Background CXCL4 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.
Objectives To 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.
Methods Blood 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.
Results CXCL4 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).
Conclusions Altogether, 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.
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.
Acknowledgements This 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 grant).
Disclosure of Interest None declared