SFRP5 Enhances Wnt5a Induced-Inflammation in Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Abstract

Background: Tissue derived fibroblast-like synoviocytes (td-FLS) are key actors in pannus formation and contribute to joint destruction and inflammation during rheumatoid arthritis (RA). Several members of the Wnt family, including Wnt5a, may contribute to RA td-FLS activation and can potentially serve as therapeutic targets.

Objective: The present work aimed to investigate the expression of Wnt5a signaling elements in RA td-FLS and their potential precursors (fluid derived (fd) FLS and fibrocytes). We also studied the role of Wnt5a in RA td-FLS pro-inflammatory activity and whether the inhibitor SFRP5 could restore Wnt5a-induced synovial dysfunction in vitro.

Materials and methods: The levels of Wnt5a, SFRP5, Wnt5a receptors/coreceptors and Wnt5a pro-inflammatory targets were determined in cultured RA td-FLS, fd-FLS and fibrocytes using qPCR under basal conditions. The expression of pro-inflammatory molecules was assessed after RA td-FLS stimulation with Wnt5a and SFRP5 at different time points.

Results: Our data showed that td-FLS, fd-FLS and fibrocytes from patients with RA expressed similar levels of Wnt5a and a set of Wnt5a receptors/coreceptors. We also demonstrated that Wnt5a stimulated the expression of the pro-inflammatory targets, especially IL1β, IL8 and IL6 in RA td-FLS. Wnt5a-induced inflammation was enhanced in the presence of SFRP5. Furthermore, Wnt5a alone and in conjunction with SFRP5 inhibited the gene expression of TCF4 and the protein levels of the canonical coreceptor LRP5.

Conclusion: Wnt5a pro-inflammatory effect is not inhibited but enhanced by SFRP5 in RA td-FLS. This research highlights the importance of carefully evaluating changes in Wnt5a response in the presence of SFRP5.

Keywords: FLS; Wnt pathway; fibrocytes; inflammation; rheumatoid arthritis.

Figure 1

Comparison of Wnt5a expression in td-FLS from patients with RA and OA: Third passage RA td-FLS (A). Third passage OA td-FLS (B). Wnt5a mRNA expression in RA td-FLS (n=3) and OA td-FLS (n=3) (C). Wnt5a was expressed in RA td-FLS but not in OA td-FLS. ND, Not detected. Results are shown as the mean ± SD.

Figure 2

Light microscopic features and mesenchymal marker expression in td-FLS, fd-FLS and fibrocytes from patients with RA: Spindle-shaped adherent cells started to appear in synovial fluid cultures from patients with RA after few days of culture (C). Third passage fd-FLS showed a spindle-shaped appearance (D) similar to RA td-FLS (A, B). Fibrocytes from PBMC of patients with RA displayed some features of td-FLS, such as a fibroblast-like morphology. Fibrocytes showed cytoplasmic projections and a slim bipolar-shape (E, F) (original magnification ×100). Western blot analysis showed fibronectin, ColIA1 and GAPDH expression in fd-FLS, td-FLS and fibrocytes from patients with RA (G). The Western blots are representative of one experiment out of two.

Figure 3

Expression of Wnt5a and SFRP5 in td-FLS, fd-FLS and fibrocytes during RA. Wnt5a gene level profiles in the three cell types by quantitative RT-PCR: No significant differences were found in mRNA levels (A). SFRP5 mRNA expression: SFRP5 was expressed in td-FLS and fd-FLS but not in fibrocytes (n=3-4) (B). ND, Not detected. Results are shown as the mean ± SD.

Figure 4

Expression of Wnt5a receptors and coreceptors in td-FLS, fd-FLS and fibrocytes during RA: Relative mRNA levels of Fzd4, Ror2, Ryk and LRP5 in the three cell types: No difference was detected between the cells using qPCR analysis (A, C–E). Fzd5 was highly expressed in fibrocytes compared to td-FLS and fd-FLS (n=3-4) (B). Results are shown as the mean ± SD *Statistically significant difference p < 0.05, **statistically significant difference p < 0.01, using non-parametric Kruskal Wallis test with a post hoc test.

Figure 5

Expression of Wnt5a pro-inflammatory targets in td-FLS, fd-FLS and fibrocytes during RA. IL6 was highly expressed in fd-FLS compared to td-FLS and fibrocytes (A). IL1β and CCL2 were expressed by the three cell types (B, E). The higher mRNA levels for IL1β and CCL2 were found in fibrocytes followed by fd-FLS. IL8 and CXL10 expression profiles were greater in fibrocytes than in td-FLS or fd-FLS (C, D). Relative mRNA levels of COX2 in the three cell types (F): No difference was detected between the cells using qPCR analysis (n=3-4). Results are shown as the mean ± SD.*Statistically significant difference p < 0.05 using non-parametric Kruskal Wallis test with a post hoc test.

Figure 6

Effect of Wnt5a on the inflammatory response of RA td-FLS. RA td-FLS (passage 3) were stimulated with recombinant Wnt5a (300 ng/ml) in the presence or absence of SFRP5 (1 mg/ml) for 4 h (A) and 24h (B). Cytokine expression profiles (CCL2, IL8, IL1β, CXCL10 and COX2) were determined in cell culture using qPCR. Gene expression levels in unstimulated cells were assumed to be 1. Data are means ± SD of two independent experiments each performed in triplicates using at least two different lots of each of the recombinant Wnt proteins.

Figure 7

Effect of Wnt5a on the canonical Wnt pathway activation in RA td-FLS. RA td-FLS (passage 3) were stimulated with recombinant Wnt5a (300 ng/ ml) in the presence or absence of SFRP5 (1 mg/ml) for 4 h and/or 24h. Intracellular regulator expression profiles (β- catenin and TCF4) were determined in cell culture using qPCR (A). Gene expression levels in unstimulated cells were assumed to be 1. Results are shown as the mean ± SD of two independent experiments each performed in triplicates using at least two different lots of each of the recombinant Wnt proteins. The protein abundance of LRP5 was studied by Western blot analysis after 24h of stimulation (B). The Western blot is representative of one experiment out of two.