Background/Purpose:  Although elevated IL-6 and its soluble receptor (sIL6R) have been found in the serum and synovium of arthritic patients, the molecular mechanisms by which it contributes to the pathogenesis of rheumatoid arthritis (RA) is not fully understood. Fibroblast-like synoviocytes (FLS) are one of the key players in the chronic inflammation of RA. Despite the knowledge provided by different studies, it is not fully resolved how cytokines present in the inflammatory environment of the RA synovium and particularly IL-6, synergize and contribute to the establishment of the rheumatoid arthritis synovial fibroblast (RASF) inflammatory phenotype.

Methods: Total RNA was extracted from 8 HSF lines and 5 RASF stimulated with either TNF-α (20ng/ml), IL-6 and sIL-6R (20ng/ml each) or in combination for 24h were used for this study. The cRNA from 4 HSF lines stimulated with either TNF-α or IL-6 and sIL-6R was hybridized in Affymetrix GeneChip® PrimeView™ Human Gene Expression Array platform containing a total of 49,395 probe sets. The microarray data analysis was performed by using Expression Console and Transcriptome Analysis Console (TAC) software (Affymetrix). Additionally, differentially expressed genes were analyzed to identify potential functional pathways using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Validation of microarray data as well as synergy and priming experiments were performed by quantitative RT-PCR using specific primers.

Results:  Using microarray analysis we identified genes differentially regulated (≥2 folds vs control) by the trans-signal activation of IL-6 (522 genes) and TNF-α (2640 genes) in FLS cells in culture. Interestingly, we found a significant overlap between TNF-a and IL-6/sIL-6R regulated genes. Almost 55% (287 genes) of total IL-6/sIL-6R regulated genes are common to those of TNF-a. Simultaneous induction of FLS cells with both factors synergistically stimulated the expression of selected common genes as MCP-1 and IL-6 genes, in contrast to CCL5 or IL-8 which are only activated by TNF-a in our cellular system. These data suggest that the crosstalk between TNF-a and IL-6/sIL-6R controls specific group of genes. Furthermore, stimulation of RASF cells with both inflammatory factors further enhanced the expression of these genes up to 10 fold compared to non-pathological FLS. In addition, priming experiments stimulating FLS cells with either TNF-a or IL-6/sIL-6R, followed by induction with the counterpart factor at different time-points, demonstrated that the synergistic effect requires the constant presence of both factors. These data suggest that the mechanism of crosstalk between TNF-a and IL-6/sIL-6R more likely occurs through regulation of signaling and/or transcriptional mediators rather than at a post-transcriptional level.

Conclusion:  Our study suggests that despite the differential IL-6 and TNF-a intracellular signaling, there is significant overlap between the transcriptional response induced by both factors. In addition, a potent synergistic effect was confirmed for some target genes, pointing to a relevant interaction of both cytokines in the RASF pro-inflammatory effector response.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/crosstalk-between-il-6-and-tnf-alpha-signaling-pathway-in-rheumatoid-arthritis-synovial-fibroblasts/