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Identification of putative master regulators in rheumatoid arthritis synovial fibroblasts using gene expression data and network inference.

ABSTRACT: Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects the synovial joints of the body. Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) are central players in the disease pathogenesis, as they are involved in the secretion of cytokines and proteolytic enzymes, exhibit invasive traits, high rate of self-proliferation and an apoptosis-resistant phenotype. We aim at characterizing transcription factors (TFs) that are master regulators in RA FLS and could potentially explain phenotypic traits. We make use of differentially expressed genes in synovial tissue from patients suffering from RA and osteoarthritis (OA) to infer a TF co-regulatory network, using dedicated software. The co-regulatory network serves as a reference to analyze microarray and single-cell RNA-seq data from isolated RA FLS. We identified five master regulators specific to RA FLS, namely BATF, POU2AF1, STAT1, LEF1 and IRF4. TF activity of the identified master regulators was also estimated with the use of two additional, independent software. The identified TFs contribute to the regulation of inflammation, proliferation and apoptosis, as indicated by the comparison of their differentially expressed target genes with hallmark molecular signatures derived from the Molecular Signatures Database (MSigDB). Our results show that TFs influence could be used to identify putative master regulators of phenotypic traits and suggest novel, druggable targets for experimental validation.

SUBMITTER: Zerrouk N

PROVIDER: S-EPMC7529794 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Identification of putative master regulators in rheumatoid arthritis synovial fibroblasts using gene expression data and network inference.

Zerrouk Naouel N Miagoux Quentin Q Dispot Aurelien A Elati Mohamed M Niarakis Anna A

Scientific reports 20201001 1

Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects the synovial joints of the body. Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) are central players in the disease pathogenesis, as they are involved in the secretion of cytokines and proteolytic enzymes, exhibit invasive traits, high rate of self-proliferation and an apoptosis-resistant phenotype. We aim at characterizing transcription factors (TFs) that are master regulators in RA FLS and could potentially expl ...[more]

PMID: 33004899

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Project description:BACKGROUND: Network inference of gene expression data is an important challenge in systems biology. Novel algorithms may provide more detailed gene regulatory networks (GRN) for complex, chronic inflammatory diseases such as rheumatoid arthritis (RA), in which activated synovial fibroblasts (SFBs) play a major role. Since the detailed mechanisms underlying this activation are still unclear, simultaneous investigation of multi-stimuli activation of SFBs offers the possibility to elucidate the regulatory effects of multiple mediators and to gain new insights into disease pathogenesis. METHODS: A GRN was therefore inferred from RA-SFBs treated with 4 different stimuli (IL-1 ?, TNF- ?, TGF- ?, and PDGF-D). Data from time series microarray experiments (0, 1, 2, 4, 12 h; Affymetrix HG-U133 Plus 2.0) were batch-corrected applying 'ComBat', analyzed for differentially expressed genes over time with 'Limma', and used for the inference of a robust GRN with NetGenerator V2.0, a heuristic ordinary differential equation-based method with soft integration of prior knowledge. RESULTS: Using all genes differentially expressed over time in RA-SFBs for any stimulus, and selecting the genes belonging to the most significant gene ontology (GO) term, i.e., 'cartilage development', a dynamic, robust, moderately complex multi-stimuli GRN was generated with 24 genes and 57 edges in total, 31 of which were gene-to-gene edges. Prior literature-based knowledge derived from Pathway Studio or manual searches was reflected in the final network by 25/57 confirmed edges (44%). The model contained known network motifs crucial for dynamic cellular behavior, e.g., cross-talk among pathways, positive feed-back loops, and positive feed-forward motifs (including suppression of the transcriptional repressor OSR2 by all 4 stimuli. CONCLUSION: A multi-stimuli GRN highly concordant with literature data was successfully generated by network inference from the gene expression of stimulated RA-SFBs. The GRN showed high reliability, since 10 predicted edges were independently validated by literature findings post network inference. The selected GO term 'cartilage development' contained a number of differentiation markers, growth factors, and transcription factors with potential relevance for RA. Finally, the model provided new insight into the response of RA-SFBs to multiple stimuli implicated in the pathogenesis of RA, in particular to the 'novel' potent growth factor PDGF-D.

Project description:Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Dataset Information

Identification of putative master regulators in rheumatoid arthritis synovial fibroblasts using gene expression data and network inference.

Publications

Identification of putative master regulators in rheumatoid arthritis synovial fibroblasts using gene expression data and network inference.

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