Modulation of miR-155-5p signalling via 5-ASA for the prevention of high microsatellite instability: an in vitro study using human epithelial cell lines.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug's chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA's prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

Effect of Low Testosterone Levels on the Expression of Proliferator-Activated Receptor Alpha in Female Patients with Primary Biliary Cholangitis.

Sex-dependent patterns in chronic immune-mediated cholangiopathies, like primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), remain poorly understood. Peroxisome proliferator-activated receptor alpha (PPAR-α), expressed in immune cells, plays a key role in innate defence. In this study, the relationship between PPAR-α expression in peripheral blood mononuclear cells (PBMCs), serum androgen levels, IFNγ production, and sex-dependent tendencies during the development of PBC and PSC was investigated. We confirmed that normal cholangiocytes respond to PPAR-α and inhibit the lipopolysaccharide-induced expression of IL-6, IL-1b, and TNFα. Compared with PSC patients, PPAR-α was downregulated, while IFNγ was upregulated, in the PBMCs of PBC patients. When the analysis was conducted on females only, there was no difference in PPAR-α, but IFNγ was elevated in females with PBC compared with those with PSC. Serum testosterone concentrations in females with PBC were below the normal range (regardless of age) and correlated positively with PPAR-α and negatively with IFNγ. While PPAR-α has been reported to be a target of miR-155 and miR-21, no correlations with these microRNAs were observed in the PBMCs. However, a positive correlation between miR-21 and IFNγ was observed. Our results showed suppressed PPAR-α expression accompanied by reduced testosterone levels in women with PBC, which should elicit interest in the role of testosterone in PBC development.

The Role of hsa-miR-125b-5p Interaction with S1P/Ceramide Axis in the Potential Development of Inflammation-Associated Colon Cancer in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is characterised by the co-occurrence of inflammatory bowel diseases, particularly ulcerative colitis (UC). We investigated how the interaction of miR-125b with the sphingosine-1-phosphate (S1P)/ceramide axis may predispose patients with PSC, PSC/UC, and UC to carcinogenesis in the ascending and sigmoid colons. The overexpression of miR-125b was accompanied by the upregulation of S1P, ceramide synthases, ceramide kinases, and the downregulation of AT-rich interaction domain 2 in the ascending colon of PSC/UC, which contributed to the progression of high microsatellite instability (MSI-H) colorectal carcinoma. We also showed that the overexpression of sphingosine kinase 2 (SPHK2) and the genes involved in the glycolytic pathway in the sigmoid colon of UC led to the upregulation of Interleukin 17 (IL-17). In vitro stimulation of human intestinal epithelial cells (Caco-2, HT-29, and NCM460D) with lipopolysaccharide suppressed miR-125b and increased proinflammatory cytokines, whereas the induction of miR-125b activity by either a miR-125b mimetic or lithocholic acid resulted in the inhibition of miR-125b targets. In summary, miR-125b overexpression was associated with an imbalance in the S1P/ceramide axis that can lead to MSI-H cancer progression in PSC/UC. Furthermore, SPHK2 overexpression and a change in the cellular metabolic flux are important players in inflammation-associated colon cancer in UC.

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). METHODS: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. CONCLUSIONS: Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

TREM-2 plays a protective role in cholestasis by acting as a negative regulator of inflammation.

BACKGROUND & AIMS: Inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis. METHODS: TREM-2 expression was analyzed in the livers of patients with primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Primary cultured Kupffer cells were incubated with lipopolysaccharide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed. RESULTS: TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/- mice. This was characterized by enhanced necroptotic cell death, inflammatory responses and biliary expansion. Antibiotic treatment partially abrogated the effects observed in Trem-2-/- mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and dampened inflammatory gene transcription via a TREM-2-dependent mechanism. CONCLUSIONS: TREM-2 acts as a negative regulator of inflammation during cholestasis, representing a novel potential therapeutic target. LAY SUMMARY: Cholestasis (the reduction or cessation of bile flow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors (specifically Toll-like receptors or TLRs) on liver-resident immune cells, promoting inflammation. Herein, we show that the anti-inflammatory receptor TREM-2 dampens TLR-mediated signaling and hence protects against cholestasis-induced liver injury. Thus, TREM-2 could be a potential therapeutic target in cholestasis.

Modulation of Mismatch Repair and the SOCS1/p53 Axis by microRNA-155 in the Colon of Patients with Primary Sclerosing Cholangitis.

Deficient mismatch repair (MMR) proteins may lead to DNA damage and microsatellite instability. Primary sclerosing cholangitis (PSC) is a risk factor for colitis-associated colon cancer. MiR-155 is suggested to act as a key regulating node, linking inflammation and tumorigenesis. However, its involvement in the chronic colitis of PSC-UC patients has not been examined. We investigated the involvement of miR-155 in the dysregulation of MMR genes and colitis in PSC patients. Colon tissue biopsies were obtained from patients with PSC, PSC with concomitant ulcerative colitis (PSC-UC), uncomplicated UC, and healthy controls (n = 10 per group). In the ascending colon of PSC and PSC-UC patients, upregulated miR-155 promoted high microsatellite instability and induced signal transducer and activator of transcription 3 (STAT-3) expression via the inhibition of suppressors of cytokine signalling 1 (SOCS1). In contrast, the absence of miR-155 overexpression in the sigmoid colon of PSC-UC patients activated the Il-6/S1PR1 signalling pathway and imbalanced the IL17/FOXP3 ratio, which reinforces chronic colitis. Functional studies on human intestinal epithelial cells (HT-29 and NCM460D) confirmed the role of miR-155 over-expression in the inhibition of MMR genes and the modulation of p53. Moreover, those cells produced more TNFα upon a lipopolysaccharide challenge, which led to the suppression of miR-155. Additionally, exposure to bile acids induced upregulation of miR-155 in Caco-2 cell lines. Thus, under different conditions, miR-155 is involved in either neoplastic transformation in the ascending colon or chronic colitis in the sigmoid colon of patients with PSC. New insight into local modulation of microRNAs, that may alter the course of the disease, could be used for further research on potential therapeutic applications.

DHEA Protects Human Cholangiocytes and Hepatocytes against Apoptosis and Oxidative Stress.

Primary biliary cholangitis (PBC) is a rare chronic cholestatic and immune-mediated liver disease of unknown aetiology that targets intrahepatic bile duct cells (cholangiocytes) and primarily affects postmenopausal women, when their estrogen levels sharply decrease. An impaired cholangiocyte response to estrogen characterizes the terminal stage of the disease, as this is when an inefficiency of cholangiocyte proliferation, in balancing the loss of intrahepatic bile ducts, is observed. Here, we report that the estrogen precursor dehydroepiandrosterone (DHEA) and its sulfate metabolites, DHEA-S and 17 ß-estradiol, enhance the proliferation of cholangiocytes and hepatocytes in vitro. Flow cytometry analysis showed that DHEA and DHEA-S decreased glyco-chenodeoxycholic acid (GCDC)-driven apoptosis in cholangiocytes. Cell viability assay (MTT) indicated that ER-α, -ß, and the G-protein-coupled estrogen receptor, are involved in the protection of DHEA against oxidative stress in cholangiocytes. Finally, immunoblot analysis showed an elevated level of steroid sulfatase and a reduced level of sulfotransferase 1E1 enzymes, involved in the desulfation/sulfation process of estrogens in cirrhotic PBC, and primary sclerosis cholangitis (PSC) liver tissues, another type of chronic cholestatic and immune-mediated liver disease. Taken together, these results suggest that DHEA can prevent the deleterious effects of certain potentially toxic bile acids and reactive oxygen species, delaying the onset of liver disease.

p-STAT3 is a PDC-E2 interacting partner in human cholangiocytes and hepatocytes with potential pathobiological implications.

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury.

BACKGROUND & AIMS: Mitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored. METHODS: We studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury. RESULTS: Elevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics. CONCLUSIONS: We demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis. LAY SUMMARY: In this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Role of miR-506 in ulcerative colitis associated with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is commonly accompanied by ulcerative colitis (UC). MicroRNA-506 modulates expression of genes which are essential for sphingosine-mediated signaling pathway and intestinal mucosa protection. We investigated whether miR-506 and its target genes are involved in phenotypic presentations of colonic inflammation and/or neoplasia. We analyzed serum and colon tissue samples collected from patients with PSC, PSC with concurrent UC (PSC + UC), UC alone, and healthy controls (n = 10 each). MiR-506 was substantially upregulated in ascending colons of PSC and PSC + UC patients, in contrast to sigmoid colons of PSC and UC patients. Upregulation of miR-506 was associated with inhibition of SPHK1, AE2, InsP3R3, and p53. Colonic suppression of miR-506 presented in UC was accompanied by substantially increased DNMT1, SPHK1, and S1P lyase expressions. A functional in vitro analysis in Caco-2 cells showed that the induction of miR-506 activity by miR-506 mimic or GDCDA bile acid suppressed, whereas inhibition of miR-506 by miR-506 inhibitor or lipopolysaccharide (LPS) upregulated the expression of the examined target genes. A different phenotypic presentation of colitis may be related to miR-506 expression. In ascending colons with PSC + UC, upregulation of miR-506 may result in failure of bicarbonate secretion and inhibition of p53, which predisposes to pro-tumorigenic transformation. In contrast, downregulation of miR-506 enhances S1P production, leading to pro-inflammatory signaling.

Anti-glycoprotein 2 (anti-GP2) IgA and anti-neutrophil cytoplasmic antibodies to serine proteinase 3 (PR3-ANCA): antibodies to predict severe disease, poor survival and cholangiocarcinoma in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is associated with progressive liver disease and cholangiocarcinoma. Although risk stratification is crucial for making clinical decisions, it is hindered by a scarcity of proven prognostic markers. AIMS: To assess the value of novel anti-glycoprotein 2 (anti-GP2) and anti-neutrophil cytoplasmic antibodies to serine proteinase 3 (PR3-ANCA) in combination with PSC-specific clinical and laboratory markers as predictors of quality of life, disease severity, and cholangiocarcinoma in two large, independent cohorts of PSC patients METHODS: Discovery (338 Polish patients) and validation (178 German patients) cohorts with PSC were evaluated. Anti-GP2 (isoforms 1/4) was detected by ELISAs and PR3-ANCA by chemiluminescence immunoassay. Clinical and laboratory data were collected and analysed. The outcome was defined as liver transplantation-free survival and occurrence of cholangiocarcinoma during follow-up. RESULTS: In the discovery group, anti-GP21/4 IgA and PR3-ANCA were associated with liver dysfunction, anti-GP21/4 IgA with risk scores for PSC and anti-GP24 IgA with cirrhosis. All cholangiocarcinoma patients were positive for PR3-ANCA and/or anti-GP24 IgA. The association between anti-GP2 IgA and liver biochemistry, risk scores, cirrhosis, impaired survival, and cholangiocarcinoma was confirmed in the validation cohort. Cox proportional-hazards regression indicated anti-GP21 IgA as an independent variable of poor outcome in both study cohorts. Analysis of the combined data showed that anti-GP24 IgA and PR3-ANCA were independent predictors for cholangiocarcinoma, while anti-GP21 IgA and PR3-ANCA were indicators for poor survival. CONCLUSIONS: Anti-GP2 and PR3-ANCA are prognostic antibodies in PSC as they identify patients at risk of severe disease, poor survival and biliary cancer.

Melatonin Protects Cholangiocytes from Oxidative Stress-Induced Proapoptotic and Proinflammatory Stimuli via miR-132 and miR-34.

Biosynthesis of melatonin by cholangiocytes is essential for maintaining the function of biliary epithelium. However, this cytoprotective mechanism appears to be impaired in primary biliary cholangitis (PBC). MiR-132 has emerged as a mediator of inflammation in chronic liver diseases. The effect of melatonin on oxidative stress and bile acid-induced apoptosis was also examined in cholangiocyes overexpressing miR506, as a PBC-like cellular model. In PBC patients the serum levels of melatonin were found increased in comparison to healthy controls. Whereas, in cholangiocytes within cirrhotic PBC livers the melatonin biosynthetic pathway was substantially suppressed even though the expressions of melatonin rate-limiting enzyme aralkylamine N-acetyltransferase (AANAT), and CK-19 (marker of cholangiocytes) were enhanced. In cholangiocytes exposed to mitochondrial oxidative stress melatonin decreased the expression of proapoptotic stimuli (PTEN, Bax, miR-34), which was accompanied by the inhibition of a pivotal mediator of inflammatory response Nf-κB-p65 and the activation of antiapoptotic signaling (miR-132, Bcl2). Similarly, melatonin reduced bile acid-induced proapoptotic caspase 3 and Bim levels. In summary, the insufficient hepatic expression of melatonin in PBC patients may predispose cholangiocytes to oxidative stress-related damage. Melatonin, via epigenetic modulation, was able to suppress NF-κB signaling activation and protect against biliary cells apoptotic signaling.

Oncomir MicroRNA-346 Is Upregulated in Colons of Patients With Primary Sclerosing Cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a cholestatic liver disorder that is frequently associated with ulcerative colitis (UC). Patients with PSC and UC (PSC-UC) have a higher risk of colorectal neoplasia compared with patients with UC. The oncogenic properties of microRNA-346 (miR-346) have been recently reported. We investigated the expression of miR-346 and its 2 target genes, the receptor of vitamin D (VDR), and the tumor necrosis factor-α (TNF-α), which are known to modulate carcinogenesis. METHODS: Ascending and sigmoid colon biopsies were obtained from patients with PSC, PSC and UC (PSC-UC), UC, and healthy controls (n = 10 in each group). Expressions of VDR, TNF-α, 18S RNA, p27, miR-346, and reference microRNA, miR-191, were evaluated by real-time PCR using human TaqMan Gene Expression and TaqMan MicroRNA Assays. Functional studies with miR-346 mimic and inhibitor were conducted in HepG2 and Caco-2 cells. The effect of ursodeoxycholic acid on miR-346 expression was examined in Caco-2 cells. RESULTS: An increased expression of miR-346 in the ascending colon of PSC-UC was observed (P < 0.001 vs all groups). In patients with UC, an exceptionally low colonic expression of miRNA-346 was accompanied by the extensive upregulation of VDR and TNF-α genes. A functional in vitro analysis demonstrated that inhibition of miR-346 resulted in the upregulation of VDR and TNF-α, whereas the induction of miR-346 activity suppressed VDR, TNF-α, and p27. DISCUSSION: The upregulation of miRNA-346 in the colon of patients with PSC may be responsible for the disturbance of VDR and TNF-α signaling pathway, which could result in an inadequate suppression of neoplasia.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

The Epidermal Growth Factor Receptor Ligand Amphiregulin Protects From Cholestatic Liver Injury and Regulates Bile Acids Synthesis.

Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.

Effect of S-adenosyl-L-methionine on liver biochemistry and quality of life in patients with primary biliary cholangitis treated with ursodeoxycholic acid. A prospective, open label pilot study.

BACKGROUND AND AIMS: Chronic liver disease induces an acquired deficiency of S-adenosyl-L-methionine (SAMe) leading to impairment of detoxifying processes in the liver. Ursodeoxycholic acid (UDCA) represents the standard treatment in primary biliary cholangitis (PBC). As both compounds exert their hepatoprotective effects by different mechanisms, it is conceivable that when used together their effect might be additive. The aim of this study was to analyse the effect of SAMe supplementation on liver biochemistry and health-related quality of life (HRQoL) in patients with PBC, treated with UDCA. METHODS: In this prospective pilot, proof of the principle, non-randomized and open label study we enrolled 24 patients with PBC treated with UDCA for at least 6 months. They had received both UDCA in a standard dose of 13-15 mg/kg b.w. and SAMe in the dose of 1200 mg daily over a period of 6 months. A group of 24 patients with PBC treated with UDCA served as control for liver biochemistry (Study registered on the platform ClinicalTrials.gov under ID: NCT02557360). RESULTS: We observed a significant decrease of ALP, GGT and total cholesterol in non-cirrhotic patients treated with SAMe. There was also a significant improvement of fatigue and pruritus in PBC-40 questionnaire and amelioration of anxiety in STAI 2 questionnaire in the SAMe group. Treatment with SAMe neither increased sulfation capacity of the liver nor had an effect on fibroblast growth factor-19 serum levels. CONCLUSIONS: Our pilot study demonstrates a positive effect of adding SAMe to UDCA in non-cirrhotic patients with PBC.

Genetic Risk Factors for Autoimmune Thyroid Disease might Affect the Susceptibility to and Modulate the Progression of Primary Biliary Cholangitis.

BACKGROUND AND AIMS: Patients with primary biliary cholangitis (PBC) frequently suffer from extrahepatic autoimmune conditions, of which autoimmune thyroid disease (AITD) is one of the most common. Previous studies identified several genetic variants increasing the odds of developing AITD. Here we investigate whether AITD-associated polymorphisms might also play a role in the development and clinical course of PBC and PBC associated with AITD (PBC-AITD). METHODS: To this end, we prospectively recruited 230 patients with PBC and 421 healthy controls. Among recruited patients, 64 (30.9%) had PBC-AITD as diagnosed by elevated serum TPO-antibodies. In all subjects we genotyped 10 variants previously associated with AITD. RESULTS: We detected significant associations between the PTPN22 polymorphism and risk of developing PBC (rs2476601, OR=1.43, P=0.035) as well as PBC-AITD (OR=1.74, P=0.028). The IL2RA polymorphism was associated with liver cirrhosis (rs41295061, OR=1.76, P=0.033) whereas the MMEL1 polymorphism increased the risk of requiring liver transplantation (rs2843403, OR=1.70, P=0.023). Although no significant differences in clinical or biochemical characteristics between patients with PBC and PBC-AITD were seen (all P>0.05), liver function tests and metabolic traits in PBC patients were significantly (all P<0.05) affected by the CTLA4 (rs3087243), MMEL1 (rs2843403), PTPN22 (rs2476601) and RNASET2 (rs9355610) variants. CONCLUSION: Our study demonstrates the existence of a genetic overlap between PBC and AITD. Apparently, genetic variants known to increase the AITD risk might affect the clinical course of PBC. On the other hand, AITD per se does not seem to significantly influence the natural history of PBC.

Does transient elastography correlate with liver fibrosis in patients with PSC? Laennec score-based analysis of explanted livers.

BACKGROUND AND AIMS: Previous studies demonstrated a close correlation between transient elastography (TE) and liver histology in chronic liver diseases. Data on the accuracy of TE in primary sclerosing cholangitis (PSC) remains scarce. Here, we investigated the association between TE, serum marker of liver injury and histology of explanted livers in PSC patients. METHODS: Thirty patients were prospectively recruited. TE (Fibroscan®) and blood sampling were performed during evaluation for liver transplantation (LT); the second blood sampling was performed on the day of LT. Fibrosis of explanted livers according to the seven-point Laennec staging system and liver collagen contents were measured. RESULTS: TE correlated with Laennec stages of fibrosis (p = .001), collagen contents (p < .001) and with diameter of thickest septa (p = .034) in explanted livers. It also correlated with serum indices of liver injury, namely AST, bilirubin as well as FIB-4 and APRI scores (all p < .05). In a multivariate model, only liver fibrosis, according to either Laennec score (p = .035) or collagen contents (p = .005), was significantly associated with TE. Finally, patients with cirrhosis had increased liver stiffness (p = .002) and the TE cut-off of 13.7 kPa showed the best predictive value (AUC = .90, 95% CI: 0.80-1.00, p < .001) for detecting cirrhosis. CONCLUSIONS: TE correlates with liver fibrosis and markers of liver injury in patients with PSC. However, liver fibrosis seems to be the strongest predictor of liver stiffness assessed with TE. Hence, we postulate that TE is a reliable tool for non-invasive monitoring of PSC.

Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in Primary Biliary Cholangitis.

In response to oxidative stress, nuclear factor (erythroid-derived 2)-like2 (Nrf2) induces expression of cytoprotective genes. The Nrf2 pathway is controlled by microRNAs and Kelch-like ECH-associated protein1 (Keap1). Nrf2 is stabilized when Keap1 is degraded through the autophagy pathway in a p62-dependent manner. The inhibition of autophagy causes protein accumulation, and Keap1 is inactivated by binding to p62. We investigated the role of the Nrf2/Keap1 axis in the amelioration of oxidative stress in primary biliary cholangitis (PBC). Liver specimens from patients with PBC, with (n = 24) or without cirrhosis (n = 14), and from controls (n = 16) were used for molecular analyses. We found that Nrf2 protein levels were elevated in PBC compared to controls, but Nrf2 gene expression was significantly reduced in cirrhotic PBC. Nrf2 target gene products, HO-1 and GCLC proteins, were reduced compared to controls and reduction of Nrf2 gene expression was associated with elevated levels of microRNA-132 and microRNA-34a. Both Keap1 and p62 protein levels were substantially increased in PBC compared to controls. PBC was associated with reduced Nrf2 expression and autophagy deterioration and these impairments were more advanced in patients with cirrhosis. Aberrant Nrf2/Keap1 system integrity may affect self-defence mechanisms against oxidative stress in PBC.

Impaired Hepatic Adaptation to Chronic Cholestasis induced by Primary Sclerosing Cholangitis.

Pathogenesis of primary sclerosing cholangitis (PSC) may involve impaired bile acid (BA) homeostasis. We analyzed expressions of factors mediating enterohepatic circulation of BA using ileal and colonic (ascending and sigmoid) biopsies obtained from patients with PSC with and without ulcerative colitis (UC) and explanted PSC livers. Two-fold increase of BA-activated farnesoid X receptor (FXR) protein levels were seen in ascending and sigmoid colon of PSC patients with correspondingly decreased apical sodium-dependent BA transporter (ASBT) gene expression. This was associated with increased OSTß protein levels in each part of analyzed gut. An intestinal fibroblast growth factor (FGF19) protein expression was significantly enhanced in ascending colon. Despite increased hepatic nuclear receptors (FXR, CAR, SHP), and FGF19, neither CYP7A1 suppression nor CYP3A4 induction were observed. The lack of negative regulation of BA synthesis may be accountable for lower levels of cholesterol observed in PSC in comparison to primary biliary cholangitis (PBC). In conclusion, chronic cholestasis in PSC induces adaptive changes in expression of BA transporters and FXR in the intestine. However hepatic impairment of expected in chronic cholestasis downregulation of CYP7A1 and upregulation of CYP3A4 may promote BA-induced liver injury in PSC.