Dataset Information

Matrix-mediated activation of fibroblasts in rheumatoid arthritis

ABSTRACT: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting synovial joints and leading to cartilage damage and bone loss. This destruction is promoted by activated fibroblast-like synoviocytes (FLS) that show an invasive and migratory phenotype. The mechanisms of FLS activation are unknown, but evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is highly increased in the synovium of RA patients and hTNFtg mice, an RA mouse model. Since TNFα is the major cytokine induced in RA, we treated murine chondrocytes with TNFα to produce a damaged, RA-like matrix. Comparison to healthy chondrocyte matrix revealed decreased ECM proteins, including several collagens and proteoglycans, increased matrix-degrading proteins and elevated levels of inflammatory cytokines. FLS responded to those differences in the damaged chondrocyte matrix with a matrix-remodeling and pro-inflammatory phenotype characterized by expressing genes involved in matrix degradation and increased production of CLL11 and CCL19. Damaged chondrocyte matrix induced increased Itga11 expression in FLS, which correlates with the increased α11β1 amounts in RA patients. FLS deficient in integrin α11β1 released lower amounts of inflammation-associated cytokines but did not reveal significant differences from the response of wild type FLS to a damaged, RA-like matrix. Our results demonstrate differences in healthy and RA-like chondrocyte ECM and distinctly different responses of wt FLS to damaged versus healthy ECM.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Luisa Marie Schmidt

LAB HEAD: Beate Eckes

PROVIDER: PXD049800 | Pride | 2025-01-14

REPOSITORIES: Pride

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			Action	DRS
	20230220_E1_LC6colID540_LS2434_PEC_tgf_decell_1.raw	Raw
	20230220_E1_LC6colID540_LS2435_PEC_tgf_decell_2.raw	Raw
	20230220_E1_LC6colID540_LS2436_PEC_TNF_decell_1.raw	Raw
	20230220_E1_LC6colID540_LS2437_PEC_TNF_decell_2.raw	Raw
	20230220_E1_LC6colID540_LS2438_PEC_tgf_decell_n1.raw	Raw

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Publications

Matrix-mediated activation of murine fibroblast-like synoviocytes.

Zeinert Isabel I Schmidt Luisa L Baar Till T Gatto Giulio G De Giuseppe Anna A Korb-Pap Adelheid A Pap Thomas T Mahabir Esther E Zaucke Frank F Brachvogel Bent B Krüger Marcus M Krieg Thomas T Eckes Beate B

Experimental cell research 20250105 1

Fibroblast-like synoviocytes (FLS) are key cells promoting cartilage damage and bone loss in rheumatoid arthritis (RA). They are activated to assume an invasive and migratory phenotype. While mechanisms of FLS activation are unknown, evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is increased in RA patients and hTNFtg mice, an RA mouse model. We treated murine chondrocytes with T ...[more]

PMID: 39765309

Similar Datasets

Project description:Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLS) and synovial macrophages (SM), are crucial for the progression of rheumatoid arthritis (RA). Particularly, FLS of RA patients (RA-FLS) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone, although it remains unresolved how RA-FLS exhibit invasive phenotype. RA-FLS and SM originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. Presently, we performed global transcriptome profiling of FLS and SM obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLS and pro-inflammatory properties of RA synovial macrophages (RA-SM), respectively. Interestingly, under interleukin1β-stimulated condition, RA-FLS newly acquired pro-inflammatory signature mimicking RA-SM without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS-dominant (invasive), and RA-SM-dominant (inflammatory) processes. From the network model, we selected 13 genes, including POSTN and TWIST1, as novel regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLS and were further instigated by interleukin1β. In vitro functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLS stimulated with interleukin1β. Taken together, our systems approach to rheumatoid synovitis provides a basis for identifying novel regulators responsible for pathological features of RA-FLS and RA-SM, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions. To identify molecular signatures of FLS and MLS in RA joints, we isolated FLS from synovial tissues of RA and osteoarthritis (OA) patients, obtained synovial macrophages from synovial fluid of RA patients, and differentiated control macrophages from peripheral blood of healthy subjects. Also, we stimulated FLS with IL1β, and then analyzed gene expression profiles of both IL1β-stimulated RA-FLS and OA-FLS

Project description:Objectives: Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed. Methods: Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini–Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. Results: RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3. Hypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern. Conclusions: Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis. Fibroblast-like synoviocyte cell-lines from osteoarthritis (OA) and rheumatoid arthritis (RA) patients.

Project description:Rheumatoid arthritis (RA) leads to progressive destruction of articular structures. Despite recent progress in controlling inflammation and pain, little cartilage repair has yet been observed. This in vitro study aims to determine the role of chondrocytes in RA-related cartilage destruction and antirheumatic drug-related regenerative processes. Human chondrocytes were three-dimensionally cultured in alginate beads. To determine the RA-induced gene expression pattern, human chondrocytes were stimulated with supernatant of RA synovial fibroblasts (RASF) and normal donor synovial fibroblasts (NDSF), respectively. To examine antirheumatic drug response signatures, human chondrocytes were stimulated with supernatant of RASF that have been treated with disease-modifying antirheumatic drugs (DMARD; azathioprine, sodium aurothiomalate, chloroquine phosphate, methotrexate), non-steroidal anti-inflammatory drugs (NSAID; piroxicam, diclofenac) or steroidal anti-inflammatory drugs (SAID; methylprednisolone, prednisolone). Genome-wide expression profiling with oligonucleotide microarrays was used to determine differentially expressed genes. Real-time RT-PCR and ELISA were performed for validation of microarray data. Following antirheumatic treatment, microarray analysis disclosed a reverted expression of 94 RA-induced chondrocyte genes involved in inflammation/NF-ÎºB signalling, cytokine/chemokine activity, immune response, proliferation/differentiation and matrix remodelling. Hierarchical clustering analysis showed that treatment of RASF with the DMARD azathioprine, gold sodium thiomalate and methotrexate resulted in chondrocyte gene expression signatures that were closely related to the âhealthyâ pattern. Treatment with the SAID methylprednisolone and prednisolone strongly reverted the RA-related chondrocyte gene expression, in particular the expression of genes involved in inflammation/NF-ÎºB and cytokine/chemokine activity. The NSAID piroxicam and diclofenac and the DMARD chloroquine phosphate had only moderate to marginal effects. Pathway analysis determined major mechanisms of drug action, for example pathways of cytokine-cytokine receptor interaction, TGF-Î²/TLR/Jak-STAT signalling and ECM-receptor interaction were targeted. This in vitro study provides a comprehensive molecular insight into the antirheumatic drug response signatures in human chondrocytes, thereby revealing potential molecular targets, pathways and mechanisms of drug action involved in chondrocyte regeneration. Thus, the present study may contribute to the development of novel therapeutic chondro-protective compounds and strategies. Experiment Overall Design: Drug-related suppression of gene expression in activated chondrocytes was determined by genome-wide microarray analysis. Chondrocytes were stimulated with supernatant of RASF and NDSF. Effect of treatment with DMARDs, NSAIDs and glucocorticoids was tested by treating RASF prior to collection of supernatant. Two RNA pools were analyzed for each group (RASF-stimulated NDSF stimulated and RASF-treated), each pool consisting of equal amounts of RNA from three different donors.