Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation.

BACKGROUND AND AIMS: Inflammatory response is crucial for bile acid (BA)-induced cholestatic liver injury, but molecular mechanisms remain to be elucidated. Solute Carrier Family 35 Member C1 (SLC35C1) can transport Guanosine diphosphate-fucose into the Golgi to facilitate protein glycosylation. Its mutation leads to the deficiency of leukocyte adhesion and enhances inflammation in humans. However, little is known about its role in liver diseases. APPROACH AND RESULTS: Hepatic SLC35C1 mRNA transcripts and protein expression were significantly increased in patients with obstructive cholestasis and mouse models of cholestasis. Immunofluorescence revealed that the upregulated SLC35C1 expression mainly occurred in hepatocytes. Liver-specific ablation of Slc35c1 ( Slc35c1 cKO ) significantly aggravated liver injury in mouse models of cholestasis induced by bile duct ligation and 1% cholic acid-feeding, evidenced by increased liver necrosis, inflammation, fibrosis, and bile ductular proliferation. The Slc35c1 cKO increased hepatic chemokine Ccl2 and Cxcl2 expression and T cell, neutrophil, and F4/80 macrophage infiltration but did not affect the levels of serum and liver BA in mouse models of cholestasis. Liquid chromatography with tandem mass spectrometry analysis revealed that hepatic Slc35c1 deficiency substantially reduced the fucosylation of cell-cell adhesion protein CEACAM1 at N153. Mechanistically, cholestatic levels of conjugated BAs stimulated SLC35C1 expression by activating the STAT3 signaling to facilitate CEACAM1 fucosylation at N153, and deficiency in the fucosylation of CEACAM1 at N135 enhanced the BA-stimulated CCL2 and CXCL2 mRNA expression in primary mouse hepatocytes and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5- ASBT cells. CONCLUSIONS: Elevated hepatic SLC35C1 expression attenuates cholestatic liver injury by enhancing CEACAM1 fucosylation to suppress CCL2 and CXCL2 expression and liver inflammation.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

BACKGROUND AND AIMS: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. APPROACH AND RESULTS: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. CONCLUSIONS: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

Outcome of COVID-19 in Patients With Autoimmune Hepatitis: An International Multicenter Study.

BACKGROUND AND AIMS: Data regarding outcome of COVID-19 in patients with autoimmune hepatitis (AIH) are lacking. APPROACH AND RESULTS: We performed a retrospective study on patients with AIH and COVID-19 from 34 centers in Europe and the Americas. We analyzed factors associated with severe COVID-19 outcomes, defined as the need for mechanical ventilation, intensive care admission, and/or death. The outcomes of patients with AIH were compared to a propensity score-matched cohort of patients without AIH but with chronic liver diseases (CLD) and COVID-19. The frequency and clinical significance of new-onset liver injury (alanine aminotransferase > 2 × the upper limit of normal) during COVID-19 was also evaluated. We included 110 patients with AIH (80% female) with a median age of 49 (range, 18-85) years at COVID-19 diagnosis. New-onset liver injury was observed in 37.1% (33/89) of the patients. Use of antivirals was associated with liver injury (P = 0.041; OR, 3.36; 95% CI, 1.05-10.78), while continued immunosuppression during COVID-19 was associated with a lower rate of liver injury (P = 0.009; OR, 0.26; 95% CI, 0.09-0.71). The rates of severe COVID-19 (15.5% versus 20.2%, P = 0.231) and all-cause mortality (10% versus 11.5%, P = 0.852) were not different between AIH and non-AIH CLD. Cirrhosis was an independent predictor of severe COVID-19 in patients with AIH (P < 0.001; OR, 17.46; 95% CI, 4.22-72.13). Continuation of immunosuppression or presence of liver injury during COVID-19 was not associated with severe COVID-19. CONCLUSIONS: This international, multicenter study reveals that patients with AIH were not at risk for worse outcomes with COVID-19 than other causes of CLD. Cirrhosis was the strongest predictor for severe COVID-19 in patients with AIH. Maintenance of immunosuppression during COVID-19 was not associated with increased risk for severe COVID-19 but did lower the risk for new-onset liver injury during COVID-19.

Role of Biliary Organoids in Cholestasis Research and Regenerative Medicine.

Translational studies in human cholestatic diseases have for years been hindered by various challenges, including the rarity of the disorders, the difficulty in obtaining biliary tissue from across the spectrum of the disease stage, and the difficulty culturing and maintaining primary cholangiocytes. Organoid technology is increasingly being viewed as a technological breakthrough in translational medicine as it allows the culture and biobanking of self-organizing cells from various sources that facilitate the study of pathophysiology and therapeutics, including from individual patients in a personalized approach. This review describes current research using biliary organoids for the study of human cholestatic diseases and the emerging applications of organoids to regenerative medicine directed at the biliary tree. Challenges and possible solutions to the current hurdles in this emerging field, particularly the need for standardization of terminology and clarity on source materials and techniques, are also discussed.

Hepatic NFAT signaling regulates the expression of inflammatory cytokines in cholestasis.

BACKGROUND & AIMS: The nuclear factor of activated T-cells (NFAT) plays an important role in immune responses by regulating the expression of inflammatory genes. However, it is not known whether NFAT plays any role in the bile acid (BA)-induced hepatic inflammatory response. Thus, we aimed to examine the functional role of NFATc3 in cholestatic liver injury in mice and humans. METHODS: Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with primary biliary cholangitis [PBC] or primary sclerosing cholangitis [PSC]) by quantitative PCR, western blot and immunohistochemistry. Specific NFAT inhibitors were used in vivo and in vitro. Gene reporter assays and ChIP-PCR were used to determine promoter activity. RESULTS: NFAT isoforms c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BAs, nuclear translocation of NFATc3 was increased in both human and mouse hepatocytes and was associated with elevated mRNA levels of IL-8, CXCL2, and CXCL10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-driven induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4-/- mice) and in patients with PBC and PSC in association with elevated tissue levels of Cxcl2 (mice) or IL-8 (humans). Gene reporter assays and ChIP-PCR demonstrated that the NFAT response element in the IL-8 promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation in vivo in Abcb4-/- mice reduced cholestatic liver injury. CONCLUSIONS: NFAT plays an important role in BA-stimulated hepatic cytokine expression in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans. LAY SUMMARY: Bile acid induces liver injury by stimulating the expression of inflammatory genes in hepatocytes through activation of the transcription factor NFAT. Blocking this activation in vitro (in hepatocyte cultures) and in vivo (in cholestatic mice) decreased the expression of inflammatory genes and reduced liver injury.

Liver Complications Following Treatment of Hematologic Malignancy With Anti-CD22-Calicheamicin (Inotuzumab Ozogamicin).

Treatment of hematological malignancy with antibody-drug conjugates (ADCs) may cause liver injury. ADCs deliver a toxic moiety into antigen-expressing tumor cells, but may also injure hepatic sinusoids (sinusoidal obstruction syndrome; SOS). We studied patients who received an anti-CD22/calicheamicin conjugate (inotuzumab ozogamicin; InO) to gain insight into mechanisms of sinusoidal injury, given that there are no CD22+ cells in the normal liver, but nonspecific uptake of ADCs by liver sinusoidal endothelial cells (LSECs). Six hundred thirty-eight patients (307 with acute lymphocytic leukemia [ALL], 311 with non-Hodgkin's lymphoma [NHL]) were randomized to either InO or standard chemotherapy (controls). While blinded to treatment assignment, we reviewed all cases with hepatobiliary complications to adjudicate the causes. Frequency of SOS among patients who received InO was 5 of 328 (1.5%), compared to no cases among 310 control patients. Drug-induced liver injury (DILI) developed in 26 (7.9%) InO recipients and 3 (1%) controls. Intrahepatic cholestasis (IHC) was observed in 4.9% of InO recipients and in 5.5% of controls. Subsequent to the randomization study, 113 patients with ALL underwent allogeneic hematopoietic cell transplantation (HCT); frequency of SOS in those previously exposed to InO was 21 of 79 (27%) versus 3 of 34 (9%) in controls. An exploratory multivariate model identified a past history of liver disease and thrombocytopenia before conditioning therapy as dominant risk factors for SOS after transplant. Conclusion: Frequencies of SOS and DILI after inotuzumab ozogamicin treatment were 1.5% and 7.9%, respectively, compared to none and 1% among controls who received standard chemotherapy. These data suggest that ADCs that do not target antigens present in the normal liver have a relatively low frequency of SOS, but a relatively high frequency of DILI.

Bile-Derived Organoids From Patients With Primary Sclerosing Cholangitis Recapitulate Their Inflammatory Immune Profile.

Primary sclerosing cholangitis (PSC) is a heterogeneous and progressive fibroinflammatory cholangiopathy with no known etiology or effective treatment. Studies of PSC are limited due to difficulty in accessing the cholangiocyte, the small percentage of these cells in the liver, instability of in vitro culture systems, and reliance on samples from end-stage disease. Here, we demonstrate that stem cells can be isolated from the bile of PSC patients undergoing endoscopic retrograde cholangiopancreatography earlier in their clinical course and maintained long term in vitro as three-dimensional (3D) organoids that express a biliary genetic phenotype. Additionally, bile-derived organoids (BDOs) can be biobanked and samples obtained longitudinally over the course of the disease. These BDOs express known cholangiocyte markers including gamma glutamyl transferase, cytokeratin 19, epithelial cellular adhesion molecule, cystic fibrosis transmembrane conductance regulator, and anion exchanger 2. RNA sequence analysis identified 39 genes whose expression differed in organoids from PSC patients compared to non-PSC controls, including human leukocyte antigen DM alpha chain and chemokine (C-C motif) ligand 20 (CCL20), immune-related genes previously described in genome-wide association studies of PSC. Incubation of these BDOs with interleukin 17A or tumor necrosis factor alpha led to an immune-reactive phenotype with a significant increase in secretion of proinflammatory mediators, including CCL20, a T-cell chemoattractant. Conclusion: This study demonstrates that bile can be used as a source of biliary-like cells that can be maintained long term in vitro as 3D organoids; these BDOs retain features of cholangiopathies, including the ability to react to inflammatory stimuli by secreting chemokines and propagating an immune-reactive phenotype reflective of the pathogenesis of these diseases; thus, BDOs represent a platform for the study of the pathogenesis and therapy of cholangiopathies, particularly PSC.

Solute Carrier Organic Anion Transporter Family Member 3A1 Is a Bile Acid Efflux Transporter in Cholestasis.

BACKGROUND & AIMS: Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. METHODS: We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. RESULTS: Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. CONCLUSIONS: We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.

Cenicriviroc, a cytokine receptor antagonist, potentiates all-trans retinoic acid in reducing liver injury in cholestatic rodents.

BACKGROUND & AIMS: Cholestatic liver injury is mediated by bile acid-induced inflammatory responses. We hypothesized that superior therapeutic effects might be achieved by combining treatments that reduce the bile acid pool size with one that blocks inflammation. METHODS: Bile duct-ligated (BDL) rats and Mdr2(Abcb4)-/- mice were treated with all-trans retinoic acid (atRA), a potent inhibitor of bile acid synthesis, 5 mg/kg/d by gavage, or Cenicriviroc (CVC), a known antagonist of CCR2 and CCR5, 50 mg/kg/d alone or in combination for 14 days and 1 month respectively. RESULTS: All-trans retinoic acid alone reduced bile acid pool size and liver necrosis in BDL rats. However, the combination with CVC further reduced liver to body weight ratio, bile acid pool size, plasma liver enzyme, bilirubin, liver necrosis and fibrosis when compared to the atRA treatment. The assessment of hepatic hydroxyproline content further confirmed the reduced liver injury concurrent with reduction of pro-inflammatory cytokines emphasizing the synergistic effects of these two agents. Profiling of hepatic inflammatory cells revealed that combination therapy reduced neutrophils and T cells but not macrophages. The superior therapeutic effects of combination treatment were also confirmed in Mdr2-/- mice where a significant reduction in plasma liver enzymes, bilirubin, liver fibrosis, bile duct proliferation and hepatic infiltration of neutrophils and T cells and expression of cytokines were found. CONCLUSIONS: Multitargeted therapy is an important paradigm for treating cholestatic liver injury. The combination of CVC with atRA or other FXR activators may warrant a clinical trial in patients with cholestatic liver disease.