Intracellular Trafficking Defects in Congenital Intestinal and Hepatic Diseases.

Enterocytes and liver cells fulfill important metabolic and barrier functions and are responsible for crucial vectorial secretive and absorptive processes. To date, genetic diseases affecting metabolic enzymes or transmembrane transporters in the intestine and the liver are better comprehended than mutations affecting intracellular trafficking. In this review, we explore the emerging knowledge on intracellular trafficking defects and their clinical manifestations in both the intestine and the liver. We provide a detailed overview including more investigated diseases such as the canonical, variant and associated forms of microvillus inclusion disease, as well as recently described pathologies, highlighting the complexity and disease relevance of several trafficking pathways. We give examples of how intracellular trafficking hubs, such as the apical recycling endosome system, the trans-Golgi network, lysosomes, or the Golgi-to-endoplasmic reticulum transport are involved in the pathomechanism and lead to disease. Ultimately, understanding these processes could spark novel therapeutic approaches, which would greatly improve the quality of life of the affected patients.

Abcb4-defect cholangitis mouse model with hydrophobic bile acid composition by in vivo liver-specific gene deletion.

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with both human-like bile acid composition and Abcb4-defect exhibit severe cholestatic liver injury and are useful for studying human cholestatic diseases and developing new treatments.

Proteomics Defines Plasma Biomarkers for the Early Diagnosis of Biliary Atresia.

Early diagnosis of biliary atresia (BA) is crucial for improving the chances of survival and preserving the liver function of pediatric patients with BA. Herein, we performed proteomics analysis using data-independent acquisition (DIA) and parallel reaction monitoring (PRM) to explore potential biomarkers for the early diagnosis of BA compared to other non-BA jaundice cases. Consequently, we detected and validated differential protein expression in the plasma of patients with BA compared to the plasma of patients with intrahepatic cholestasis. Bioinformatics analysis revealed the enriched biological processes characteristic of BA by identifying the differential expression of specific proteins. Signaling pathway analysis revealed changes in the expression levels of proteins associated with an alteration in immunoglobulin levels, which is indicative of immune dysfunction in BA. The combination of polymeric immunoglobulin receptor expression and immunoglobulin lambda variable chain (IGL c2225_light_IGLV1-47_IGLJ2), as revealed via machine learning, provided a useful early diagnostic model for BA, with a sensitivity of 0.8, specificity of 1, accuracy of 0.89, and area under the curve value of 0.944. Thus, our study identified a possible effective plasma biomarker for the early diagnosis of BA and could help elucidate the underlying mechanisms of BA.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids.

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.

L-Glutamine mitigates bile acid-induced inhibition of growth factor activity in rat hepatocyte cultures.

Bile acid-induced hepatotoxicity is inevitable in Cholestasis pathogenesis and L-Glutamine (L-Gln) has been reported to prevent total parenteral nutrition (TPN)-induced cholestasis in premature neonates. While mechanisms remain unknown, we hypothesize that bile acids impair growth factor (GF) function in hepatocytes which L-glutamine prevents through NAPDH oxidase (NOX) modulation. Glycochenodeoxycholic acid (GCDC, 0-100 µM) when added to primary hepatocyte cultures significantly (p < 0.01) decreased the FBS-induced BrdU incorporation, however inhibition of Fibroblast Growth factor (FGF)- or Hepatocyte growth factor (HGF)-induced DNA synthesis was more pronounced (p < 0.001). L-Gln markedly attenuated GCDC-mediated inhibition of DNA synthesis in both FBS and GF-treated cells. GCDC significantly increased the NADPH oxidase activity and NOX-1 protein expression that were markedly reduced by L-Gln and protein kinase c (PKC) inhibitor, LY-333531. Apocynin (APCN) and diphenyliodonium (DPI) significantly blocked the GCDC-mediated inhibition of GF-induced DNA synthesis. This study demonstrates that bile acid-induced hepatotoxicity involves dysfunction of certain growth factors via protein kinase c (PKC)- mediated NOX modulation which can be corrected, at least partly, by L-glutamine.

Inhibition of the renal apical sodium dependent bile acid transporter prevents cholemic nephropathy in mice with obstructive cholestasis.

BACKGROUND & AIMS: Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. METHODS: Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. RESULTS: Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. CONCLUSIONS: BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS: Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN.

Identification and functional validation of miR-190b-5p and miR-296-3p as novel therapeutic attenuators of liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis and its end-stage form known as cirrhosis contributes to millions of deaths annually. The lack of robust anti-fibrotic molecules is in part attributed to absence of any functional screens to identify molecular regulators using patient-derived primary human hepatic myofibroblasts, which are key drivers of fibrosis. METHODS: Here, to identify robust regulators of fibrosis, we performed functional microRNA screenings in primary human hepatic myofibroblasts followed by in vivo validation in three independent mouse models of fibrosis (toxin, cholestasis and MASH). RESULTS: We identified miR-190b-5p and miR-296-3p as robust anti-fibrotic miRNAs that suppress liver fibrosis. Notably, the expression of miR-190b-5p and miR-296-3p was found significantly reduced in human livers with fibrosis. Mechanistically, we discovered hyaluronan synthase 2 (HAS2) and integrin alpha-6 (ITGA6) as novel targets of miR-190b-5p and miR-296-3p, respectively. Furthermore, we demonstrated that the anti-fibrotic properties of miR-190b-5p and miR-296-3p are, at least in part, dependent on HAS2 and ITGA6. Finally, we showed the anti-fibrotic function of both miRNAs in a human liver bud model, which mimics multiple features of human liver. CONCLUSIONS: Collectively, in our study we discovered miR-190b-5p and miR-296-3p as two novel anti-fibrotic miRNAs, and that HAS2 and ITGA6 contribute to miR-190b-5p- and miR-296-3p-mediated inhibition of liver fibrosis. These results provide a foundation for future research to explore the clinical utility of miR-190b-5p and miR-296-3p in liver injuries with fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis and cirrhosis contribute to millions of deaths world-wide and, till date, remain as unmet medical needs. In this study, we discovered two microRNAs, miR-190b-5p and miR-296-3p, which suppress liver fibrosis in preclinical mouse models and a human liver bud model. Our promising results encourage further studies that aim to develop both miRNAs for the treatment of liver fibrosis in patients.

Hepatic bile acid accretion correlates with cholestatic liver injury and therapeutic response in Cyp2c70 knockout mice with a humanized bile acid composition.

Cyp2c70 knockout (KO) mice lack the liver enzyme responsible for synthesis of 6-hydroxylated muricholate bile acid species and possess a more hydrophobic human-like bile acid composition. Cyp2c70 KO mice develop cholestatic liver injury that can be prevented by administration of an ileal bile acid transporter (IBAT) inhibitor. In this study, we investigated the potential of an ileal bile acid transporter (IBAT) inhibitor (SC-435) and steroidal FXR agonist (cilofexor) to modulate established hepatobiliary injury and the consequent relationship of intrahepatic bile acid content and hydrophobicity to the cholestatic liver injury phenotype. Oral administration of SC-435, cilofexor, or combined treatment for 2 weeks markedly reduced serum markers of liver injury and improved histological and gene expression markers of fibrosis, liver inflammation, and ductular reaction in male and female Cyp2c70 KO mice, with greatest benefit in the combination treatment group. The IBAT inhibitor and FXR agonist significantly reduced intrahepatic bile acid content but not hepatic bile acid pool hydrophobicity, and markers of liver injury were strongly correlated with intrahepatic total bile acid and taurochenodeoxycholic acid accretion. Biomarkers of liver injury increased linearly with similar hepatic thresholds for pathological accretion of hydrophobic bile acids in male and female Cyp2c70 KO mice. These findings further support targeting intrahepatic bile acid retention as a component of treatments for cholestatic liver disease.

Parenteral nutrition results in peripheral ileal to hepatic circadian discordance in mice.

BACKGROUND: We have developed a mouse model of Parenteral Nutrition Associated Liver Disease in which PN infusion results in cholestatic liver injury. In the liver, the master circadian genes Arntl/Bmal drive rhythmic gene expression and regulate circadian expression of hepatic functions including bile acid synthesis. The aim of this study was to examine the effect of continuous PN on ileal and hepatic expression of circadian regulatory (CR) genes, FXR signaling and bile acid synthesis in mice. METHODS: WT mice were exposed to continuous soy oil lipid emulsion-based PN infusion through a central venous catheter for 4 days (PN). Water was provided ad libitum, but no nutrients were provided enterally. On d4, mice were sacrificed every 6 hours (7AM, 1PM, 7PM and 1AM), and ileal, hepatic tissue and serum harvested. From tissue samples, the relative expression of circadian transcription factors and FXR signaling was assessed. RESULTS: Administration of 4d PN increased hepatic injury, inflammatory cytokine expression and gut permeability. In the ileum, PN activated FXR and induced expression of Fgf15 and Nr0b2. In the liver, expression of FXR-downstream targets was dysregulated. PN administrations impacted hepatic and ileal circadian transcription factor mRNA expression which was discordant between the two organs. CONCLUSIONS: Dysregulation of circadian regulatory machinery is in part due to discordance of the gut-liver axis during PN. Pharmacologic targeting of CR as a therapeutic strategy for PNALD thus deserves further investigation.

A Medium-Chain Fatty Acid Analogue Prevents Intestinal Failure-Associated Liver Disease in Preterm Yorkshire Piglets.

BACKGROUND & AIMS: At least 20%-30% of patients with intestinal failure receiving long-term parenteral nutrition will develop intestinal failure-associated liver disease (IFALD), for which there are few therapeutic options. SEFA-6179 is a first-in-class structurally engineered medium-chain fatty acid analogue that acts through GPR84, PPARα, and PPARγ agonism. We hypothesized that SEFA-6179 would prevent biochemical and histologic liver injury in a preterm piglet model of IFALD. METHODS: Preterm Yorkshire piglets were delivered by cesarean section, and parenteral nutrition was provided for 14 days via implanted central venous catheters. Animals were treated with either medium-chain triglyceride vehicle control or SEFA-6179. RESULTS: Compared to medium-chain triglyceride vehicle at day of life 15, SEFA-6179 prevented biochemical cholestasis (direct bilirubin: 1.9 vs <0.2 mg/dL, P = .01; total bilirubin: 2.7 vs 0.4 mg/dL, P = .02; gamma glutamyl transferase: 172 vs 30 U/L, P = .01). SEFA-6179 also prevented steatosis (45.6 vs 13.9 mg triglycerides/g liver tissue, P = .009), reduced bile duct proliferation (1.6% vs 0.5% area cytokeratin 7 positive, P = .009), and reduced fibrosis assessed by a masked pathologist (median Ishak score: 3 vs 1, P = 0.007). RNA sequencing of liver tissue demonstrated that SEFA-6179 broadly impacted inflammatory, metabolic, and fibrotic pathways, consistent with its in vitro receptor activity (GPR84/PPARα/PPARγ agonist). CONCLUSIONS: In a preterm piglet model of IFALD, SEFA-6179 treatment prevented biochemical cholestasis and steatosis and reduced bile duct proliferation and fibrosis. SEFA-6179 is a promising first-in-class therapy for the prevention and treatment of IFALD that will be investigated in an upcoming phase II clinical trial.

Baicalein alleviates intrahepatic cholestasis by regulating bile acid metabolism via an FXR-dependent manner.

Cholestasis is characterized by impaired bile secretion and flow, leading to the accumulation of toxic bile acids in the liver, further causing inflammatory reaction, fibrosis, and ultimately liver transplantation. Although first-line clinical agents such as Ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) are available, serious side effects still exist. Therefore, pharmacologic treatment of cholestatic liver disease remains challenging. Here, we used a murine model of cholestasis treated with or without intraperitoneal injection of baicalein and found that baicalein could attenuate 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced inflammatory response, ductular reaction, liver fibrosis, and bile acid metabolism disorders. Furthermore, the therapeutic effect of baicalein was hampered in the presence of Guggulsterone (GS), an Farnesoid X receptor (FXR) antagonist. These results indicated that baicalein alleviated DDC diet-induced cholestatic liver injury in an FXR-dependent manner.

Loss of the DNA-binding domain of the farnesoid X receptor gene causes severe liver and kidney injuries.

Farnesoid X receptor (FXR) regulates bile acid synthesis, lipid metabolism, and glucose homeostasis in metabolic organs. FXR-knockout (FXR-KO) mice lacking the last exon of the FXR gene develop normally and display no prenatal and early postnatal lethality, whereas human patients with mutations in the DNA-binding domain of the FXR gene develop severe hepatic dysfunction. In this study, we generated novel FXR-KO mice lacking the DNA-binding domain of the FXR gene using CRISPR-Cas9 technology and evaluated their phenotypes. Similar to the aforementioned FXR-KO mice, our novel mice showed elevated serum levels of total bile acids and total cholesterol. However, they were obviously short-lived, showing severe liver and renal pathologies at an early age. These results indicate that FXR, including its unknown isoforms, has more significant functions in multiple organs than previously reported. Thus, the novel FXR-KO mice could lead to a new aspect that requires reworking of previous knowledge of FXR in the liver and renal function.

Targeting the FGF19-FGFR4 pathway for cholestatic, metabolic, and cancerous diseases.

Human fibroblast growth factor 19 (FGF19, or FGF15 in rodents) plays a central role in controlling bile acid (BA) synthesis through a negative feedback mechanism. This process involves a postprandial crosstalk between the BA-activated ileal farnesoid X receptor and the hepatic Klotho beta (KLB) coreceptor complexed with fibrobalst growth factor receptor 4 (FGFR4) kinase. Additionally, FGF19 regulates glucose, lipid, and energy metabolism by coordinating responses from functional KLB and FGFR1-3 receptor complexes on the periphery. Pharmacologically, native FGF19 or its analogs decrease elevated BA levels, fat content, and collateral tissue damage. This makes them effective in treating both cholestatic diseases such as primary biliary or sclerosing cholangitis (PBC or PSC) and metabolic abnormalities such as nonalcoholic steatohepatitis (NASH). However, chronic administration of FGF19 drives oncogenesis in mice by activating the FGFR4-dependent mitogenic or hepatic regenerative pathway, which could be a concern in humans. Agents that block FGF19 or FGFR4 signaling have shown great potency in preventing FGF19-responsive hepatocellular carcinoma (HCC) development in animal models. Recent phase 1/2 clinical trials have demonstrated promising results for several FGF19-based agents in selectively treating patients with PBC, PSC, NASH, or HCC. This review aims to provide an update on the clinical development of both analogs and antagonists targeting the FGF19-FGFR4 signaling pathway for patients with cholestatic, metabolic, and cancer diseases. We will also analyze potential safety and mechanistic concerns that should guide future research and advanced trials.

Serum matrix metalloproteinase-7 for discriminating biliary atresia: a diagnostic accuracy and validation study.

BACKGROUND: Prompt and precise differential diagnosis of biliary atresia (BA) among cholestatic patients is of great importance. Matrix metalloproteinase-7 (MMP-7) holds great promise as a diagnostic marker for BA. This study aimed to investigate the accuracy of age-specific serum MMP-7 for discriminating BA from other cholestatic pediatric patients. METHODS: This was a single center diagnostic accuracy and validation study including both retrospective and prospective cohorts. Serum MMP-7 concentrations were measured using an ELISA kit, the trajectory of which with age was investigated in a healthy infants cohort aged 0 to 365 days without hepatobiliary diseases (n = 284). Clinical BA diagnosis was based on intraoperative cholangiography and subsequent histological examinations. The diagnostic accuracy of age-specific cutoffs of serum MMP-7 were assessed in a retrospective cohort of cholestatic patients (n = 318, with 172 BA) and validated in a prospective cohort (n = 687, including 395 BA). RESULTS: The MMP-7 concentration declines non-linearly with age, showing higher levels in healthy neonates as well as higher cutoff value in neonatal cholestasis. The area under the ROC curve (AUROC) was 0.967 (95% confidence interval [CI]: 0.946-0.988) for the retrospective cohort, and the cutoff of 18 ng/mL yielded 93.0% (95%CI: 88.1-96.3%), 93.8% (95%CI: 88.6-97.1%), 94.7% (95%CI: 90.1-97.5%), and 91.9% (95%CI: 86.4-95.8%) for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. The performance of MMP-7 was successfully validated in the larger prospective cohort, resulting in a diagnostic sensitivity of 95.9% (379/395; 95% CI: 93.5-97.7%), a specificity of 87.3% (255/292; 95% CI: 83.0-90.9%), a PPV of 91.1% (379/416; 95% CI: 87.9-93.7%), and a NPV of 94.1% (255/271; 95% CI: 90.6-96.6%), respectively. Besides, higher cutoff value of 28.1 ng/mL achieved the best sensitivity, specificity, PPV, and NPV for infants aged 0-30 days, which was 86.4% (95% CI: 75.0-94.0%), 95.5% (95% CI: 77.2-99.9%), 98.1% (95% CI: 89.7-100%), and 72.4% (95% CI: 52.8-87.3%), respectively. CONCLUSIONS: The serum MMP-7 is accurate and reliable in differentiating BA from non-BA cholestasis, showing its potential application in the diagnostic algorithm for BA and significant role in the future research regarding pathogenesis of BA.

Large-scale genomic investigation of pediatric cholestasis reveals a novel hepatorenal ciliopathy caused by PSKH1 mutations.

PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.

Novel PLEC variants associated with infantile cholestasis.

Plectin is a cytoskeletal linker of intermediate filaments, encoded by the PLEC gene. Recently, plectin mutations have been identified in a pair of siblings with progressive familial intrahepatic cholestasis. Here, we reported two unrelated infants with plectinopathy causing cholestatic jaundice with novel variants in the PLEC gene. Trio exome sequencing identified compound heterozygous variants in the PLEC gene for each patient: c.71-11768C>T and c.4331G>T (p.Arg1444Leu) in Patient 1, and c.592C>T (p.Arg198Trp) and c.4322G>A (p.Arg1441His) in Patient 2. Immunofluorescence staining of liver samples from both patients revealed scattered signals of plectin in the cytoplasm of hepatocytes and reduced colocalization of plectin and cytokeratin 8. This study not only underscores the involvement of plectin in cholestasis but also highlights the utility of exome sequencing as a powerful diagnostic tool in identifying genetic underpinnings of infantile cholestasis.

Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

CLDN1 Arg81His founder variant causes ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome in Moroccan Jews.

Neonatal ichthyosis and sclerosing cholangitis syndrome (NISCH), also known as ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC), is an extremely rare disease of autosomal recessive inheritance, resulting from loss of function of the tight junction protein claudin-1. Its clinical presentation is highly variable, and is characterized by liver and ectodermal involvement. Although most ILVASC cases described to date were attributed to homozygous truncating variants in CLDN1, a single missense variant CLDN1 p.Arg81His, associated with isolated skin ichthyosis phenotype, has been recently reported in a family of Moroccan Jewish descent. We now describe seven patients with ILVASC, originating from four non consanguineous families of North African Jewish ancestry (including one previously reported family), harboring CLDN1 p.Arg81His variant, and broaden the phenotypic spectrum attributed to this variant to include teeth, hair, and liver/bile duct involvement, characteristic of ILVASC. Furthermore, we provide additional evidence for pathogenicity of the CLDN1 p.Arg81His variant by transmission electron microscopy of the affected skin, revealing distorted tight junction architecture, and show through haplotype analysis in the vicinity of the CLDN1 gene, that this variant represents a founder variant in Jews of Moroccan descent with an estimated carrier frequency of 1:220.

Liver-specific deletion of microRNA-34a alleviates ductular reaction and liver fibrosis during experimental cholestasis.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammatory responses and fibrotic scar formation leading to cholestasis. Ductular reaction and liver fibrosis are typical liver changes seen in human PSC and cholestasis patients. The current study aimed to clarify the role of liver-specific microRNA-34a in the cholestasis-associated ductular reaction and liver fibrosis. We demonstrated that miR-34a expression was significantly increased in human PSC livers along with the enhanced ductular reaction, cellular senescence, and liver fibrosis. A liver-specific miR-34a knockout mouse was established by crossing floxed miR-34a mice with albumin-promoter-driven Cre mice. Bile duct ligation (BDL) induced liver injury characterized by necrosis, fibrosis, and immune cell infiltration. In contrast, liver-specific miR-34a knockout in BDL mice resulted in decreased biliary ductular pathology associated with the reduced cholangiocyte senescence and fibrotic responses. The miR-34a-mediated ductular reactions may be functioning through Sirt-1-mediated senescence and fibrosis. The hepatocyte-derived conditioned medium promoted LPS-induced fibrotic responses and senescence in cholangiocytes, and miR-34a inhibitor suppressed these effects, further supporting the involvement of paracrine regulation. In conclusion, we demonstrated that liver-specific miR-34a plays an important role in ductular reaction and fibrotic responses in a BDL mouse model of cholestatic liver disease.

Cholestasis-induced phenotypic transformation of neutrophils contributes to immune escape of colorectal cancer liver metastasis.

BACKGROUND: Cholestasis is a common yet severe complication that occurs during the advancement of liver metastasis. However, how cholestasis impacts the development, treatment, and tumor microenvironment (TME) of liver metastasis remains to be elucidated. METHODS: Extrahepatic and intrahepatic cholestatic mouse models with liver metastasis were established to detect the differential expression levels of genes, infiltration of immune cells and change in bile acid-associated metabolites by using RNA-Sequencing, flowcytometry, and liquid chromatography and mass spectrometry. Western blot was applied to neutrophils under the stimulation of primary bile acids (BAs) in vitro to study the mechanism of phenotypic alteration. In vitro coculture of BA-treated neutrophils with CD8+ T cells were performed to study the immune-suppressive effect of phenotypic-altered neutrophils. Clinical samples collected from colorectal cancer patients with liver metastasis and cholestasis were applied to RNA-Seq. RESULTS: Compared to non-cholestatic mice, the progression of liver metastasis of cholestatic mice was significantly accelerated, which was associated with increased neutrophil infiltration and T-cell exclusion. Both neutrophils and T cells expressed higher immunosuppressive markers in the cholestatic mouse model, further indicating that an immunosuppressive tumor microenvironment was induced during cholestasis. Although neutrophils deletion via anti-Ly6G antibody partially hindered liver metastasis progression, it reduced the overall survival of mice. Tauro-ß-muricholic acid (Tß-MCA) and Glycocholic acid (GCA), the two most abundant cholestasis-associated primary BAs, remarkably promoted the expression of Arg1 and iNOS on neutrophils via p38 MAPK signaling pathway. In addition, BAs-pretreated neutrophils significantly suppressed the activation and cytotoxic effects of CD8+ T cells, indicating that the immunosuppressive phenotype of neutrophils was directly induced by BAs. Importantly, targeting BA anabolism with Obeticholic acid (OCA) under cholestasis effectively suppressed liver metastasis progression, enhanced the efficacy of immune checkpoint blockade, and prolonged survival of mice. CONCLUSIONS: Our study reveals the TME of cholestasis-associated liver metastasis and proposes a new strategy for such patients by targeting bile acid anabolism.