Bettina Langhans

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by progressive inflammation of the intra- and extrahepatic bile ducts and a high association with inflammatory bowel disease. Since bacterial and viral stimuli are thought to contribute to the pathogenesis of autoimmune diseases, our study aims to investigate the effects of synthetic pathogenic agonists on the cytotoxic function of cluster of differentiation (CD)56+ natural killer (NK) cells and CD8+ T cells in PSC. A total of 17 PSC patients, 18 autoimmune hepatitis (AIH) patients, and 14 healthy controls (HCs) were included in this study. Using multicolor flow cytometry, we analyzed cytotoxic activity (CD107a assay) and secretion of interferon-gamma and tumor necrosis factor-alpha in peripheral CD56+ NK-cell subsets and CD8+ T cells after in vitro stimulation with synthetic bacterial (Pam3CSK, lipopolysaccharide [LPS], CpG-ODN-2216, and flagellin) and viral agonists (polyinosinic: polycytidylic acid [poly(I: C)], poly(I: C)-high molecular weight/LyoVec™, 5’ppp-dsRNA/LyoVec™, and R837). Compared with AIH patients and HCs, CD56+ NK-cell subsets from PSC patients showed reduced cytotoxicity both at baseline and upon in vitro stimulation with synthetic bacterial/viral agonists. Notably, the percentage of unstimulated CD107a+CD56dimCD16+ NK cells positively correlated with clinical serum parameters in PSC. In addition, the percentage of CD107a+CD8+ cells was significantly reduced after synthetic bacterial/viral stimulation in PSC patients. Finally, in PSC patients, production of pro-inflammatory cytokines was markedly reduced in CD56highCD16− NK cells upon in vitro stimulation with Pam3CSK and LPS, respectively. This hyporesponsiveness of CD56+ NK cells and CD8+ T cells may contribute to the development of hepatic inflammation, representing a hallmark of PSC pathogenesis.

Background/Objectives: Primary sclerosing cholangitis (PSC) is a rare, incurable liver disease characterized by chronic biliary inflammation and fibrosis. PSC is a significant risk factor for biliary tract cancer (BTC). This study aims to evaluate STAT3 expression in BTC and its prognostic significance as well as explore the potential of organoids derived from PSC and liver tumor patients as an in vitro model for testing novel therapeutic strategies in both PSC and BTC. Methods: Fresh tissue samples obtained from 10 PSC patients through targeted endoscopic retrograde cholangiography (ERC) and biopsy samples from liver tumor patients were used to establish organoid cultures. Organoids were treated with different agents and the therapeutic effect was measured by CellTiterGlo. Treatment with the JAK inhibitor baricitinib was followed by the measurement of cytokine concentrations in the supernatant. Archived formalin-fixed paraffin-embedded (FFPE) samples from 55 surgically resected BTC tumors were analyzed for STAT3 expression using immunohistochemistry. Results: We successfully established organoid cultures from all ERC samples. STAT3 protein expression was detected in 56% of tumor samples and 69% of the immune microenvironment. STAT3 positivity in the immune cell compartment was associated with longer disease-free survival, although the multivariate analysis could not confirm its value as an independent prognostic factor. Chemotherapy testing on liver tumor organoids showed various degrees of decreases in viability after treatment with gemcitabine, cisplatin, and cabozantinib. Baricitinib treatment significantly reduced IL-6 and MCP-1 secretion in cholangiocarcinoma Conclusions: The patient-derived organoid model of PSC and liver tumors is a valuable tool for testing novel and established therapeutic strategies, including JAK inhibitors and chemotherapy regimens. STAT3 expression in the immune microenvironment of BTC may serve as a prognostic marker. Further studies are needed to explore the integration of co-cultured organoid systems with stromal and immune components to improve physiological relevance.

Background: Aging is characterized by the progressive decline of physiological functions and is associated with an increasing risk for developing multiple age‐related diseases. UDP‐glucuronosyltransferase (UGT)1A enzymes detoxify a variety of endo‐ and xenobiotic reactive metabolites, thereby acting as indirect antioxidants. A common genetic UGT1A haplotype was shown to affect redox balance in humanized transgenic (htg) UGT1A mice. Since oxidative stress is a main activator of cellular senescence, we aimed to investigate the role of genetic UGT1A variants in the process of aging. Methods: Htg UGT1A ‐WT and htg UGT1A ‐SNP mice were harvested at the age of either 12 weeks (young) or 18 months (aged). The effect of aging was examined by analyzing UGT1A expression and activity, expression of senescence markers, and senescence‐associated secretory phenotype (SASP) factors, as well as blood counts, serum parameter, and histological staining. Results: In comparison to aged htg UGT1A ‐WT mice, hepatic UGT1A mRNA and protein expression as well as UGT activity were significantly reduced in aged htg UGT1A ‐SNP mice. Moreover, elderly htg UGT1A ‐SNP mice exhibited increased levels of oxidative stress, senescence markers, SASP factors, and peripheral leukocyte counts compared to the respective htg UGT1A ‐WT mice. Consistent with these findings, we observed higher amounts of collagen and amyloid fibrils as well as an elevated senescence‐associated β‐galactosidase (SA‐β‐gal) activity in histological sections of the liver obtained from aged htg UGT1A ‐SNP mice. Conclusion: Our data suggest an accelerated aging process caused by a common UGT1A haplotype. Moreover, elderly individuals carrying the UGT1A haplotype might exhibit an altered metabolism of drugs, which could necessitate dose adjustments.

Introduction: Regulatory CD4⁺ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK‐STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines.Methods: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3⁺CD25⁺CD127^lowCD4⁺ Tregs, their expression of ectonucleotidase CD39, and cytokine‐induced phosphorylation of STAT1, 3, 5, and 6 using phospho‐flow cytometry. In parallel, we measured cytokines IFN‐gamma, interleukin (IL)‐6, IL‐2, and IL‐4 in serum via bead‐based immunoassays.Results: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN‐gamma, IL‐6, IL‐2, and IL‐4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL‐6 induced increased phosphorylation of STAT3 in Tregs of PSC‐patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts.Conclusions: In PSC, we observed enhanced STAT3 responsiveness of CD4⁺ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

Background: The level of type-I interferons (IFNs) in primary sclerosing cholangitis (PSC) was investigated to evaluate its association with disease activity and progression. Methods: Bioactive type-I IFNs were evaluated in a murine model of PSC and human patients’ sera using a cell-based reporter assay and ELISA techniques. In total, 57 healthy participants, 71 PSC, and 38 patients with primary biliary cholangitis were enrolled in this study. Results: Bioactive type-I IFNs were elevated in the liver and serum of multidrug resistance protein 2–deficient animals and showed a correlation with the presence of CD45⁺ immune cells and serum alanine transaminase levels. Concordantly, bioactive type-I IFNs were elevated in the sera of patients with PSC as compared to healthy controls (sensitivity of 84.51%, specificity of 63.16%, and AUROC value of 0.8267). Bioactive IFNs highly correlated with alkaline phosphatase (r=0.4179, pr=0.4704, pr=0.6629, p<0.0001) but not with serum bilirubin. In addition, patients with PSC with advanced fibrosis demonstrated significantly higher type-I IFN values. Among the type-I IFN subtypes IFNα, β and IFNω could be detected in patients with PSC with IFNω showing the highest concentration among the subtypes and being the most abundant among patients with PSC. Conclusions: The selectively elevated bioactive type-I IFNs specifically the dominating IFNω could suggest a novel inflammatory pathway that might also have a hitherto unrecognized role in the pathomechanism of PSC.

Background: Development of cirrhosis and hepatocellular carcinoma (HCC) in patients with high alcohol intake is modulated by genetic predispositions. Genetic variation in angiotensin II type 1 receptor (AGTR1) has been described as a risk factor for non-alcoholic fatty liver disease in Asian patients. Methods: We analysed Caucasian patients with alcohol–associated cirrhosis without (n = 238) and with (n = 339) HCC, healthy controls (n = 200), and HCV–infected cirrhotic patients with and without HCC (n = 263) for association with the polymorphisms rs3772622 and rs2276736 in AGTR1. Results: Rs2276736 in AGTR1 was associated with both low–density lipoprotein (LDL) cholesterol levels and hepatic steatosis in patients with alcohol–associated liver disease. The distribution of genotypes for both rs3772622 and rs2276736 in AGTR1 were comparable between controls, cirrhosis patients, and those with HCC. Minor allele frequencies were 32% (44%) in healthy controls, 35%/34% (46%/45%) in alcohol–associated liver disease without/with HCC and 31%/38% (43%/39%) in HCV cirrhosis and HCV HCC, respectively. The genotype of the most important genetic risk factor for fatty liver disease, PNPLA3 I148M, did not interact with the AGTR1 polymorphisms. Conclusion: Genetic variation in AGTR1, although associated with blood lipid levels and hepatic steatosis, is not a risk factor for alcohol–associated cirrhosis or HCC in Caucasians.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6–dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. METHODS: We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. RESULTS: In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4⁺ T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3–positive CD4⁺ T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3–positive cells were enriched in the peribiliary liver stroma and represented CD4⁺ T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. DISCUSSION: Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.

Background and Aims: Human innate lymphoid cells (ILCs) are critically involved in the modulation of homeostatic and inflammatory processes in various tissues. However, only little is known about the composition of the intrahepatic ILC pool and its potential role in chronic liver disease. Here, we performed a detailed characterization of intrahepatic ILCs in both healthy and fibrotic livers. Approach and Results: A total of 50 livers (nonfibrotic = 22, and fibrotic = 29) were analyzed and compared with colon and tonsil tissue (each N = 14) and peripheral blood (N = 32). Human intrahepatic ILCs were characterized ex vivo and on stimulation using flow cytometry and single-cell RNA sequencing. ILC differentiation and plasticity were analyzed by both bulk and clonal expansion experiments. Finally, the effects of ILC-derived cytokines on primary human HSteCs were studied. Unexpectedly, we found that an “unconventional” ILC3-like cell represented the major IL-13-producing liver ILC subset. IL-13⁺ ILC3-like cells were specifically enriched in the human liver, and increased frequencies of this cell type were found in fibrotic livers. ILC3-derived IL-13 production induced upregulation of proinflammatory genes in HSteCs, indicating a potential role in the regulation of hepatic fibrogenesis. Finally, we identified KLRG1-expressing ILC precursors as the potential progenitor of hepatic IL-13⁺ ILC3-like cells. Conclusions: We identified a formerly undescribed subset of IL-13–producing ILC3-like cells that is enriched in the human liver and may be involved in the modulation of chronic liver disease.

Hepatocellular carcinoma (HCC) is a severe complication of advanced alcoholic liver disease, which is modulated by genetic predisposition. Identifying new genetic loci might improve screening. Genetic variation of SAMM50 was linked to HCC. We aimed to validate this finding in a large cohort of patients with advanced alcoholic liver disease (ALD). A large, well-characterised cohort of patients with alcoholic cirrhosis without (n = 674) and with (n = 386) HCC, as well as controls with HCC due to viral hepatitis (n = 134), controls with heavy alcohol abuse without liver disease (n = 266) and healthy subjects (n = 237), were genotyped for SAMM50 rs3827385 and rs3761472 and for PNPLA3 rs738409. Genotype frequencies were compared between patients with alcohol-associated cirrhosis with and without HCC by uni- and multivariate analysis. Minor variants in both SAMM50 rs3827385 and rs3761472 were significantly more frequent in patients with alcoholic HCC versus alcoholic cirrhosis and versus the control cohorts. An even stronger association was noted for PNPLA3 rs738409. The univariate analysis resulted in an odds ratio (OR) of 1.8 for carriers of at least one minor variant of SAMM50 rs3827385 and rs3761472 (each p < 0.001), but this association was lost in multivariate analysis with age (OR 1.1/year), male sex (OR 3.2), diabetes (OR 1.9) and carriage of PNPLA3 148M (OR 2.1) remaining in the final model. Although minor variants of both SAMM50 loci are strongly associated with alcoholic HCC, this association is not independent of carriage of the well-known risk variant PNPLA3 148M.

HLA-B*57 affects the course of HIV infection. Under antiretroviral therapy, its effects cannot be explained by outstandingly efficient T cell responses alone but may also involve cells of innate immunity. Studying in vitro stimulation with Pam3CSK4, E. coli LPS-B5 and CpG-ODN-2216, we observed greater induction of IL-6/IL-1beta double-positive CD14⁺CD16⁺⁺ monocytes as well as IFN-gamma-positive cytotoxic CD56^highCD16^neg NK cells in HLA-B*57- versus HLA-B*44-positive HIV patients, while TNF-alpha induction remained unchanged. Differences were not seen in the other monocyte and NK cell subsets or in HLA-matched healthy controls. Our findings show that, in virally suppressed HIV infection, HLA-B*57 is associated with enhanced responsiveness of inflammatory innate immune cells to TLR ligands, possibly contributing to increased vulnerability in sepsis. Key messages: • HLA-B*57 is a host factor affecting clinical outcomes of HIV infection. • HLA-B*57 modifies inflammatory subsets of NK cells and monocytes in HIV infection. • In HLA-B*57-positive HIV patients TLR agonists induce enhanced IL-6/IL-1beta in monocytes. • NK cells from HLA-B*57 HIV patients release more IFN-gamma upon TLR costimulation. • HLA-B*57 is linked to enhanced inflammatory responsiveness to TLR ligands.