Gores, Gregory J.

Fatty acid‐induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition is prevalent in tumor necrosis factor receptor antagonism, but recent studies suggest a compelling alternative: non‐competitive inhibition through perturbation of receptor conformation. We used a yeast‐displayed combinatorial library to identify a synthetic affibody that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor‐receptor interaction. Live‐cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL‐ or palmitate‐treated Huh‐7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease. Support or Funding Information National Institutes of Health Grants R01EB023339, R01DK41876, R35GM131814 and R01EB028274.

Background and Aims Alcoholic hepatitis (AH) is diagnosed by clinical criteria, although several objective scores facilitate risk stratification. Extracellular vesicles (EVs) have emerged as biomarkers for many diseases and are also implicated in the pathogenesis of AH. Therefore, we investigated whether plasma EV concentration and sphingolipid cargo could serve as diagnostic biomarkers for AH and inform prognosis to permit dynamic risk profiling of AH subjects. Approach and Results EVs were isolated and quantified from plasma samples from healthy controls, heavy drinkers, and subjects with end‐stage liver disease (ESLD) attributed to cholestatic liver diseases and nonalcoholic steatohepatitis, decompensated alcohol‐associated cirrhosis (AC), and AH. Sphingolipids were quantified by tandem mass spectroscopy. The median plasma EV concentration was significantly higher in AH subjects (5.38 × 1011/mL) compared to healthy controls (4.38 × 1010/mL; P < 0.0001), heavy drinkers (1.28 × 1011/mL; P < 0.0001), ESLD (5.35 × 1010/mL; P < 0.0001), and decompensated AC (9.2 × 1010/mL; P < 0.0001) disease controls. Among AH subjects, EV concentration correlated with Model for End‐Stage Liver Disease score. When EV counts were dichotomized at the median, survival probability for AH subjects at 90 days was 63.0% in the high‐EV group and 90.0% in the low‐EV group (log‐rank P value = 0.015). Interestingly, EV sphingolipid cargo was significantly enriched in AH when compared to healthy controls, heavy drinkers, ESLD, and decompensated AC (P = 0.0001). Multiple sphingolipids demonstrated good diagnostic and prognostic performance as biomarkers for AH. Conclusions Circulating EV concentration and sphingolipid cargo signature can be used in the diagnosis and differentiation of AH from heavy drinkers, decompensated AC, and other etiologies of ESLD and predict 90‐day survival permitting dynamic risk profiling.

Abstract The extracellular matrix is a highly reactive scaffold formed by a wide array of multifunctional molecules, encompassing collagens and noncollagenous glycoproteins, proteoglycans, glycosaminoglycans, and polysaccharides. Besides outlining the tissue borders, the extracellular matrix profoundly regulates the behavior of resident cells by transducing mechanical signals, and by integrating multiple cues derived from the microenvironment. Evidence is mounting that changes in the biostructure of the extracellular matrix are instrumental for biliary repair. Following biliary damage and eventually, malignant transformation, the extracellular matrix undergoes several quantitative and qualitative modifications, which direct interactions among hepatic progenitor cells, reactive ductular cells, activated myofibroblasts and macrophages, to generate the ductular reaction. Herein, we will give an overview of the main molecular factors contributing to extracellular matrix remodeling in cholangiopathies. Then, we will discuss the structural alterations in terms of biochemical composition and physical stiffness featuring the “desmoplastic matrix” of cholangiocarcinoma along with their pro-oncogenic effects.

Background and Aims In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug‐resistant 2 (Mdr2−/− ) double knockout (DKO) mice lacking tumor necrosis factor–related apoptosis‐inducing ligand receptor (Tr−/− ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2−/− mice. This observation suggests that the magnitude of the DR‐cell population may be regulated by apoptosis. Approach and Results To examine this concept, we cultured epithelial cell adhesion molecule–positive reactive cholangioids (ERCs) obtained from wild‐type (WT), Tr−/− , Mdr2−/− and DKO mice. Single‐cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR‐cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3‐mimetic therapy, significantly decreased DKO and Mdr2−/− ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR‐cell population and markers of inflammation and liver fibrosis in Mdr2−/− and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high‐definition mass cytometry by time‐of‐flight. Coculture of bone marrow–derived macrophages with ERCs from DKO mouse livers up‐regulated expression of the B cell–directed chemokine (C‐C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl‐2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. Conclusions DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR‐cell and B‐cell populations and hepatic fibrosis.

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L‐MSCs) are superior immunomodulators and contribute to liver‐mediated tolerance. L‐MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs). Trilineage differentiation of L‐MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L‐MSCs are different than the A‐MSCs and BM‐MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L‐MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L‐MSCs (versus A‐MSCs/BM‐MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ–producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L‐MSCs was not dependent on cell‐to‐cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3‐dioxygenase for complete reversal. In conclusion, L‐MSCs appear to be uniquely well‐equipped immunomodulatory cells, and they are more potent than A‐MSCs and BM‐MSCs in that capacity, which suggests that they may contribute to liver‐induced systemic tolerance.

Background and Aims Presence of malignant regional lymph nodes (MRLNs) precludes curative oncological resection or liver transplantation for cholangiocarcinoma (CCA). Limited data support the utility of endoscopic ultrasound (EUS)/fine needle aspiration (FNA) for detection of MRLNs in extrahepatic CCA, but there are no data for its role in intrahepatic CCA (iCCA). The aim of this study is to evaluate the staging impact of EUS for CCA, including analysis by subtype. Approach and Results We identified consecutive patients with CCA who underwent EUS staging at a single tertiary care center from October 2014 to April 2018. Among this cohort, we abstracted clinical demographic, radiographical, procedural, cytopathological, and surgical data. STATA 15 software was used for comparative analysis calculations (StataCorp LP, College Station, TX). The study cohort included 157 patients; 24 (15%), 124 (79%), and 9 (6%) with intrahepatic, perihilar, and distal CCA, respectively. EUS was able to identify regional lymph nodes (RLNs) in a higher percentage of patients compared to cross‐sectional imaging (86% vs. 47%; P < 0.001). FNA was performed in 133 (98.5%) patients with RLNs, with a median of three passes per node. EUS‐FNA identified MRLN in 27 of 31 (87.1%) patients ultimately found to have MRLNs. For iCCA, EUS detected a higher percentage of RLN compared to cross‐sectional imaging (83% vs. 50%; P = 0.048), with MRLNs identified in 4 (17%) patients. Among the entire cohort, identification of at least one MRLN by EUS was associated with lower median survival (353 vs. 1,050 days; P < 0.001) and increased risk of death (hazard ratio = 4.1; P < 0.001). Conclusions EUS‐FNA is effective for identifying MRLN in patients with CCA, and should be routinely incorporated into staging of all CCA subtypes given the impact of MRLN on prognosis and management decisions.

Cholangiocytes are the target of a group of chronic liver diseases termed the “cholangiopathies,” in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.