Intestinal microbiota and biliary system diseases.

Introduction: The incidence of biliary system diseases has been continuously increasing in the past decade. Biliary system diseases bring a heavy burden to humanity and society. However, the specific etiology and pathogenesis are still unknown. The biliary system, as a bridge between the liver and intestine, plays an indispensable role in maintaining the physiological metabolism of the body. Therefore, prevention and treatment of biliary diseases are crucial. It is worth noting that the microorganisms participate in the lipid metabolism of the bile duct, especially the largest proportion of intestinal bacteria. Methods: We systematically reviewed the intestinal microbiota in patients with gallstones (GS), non-calculous biliary inflammatory, and biliary tract cancer (BTC). And searched Pubmed, Embase and Web of science for research studies published up to November 2023. Results: We found that the abundance of Faecalibacterium genus is decreased in GS, primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and BTC. Veillonella, Lactobacillus, Streptococcus and Enterococcus genus were significantly increased in PSC, PBC and BTC. Interestingly, we found that the relative abundance of Clostridium was generally reduced in GS, PBC and BTC. However, Clostridium was generally increased in PSC. Discussion: The existing research mostly focuses on exploring the mechanisms of bacteria targeting a single disease. Lacking comparison of multiple diseases and changes in bacteria during the disease process. We hope to provide biomarkers forearly diagnosis of biliary system diseases and provide new directions for the mechanism of intestinal microbiota in biliary diseases.

Enteroblastic cholangiocarcinoma: An uncommon, underrecognized subtype of bile duct cancer.

Enteroblastic carcinoma is clinically characterized by an elevated serum level of alpha-fetoprotein (AFP) and is histologically characterized by cancer cells with a clear cytoplasm and 'blastic' coarse chromatin. It sometimes has an element of hepatoid carcinoma; therefore, these two neoplasms are often regarded as sister entities. Although hepatoid carcinoma in the biliary tree has been reported, enteroblastic cholangiocarcinoma is extremely uncommon. In the present study, four cases of enteroblastic cholangiocarcinoma were examined. Tumors were located inside the liver (n = 2) or common bile duct (n = 2). The two intrahepatic cases had a history of primary sclerosing cholangitis, and serum AFP levels were elevated in both. One unresectable case was diagnosed by needle liver biopsy, while the remaining three underwent surgical resection. Histologically, all cases showed similar microscopic features. Cuboidal or polygonal cancer cells with the characteristic clear cytoplasm and subnuclear vacuoles were arranged in a papillary, micropapillary, tubular, or solid architecture. One case had an element of pancreatobiliary-type adenocarcinoma, while a hepatoid carcinoma element was not observed in any cases. All cases were positive for AFP, glypican 3, and SALL4, with SALL4 being the most widely expressed. Heppar-1 and arginase-1 were negative, except for one case, which was positive for Heppar-1. In conclusion, enteroblastic cholangiocarcinoma is an uncommon subtype of biliary tract malignancy. These cases may have been categorized as 'clear cell' cholangiocarcinoma. Although enteroblastic cholangiocarcinoma seems to occur more commonly in extrahepatic regions, including the gallbladder, it may also develop in the liver, particularly in patients with primary sclerosing cholangitis.

Mimickers of immunoglobulin G4-related hepatobiliary disease on biopsy.

With the growing recognition of IgG4-related hepatobiliary disease, establishing a definitive diagnosis relies mainly on a combination of clinical findings, serological markers, and imaging modalities. However, the role of histopathological evaluation remains indispensable, particularly in cases necessitating differential diagnosis or malignancy exclusion. While diagnosing IgG4-related hepatobiliary disease through surgical resection specimens is often straightforward, pathologists encounter substantial challenges when evaluating biopsies. The increasing rarity of surgical interventions exacerbates this due to improved disease recognition and suspicion. Numerous confounding factors, including the absence of the characteristic histologic features, limited tissue sample size, biopsy artifacts, and the limited value of IgG4 counts, further complicate the diagnostic process. Additionally, many other disorders exhibit clinical and histological features that overlap with IgG4-related disease, intensifying the complexity of interpreting biopsy specimens. This article explores the clinical and histomorphologic features of IgG4-related hepatobiliary disease and its potential mimickers. It offers valuable insights for pathologists and clinicians when confronted with biopsy specimens from hepatobiliary organs.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

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

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

Increased expression of TNFRSF14 and LIGHT in biliary epithelial cells of patients with primary sclerosing cholangitis.

BACKGROUND AND AIMS: There is a lack of biliary epithelial molecular markers for primary sclerosing cholangitis (PSC). We analyzed candidates from disease susceptibility genes identified in recent genome-wide association studies (GWAS). METHODS: Expression levels of GWAS genes were analyzed in archival liver tissues of patients with PSC and controls. Immunohistochemical analysis was performed to evaluate expression levels in the biliary epithelia of PSC (N = 45) and controls (N = 12). Samples from patients with primary biliary cholangitis (PBC) were used as disease controls (N = 20). RESULTS: Hepatic expression levels of ATXN2, HHEX, PRDX5, MST1, and TNFRSF14 were significantly altered in the PSC group. We focused on the immune-related receptor, TNFRSF14. Immunohistochemistry revealed that high expression of TNFRSF14 in biliary epithelial cells was observed only in the PSC group. In addition, the expression of LIGHT, which encodes a TNFRSF14-activating ligand, was increased in PSC liver. Immunohistochemistry showed that high expression of LIGHT was more common in PSC biliary epithelia (53%) than in the PBC (15%) or control (0%) groups; moreover, it was positively associated with fibrotic progression, although it was not an independent prognostic factor. CONCLUSIONS: TNFRSF14 and LIGHT are promising candidate markers for PSC.

Beta-lapachone ameliorates the progression of primary sclerosing cholangitis pathogenesis in rodent models.

AIMS: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by chronic inflammation and severe fibrosis for which effective treatment options are currently lacking. In this study, we explored the potential of beta-lapachone (ßL) as a drug candidate for PSC therapy. MATERIALS AND METHODS: We employed an animal model fed a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to assess the preventive and therapeutic effects of ßL. The beneficial effects of ßL on PSC pathogenic characteristics, including blood biomarkers, inflammation, and fibrosis, were determined by assessing relevant parameters. Differential gene expression between each group was analyzed by RNA sequencing of liver tissues. Mdr2-/- mice were utilized to explore the involvement of Abcb4 in the ßL-induced improvement of PSC pathogenesis. KEY FINDINGS: ßL effectively inhibited key features of PSC pathogenesis, as demonstrated by reduced blood biomarkers and improved pathogenic characteristics. Treatment with ßL significantly mitigated DDC-induced apoptosis, cell proliferation, inflammation, and fibrosis. Analysis of differential gene expression confirmed a new insight that ßL could stimulate the expression of genes related to NAD synthesis and Abcb4. Indeed, ßL-induced NAD exhibited effective functioning, as evidenced by enhanced sirt1/3 and acetyl-lysine levels, leading to improved mitochondrial stability. The role of Abcb4 in response to ßL was confirmed in Mdr2/Abcb4 KO mice, where the beneficial effects of ßL were abolished. SIGNIFICANCE: This study provided a new concept for PSC treatment, suggesting that pharmacological stimulation of the NAD synthetic pathway and Abcb4 via ßL ameliorates PSC pathogenesis.

Human vascularized bile duct-on-a chip: a multi-cellular micro-physiological system for studying cholestatic liver disease.

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity ofin vitromodels that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured inex vivosystems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

Telomere dysfunction promotes cholangiocyte senescence and biliary fibrosis in primary sclerosing cholangitis.

Cellular senescence and biliary fibrosis are prototypical features of obliterative cholangiopathies, such as primary sclerosing cholangitis (PSC). Telomere dysfunction can lead to senescence either through telomere erosion or damaged telomeres. Our goal was to investigate a mechanistic relationship between telomere damage and biliary fibrosis in PSC. Telomere attrition was observed in the bile ducts of patients with PSC along with a reduction in telomerase reverse transcriptase (TERT) expression, compared with that in normal livers. Similarly, liver tissue from mouse models of biliary fibrosis showed telomere attrition with increased damage at telomeres measured as telomere-associated foci (TAF). Cellular models of senescence induction increased the TAF in cholangiocytes. This coincided with decreased TERT expression and increased senescence, which was rescued by modulating TERT levels. Epigenetic analysis revealed increased acquisition of repressive histone methylation at the TERT promoter, which correlated with decreased TERT transcription. Cholangiocyte-selective deletion of TERT in mice exacerbated fibrosis, whereas androgen therapy toward telomerase rescued liver fibrosis and liver function in a genetic mouse model of PSC. Our results demonstrate a mechanistic role for telomere dysfunction in cellular senescence and fibrosis that characterize PSC. This suggests that PSC may be, in part, a telomere biology disorder, and identifies TERT as a potential therapeutic target.

Independent of Primary Sclerosing Cholangitis and Cirrhosis, Early Adulthood Obesity Is Associated with Cholangiocarcinoma.

BACKGROUND: It is estimated that 6% to 20% of all cholangiocarcinoma (CCA) diagnoses are explained by primary sclerosing cholangitis (PSC), but the underlying risk factors in the absence of PSC are unclear. We examined associations of different risk factors with intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) in the United States. METHODS: We conducted a case-control study of 121 patients with ECC and 308 patients with ICC treated at MD Anderson Cancer Center between May 2014 and March 2020, compared with 1,061 healthy controls. Multivariable logistic regression analysis was applied to estimate the adjusted OR (AOR) and 95% confidence interval (CI) for each risk factor. RESULTS: Being Asian, diabetes mellitus, family history of cancer, and gallbladder stones were associated with higher odds of developing ICC and ECC. Each 1-unit increase in body mass index in early adulthood (ages 20-40 years) was associated with a decrease in age at diagnosis of CCA (6.7 months, P < 0.001; 6.1 months for ICC, P = 0.001; 8.2 months for ECC, P = 0.007). A family history of cancer was significantly associated with the risk of ICC and ECC development; the AORs (95% CI) were 1.11 (1.06-1.48) and 1.32 (1.01-2.00) for ICC and ECC, respectively. CONCLUSIONS: In this study, early adulthood onset of obesity was significantly associated with CCA and may predict early diagnosis at younger age than normal weight individuals. IMPACT: The study highlights the association between obesity and CCA, independent of PSC. There is a need to consider the mechanistic pathways of obesity in the absence of fatty liver and cirrhosis.

Primary Sclerosing Cholangitis, Small Duct Primary Sclerosing Cholangitis, IgG4-Related Sclerosing Cholangitis, and Ischemic Cholangiopathy: Diagnostic Challenges on Biopsy.

Pathologists face many challenges when diagnosing sclerosing biliary lesions on liver biopsy. First, histologic findings tend to be nonspecific with similar to identical features seen in numerous conditions, from benign to outright malignant. In addition, the patchy nature of many of these entities amplifies the inherent limitations of biopsy sampling. The end result often forces pathologists to issue descriptive sign outs that require careful clinical correlation; however, certain clinical, radiologic, and histologic features may be of diagnostic assistance. In this article, we review key elements of four sclerosing biliary processes whose proper identification has significant prognostic and therapeutic implications.

Distinct immune surveillance in primary biliary cholangitis and primary sclerosing cholangitis is linked with discrete cholangiocarcinoma risk.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are 2 major liver autoimmune diseases. PBC is common in women and primarily affects intrahepatic small bile duct epithelial cells, known as cholangiocytes. In contrast, PSC is dominant in men and primarily affects medium and big intrahepatic and extrahepatic bile duct epithelial cells. Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, and its incidence is increasing worldwide in both men and women. Numerous retrospective and clinical studies have suggested that PBC patients rarely develop CCA compared to PSC patients. CCA is accountable for the higher deaths in PSC patients due to ineffective therapies and our inability to diagnose the disease at an early stage. Therefore, it is paramount to understand the differences in immune surveillance mechanisms that render PBC patients more resistant while PSC patients are susceptible to CCA development. Here, we review several potential mechanisms contributing to differences in the susceptibility to CCA in PBC versus PSC patients.

Primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD): a condition exemplifying the crosstalk of the gut-liver axis.

The close relationship between primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) provides a good opportunity to comprehend the gut-liver axis. The gut and the liver have reciprocal interactions, including how gut inflammation influences the liver through immune cells and the microbiota and how the microbiota in the gut modifies bile acids, which are produced and secreted from the liver. PSC-IBD shows distinct clinical findings from classical IBD. In addition, a distinct genetic predisposition and unique microbiota composition suggest that PSC-IBD is an independent disease entity. Understanding the pathogenesis of PSC-IBD helps to develop novel and effective therapeutic agents. Given the high risk of malignancies associated with PSC-IBD, it is critical to identify patients at high risk and implement appropriate surveillance and monitoring strategies. In this review, we provide an overview of PSC-IBD, which exemplifies the gut-liver axis.

Antigen-driven colonic inflammation is associated with development of dysplasia in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated disease of the bile ducts that co-occurs with inflammatory bowel disease (IBD) in almost 90% of cases. Colorectal cancer is a major complication of patients with PSC and IBD, and these patients are at a much greater risk compared to patients with IBD without concomitant PSC. Combining flow cytometry, bulk and single-cell transcriptomics, and T and B cell receptor repertoire analysis of right colon tissue from 65 patients with PSC, 108 patients with IBD and 48 healthy individuals we identified a unique adaptive inflammatory transcriptional signature associated with greater risk and shorter time to dysplasia in patients with PSC. This inflammatory signature is characterized by antigen-driven interleukin-17A (IL-17A)+ forkhead box P3 (FOXP3)+ CD4 T cells that express a pathogenic IL-17 signature, as well as an expansion of IgG-secreting plasma cells. These results suggest that the mechanisms that drive the emergence of dysplasia in PSC and IBD are distinct and provide molecular insights that could guide prevention of colorectal cancer in individuals with PSC.

COVID-19 related biliary injury: A review of recent literature.

Since its emergence in 2019, it has become apparent that coronavirus 2019 (COVID-19) infection can result in multi systemic involvement. In addition to pulmonary symptoms, hepatobiliary involvement has been widely reported. Extent of hepatic involvement ranges from minor elevation in liver function tests (LFTs) to significant hepatocellular or cholestatic injury. In majority of cases, resolution of hepatic injury or improvement in LFTs is noted as patients recover from COVID-19 infection. However, severe biliary tract injury progressing to liver failure has been reported in patients requiring prolonged intensive care unit stay or mechanical ventilation. Due to the timing of its presentation, this form of progressive cholestatic injury has been referred to as COVID-19 cholangiopathy or post-COVID-19 cholangiopathy, and can result in devastating consequences for patients. COVID-19 cholangiopathy is recognized by dramatic elevation in serum alkaline phosphatase and bilirubin and radiologic evidence of bile duct injury. Cholangiopathy in COVID-19 occurs weeks to months after the initial infection and during the recovery phase. Imaging findings and pathology often resemble bile duct injury associated with primary or secondary sclerosing cholangitis. Etiology of COVID-19 cholangiopathy is unclear. Several mechanisms have been proposed, including direct cholangiocyte injury, vascular compromise, and cytokine release syndromes. This review summarizes existing data on COVID-19 cholangiopathy, including reported cases in the literature, proposed pathophysiology, diagnostic testing, and long-term implications.

DNA content abnormality frequently develops in the right/proximal colon in patients with primary sclerosing cholangitis and inflammatory bowel disease and is highly predictive of subsequent detection of dysplasia.

AIMS: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD, termed PSC-IBD) have a higher risk of harbouring nonconventional and/or invisible dysplasias, especially in the right/proximal colon, than those with IBD alone. We postulated that DNA content abnormality may be frequently detected in the right/proximal colon in PSC-IBD patients, even in the absence of dysplasia, and that this may predispose to progression to nonconventional and/or invisible dysplasias that are often associated with increased rates of aneuploidy and advanced neoplasia. METHODS AND RESULTS: DNA flow cytometry was performed on 96 morphologically benign colon biopsies taken throughout the colon from 25 PSC-IBD patients during the surveillance colonoscopy that preceded the next procedure that detected dysplasia. Thirty (31%) of the 96 benign colon biopsies in this dysplasia group demonstrated abnormal DNA content, with a propensity for the right/proximal colon (70%) (P < 0.001). In contrast, only one (1%) of 87 benign colon biopsies from 20 IBD patients without neoplasia (control group) demonstrated DNA content abnormality, and it was from the left colon. For analysis per patient, 48% (12 of 25) of the patients in the dysplasia group had abnormal DNA content compared with 5% (1 of 20) of the control group (P = 0.002). Of the 12 PSC-IBD patients with DNA content abnormality, invisible dysplasia was detected in 10 (83%) patients on follow-up, nine (75%) of whom had nonconventional dysplasia. CONCLUSION: PSC-IBD patients have an increased risk of developing abnormal DNA content in the right/proximal colon, predating the detection of dysplasia.

The Epigenetic Reader, Bromodomain Containing 2, Mediates Cholangiocyte Senescence via Interaction With ETS Proto-Oncogene 1.

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

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

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

Longitudinal changes in quantitative magnetic resonance imaging metrics in children and young adults with autoimmune liver disease.

PURPOSE: To assess longitudinal changes in quantitative MRI metrics in pediatric and young adult patients with autoimmune liver disease (AILD). METHODS: This prospective, IRB-approved study included 20 children and young adults (median age = 15 years) with primary sclerosing cholangitis (PSC)/autoimmune sclerosing cholangitis (ASC) and 19 (median age = 17 years) with autoimmune hepatitis (AIH). At a field strength of 1.5-T, T2*-corrected T1 mapping (cT1), 3D fast spin-echo MRCP, and 2D gradient recalled echo MR elastography (MRE) were performed at baseline, one year, and two years. cT1 and quantitative MRCP were processed using LiverMultiScan and MRCP + , respectively (Perspectum Ltd, Oxford, UK). Linear mixed models were used to assess longitudinal changes in quantitative MRI metrics. Spearman rank-order correlation was used to assess relationships between changes in quantitative MRI metrics. RESULTS: Changes in quantitative MRI metrics greater than established repeatability coefficients were measured in six (cT1) and five (MRE) patients with PSC/ASC as well as in six patients (cT1 and MRE) with AIH, although linear mixed models identified no significant changes for the subgroups as a whole. For PSC/ASC, there were positive correlations between change in liver stiffness and changes in bile duct strictures (ρ = 0.68; p = 0.005) and bile duct dilations (ρ = 0.70; p = 0.004) between baseline and Year 2. CONCLUSION: On average, there were no significant changes in quantitative MRI metrics over a two-year period in children and young adults with AILD. However, worsening cholangiopathy was associated with increasing liver stiffness by MRE in patients with PSC/ASC.