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ScRNAseq characterization of synovial stromal cells in mouse STIA

ABSTRACT: Objective: Upregulation of glycolysis and glutaminolysis in the rheumatoid arthritis fibroblast-like synoviocytes (RAFLS) has already been established by existing literature . Henceforth, this study was undertaken to determine if dual inhibition of these processes by novel phytobiologic c28MS have the potential of promising synergistic therapy for rheumatoid arthritis by targeting the underlying metabolic reprogramming as existing treatment strategies for RA do not target dual inhibition. Methods: Gene associations with HK2 and GLS expression were identified by examining murine and human available scRNA-seq. The metabolic profile of FLS cells was determined by 1H magnetic resonance spectroscopy under conditions of glycolytic and glutaminolysis inhibition and HK2 and GLS1 expression was evaluated by western blot. FLS functional analysis was conducted under similar inhibitory conditions. ELISA was employed for the quantification of pivotal markers namely IL6, CCL2 and MMP3. For arthritis experiments, mice were injected with K/BxN sera on Day 0 and treated for 10 days. Results: scRNAseq data identified... Metabolomic and functional analysis revealed that c28MS had the potential of checking the aggressive behavior of RAFLS by targeting upregulated glycolysis and glutaminolysis thereby inhibiting them. Target expression remained unaffected by the action of the dual inhibitor suggesting only potential blockage. Furthermore, the compound was able to inhibit inflammatory arthritis by decreasing the clinical score and paw inflammation in the K/BxN model. Conclusion: Our findings imply that dual inhibition of glycolysis and glutaminolysis could be an effective strategy in the treatment of RA with chances of greater remission. It also suggests that targeting more than one pathway in the metabolome can be a novel treatment approach in non-cancer scenarios.

ORGANISM(S): Mus musculus

PROVIDER: GSE230145 | GEO | 2023/04/20

REPOSITORIES: GEO

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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.

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ScRNAseq characterization of synovial stromal cells in mouse STIA

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