Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

Chronic liver diseases are marked by persistent inflammation and can evolve into liver fibrosis, cirrhosis, and hepatocellular carcinoma. In an affected liver, hepatic stellate cells (HSCs) transition from a quiescent to an activated state and adopt a myofibroblast-like cell phenotype. While these activated cells play a role in supporting liver regeneration, they can also have detrimental effects on liver function as the disease progresses to fibrosis and cirrhosis. These findings highlight the dynamic switching between different signaling pathways involving Ras, Rho GTPases, and Notch signaling. Notably, two specific members of the Ras and Rho GTPases, Eras and Rnd3, are predominantly expressed in quiescent HSCs, while Mras and Rhoc are more abundant in their activated forms. In addition, this study highlights the critical role of cytosolic Notch1 in quiescent HSCs and Rock in activated HSCs. We hypothesize that distinct yet interdependent intracellular signaling networks regulate HSC fate decisions in two key ways: by maintaining HSC quiescence and homeostasis and by facilitating HSC activation, thereby influencing processes such as proliferation, transdifferentiation, and mesenchymal transition. The proposed signaling model, combined with specific methodological tools for maintaining HSCs in a quiescent state, will deepen our understanding of the mechanisms underlying chronic liver disease and may also pave the way for innovative therapies. These therapies could include small molecule drugs targeting Ras- and Rho-dependent pathways.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) at high doses damages the liver and disrupts gut microbiota in mice. We investigated whether very low TCDD doses also cause dysbiosis and if sex-specific differences exist. Male and female C57BL/6 mice received 1 µg TCDD/kg body weight initially and maintenance doses biweekly for 12 weeks; controls received DMSO/corn oil. Fecal pellets were collected throughout, and cecal, liver, and gut samples at euthanasia. Mice did not suffer weight loss, and the liver weight to body weight ratio remained normal in all groups. Barrier genes’ expression was unaffected in all groups. 16S sequencing of fecal pellets revealed an increase in α-diversity over the 12-week treatment period, regardless of TCDD exposure or sex. No significant differences in α-diversity were observed between control and TCDD-treated groups. Analysis of β-diversity indicated differences in microbiota composition between male and female mice, as well as over time. However, probably due to low sample numbers, we could not detect significant differences using UniFrac or other ß-diversity metrics. However, our flow cytometry-based analysis tool, FlowSoFine™, identified significantly distinct microbial signatures in TCDD-treated female mice compared to control females. We hypothesized that this could be due to the formation of bacterial clusters or to different glycosylation patterns, both undetectable by sequencing. We therefore stained the cecal material with a panel of seven fluorescent lectins, which detect typical bacterial sugar structures, i.e., glycans. Significant changes were detected for concanavalin A, soybean agglutinin, and Dolichos biflorus agglutinin binding in the gut microbiota in female but not in male mice. We isolated bacteria by fluorescence-activated cell sorting (FACS), which differed between TCDD-treated and control female mice. 16S sequencing of these bacteria revealed a high frequency of Lachnospiraceae and reduced amount of Lactobacillaceae, CAG-508, and Oscillospiraceae in the TCDD-exposed samples. Glycoconjugates govern biofilm formation, infectious behavior of bacteria, and host immune responses, but little is known about diet or xenobiotic induced changes in glycosylation. We conclude that assessing glycosylation parameters in studies of dysbiosis is relevant, as well as stratifying for sex-specific differences.

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. Much less is known regarding their role and function in skin health and disease than regarding the roles of murine skin-resident γδT cells. In this study, we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents. We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the skin equivalents. Plated on top of the dermal equivalents, almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the dermal equivalents and the epithelium. Vδ1 T cells contributed to epidermal differentiation of HaCaT cells as indicated by histology, expression of epidermal differentiation markers, and RNA-sequencing expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin equivalents and skin carcinoma equivalents and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.