Background/Purpose: Chronic exposure to IL-6 trans-signaling has been shown to significantly impact the characteristics of rheumatoid arthritis synovial fibroblasts (RASFs). Previous studies have demonstrated that this exposure increases heterogeneity markers, upregulates reprogramming factors, and transforms RASFs into cells with stem cell-like characteristics. Moreover, the transcription factor Ets2 has been identified as a crucial player in these effects, as it is upregulated and translocated to the nucleus of RASFs upon IL-6 trans-signaling stimulation.

Methods: To further explore the role of Ets2 in IL-6-induced inflammation, comprehensive analyses were performed. RNA-seq analysis was conducted on RASFs treated with IL-6 + IL6R, either alone or in combination with Ets2 knockdown using siRNA. Mass spectrometry-based untargeted phospho-proteomics analysis was utilized to evaluate global protein changes in RASFs. Additional RNA sequencing was carried out, and the data were evaluated using ELISA, quantitative RT-PCR, and Western blot analyses. The experiments involved multiple RASF donor lines, with statistical significance set at p< 0.05.

Results: Chronic IL-6 trans-signaling promotes stem cell-like characteristics in rheumatoid arthritis synovial fibroblasts (RASFs), as evidenced by the upregulation of Nanog, OCT4, Sox2, and Myc. This effect is also observed in normal synovial fibroblasts (NSFs), indicating a broader influence on cell differentiation. Proteomics analysis confirmed the upregulation of stemness markers and metabolic intermediates associated with glycolysis in IL-6 trans-stimulated RASFs. Inhibition of the Ets2 transcription factor resulted in the downregulation of Nanog, Myc, and Sox2, suggesting that Ets2 drives the reprogramming of RASFs to a stem cell-like phenotype (N=3, p< 0.05). IL-6 trans-signaling induced the upregulation of CD90/Thy1, a marker of mesenchymal stem cells, which was reversed by Ets2 knockdown (N=3, p< 0.05). The downregulation of heterogeneity markers Thy1 and PDPN without Ets2 implies that IL-6 employs Ets2 to drive both inflammatory and differentiation functions in RASFs. RNA sequencing identified 506 differentially regulated genes (N=3, p< 0.05), with the enrichment of stem cell factors in the transcriptomics data. These findings shed light on the impact of chronic IL-6 trans-signaling on RASFs and highlight the role of Ets2 in regulating inflammatory and differentiation processes. Understanding these molecular events could lead to novel therapeutic strategies for rheumatoid arthritis.

Conclusion: To further explore the molecular changes induced by chronic IL-6 trans-signaling, we performed RNA sequencing on RASFs that were continuously stimulated with IL-6 + sIL6R. Our analysis identified 506 differentially regulated genes, with stem cell factors like Sox2, Myc, Oct4, Nanog, among others, showing enrichment in the transcriptomics data. The role of Ets2 as a key mediator in this process highlights its significance in regulating both inflammatory and differentiation functions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/chronic-il-6-trans-signaling-enhances-stem-cell-like-characteristics-of-rheumatoid-arthritis-synovial-fibroblasts/