Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Podoplanin/gp38⁺ stromal cells present in lymphoid organs play a central role in the formation and reorganization of the extracellular matrix and in the functional regulation of immune responses. Gp38⁺ cells are present during embryogenesis and in human livers of primary biliary cirrhosis. Since little is known about their function, we studied gp38⁺ cells during chronic liver inflammation in models of biliary and parenchymal liver fibrosis and steatohepatitis. Gp38⁺ cells were analyzed using flow cytometry and confocal microscopy, and the expression of their steady state and inflammation-associated genes was evaluated from healthy and inflamed livers. Gp38⁺ cells significantly expanded in all three models of liver injury and returned to baseline levels during regression of inflammation. Based on CD133 and gp38 expression in the CD45⁻CD31⁻Asgpr1⁻ liver cell fraction, numerous subsets could be identified that were negative for CD133 (gp38^hiCD133⁻, gp38^lowCD133⁻, and gp38⁻CD133⁻). Moreover, among the CD133⁺ cells, previously identified as progenitor population in injured liver, two subpopulations could be distinguished based on their gp38 expression (gp38⁻CD133⁺ and CD133⁺gp38⁺). Importantly, the distribution of the identified subsets in inflammation illustrated injury-specific changes. Moreover, the gp38⁺CD133⁺ cells exhibited liver progenitor cell characteristics similar to the gp38⁻CD133⁺ population, thus representing a novel subset within the classical progenitor cell niche. Additionally, these cells expressed distinct sets of inflammatory genes during liver injury. Our study illuminates a novel classification of the stromal/progenitor cell compartment in the liver and pinpoints a hitherto unrecognized injury-related alteration in progenitor subset composition in chronic liver inflammation and fibrosis.

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, p<0.001), alanine transaminase (r=0.4704, p<0.0001), and gamma-glutamyl transpeptidase activities (r=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.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of...