Biology subjects

Weiskirchen, R.

Publications and source records attributed to Weiskirchen, R..

5 recordsLinked to original sources

bioRxiv2025

SMAD2 sets divergent thresholds for TGF-β-induced SMAD1/5 signaling and IgE-mediated pro-inflammatory activation of mast cells

ObjectiveTGF-{beta}/SMAD signaling controls mast cell (MC) development and exerts anti-inflammatory functions, while antigen (Ag)-triggered Fc{varepsilon}RI/MAPK activation commands pro-inflammatory processes. SMAD2 is a signaling hub integrating both, TGF-{beta}/ALK5-mediated SMAD2 C-terminal-and ERK-mediated SMAD2 linker-phosphorylation. Here, we analyzed the role of SMAD2 in TGF-{beta}-and Ag-stimulated signaling, and their cross-talk which regulate MC responses. MethodsThe CRISPR-Cas method was used to disable SMAD2 expression in the PMC-306 cell line. The transcriptome of the corresponding cells was analyzed by NGS in homeostatic and stimulated conditions. Activation of signaling intermediates, target gene responses and effector function were analyzed by qPCR, Western blot and ELISAs. The causal association of SMAD2 with the found effects was proven by re-introduction of a tagged SMAD2 in knock out cells. ResultsThe absence of SMAD2 led to increased proliferation and survival, and decreased transcription of target genes like Smad7 and Jun in steady state and upon TGF-{beta} treatment. Intriguingly, SMAD2 was found to regulate TGF-{beta}-mediated SMAD1/5 activation, resulting in augmented expression of Id3 in SMAD2-deficient MCs. Unexpectedly, we found that SMAD2 is indispensable for Ag-triggered production of pro-inflammatory cytokines, such as IL-6 and TNF. Re-introducing SMAD2 restores these events with varying sensitivity depending on the receptor system triggered. ConclusionsOur findings highlight a previously unrecognized role of SMAD2 as a decisive hub in TGF-{beta}-ALK5-SMAD1/5 and Ag-Fc{varepsilon}RI-MAPK signaling, suggesting that pharmacological modulation of SMAD2 activity could be leveraged as regulator of both suppressive and inflammatory MC functions.

immunology↗

bioRxiv2025

Identification, Purification and Characterization of Mast Cells in Murine Liver Fibrosis: Novel Methods, Expression Signatures and Correlation with Disease Severity

Mast cells (MCs) are myeloid cells of the innate immune system. As a first line of defense, they fulfill effector functions and immune modulatory properties. Upon activation, they release pro-inflammatory mediators such as cytokines and proteases. It has been suggested that MCs may contribute to the development of liver fibrosis. However, investigating hepatic MC biology in mice is challenging due to low MC numbers and a lack of suitable detection techniques relying on MC proteins and their modifications. Here, we evaluated whether the expression strength of MC markers correlates with the degree of liver fibrosis in mice and aimed to determine the frequency and localization of hepatic MCs. We applied both a toxic (DEN/CCl4 treatment) and a genetic (Mdr2-/- mice) liver fibrosis model in C57BL/6 mice and found significant correlations between fibrosis grade and the expression of several established MC markers. These correlations were further supported by data analyses from patients with fibrosis and hepatocellular carcinoma (HCC) using publicly available transcriptomics datasets. We used FACS to purify and isolate MCs from fibrotic mouse livers and verified MC signatures by qPCR analysis of MC-specific gene expression. Hepatic MCs were predominantly negative for Mast Cell Protease 5 (Mcpt5) and occurred at a low frequency (approximately 1-2% of leukocytes). Using Molecular CartographyTM of fibrotic liver sections, we determined the spatial localization, expression signature, abundance (approximately 2 cells/mm2) and cellular environment of murine hepatic MCs. In summary, we demonstrated the existence of MCs in murine fibrotic livers and defined MC expression signatures that correlate with the strength of liver fibrosis. These findings will help to study MC biology in murine models of liver disease more effectively in the future.

cell biology↗

bioRxiv2024

SIngle cell level Genotyping Using scRna Data (SIGURD)

MotivationBy accounting for variants within measured transcripts, it is possible to evaluate the status of somatic variants using single-cell RNA-sequencing (scRNA-seq) and to characterize their clonality. However, the sparsity (very few reads per transcript) or bias in protocols (favoring 3 ends of the transcripts) makes the chance of capturing somatic variants very unlikely. This can be overcome by targeted sequencing or the use of mitochondrial variants as natural barcodes for clone identification. Currently, available computational tools focus on genotyping, but do not provide functionality for combined analysis of somatic and mitochondrial variants and functional analysis such as characterization of gene expression changes in detected clones. ResultsHere, we propose SIGURD, which is an R-based pipeline for the clonal analysis of single-cell RNA-seq data. This allows the quantification of clones by leveraging both somatic and mitochondrial variants. SIGURD also allows for functional analysis after clonal detection: association of clones with cell populations, detection of differentially expressed genes across clones and association of somatic and mitochondrial variants. Here, we demonstrate the power of SIGURD by analyzing single-cell data of colony-forming cells derived from patients with myeloproliferative neoplasms. AvailabilityCode and tutorial of SIGURD are available at GitHub https://github.com/CostaLab/sigurd. Contactivan.costa@rwth-aachen.de, skoschmieder@ukaachen.de Supplementary InformationSupplementary data are available online.

bioinformatics↗

bioRxiv2023

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗

bioRxiv2023

Transformation of primary murine peritoneal mast cells by constitutive KIT activation as a result of lost Cdkn2a/Arf expression

Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on IL-3 and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The reason for immortalization most likely is the loss of the two critical cell cycle regulators Cdkn2a/INK4A and Arf/p19, respectively. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by SCF supplementation strongly arguing for an involvement of KIT activation in the transformation process. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.

immunology↗