RESUMEN
BACKGROUND & AIMS: Chronic inflammation surrounding bile ducts contributes to the disease pathogenesis of most cholangiopathies. Poor efficacy of immunosuppression in these conditions suggests biliary-specific pathologic principles. Here we performed biliary niche specific functional interpretation of a causal mutation (CD100 K849T) of primary sclerosing cholangitis (PSC) to understand related pathogenic mechanisms. METHODS: Biopsy specimens of explanted livers and endoscopy-guided sampling were used to assess the CD100 expression by spatial transcriptomics, immune imaging, and high-dimensional flow cytometry. To model pathogenic cholangiocyte-immune cell interaction, splenocytes from mutation-specific mice were cocultured with cholangiocytes. Pathogenic pathways were pinpointed by RNA sequencing analysis of cocultured cells and cross-validated in patient materials. RESULTS: CD100 is mainly expressed by immune cells in the liver and shows a unique pattern around PSC bile ducts with RNA-level colocalization but poor detection at the protein level. This appears to be due to CD100 cleavage as soluble CD100 is increased. Immunophenotyping suggests biliary-infiltrating T cells as the major source of soluble CD100, which is further supported by reduced surface CD100 on T cells and increased metalloproteinases in cholangiocytes after coculturing. Pathogenic T cells that adhered to cholangiocytes up-regulated genes in the T-helper 17 cell differentiation pathway, and the CD100 mutation boosted this process. Consistently, T-helper 17 cells dominate biliary-resident CD4 T cells in patients. CONCLUSIONS: CD100 exerts its functional impact through cholangiocyte-immune cell cross talk and underscores an active, proinflammatory role of cholangiocytes that can be relevant to novel treatment approaches.
Asunto(s)
Sistema Biliar , Colangitis Esclerosante , Colangitis , Humanos , Animales , Ratones , Hígado/patología , Conductos Biliares/patología , Sistema Biliar/patología , Células Epiteliales/patología , Diferenciación Celular , Colangitis Esclerosante/patologíaRESUMEN
BACKGROUND: Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. METHODS: In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-ß. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. RESULTS: The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-ß-induced activation-related transcriptome. However, TGF-ß-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-ß. CONCLUSIONS: scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-ß-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.