Ab0077 semaphorin4b Is Upregulated in Rheumatoid Arthritis Patients and Induces Expression of Inflammatory Mediators by Macrophages and Fibroblast-Like Synoviocytes

Abstract

BackgroundSeveral studies have shown that different semaphorin family members are involved in the pathogenesis of rheumatoid arthritis (RA). On one hand, our group has demonstrated that (Sema)phorin 3B and Sema3F are reduced in RA patients and play a protective role. On the other hand, Sema4A and Sema4D are increased in RA patients and are associated with inflammatory processes (Garcia, 2019; Igea, 2022).ObjectivesThe aim of this study is to determine the role of Sema4B in the pathogenesis of RA.MethodsGene expression of Sema4B was obtained from the gene expression array available in Gene Expression Omnibus-NCBI (GSE77298). Fibroblasts-like synoviocytes from RA patients (RA-FLS) (n=8) were stimulated 4 and 24 h with Sema4B (200ng/mL), TNF (10 ng/mL) or the combination of both. Peripheral blood monocytes from RA patients (n=12) were differentiated into M1 macrophages by culturing in the presence of IFN-γ (10 ng/mL) for 6 days. Afterwards, macrophages were stimulated 24 h with Sema4B (200ng/mL), LPS (10 ng/mL) or the combination of both. The expression of inflammatory mediators was determined by quantitative PCR (qPCR) and ELISA. Viability and migration of FLS were determined using calcein assays and wound closure assays, respectively.ResultsSema4B expression was significantly higher in the synovial tissue and FLS of RA patients compared to healthy controls (HC) and osteoarthritis patients (OA), respectively. A significantly higher expression ofSEMA4Bin the synovium and FLS of RA patients compared to, respectively, was found. Interestingly, TNF stimulation induced the expression ofSEMA4Bby RA-FLS. Functional studies showed that Sema4B did not affect the viability of FLS but increased their migratory capacity. Moreover, Sema4B alone did not induce the expression of inflammatory mediators (data non shown), but significantly enhanced the TNF-induced expression ofIL6, IL8, TNF, CCL2andMMP3(Figure 1A) and the secretion of TNF. Finally, Sema4B alone did not modulate the expression of inflammatory mediators in macrophages, but significantly enhanced the LPS-mediated expression ofTNF,CCL2, andMMP1(Figure 1B), as well as the TNF protein secretion.Figure 1.Sema4B enhances pro-inflammatory activity in FLS (n=6) and macrophages (n=10) of RA patients. (A) Significative increment of the genetic expression of pro-inflammatory mediators in RA-FLS stimulated with Sema4B (200 ng/ml), TNF (10 ng/mL) and their combination for 24 hours. (B) Significative increase of the genetic expression of pro-inflammatory mediators in M1 macrophages. Macrophages differentiated from monocytes of RA patients during 6 days (IFN-γ 10 ng/mL) and stimulated with Sema4B (200 ng/ml), LPS (10 ng/mL) and their combination for 24 hours. Symbols represent individual patients; bars show the mean ± SEM. *P < 0.05.ConclusionOur data demonstrate that, in an inflammatory context, Sema4B induces FLS migration and the production of inflammatory mediators by FLS and macrophages. These results suggest that Sema4B is involved in inflammatory processes observed in the RA synovium and might be a potential therapeutic target in the treatment of RA.References[1]Garcia S. Role of Semaphorins in Immunopathologies and Rheumatic Diseases.Int J Mol Sci. 2019;20(2):374. Published 2019 Jan 16. doi:10.3390/ijms20020374[2]Igea A, Carvalheiro T, Malvar-Fernández B, et al. Central Role of Semaphorin 3B in a Serum-Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis.Arthritis Rheumatol. 2022;74(6):972-983. doi:10.1002/art.42065AcknowledgementsAll patients involved in this study.Disclosure of InterestsNone Declared.