The journal retracts the article titled “The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling” [...]

Stimulator of interferon genes (STING) is positively correlated with the degrees of liver inflammation in human metabolic dysfunction-associated steatotic liver disease (MASLD). In addition, STING disruption alleviates MASLD in mice fed a high-fat diet (HFD) for 3 months (3-m-HFD). Here we investigated the role of the duration of dietary feeding in regulating MASLD in mice and explored the involvement of STING in sex differences in MASLD. Both male and female STING-disrupted (STINGgt) and wild-type C57BL/6J mice were fed an HFD for 3 or 7 months (7-m-HFD). Additionally, female STINGgt mice upon ovariectomy (OVX) and 3-m-HFD were analyzed for MASLD. Upon 3-m-HFD, STINGgt mice exhibited decreased severity of MASLD compared to control. However, upon 7-m-HFD, STINGgt mice were comparable with wild-type mice in body weight, fat mass, and MASLD. Regarding regulating the liver RNA transcriptome, 7-m-HFD increased the expression of genes indicating proinflammatory activation of various liver cells. Interestingly, the severity of MASLD in female mice was much lighter than in male mice, regardless of STING disruption. Upon OVX, female STINGgt mice showed significantly increased severity of MASLD relative to sham control but were comparable with male STINGgt mice. Upon treatment with 17-beta estradiol (E2), hepatocytes revealed decreased fat deposition while macrophages displayed decreases in lipopolysaccharide-induced phosphorylation of Nfkb p65 and Jnk p46 independent of STING. These results suggest that 7-m-HFD, without altering female sex-based protection, abolishes STING disruption-driven protection of MASLD, likely through causing proinflammatory activation of multiple types of liver cells to offset the effect of STING disruption.

BACKGROUND AND AIMS:NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling.APPROACH AND RESULTS:4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro.CONCLUSION:Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

The liver has a vital role in many metabolic and regulatory processes in the body. Primary biliary cholangitis (PBC), pre-viously known as primary biliary cirrhosis, is a chronic choles-tatic autoimmune disease of the intrahepatic bile ducts asso-ciated with loss of tolerance to mitochondrial antigens. At this time there is no definitive cure for PBC; however, ursodeoxy-cholic acid (UDCA) has been shown to reduce injury when ad-ministered as the first line of treatment. Additional therapeu-tics can be given concurrently or as an alternative to UDCA to manage the symptoms and further curb disease progression. Currently, a liver transplant is the only potentially curative option when the patient has developed end-stage liver dis-ease or intractable pruritus. This review aims to delineate the pathogenesis of primary biliary cholangitis and shed light on current therapeutic strategies in the treatment of PBC.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is highly associated with obesity and progresses to nonalcoholic steatohepatitis when the liver develops overt inflammatory damage. While removing adenosine in the purine salvage pathway, adenosine kinase (ADK) regulates methylation reactions. We aimed to study whether hepatocyte ADK functions as an obesogenic gene/enzyme to promote excessive fat deposition and liver inflammation. METHODS: Liver sections of human subjects were examined for ADK expression using immunohistochemistry. Mice with hepatocyte-specific ADK disruption or overexpression were examined for hepatic fat deposition and inflammation. Liver lipidomics, hepatocyte RNA sequencing (RNA-seq), and single-cell RNA-seq for liver non-parenchymal cells were performed to analyze ADK regulation of hepatocyte metabolic responses and hepatocyte-nonparenchymal cells crosstalk. RESULTS: Whereas patients with nonalcoholic fatty liver disease had increased hepatic ADK levels, mice with hepatocyte-specific ADK disruption displayed decreased hepatic fat deposition on a chow diet and were protected from diet-induced excessive hepatic fat deposition and inflammation. In contrast, mice with hepatocyte-specific ADK overexpression displayed increased body weight and adiposity and elevated degrees of hepatic steatosis and inflammation compared with control mice. RNA-seq and epigenetic analyses indicated that ADK increased hepatic DNA methylation and decreased hepatic Ppara expression and fatty acid oxidation. Lipidomic and single-cell RNA-seq analyses indicated that ADK-driven hepatocyte factors, due to mitochondrial dysfunction, enhanced macrophage proinflammatory activation in manners involving increased expression of stimulator of interferon genes. CONCLUSIONS: Hepatocyte ADK functions to promote excessive fat deposition and liver inflammation through suppressing hepatocyte fatty acid oxidation and producing hepatocyte-derived proinflammatory mediators. Therefore, hepatocyte ADK is a therapeutic target for managing obesity and nonalcoholic fatty liver disease.

Background Alcohol-related liver disease (ALD) is characterized by ductular reaction (DR), liver inflammation, steatosis, fibrosis, and cirrhosis. The secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes) regulates liver phenotypes in cholestasis. We evaluated the role of Sct signaling on ALD phenotypes. Methods We used male wild-type and Sct −/− mice fed a control diet (CD) or ethanol (EtOH) for 8 wk. Changes in liver phenotypes were measured in mice, female/male healthy controls, and patients with alcoholic cirrhosis. Since Cyp4a10 and Cyp4a11/22 regulate EtOH liver metabolism, we measured their expression in mouse/human liver. We evaluated: (i) the immunoreactivity of the lipogenesis enzyme elongation of very-long-chain fatty acids 1 (Elovl, mainly expressed by hepatocytes) in mouse/human liver sections by immunostaining; (ii) the expression of miR-125b (that is downregulated in cholestasis by Sct) in mouse liver by q PCR; and (iii) total bile acid (BA) levels in mouse liver by enzymatic assay, and the mRNA expression of genes regulating BA synthesis (cholesterol 7a-hydroxylase, Cyp27a1, 12a-hydroxylase, Cyp8b1, and oxysterol 7a-hydroxylase, Cyp7b11) and transport (bile salt export pump, Bsep, Na + -taurocholate cotransporting polypeptide, NTCP, and the organic solute transporter alpha (OSTa) in mouse liver by q PCR. Results In EtOH-fed WT mice there was increased biliary and liver damage compared to control mice, but decreased miR-125b expression, phenotypes that were blunted in EtOH-fed Sct −/− mice. The expression of Cyp4a10 increased in cholangiocytes and hepatocytes from EtOH-fed WT compared to control mice but decreased in EtOH-fed Sct −/− mice. There was increased immunoreactivity of Cyp4a11/22 in patients with alcoholic cirrhosis compared to controls. The expression of miR-125b decreased in EtOH-fed WT mice but returned at normal values in EtOH-fed Sct −/− mice. Elovl1 immunoreactivity increased in patients with alcoholic cirrhosis compared to controls. There was no difference in BA levels between WT mice fed CD or EtOH; BA levels decreased in EtOH-fed Sct −/− compared to EtOH-fed WT mice. There was increased expression of Cyp27a1, Cyp8b1, Cyp7b1, Bsep, NTCP and Osta in total liver from EtOH-fed WT compared to control mice, which decreased in EtOH-fed Sct −/− compared to EtOH-fed WT mice. Conclusions Targeting Sct/SR signaling may be important for modulating ALD phenotypes.

ABSTRACTOrganoids are novelin vitromodels to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using ‘primary’ liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate ‘primary’ cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31−/CD68−/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGFβ1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-α) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as anin vitromodel in mimicking PSCin vivophenotypes compared to 2D cell culture, which can be used in PSC disease-related research.

The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce the in vitro formation of complex biliary networks using encapsulated immortalized mouse small biliary epithelial cells (cholangiocytes). This phenomenon is not observed using immortalized mouse large cholangiocytes, or with purified collagen 1 gels or Matrigel. We also show phenotypic stability via immunostaining for specific cholangiocyte markers. Moreover, tight junction formation and maturation was observed to occur between cholangiocytes, exhibiting polarization and transporter activity. To better define the mechanism of duct formation, we utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, radiating from a single nucleation point, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.

Background & Aims: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. Methods: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late -stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. Results: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA -sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late -stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. Conclusion: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature chol-angiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. (c) 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Interleukin-22 (IL-22) has been demonstrated as a critical regulator of epithelial homeostasis and repair; it showed an anti-inflammatory effect against ulcerative colitis. Local microinjection of IL-22 cDNA vector has been shown to be effective in treating ulcerative colitis in mouse models. However, microinjection comes with multiple technical challenges for routine colon-targeted drug delivery. In contrast, oral administration can get around these challenges and provide comparable efficacy. We showed in previous studies that oral administration of new lipid nanoparticles (nLNP)-encapsulated IL-22 mRNA targets the colon region and efficiently ameliorates colitis. This protocol describes the details of preparing and characterizing the nLNP-encapsulated IL-22 mRNA using three major lipids that mimic the natural ginger-derived nanoparticles. It provides an nLNP platform that can be used to orally deliver other types of nucleic acids to the colon.

BackgroundPrimary biliary cholangitis (PBC) is characterized by increased biliary damage, inflammation and liver fibrosis. Early stage PBC is marked by biliary proliferation, whereas late stage PBC shows ductopenia. We have shown that dominant‐negative transforming growth factor b receptor II (dnTGFβRII) mice at 12 wk of age mimic early stage PBC. We have found that melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in models of cholestasis. However, the impact of melatonin or dark therapy on PBC‐related injury is unknown. The aim of our study was to evaluate the effects of melatonin or dark therapy in a mouse model of early stage PBC.MethodsWe used background‐matched, male wild‐type (WT) and dnTGFβRII at 12 wk of age that were given drinking water containing melatonin (0.03%) or placed in complete darkness for 1 wk along with the related controls. Liver damage was evaluated by H&E. Intrahepatic bile duct mass (IBDM) was measured by CK‐19 staining. Biliary senescence was evaluated by staining for p16 and p21, and SA‐β‐galactosidase activity. Biliary and liver inflammation were determined by IL‐6 and F4/80 (Kupffer cell marker) staining. Liver fibrosis was determined by Sirius Red staining and immunofluorescence for collagen type‐1a. Hepatic stellate cell (HSC) activation was shown by SYP‐9 and α‐SMA staining. Human control and early stage PBC serum samples were obtained, and serum melatonin levels were measured by EIA.ResultsdnTGFβRII at 12 wk of age (early stage PBC mouse model) treated with melatonin or complete darkness had decreased (i) IBDM, (ii) biliary senescence, (iii) liver fibrosis, (iv) biliary and liver inflammation, and (v) HSC activation/liver fibrosis. Human early stage PBC samples had decreased serum melatonin levels.ConclusionInhibition of melatonin signaling perpetuates biliary damage associated with PBC. Restoration of melatonin‐dependent signaling via melatonin treatment or dark therapy may be therapeutic for patients with early stage PBC.Support or Funding InformationNIH NIDDK R01, VA MeritThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

See counterpoint on page 806. See counterpoint on page 806. Ductular reaction is referred to as expansion of proliferative and reactive cells that express biliary markers, such as cytokeratin (CK)-7 and CK-19.1Sato K. Marzioni M. Meng F. et al.Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (209) Google Scholar Ductular reaction is identified by histologic staining in liver specimen of patients with various liver diseases including cholangiopathies, viral hepatitis, and alcoholic and nonalcoholic liver diseases.1Sato K. Marzioni M. Meng F. et al.Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (209) Google Scholar Ductular reaction is clinically recognized as bile duct hyperplasia; however, the term "ductular reaction" is more commonly used in recent studies to define enhanced intrahepatic bile duct mass that can be attributed to the proliferation of cholangiocytes and/or transdifferentiation of various hepatic cells.1Sato K. Marzioni M. Meng F. et al.Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (209) Google Scholar It is known that ductular reaction is associated with liver fibrosis and thought to be caused by expansion of hepatic progenitor cells (HPCs).2Williams M.J. Clouston A.D. Forbes S.J. Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion.Gastroenterology. 2014; 146: 349-356Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Accumulating evidence indicates that ductular reaction (ie, expansion of CK-7+ or CK-19+ cells) can be caused not only by HPCs, but also other hepatic cells. Bile duct epithelial cells, cholangiocytes, express CK-7 and CK-19 and proliferate during bile duct injury, which is identified as ductular reaction.1Sato K. Marzioni M. Meng F. et al.Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (209) Google Scholar Furthermore, in certain pathologic conditions, hepatocytes can transdifferentiate into cholangiocyte-like cells expressing CK-7 or CK-19 to compensate the loss of cholangiocyte numbers and functions.3Nejak-Bowen K. If it looks like a duct and acts like a duct: on the role of reprogrammed hepatocytes in cholangiopathies.Gene Expr. 2020; 20: 19-23Crossref PubMed Scopus (5) Google Scholar Ductular reaction can be induced by expansion of HPCs, cholangiocytes, or hepatocytes, and functional roles of ductular reaction may differ depending on liver diseases or origins of expanding cells. This section discusses the pathophysiological roles of ductular reaction in liver diseases. Hepatic cells communicate with each other to coordinate pathophysiological responses against liver injury via secreting cytokines, chemokines, or extracellular vesicles.4Sato K. Kennedy L. Liangpunsakul S. et al.Intercellular communication between hepatic cells in liver diseases.Int J Mol Sci. 2019; 20: 2180Crossref PubMed Scopus (33) Google Scholar In the portal area and the HPC niche, HPCs, hepatic stellate cells, and macrophages secrete and receive cytokines and growth factors leading to liver inflammation and regeneration.2Williams M.J. Clouston A.D. Forbes S.J. Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion.Gastroenterology. 2014; 146: 349-356Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Ductular reaction is associated with portal inflammation and neutrophil infiltration in alcoholic hepatitis, and the cells in ductular reaction (CK-7+ cells) express elevated neutrophil recruiting chemokines and HPC markers.5Aguilar-Bravo B. Rodrigo-Torres D. Arino S. et al.Ductular reaction cells display an inflammatory profile and recruit neutrophils in alcoholic hepatitis.Hepatology. 2019; 69: 2180-2195Crossref PubMed Scopus (45) Google Scholar In nonalcoholic fatty liver disease, portal inflammation is strongly correlated with liver fibrosis levels and ductular reaction with increased portal infiltration of macrophages and lymphocytes.6Gadd V.L. Skoien R. Powell E.E. et al.The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease.Hepatology. 2014; 59: 1393-1405Crossref PubMed Scopus (296) Google Scholar Ductular reaction and increased portal macrophage infiltration can also be identified in cholestatic liver injury.7Chen L. Zhou T. Wu N. et al.Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis.Biochim Biophys Acta Mol Basis Dis. 2019; 1865: 1525-1539Crossref PubMed Scopus (17) Google Scholar Cholangiocytes become senescent during cholestatic liver injury, including primary sclerosing cholangitis (PSC).8Tabibian J.H. O'Hara S.P. Splinter P.L. et al.Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.Hepatology. 2014; 59: 2263-2275Crossref PubMed Scopus (179) Google Scholar Senescent cholangiocytes function as a senescence-associated secretory phenotype secreting various cytokines, such as interleukin-6 and transforming growth factor-β1.8Tabibian J.H. O'Hara S.P. Splinter P.L. et al.Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.Hepatology. 2014; 59: 2263-2275Crossref PubMed Scopus (179) Google Scholar Interleukin-6 promotes cholangiocyte proliferation and activates macrophages leading to ductular reaction and liver inflammation.9Sato K. Meng F. Giang T. et al.Mechanisms of cholangiocyte responses to injury.Biochim Biophys Acta. 2018; 1864: 1262-1269Crossref PubMed Scopus (51) Google Scholar These findings indicate the close relationship between ductular reactive cells (ie, HPCs and cholangiocytes) and portal infiltration and inflammation. Liver fibrosis is observed in various liver diseases, and the association of expansion of the HPC niche (ie, ductular reaction) with liver fibrosis has been identified.2Williams M.J. Clouston A.D. Forbes S.J. Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion.Gastroenterology. 2014; 146: 349-356Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Liver fibrosis is a hallmark in cholangiopathies, such as PSC, primary biliary cholangitis, and biliary atresia, and ductular reaction is identified along with liver fibrosis.1Sato K. Marzioni M. Meng F. et al.Ductular reaction in liver diseases: pathological mechanisms and translational significances.Hepatology. 2019; 69: 420-430Crossref PubMed Scopus (209) Google Scholar Expansion of CK-7+ or CK-19+ cells is found in liver sections of patients with hepatitis B or C virus infection, and ductular reaction levels are correlated with liver fibrosis levels.10Prakoso E. Tirnitz-Parker J.E. Clouston A.D. et al.Analysis of the intrahepatic ductular reaction and progenitor cell responses in hepatitis C virus recurrence after liver transplantation.Liver Transpl. 2014; 20: 1508-1519Crossref PubMed Scopus (21) Google Scholar Ductular reaction levels also correlate with poor prognosis of patients with alcoholic hepatitis and can be used to predict survival rates of patients in severe conditions.11Atkinson S.R. Aly M. Remih K. et al.Serum keratin 19 (CYFRA21-1) is a prognostic biomarker in severe alcoholic hepatitis.Liver Int. 2022; 42: 1049-1057Crossref PubMed Scopus (1) Google Scholar A higher grade of ductular reaction determined by histologic CK-7 staining is correlated with higher fibrosis stages in patients with nonalcoholic fatty liver disease.6Gadd V.L. Skoien R. Powell E.E. et al.The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease.Hepatology. 2014; 59: 1393-1405Crossref PubMed Scopus (296) Google Scholar It is also suggested that ductular reaction is associated with hepatocellular carcinoma. Peritumoral ductular reaction significantly correlates with hepatic inflammation, liver fibrosis, TNM stages, and poor prognosis,12Xu M. Xie F. Qian G. et al.Peritumoral ductular reaction: a poor postoperative prognostic factor for hepatocellular carcinoma.BMC Cancer. 2014; 14: 65Crossref PubMed Scopus (20) Google Scholar indicating the association of ductular reaction with the pathophysiology of various liver diseases. Liver fibrosis is caused by accumulated extracellular matrix (ECM) secreted from activated hepatic stellate cells or myofibroblasts, major sources of ECM secretion. During PSC, senescent cholangiocytes secrete transforming growth factor-β1, which promotes hepatic stellate cell proliferation and activation leading to ECM accumulation and liver fibrosis.13Wu N. Meng F. Zhou T. et al.The secretin/secretin receptor axis modulates ductular reaction and liver fibrosis through changes in transforming growth factor-beta1-mediated biliary senescence.Am J Pathol. 2018; 188: 2264-2280Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Because ductular reaction and liver fibrosis are closely associated and mediated by ductular reactive cells, including cholangiocytes, these cells can be a therapeutic target for liver fibrosis. Inhibition of biliary senescence by p16 Vivo-Morpholino administration downregulated cholangiocyte senescence-associated secretory phenotype secretion with attenuated ductular reaction and liver fibrosis in PSC mouse models.14Kyritsi K. Francis H. Zhou T. et al.Downregulation of p16 decreases biliary damage and liver fibrosis in the Mdr2-/- mouse model of primary sclerosing cholangitis.Gene Expr. 2020; 20: 89-103Crossref PubMed Scopus (19) Google Scholar Cholangiocytes undergo epithelial-to-mesenchymal transition and transform into myofibroblast-like profibrogenic phenotypes secreting robust ECM components contributing to hepatic fibrogenesis in cholestatic liver injury.15Zhou T. Kyritsi K. Wu N. et al.Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2-/- mouse model of primary sclerosing cholangitis (PSC).EBioMedicine. 2019; 48: 130-142Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Inhibition of cholangiocyte epithelial-to-mesenchymal transition by vimentin Vivo-Morpholino attenuated ductular reaction and liver fibrosis in PSC mouse models.15Zhou T. Kyritsi K. Wu N. et al.Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2-/- mouse model of primary sclerosing cholangitis (PSC).EBioMedicine. 2019; 48: 130-142Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar Vimentin knockdown also ameliorated liver inflammation and serum transforming growth factor-β1 levels in PSC mice.15Zhou T. Kyritsi K. Wu N. et al.Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2-/- mouse model of primary sclerosing cholangitis (PSC).EBioMedicine. 2019; 48: 130-142Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar These findings support the close association of ductular reaction and liver fibrosis, and inhibition of ductular reaction can be therapeutic in liver diseases. Current studies show the pathophysiological roles of ductular reaction and its close association with hepatic inflammation and liver fibrosis in various liver diseases (Figure 1). In PSC, inhibiting cholangiocyte senescence or epithelial-to-mesenchymal transition could be a novel therapeutic approach to improve liver conditions by attenuating ductular reaction and liver fibrosis, showing the potentials of cholangiocytes as a promising target of liver diseases. However, not only cholangiocytes but also HPCs and hepatocytes are involved in ductular reaction. The HPC niche is associated with liver fibrosis,2Williams M.J. Clouston A.D. Forbes S.J. Links between hepatic fibrosis, ductular reaction, and progenitor cell expansion.Gastroenterology. 2014; 146: 349-356Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar and elevated HPC marker expression (ie, expansion of the HPC niche) is associated with neutrophil infiltration and liver inflammation.5Aguilar-Bravo B. Rodrigo-Torres D. Arino S. et al.Ductular reaction cells display an inflammatory profile and recruit neutrophils in alcoholic hepatitis.Hepatology. 2019; 69: 2180-2195Crossref PubMed Scopus (45) Google Scholar Hepatocytes transdifferentiate into biliary-like phenotypes during cholestatic liver injury.3Nejak-Bowen K. If it looks like a duct and acts like a duct: on the role of reprogrammed hepatocytes in cholangiopathies.Gene Expr. 2020; 20: 19-23Crossref PubMed Scopus (5) Google Scholar Although it is not fully elucidated how HPCs and hepatocytes contribute to the pathophysiology of ductular reaction and liver fibrosis, targeting HPCs and hepatocytes might be another therapeutic approach in liver diseases. In conclusion, ductular reaction is closely related to liver inflammation and fibrosis and the pathophysiology of liver diseases. Understanding the full dynamic role of ductular reaction during liver injury and identification of potential targets associated with ductular reaction are of paramount significance. Ductular Reaction and Liver Regeneration: Fulfilling the Prophecy of Prometheus!Cellular and Molecular Gastroenterology and HepatologyVol. 15Issue 3PreviewMany acute and chronic liver injuries exhibit histologically as proliferating cholangiocytes, commonly referred to as ductular reaction. The origin of the cells comprising the ductular reaction is dependent on the injury (hepatocyte vs cholangiocyte) and capability of these 2-liver epithelial or "hepithelial" cells to divide and replace injured cells. The role of ductular reaction in hepatobiliary injury versus repair remains debated. Although ductular reaction has been shown to be a source of proinflammatory and profibrogenic factors, it has also been shown to contribute toward maintaining hepatobiliary function during injury. Full-Text PDF Open Access

Biliary epithelium (i.e., cholangiocytes) is a heterogeneous population of epithelial cells in the liver, which line small and large bile ducts and have individual responses and functions dependent on size and location in the biliary tract. We discuss the recent findings showing that the intrahepatic biliary tree is heterogeneous regarding (1) morphology and function, (2) hormone expression and signaling (3), response to injury, and (4) roles in liver regeneration. This review overviews the significant characteristics and differences of the small and large cholangiocytes. Briefly, it outlines the in vitro and in vivo models used in the heterogeneity evaluation. In conclusion, future studies addressing biliary heterogeneity's role in the pathogenesis of liver diseases characterized by ductular reaction may reveal novel therapeutic approaches.

Background and Aims: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5–27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 −/− [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5–27 treatment in Mdr2 −/− mice. Approach and Results: In vivo studies were performed in male wild-type and Mdr2 −/− mice treated with saline or SCT 5–27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 −/− mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-β1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 −/− mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 −/− compared with wild-type mice, which was reversed by long-term SCT 5–27 treatment. In vitro , SCT 5–27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-β1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5–27–induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. Conclusions: Modulating the SCT/SR axis may be critical for managing PSC.

Fibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR‐16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR‐16, are unknown. In vivo studies were performed in male bile duct–ligated (BDL, 12‐week‐old) mice, multidrug resistance 2 knockout (Mdr2−/−) mice (10‐week‐old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti‐FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR‐16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR‐16, while treatment with AZD4547 or anti‐FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2−/− mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR‐16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR‐16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR‐16 axis may provide therapeutic options in treating cholangiopathies such as PSC.

Background & AimsPrimary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC.MethodsLiver biopsies were collected from naïve people with PBC (N = 87). Clinical–serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2−/− mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence.ResultsDuctular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals’ estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular–canalicular junctions.ConclusionsExtensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis.Lay summaryIn people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at diagnosis correlates with the lack of response to first-line therapy with ursodeoxycholic acid and serves to restore ductular–canalicular junctions in people with PBC. Assessing ductular reaction extension at diagnosis may add valuable information for clinicians.

Indole is a microbiota metabolite that functions to protect against obesity-associated non-alcoholic fatty liver disease. The present study examined the extent to which indole supplementation alleviates the severity of non-alcoholic steatohepatitis (NASH), which is the advanced form of non-alcoholic fatty liver disease. In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregations of macrophages in the liver compared with control diet-fed mice. Upon indole supplementation, the severity of MCD-induced hepatic steatosis and inflammation, as well as liver fibrosis, was significantly decreased compared with that of MCD-fed and control-treated mice. In vitro, indole treatment caused significant decreases in lipopolysaccharide-induced proinflammatory responses in hepatocytes incubated with either basal or MCD-mimicking media. However, indole treatment only significantly decreased lipopolysaccharide-induced proinflammatory responses in bone marrow-derived macrophages incubated with basal, but not MCD-mimicking media. These differential effects suggest that, relative to the responses of macrophages to indole, the responses of hepatocytes to indole appeared to make a greater contribution to indole alleviation of NASH, in particular liver inflammation. While indole supplementation decreased liver expression of desmin in MCD-fed mice, treatment of LX2 cells (a line of hepatic stellate cells) with indole also decreased the expression of various markers of hepatic stellate cell fibrogenic activation. Lastly, indole supplementation decreased intestinal inflammation in MCD-fed mice, suggesting that decreased intestinal inflammation also was involved in indole alleviation of NASH. Collectively, these results demonstrate that indole supplementation alleviates MCD-induced NASH, which is attributable to, in large part, indole suppression of hepatocyte proinflammatory responses and hepatic stellate cell fibrogenic activation, as well as intestinal proinflammatory responses.