IntroductionThe FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.MethodsThe levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.ResultsFlt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.ConclusionsThe Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.

ObjectiveComprehending the mechanisms that regulate activation of autoreactive T cells and B cell antibody production is fundamental for understanding the breakdown in self-tolerance and development of autoimmunity. Here we studied the role of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling in the pathogenesis of collagen-induced arthritis (CIA).MethodsCIA was induced in mice lacking Flt3L (Flt3L−/−) and wild-type (WT) littermates (C57/BL6, 8–10 weeks old). Mice were killed in the initial phase (acute phase: experiment 1) and late phase (chronic phase: experiment 2) of the disease. Arthritis severity was assessed using a semiquantitative scoring system (0–4), and histological analysis of cellular infiltration, cartilage destruction and peptidoglycan loss was performed. Phenotypic and functional analysis of T and B cells, FoxP3 expression, activation and lymphocyte costimulatory markers, and cytokine production were performed ex vivo by flow cytometry in lymph nodes. Serum collagen type II (CII)-specific antibodies were measured by ELISA.ResultsFlt3L−/− mice showed a marked decrease in clinical arthritis scores and incidence of arthritis in both acute and chronic phases of CIA compared with WT mice. Moreover, decreased synovial inflammation and joint destruction was observed. Both the magnitude and quality of T cell responses were altered in Flt3L−/−. In the acute phase, the amount of CII-specific IgG2a antibodies was lower in Flt3L−/− than WT mice.ConclusionsThese results strongly suggest a role for Flt3L signalling in the development of arthritis.

Background Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. Traditional approaches to treat autoimmune diseases have focused on direct inhibition of autoreactive T cells. A key requirement for tolerance is the presentation of antigens in a correct context. Dendritic cells (DCs) are the central antigen-presenting cells (APCs) for the initiation of T cell responses. In this context, stimulation of the Flt3 via Flt3L is known to drive expansion and differentiation of DCs. Moreover, mice lacking Flt3L are have reduced of DC numbers Objectives In the present study, we examined the targeted inhibition of APCs as a mean to downregulate/prevent autoimmune disease in a mouse model for rheumatoid arthritis. Methods Collagen-induced arthritis (CIA) was induced in mice lacking Flt3L (Flt3L-/-) and WT littermates (C57/BL6 background, 9-10 weeks old). The severity of the arthritis was assessed using an established semiquantitative scoring system (0–4). After 60 days, serum, spleen, lymph nodes (LN) and hind paws were collected. Collagen type II (CII) specific antibodies were measured by ELISA. Histological analysis (H&E, Toluidine blue, Safranin O and Trap) was performed on hind paws and phenotypical and functional analysis of spleen and LN was performed: T and B cell markers, FoxP3 expression, activation and co-stimulatory markers and intracellular cytokine staining (after PMA/Ionomycin stimulation). Results Histological analysis of paws showed increased synovial infiltration and joint destruction in WT mice while Flt3L-/- mice showed mild infiltration without inflammation (H&E staining). Cartilage destruction (Safranin O staining) and the number of osteoclast were higher in WT compared with Flt3L-/- mice. Importantly, in steady-state (no CIA induced), Flt3L-/- mice show reduced celularity in both spleen (p=0.007) and LN (p=0.01) and reduced T and B cell numbers compared with WT. CIA induction in Flt3L-/- led to decreased disease incidence and severity (AUC p=0.001) compared with WT littermates. In addition, Flt3L-/- mice showed reduced spleen and LN cellularity (p<0.0001) but also reduced percentage of CD4+CD25+T cells compared with WT (p=0.03). Flt3L-/- CD4+ T cells produced significantly less IL-17 (p=0.016) and TNF-a (p=0.010) while CD8+ T cells produced less IFN-g (p=0.029) compared with WT. Conclusions Mice lacking Flt3L are protected from CIA. CIA induction in mice that have reduced numbers of DC influenced not only the magnitude (cell numbers) but also the quality (CD25 expression) of T cell responses. Stimulation of lymphocytes by different types of DC, DC at different stages of maturity and producing or responding to different growth factors might contribute for this change in T cell numbers and/or effector functions in Flt3L-/- mice. Targeting this signaling pathway might be considered as a good therapeutic strategy in RA. Disclosure of Interest None Declared