Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disease with a complex interplay of synovial cells and soluble factors that create the signatures of chronic inflammation and bone destruction. IL-6 drives the sustained inflammation in the RA synovial microenvironment and contributes to the invasiveness of synovial fibroblasts (RASFs) as well as the synovial tissue heterogeneity. Molecular mechanisms through which IL-6 propels the synovial fibroblasts invasiveness in RA synovium are yet to be fully characterized. The present study evaluated the effect of IL-6 signaling and induction of Ets2 (ETS Proto-Oncogene 2) transcription factor activity in human RASFs reprogramming to invasive phenotype.

Methods: Human synovial tissue derived RASFs were treated with recombinant IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) for 10-12 days to study invasiveness of RASFs. Secretome studies from culture supernatants were performed using an untargeted proteomics approach. RNA Sequencing (RNA-seq) was performed on an Ion P1 semiconductor sequencing chip using an Ion Proton System to examine global transcriptomics changes. Effects of IL-6 trans-signaling were further examined by ELISA, immunofluorescence, flow cytometry, chromatin immunoprecipitation assay (ChIP) and transient small-interfering RNA (siRNA). Proteomics and genomics data were subjected to gene ontology for further analysis.

Results: Treatment of RASFs with IL-6/IL-6R for 12 days resulted in phenotypic changes that were confirmed by TRAP staining (p< 0.05; n=3) and increased expression of the invasion markers Podoplanin (PDPN) and Thy1. Untargeted proteomics from RASF supernatants treated with IL-6/IL-6R identifies various unique proteins produced by RASFs in response to IL-6 trans-signaling (N=3 RASFs) including RANKL, Cathepsins K and Cathepsin B.RNA sequencing from chronic exposure IL6/IL-6R revealed 506 genes differentially regulated gene expression changes affecting cellular metabolic processes, VEGFA-VEGFR2 signaling, HIF and AP1, macrophage activation and receptor tyrosine kinase pathways. Global untargeted proteomics of IL-6/IL-6R exposure identified changes in metabolic pathways including carboxylic acid, oxidation and glycolysis metabolism. Further validation using Western blotting confirms chronic IL-6 exposure affects the stemness of RASFs differentiation by upregulating Nanog and Oct4 expression (p< 0.05; N-=3). The knockdown of Ets2 protein in RASFs during the chronic IL-6/IL-6R exposure using siRNA approach shows abrogation of TRAP-positive RASFs phenotype (p< 0,05; N=3). Ets2 knockdown also reduced PDPN expression. ChIP experiment using Ets2 antibody reveals IL-6/IL-6R utilizes Ets2 for cathepsin expression, as a transient knockdown of Ets2 abrogated the IL-6/IL-6R-induced cathepsin expression (p< 0.05; N=3).

Conclusion: IL-6 uniquely induces an invasive phenotype in human RASFs through cellular reprogramming evidenced by the expression of markers of invasiveness and causing metabolic changes in RASFs through Ets2.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-6-trans-signaling-induces-synovial-fibroblast-invasiveness-by-utilizing-ets2-protein/