ARTHRITIC SYNOVIAL FLUID INFLUENCES EXPRESSION OF PRO- AND ANTI-INFLAMMATORY MEDIATORS IN MACROPHAGES

Purpose: Synovitis is a common trait of Osteoarthritis (OA) and Rheumatoid Arthritis (RA), with the synovial membrane showing a consistent infiltration of immune cells. Macrophages are recruited to the joint where they induce and maintain the inflammatory state by secreting pro-inflammatory mediators. Given the key role of macrophages in arthritic pathologies, the aim of this study was to investigate how macrophages react to the stimuli typical of the diseased joint environment. Therefore, we evaluated the effect of OA and RA synovial fluid (SF) on the transcriptional expression of pro- and anti-inflammatory mediators in different macrophage phenotypes. Methods: SF samples of 6 donors with no joint disease (Ctrl SF) were bought. SF was also obtained from 10 donors with end-stage knee osteoarthritis (OA SF) and from 10 donors with rheumatoid arthritis (RA SF). Monocytes were isolated from the buffy coats of 3 healthy donors with CD14 positive selection and pooled together. Immediately after isolation, adherent macrophages were either cultured in non-activating conditions (M0), stimulated with IFNγ and TNFα towards a pro-inflammatory phenotype (M1 activation), or with IL4 towards an anti-inflammatory phenotype (M2 activation). After 12 hours of culture in M0, M1, or M2 conditions, macrophages were treated with or without the different types of SF (50% V/V). All samples were cultured for an additional 12 hours and then harvested to analyze the transcriptional expression of IL6, TNFα, IL1β, IL10, CCL18, CD206 and IL1Ra. Comparison among the different types of SF was performed by One-Way ANOVA for non-parametric data (Kruskal-Wallis test) with a Dunn’s multiple comparison post hoc test.\ Results: Stimulation of macrophages with IFNγ and TNFα resulted in the upregulation of IL6, TNFα, and IL1β, and in the down-regulation of CD206 demonstrating that an M1 phenotype was induced. IL10 was also upregulated in M1-stimulated cells, probably as a feedback reaction to the pro-inflammatory stimulation. On the other hand, M2 stimulation induced the expression of CCL18, CD206 and IL1ra confirming the induction of an M2 phenotype.Compared to Ctrl SF, IL10 gene expression was decreased in response to RA SF in both M0 (Fig. A) and M2 macrophages (Fig. C). OA SF also decreased IL10, but only in M2 macrophages (Fig. C). OA SF increased IL1ra gene expression in M0, M1, and M2 macrophages (Figs. D, E, and F). RA SF also increased IL1ra but only in M0 macrophages (Fig. D). IL6 and CCL18 gene expression were decreased by RA SF in M2 macrophages (Figs. I and L) compared to Ctrl SF. No effect was seen regarding these genes by OA SF. TNFα, IL1β, and CD206 were not affected at all by any type of SF in any of the macrophage phenotypes (data not shown). Conclusions: Compared to Ctrl SF, OA SF decreased pro-inflammatory processes as shown by the decrease in IL10 (which is normally high when macrophages are in a pro-inflammatory environment) and the increase in IL1ra. This was especially seen in the M2 macrophages, indicating that OA SF enhances this phenotype. Surprisingly, RA SF also seemed to decrease pro-inflammatory processes as shown by a decrease of IL10 and an increase of IL1ra, but also by the decrease of IL6. In conclusion, SF from diseased joints does not induce pro-inflammatory processes, even not when from RA joints. Based on our results, SF from diseased joints might even induce anti-inflammatory processes.