FRI0014 Bone Marrow Regulatory CD4+FOXP3+ T Cells in Rheumatoid Arthritis (RA) Patients Share Pro- and Anti-Inflammatory Functions
Authors: M. Massalska, A. Radzikowska, E. Kuca-Warnawin, U. Musialowicz, M. Plebanczyk, U. Skalska, T. Burakowski, P. Maldyk, E. Kontny, W. Maslinski
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> Radiological observations of joint destruction in RA are preceded by inflammatory process observed in bone-marrow (BM) together with detection of autoantibodies in peripheral blood. Previous studies have supported the concept that bone marrow can actively participate in the pathogenesis of RA by TLR triggered B cell activity (1) and the presence of activated T cell subpopulations (2). However, the suppressive properties of bone-marrow-resident cells remain poorly understood. In the present work investigating suppressive properties of natural CD4<sup>+</sup>Foxp3<sup>+</sup> regulatory T cells (Tregs), we show increased production of main pro-inflammatory cytokine TNF by otherwise suppressive Tregs isolated from bone marrow of RA patients. <h3>Objectives</h3> To compare the functional activity and cytokines profile of Tregs isolated from RA and osteoarthritis (OA) bone marrow samples. <h3>Methods</h3> Bone marrow samples were obtained as a standard procedure during hip bone replacement surgery from RA and OA patients. CD4<sup>+</sup>CD25<sup>+++</sup> Tregs and CD4<sup>+</sup>CD25<sup>-</sup> responder T cells (Tresp) were isolated from bone marrow mononuclear cells (BMMC) by cells sorter (FACS Aria BD) for Tregs functional activity measurement. As a control, Treg and Tresp populations were isolated from peripheral blood of healthy volunteers. Isolated cells were cultured alone or together in 1:1 ratio and were additionally stimulated in some samples by anti-CD3/CD28 antibodies. Pro-inflammatory cytokines (TNF and IFN-gamma) and anti-inflammatory cytokines (IL-10, TGF-beta, IL-35) were estimated by specific ELISAs in the supernatants removed before addition of [<sup>3</sup>H] thymidine to proliferation assays. Data are shown as mean ± SEM. The study was approved by local Ethical Committee. <h3>Results</h3> Concentration of TNF produced by Tregs isolated from RA BM was comparable with TNF produced by responder T cells (14.2±2.7 pg/ml in Tregs vs 12.7±1.8 pg/ml in Tresp) and higher then TNF produced by Tregs isolated from OA BM (8,5±3.1 pg/ml) or healthy volunteers blood (7.7±1.9 pg/ml). However, despite high TNF production, Tregs from RA BM remain functionally active and suppress proliferation and production of TNF and IFN-gamma in coculture with Tresp (63.0±6.0%, p=0.012; 34.4±10.4%; p=0.03; 33.7±21.1%; p=0.046 respectively). Low production of IFN-gamma by Tregs was further lowered by additional TCR stimulation in all investigated groups of patients. Production of IL-10 by Treg or Tresp was negligible under investigated conditions in all investigated groups of patients (below the first standard of 4.4 pg/ml in ELISA). Similarly, no IL-35 was detected in any of investigated probes. However, we observed significant amounts of TGF-beta produced by Treg and Tresp populations. Treg from RA BM produced more TGF-beta than Tresp (116.5±32.6 pg/ml vs 77.6±49.9 pg/ml). <h3>Conclusions</h3> Natural Tregs from bone marrow of RA patients despite TNF production remain suppressive. Thus, CD4<sup>+</sup>Foxp3<sup>+</sup> Treg present in RA BM share suppressive capacities with pro-inflammatory feature, that may contribute to RA pathogenesis. <h3>References</h3> W. Rudnicka et al.; Eur J Immunol 2009; 1211-20 E. Kuca-Warnawin et al.; Ann Rheum Dis 2011; 227-33 <h3>Disclosure of Interest</h3> None declared