDCA monotherapy. Baseline IgG and ALP are the most significant predictors of fibrosis regression. The new predictive score, FRS, incorporating baseline IgG and ALP, can effectively identify individuals who would benefit from the combination therapy.

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.

The impact of PET/CT and brain MRI for metastasis detection among patients with clinical T1-category lung cancer: Findings from a large-scale cohort study.

PURPOSE: [18F]-FDG PET/CT and brain MRI are common approaches to detect metastasis in patients of lung cancer. Current guidelines for the use of PET/CT and MRI in clinical T1-category lung cancer lack risk-based stratification and require optimization. This study stratified patients based on metastatic risk in terms of the lesions' size and morphological characteristics. METHODS: The detection rate of metastasis was measured in different sizes and morphological characteristics (solid and sub-solid) of tumors. To confirm the cut-off value for discriminating metastasis and overall survival (OS) prediction, the receiver operating characteristic (ROC) analysis was performed based on PET/CT metabolic parameters (SUVmax/SUVmean/SULpeak/MTV/TLG), followed by Kaplan-Meier analysis for survival in post-operation patients with and without PET/CT plus MRI. RESULTS: 2,298 patients were included. No metastasis was observed in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm. The cut-off of PET/CT metabolic parameters on discriminating metastasis were 1.09 (SUVmax), 0.26 (SUVmean), 0.31 (SULpeak), 0.55 (MTV), and 0.81 (TLG), respectively. Patients undergoing PET/CT plus MRI exhibited longer OS compared to those who did not receive it in solid nodules ≥ 8.0 mm & sub-solid nodules ≥ 10.0 mm (HR, 0.44; p < 0.001); in solid nodules ≥ 8.0 mm (HR, 0.12; p<0.001) and in sub-solid nodules ≥ 10.0 mm (HR; 0.61; p=0.075), respectively. Compared to patients with metabolic parameters lower than cut-off values, patients with higher metabolic parameters displayed shorter OS: SUVmax (HR, 12.94; p < 0.001), SUVmean (HR, 11.33; p <0.001), SULpeak (HR, 9.65; p < 0.001), MTV (HR, 9.16; p = 0.031), and TLG (HR, 12.06; p < 0.001). CONCLUSION: The necessity of PET/CT and MRI should be cautiously evaluated in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm, however, these examinations remained essential and beneficial for patients with solid nodules ≥ 8.0 mm and sub-solid nodules ≥ 10.0 mm.

Brain-derived neurotrophic factor regulates LYN kinase-mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons.

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

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.

Genome-wide meta-analysis identifies susceptibility loci for autoimmune hepatitis type 1.

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.

A surgical Decision-making scoring model for spontaneous ventilation- and mechanical ventilation-video-assisted thoracoscopic surgery in non-small-cell lung cancer patients.

BACKGROUNDS: Spontaneous ventilation-video-assisted thoracoscopic surgery (SV-VATS) has been applied to non-small cell lung cancer (NSCLC) patients in many centers. Since it remains a new and challenging surgical technique, only selected patients can be performed SV-VATS. We aim to conduct a retrospective single-center study to develop a clinical decision-making model to make surgery decision between SV-VATS and MV (mechanical ventilation) -VATS in NSCLC patients more objectively and individually. METHODS: Four thousand three hundred sixty-eight NSCLC patients undergoing SV-VATS or MV-VATS in the department of thoracic surgery between 2011 and 2018 were included. Univariate and multivariate regression analysis were used to identify potential factors influencing the surgical decisions. Factors with statistical significance were selected for constructing the Surgical Decision-making Scoring (SDS) model. The performance of the model was validated by area under the receiver operating characteristic curve (AUC), calibration curves and decision curve analysis (DCA). RESULTS: The Surgical Decision-making Scoring (SDS) model was built guided by the clinical judgment and statistically significant results of univariate and multivariate regression analyses of potential predictors, including smoking status (p = 0.03), BMI (p < 0.001), ACCI (p = 0.04), T stage (p < 0.001), N stage (p < 0.001), ASA grade (p < 0.001) and surgical technique (p < 0.001). The AUC of the training group and the testing group were 0.72 and 0.70, respectively. The calibration curves and the DCA curve revealed that the SDS model has a desired performance in predicting the surgical decision. CONCLUSIONS: This SDS model is the first clinical decision-making model developed for an individual NSCLC patient to make decision between SV-VATS and MV-VATS.

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.

Morphological, Functional, and Tissue Characterization of Silent Myocardial Involvement in Patients With Primary Biliary Cholangitis.

BACKGROUND & AIMS: Cirrhotic cardiomyopathy is a major complication and cause of morbidity in end-stage primary biliary cholangitis (PBC). However, it is unclear whether there is clinically silent myocardial involvement at the early stage of PBC before cirrhosis and cardiac manifestations. This prospective, three-center, multi-modality cardiac imaging study on the early identification of myocardial impairment in PBC (EARLY-MYO-PBC) was designed to identify silent myocardial impairment in PBC patients without cardiac manifestations. METHODS: A total of 112 subjects (56 with PBC and 56 age- and sex-matched controls) undergoing cardiovascular magnetic resonance (CMR) were enrolled. Demographic, serologic, and cardiac imaging data were prospectively collected. All participants had no cardiac discomfort or previous heart disease and had normal electrocardiographic findings. RESULTS: Subclinical myocardial involvement, as evidenced by cardiac morphologic, functional, and tissue characterization changes on CMR, including hyperdynamic left ventricular (LV) ejection fraction (median, 75% in PBC patients vs 69% in controls, P = .029), subclinical myocardial edema by T2-short tau inversion recovery (21% vs 2% in controls, P = .001), elevated extracellular matrix indices (30% vs 26% in controls, P < .001), and impaired myocardial viability by positive late gadolinium enhancement (LGE) (36%), was detected in PBC patients. Importantly, a mid-wall "stripe" at the LV septum was identified as a PBC-specific LGE pattern that differs from other known cardiomyopathies. In multivariate analysis, gp210 positivity (odds ratio [OR] = 9.909, P = .010), lower hemoglobin (OR = 0.919, P = .004), and body mass index (OR = 0.638, P = .005) were independent predictors of cardiac abnormalities in PBC. CONCLUSIONS: This study demonstrates clinically silent cardiac impairment with specific CMR patterns in PBC, allowing optimal screening for early myocardial impairment and potentially timely therapies. (Trial registration no.: NCT03545672).

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.

Hysteresis-free MoS2 metal semiconductor field-effect transistors with van der Waals Schottky junction.

Hysteresis-free and steep subthreshold swing (SS) are essential for low-power reliable electronics. Herein, MoS2 metal semiconductor field-effect transistors are fabricated with GeSe/MoS2 van der Waals Schottky junction as a local gate, in which the rectification behavior of the heterojunction offers the modulation of channel carriers. The trap-free gate interface enables the hysteresis-free characteristics of the transistors, and promises an ideal SS of 64 mV/dec at room temperature. All the devices operate with a low threshold voltage less than -1 V with desirable saturation behavior. An OR logic gate is constructed with the dual-gated MoS2 transistors by varying the back and top gate voltage. The strategy present here is promising for the design of low-power digital electronics based on 2D materials.

Efficacy and safety of rifampicin in patients with persistent hepatocellular secretory failure.

BACKGROUND AND AIM: Persistent hepatocellular secretory failure (PHSF) is a rare condition of severe cholestasis caused by drugs, toxins, infection, or temporary biliary obstruction. Real-world data on rifampicin in cholestasis, particularly among patients with deep jaundice, are scarce. We aimed to expand the knowledge on the efficacy and safety of rifampicin treatment in PHSF patients. METHODS: Sixteen patients with PHSF who had received rifampicin treatment (150-300 mg/d) at our institution from September 2016 to July 2020 were included. Treatment efficacy was assessed by 20% improvement in serum total bilirubin (TBIL) concentration at 4 weeks. Follow-up was continued until the concentration of TBIL returned to normal. RESULTS: Among the 16 enrolled patients, 12 had predisposing factors (drugs, infection, or transient biliary obstruction). ATP8B1 or ABCB11 mutations were detected in the other four patients without trigger events. UGT1A1 mutations were found in 7/10 patients. Before rifampicin treatment, the median TBIL level was 352 µmol/L (range 171-591 µmol/L). TBIL > 20% improvement was observed in 14 patients at 4 weeks. TBIL levels of 14 patients eventually returned to normal after 6-12 weeks of rifampicin treatment. The remaining two patients who did not respond to rifampicin finally recovered after nasobiliary drainage. Except for one patient with transient drug-induced hepatitis, no other serious adverse events were observed. CONCLUSIONS: Rifampicin could be a promising option for most PHSF patients. Most PHSF patients have UGT1A1 deficiency, which may be the target of rifampicin.

Alterations of gut microbiome in autoimmune hepatitis.

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.

Cytotoxic KLRG1 expressing lymphocytes invade portal tracts in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease with an immunopathogenesis that includes highly differentiated cytotoxic T cell infiltration in portal areas. We have taken advantage of a large and well-defined cohort of patients with PBC, AIH, chronic hepatitis virus, and healthy controls to study for the presence of highly differentiated T cells which express the killer cell lectin-like receptor G1 (KLRG1). Such studies were performed using both liver and peripheral blood mononuclear cells. In particular, gene expression data (GSE79850) from 16 PBC patients stratified according to future risk of liver transplantation were analyzed for markers of highly differentiated cytotoxic T cells. Liver biopsy samples from 44 PBC patients were studied by immunohistochemistry and a separate cohort of PBC blood samples were studied by flow cytometry. Gene expression data demonstrated correlation of increased KLRG1 and cytotoxic lymphocyte molecules, such as granzyme B (GZMB) and perforin (PRF1), to disease severity as measured by future risk of liver transplantation. Immunohistochemistry demonstrated abundant infiltration of KLRG1+ cells into liver portal areas (mean of 45% of infiltrating cells, range 25-75%) positively correlated with hepatic inflammatory (r = 0.47, p = 0.001) and hepatic fibrosis (r = 0.34, p = 0.021) scores. KLRG1+ lymphocyte liver portal area infiltration was positively correlated with serum alkaline phosphatase (r = 0.45, p = 0.005) and GGT (r = 0.40, p = 0.014), and AST (r = 0.35, p = 0.033) levels. Mononuclear blood flow cytometry studies showed KLRG1+ lymphocytes had greater levels of cytotoxic molecules (granzyme B and perforin), inflammatory cytokines (IFN-γ and TNF-α) and inflammatory chemokine receptors (CCR5 and CX3CR1) than KLRG1-counterparts. However, clearly the most significant data was that found in liver with the intense portal infiltrates that are unique to PBC. Conclusion: Highly cytotoxic KLRG1+ lymphocytes have invaded PBC liver portal areas. Liver KLRG1 gene expression and the abundance of KLRG1+ lymphocytes are positively correlated with disease biomarkers used as clinical trial outcome measures (liver transplantation and serum alkaline phosphatase), suggesting the targeting of KLRG1+ lymphocytes as a rational approach for PBC therapeutic drug development.

Histologically proven AMA positive primary biliary cholangitis but normal serum alkaline phosphatase: Is alkaline phosphatase truly a surrogate marker?

BACKGROUND AND AIMS: The most highly directed and specific autoantibody in human immunopathology is the serologic hallmark of primary biliary cholangitis (PBC), antimitochondrial antibodies (AMAs). However the clinical significance of finding a positive AMA, with normal alkaline phosphatase (ALP) remains enigmatic. METHODS: We took advantage of 169 consecutive outpatients who were identified as having a positive AMA, but normal ALP levels between January 2012 and January 2018. A liver biopsy was performed on 67/169 of these AMA positive normal ALP patients. RESULTS: In all 169 patients we reconfirmed the AMA and also performed anti-gp210 and anti-sp100, liver stiffness (LSM) assessed by vibration-controlled transient elastography (VCTE), an abdominal computed tomography (CT) scan, and either a magnetic resonance imaging (MRI) or ultrasound. The liver biopsies were reviewed by two unbiased observers. 87.6% of the 169 patients were females with a mean age of 46; the median AMA titer 1:320; an elevated serum IgM was found in 53.3%. Importantly, in patients with a liver biopsy, 55（82.1%）out of 67 had varying degrees of cholangitis activity, diagnostic of PBC. CONCLUSION: In patients who were AMA-positive but had normal ALP levels, more than 80% were associated with histological classic PBC. These data emphasize the importance of a positive AMA, even with a normal ALP and also question the role of ALP as a sole surrogate marker of cholangitis.

A functional characteristic of cysteine-rich protein 61: Modulation of myeloid-derived suppressor cells in liver inflammation.

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).

The Immunobiology of Receptor Activator for Nuclear Factor Kappa B Ligand and Myeloid-Derived Suppressor Cell Activation in Immunoglobulin G4-Related Sclerosing Cholangitis.

The primary function of myeloid-derived suppressor cells (MDSCs) is reflected in their immune modulatory role in several immune-mediated diseases. In immunoglobulin G4 (IgG4)-related disease (IgG4-RD), it has been hypothesized that there are selective regulatory defects that lead to a T helper 2 (Th2) bias immune response. Herein we have taken advantage of a large cohort of patients with IgG4-related sclerosing cholangitis (IgG4-SC), the most common extrapancreatic involvement of IgG4-RD, as well as controls consisting of primary sclerosing cholangitis, autoimmune hepatitis, and healthy volunteers, to study MDSCs. We report dramatically increased levels of receptor activator for nuclear factor kappa B ligand (RANKL) expression in serum and liver from patients with IgG4-SC compared to both liver-disease and healthy controls. Moreover, in IgG4-SC liver, RANKL-secreting cells specifically colocalized with cluster of differentiation 38-positive plasma cells and MDSCs, particularly monocytic MDSCs, and express the RANKL receptor in liver. Similarly, the frequency and number of peripheral blood MDSCs were significantly increased. Importantly, serum expression levels of RANKL were inversely correlated with the serum level of gamma-glutamyltransferase but significantly positively correlated with the frequency of MDSCs. Moreover, we confirmed that RANKL induced the expansion and activation of MDSCs through the RANKL/RANK/nuclear factor kappa B signal pathway. Of note, RANKL-treated MDSCs suppressed T-cell proliferation and induced Th2 differentiation. Conclusion: Our data suggest that plasma cell-derived RANKL induces the expansion and activation of MDSCs, which suppress T-cell proliferation and contribute to the Th2-type response characteristic of IgG4-SC.