BackgroundCXCL4 regulates multiple facets of immune response and its level is highly increased in various Th17-associated rheumatic diseases, including systemic sclerosis (SSc)1–4. Recently, CXCL4 was shown to induce type I interferon production as well as endothelial activation in SSc patients1. Th17 skewing has been demonstrated in SSc5–6, however, whether CXCL4 plays a role in the induction of IL-17 production by human CD4 T cells is currently unclear.ObjectivesTo investigate the effect of CXCL4 on human CD4 T cell phenotype in particular IL-17 production in the absence or presence of antigen presenting cells.MethodsBlood was obtained from healthy donors and CD4 T cells, monocytes, dendritic cells, were isolated using magnetic-based sorting (n=20). In addition, CD4 T cells from SSc patients were isolated (n=10). CD4 T cells were activated using anti-CD3/CD28, or in co-cultures with antigen presenting cells, stimulated using superantigen Staphylococcal enterotoxin B. Exogenous recombinant human CXCL4 was added during (co-)culture in different concentrations. Cytokine production and proliferation were analyzed using Luminex immunoassays, intracellular cytokine staining, and flow cytometry.ResultsCXCL4 directly induced CD4 T cells secreting both IL-17 and IFN-γ, as well as IL-22, when costimulated with anti-CD3/CD28 (p<0.05). In many SSc patients, similar IL-17 increase upon CXCL4 treatment was observed, although this did not reach statistical significance. This might be due to the fact that CD4 T cells from SSc patients had already significant higher levels of IL-17 as compared to healthy donors (2182±722.2 vs 1053±263.6 pg/ml, mean±SEM, p<0.05). Furthermore, in co-culture system of CD4 T cells with monocytes or myeloid dendritic cells, CXCL4 treatment induced IL-17 production upon triggering by superantigen (p<0.05). Moreover, when monocyte-derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated significantly increased levels of IL-17, IFN-γ, and proliferation by CD4 T cells (all p<0.05).ConclusionsAltogether, we demonstrate that CXCL4 boosts pro-inflammatory cytokine production especially IL-17 by human CD4 T cells, either by acting directly or indirectly via antigen presenting cells. This indicates that targeting CXCL4 may potentially alleviate immune responses in Th17-mediated rheumatic diseases such as systemic sclerosis.References van Bon L, Affandi AJ, Broen J, et al. N Engl J Med 2014.Tamagawa-Mineoka R, et al. Allergol Int 2008.Vettori S, et al. Clin Rheumatol 2016.Yeo L, et al. Ann Rheum Dis 2015.Radstake TRDJ, van Bon L, et al. PLoS One 2009.Yang X, et al. Arthritis Res Ther 2014. AcknowledgementsThis study was supported by the Dutch Arthritis Association (Reumafonds) to A.J.A. and T.R.D.J.R., the Netherlands Organization for Scientific Research (NWO) to A.J.A. (Mosaic grant 017.008.014) and T.R.D.J.R. (Pre-Seed grant), the Portuguese Fundação para a Ciência e a Tecnologia to S.C.S.C. (SFRH/BD/89643/2012) and the European Research Council to T.R.D.J.R. (ERC Starting gran...

gelatinase expressed in three major forms: dimer, monomer and a complex with neutrophil gelatin-associated lipocalin (NGAL). Interleukin-(IL)-6 is a pleiotropic cytokine expressed by a variety of immune and non-immune cells. However, the mechanisms by which IL-6 contributes to the pathogenesis of chronic arthropathies are not fully understood. Objectives: The purpose of the present work was to perform a comparative study of the IL-6 production and MMP-9 activity in FLS stimulated with SF from patients with osteoarthritis (OA), rheumatoid arthritis (RA) or spondyloarthritis (SpA). In addition, the effect of IL-6 blockade on MMP-9 activity was evaluated. Methods: Primary FLS were obtained from SF of the RA patients. Furthermore, the SW982 human synovial cell line was used. The SF of patients with OA (n=11), RA (n=11) or SpA (n=9) patients were pooled. The FLS were stimulated with OA, RA or SpA SF pools and supernatants (SN) were collected after 24, 48 and 72 h. The IL-6 levels were assessed in the SN by ELISA. The gelatinase activity of the SN was determined by zymography. The IL-6 function was blocked with the anti-IL-6 receptor antagonist tocilizumab (TCZ) (200μg/ml). Results: Earlier induction of IL-6 in SW982 cell line was observed by RA and SpA SF stimulation since significant levels were detected at 24 h (p<0.001 and p<0.01 compared with non-stimulated cells, respectively), whilst OA SF induced significant IL-6 secretion at 72 h (p<0.01). Similar results were observed in primary FLS. In contrast to SF of OA patients, SF of patients with RA or SpA induced increased and sustained secretion of active MMP-9. Moreover, the molecular weight band corresponding with NGAL-MMP-9 complex, considered a protected form of MMP-9, was detected with higher intensity in the SN of FLS stimulated with RA or SpA SF compared with OA SF (p<0.001). In the presence of TCZ, significant inhibition in the gelatinase activity of all MMP-9 forms was observed at 48h of stimulation with RA or SpA SF (p<0.001 for MMP-9 dimer and NGAL-MMP-9 complex; p<0.01 for MMP-9 monomer, compared with FLS stimulated in absence of TCZ). Conclusions: We conclude that SF of patients with inflammatory arthritis recreate a differential microenvironment for FLS that impacts on early phenotypic changes of these cells. The IL-6 provokes augmented and persistent MMP-9 activity in FLS stimulated with RA or SpA SF. This work identifies TCZ as an inhibitor of all forms of MMP-9.