Citations

Abstract Introduction Primary sclerosing cholangitis (PSC) is an immune-mediated chronic fibrotic liver disease with no effective medical therapies. We showed that bile duct epithelial cells, called cholangiocytes, express IL-17C, a cytokine that modulates immune responses. Mucosal-associated invariant T cells (MAITs) have been implicated in fibrosis, but their mechanistic role in PSC remains unknown. SPARC (secreted protein acidic and rich in cysteine) is an extracellular matrix protein whose role in hepatic fibrosis is unexplored. Methods Flow cytometry, RNAscope, ELISA, RNA sequencing, immunofluorescence, immunocytochemistry, spheroid culture. Results Flow cytometry of bile duct brushings from PSC patients (n = 8) revealed that 41.32% of T cells were MAITs, indicating their proximity to cholangiocytes within the epithelial-immune niche. MAITs co-cultured with PSC patients-derived cholangiocytes showed increased activation than with normal human cholangiocytes (p = 0.018, n = 4). RNAscope on human livers identified increased IL17C expression in PSC cholangiocytes compared to controls (p = 0.064, n = 3), and IL-17C levels were elevated in PSC bile (p = 0.035, n = 25). Surprisingly, the receptor of IL-17C is enriched in MAITs in PSC (re-analysis of single cell-RNA seq, PMID: 33774059). Bulk RNA sequencing of IL-17C-treated peripheral blood MAITs revealed upregulation of T cell activation markers and pathways associated with hepatic fibrosis. Moreover, IL-17C-treated MAITs increased SPARC secretion (p = 0.025, n = 7), an effect attenuated by IL-17C blockade. Consistently, conditioned media from IL-17C—treated MAITs activated hepatic fibroblasts (HF), evidenced by increased PDGFRβ expression (p = 0.048, n = 6). SPARC directly promoted HF activation, marked by increased α-SMA (p = 0.039, n = 5). Co-culture of primary human HF, MAITs and cholangiocytes in the presence of IL-17C enhanced HF activation. Conclusion IL-17C drives MAITs toward a profibrogenic phenotype in PSC, promoting HF activation through SPARC. Funding Source C-SIG P30DK084567 Topic Categories Immune Mechanisms of Human Disease (HUM)

Mucosal-associated invariant T (MAIT) cells are abundant innate-like T lymphocytes in the human liver which can provide antimicrobial defense, amplify inflammatory processes and mediate tissue repair and fibrosis depending on microenvironmental cues. Chronic liver diseases of diverse etiologies, including viral hepatitis, metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, biliary tract disease, autoimmune hepatitis and hepatocellular carcinoma are accompanied by numerical and functional adjustments in the MAIT cell population. In this review, we integrate existing data on MAIT cell markers and functions in diverse liver diseases, comparing how these cells are similarly or differentially shaped by distinct pathogenic contexts. Finally, we propose a spatially anchored conceptual and technical framework to study MAIT cell biology in liver disease.

Abstract Description Background Primary sclerosing cholangitis (PSC) is an immune-mediated liver disease characterized by chronic inflammation and fibrosis of the bile ducts. The primary cells impacted in PSC are bile duct epithelial cells called cholangiocytes. Mucosal associated invariant T (MAIT) cells are enriched in human liver and associated with fibrosis. We hypothesize that cholangiocyte-activated MAIT cells drive fibrosis in PSC. Results Flow cytometry of PSC patient bile duct brushings show that 30% of T cells are MAIT cells, suggesting their co-existence with cholangiocytes within the peri-biliary environment. Re-analysis of single cell RNA sequencing data of PSC patient cholangiocyte organoids reveals upregulation of MR1 gene, indicating their enhanced ability to activate MAIT cells. Proteomics of human MAIT cells identify the expression of multiple profibrotic factors including secreted protein acidic and rich in cysteine (SPARC). Flow cytometry of TCR-activated MAIT cells show a significant and dose-dependent increase in a SPARClow MAIT population, indicating release of SPARC. Re-analysis of Olink proteomics on supernatant of PSC cholangiocyte organoids show IL-17C secretion. IL-17C treatment of TCR-activated MAIT cells result in significant upregulation of SPARC gene. Conclusion MAIT cells secrete profibrogenic mediators and MAIT cell profibrogenicity is enhanced by IL-17C, a novel cytokine released by cholangiocytes in PSC. Funding Sources C-SIG P30DK084567 Topic Categories Mucosal and Regional Immunology (MUC)

Cholangiocyte pathophysiological activation was examined in a model of murine cholestasis. CX3CR1 + macrophages are recruited to the periportal region, likely mediated by cholangiocyte expression of CX3CL1. Cholangiocyte transcriptomics from cholestatic mice display activation of a “pathogen-induced cytokine storm” pathway, and exhibit activation of the transcription factor NF-Y. In human cholangiocytes, NF-Y promotes expression of the profibrogenic ligand TGFβ1. These observations provide insights into the cholestatic cholangiocyte pathobiology contributing to periductal inflammation and fibrosis.

Cholangiopathies comprise a spectrum of chronic intrahepatic and extrahepatic biliary tract disorders culminating in progressive cholestatic liver injury, fibrosis, and often cirrhosis and its sequela. Treatment for these diseases is limited, and collectively, they are one of the therapeutic “black boxes” in clinical hepatology. The etiopathogenesis of the cholangiopathies likely includes disease-specific mediators but also common cellular and molecular events driving disease progression (eg, cholestatic fibrogenesis, inflammation, and duct damage). The common pathways involve cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts, which are central to the pathogenesis of these disorders. Current information suggests that cholangiocytes function as a signaling “hub” in biliary tract-associated injury. Herein, we review the pivotal role of cholangiocytes in cholestatic fibrogenesis, focusing on the crosstalk between cholangiocytes and portal fibroblasts and HSCs. The proclivity of these cells to undergo a senescence-associated secretory phenotype, which is proinflammatory and profibrogenic, and the intrinsic intracellular activation pathways resulting in the secretion of cytokines and chemokines are reviewed. The crosstalk between cholangiocytes and cells of the innate (neutrophils and macrophages) and adaptive (T cells and B cells) immune systems is also examined in detail. The information will help consolidate information on this topic and guide further research and potential therapeutic strategies for these diseases.

Selected patients with unresectable perihilar cholangiocarcinoma (pCCA) derive long-term benefits from liver transplantation. Between 1993–2019, our group at Mayo Clinic performed 237 transplants for pCCA. With this experience, we note that two distinct patient populations comprise this group of pCCA patients: those with underlying primary sclerosing cholangitis (PSC) and those without identifiable risk factors termed sporadic or de novo pCCA. Long-term survival after transplant is better in PSC patients (74% five-year survival) than in those with de novo pCCA (58% five-year survival). Herein, we review the likely clinical factors contributing to the divergence in outcomes for these two patient populations. We also offer our insights on how further advances may improve patient selection and survival, focusing on the de novo pCCA patient population.

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.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.