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Background Rheumatoid arthritis (RA) is a prototypic autoimmune disease characterised by a prominent humoral autoimmunity. Of particular relevance is the local production of autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies in the inflamed synovial tissue. The mechanisms underlying break of B cell tolerance and local autoantibody production remains poorly understood. Objectives To identify cellular and molecular pathways implicated in RA-specific humoral autoimmunity. Methods Synovial tissue samples were obtained by arthroscopy from untreated individuals with RA (n = 33) and inflammation matched SpA controls (n = 58). Gene expression profiling was performed on tissue samples of patients with established arthritis using 44K. Whole Genome Human microarrays (Agilent). Top differentially expressed genes were validated on three independent cohorts by Taqman based RT-qPCR and immunohistochemistry. Collagen-induced arthritis (CIA) and Experimental autoimmune encephalomyelitis (EAE) experiments were conducted using Bob1 knockout mice and their littermate controls. Results Microarray screening for genes differentially expressed in the inflamed synovium, the key target of the disease process in RA, revealed a prominent and disease-specific B cell/plasma cell signature with the B cell-specific transcriptional co-activator Bob1 and its transcriptional target BCMA among the most upregulated genes. Validation by RT-qPCR on two independent cohorts representing early and established arthritis confirmed microarray data and demonstrated elevated expression of Bob1 and BCMA not only in established RA, but also at the early phase of the disease. Quantitative evaluation of immunohistochemical stainings of synovial tissue with monoclonal antibody for Bob1 revealed significant increase in Bob1 positive cells in RA synovium (p − / − mice were fully resistant to CIA induction compared to their wild-type littermates. This remarkable protection from CIA is explained by decreased antigen-presentation/costimulatory capacity of B cells and by failure to produce pathogenic anti-collagen autoantibodies in the absence of Bob1. Conclusions The specific increase in Bob1 expressing cells in RA synovitis and the resistance of Bob1-deficient mice to development of CIA indicate that Bob1/BCMA axis may contribute to humoral autoimmunity in RA. The relationship between an aberrant Bob1 expression and the break of peripheral tolerance in RA is currently under investigation.

Background Macrophages and their pro-inflammatory cytokines, including TNF, are pivotal mediators of chronic synovitis in Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA). Despite similar levels of synovial macrophage infiltration and clinical responses to TNF-blockade, SpA is characterized by more pronounced infiltration with alternatively activated macrophages and ongoing osteoproliferation. Here, we investigated whether these differences were related to differential expression and/or function of TNF between both diseases. Methods Expression of membrane-bound TNF (mTNF) and soluble TNF (sTNF) was measured in IFN-g, IL-4 or IL-10 polarized macrophages obtained from healthy donors. Expression of TNF and its receptors was also measured in synovial fluid (SF) and synovial tissue (ST) of actively inflamed knee joints. Mice transgenically overexpressing mTNF were evaluated for spondylitis and arthritis. Results The expression of mTNF was increased in IL-4 and IL-10 polarized macrophages compared to IFN-g polarized macrophages. Moreover, secretion of sTNF was impaired in both alternatively polarized macrophages, indicating a shift from sTNF to mTNF. In line with these data, the sTNF SF levels were lower in SpA compared to RA (p=0.01) despite similar TNF mRNA levels in ST. This was not related to altered expression of TNF receptors as both TNF-R1 and TNF-R2 were similarly expressed in ST. mRNA levels of TACE, the enzyme responsible for the cleavage of TNF, were also similar between SpA and RA ST. To investigate the relevance of overexpressed mTNF in SpA pathophysiology, we characterized mTNF transgenic mice. As previously described, these mice develop a moderate arthritis, resulting in deformation of paws and loss of grip. Histological, the joints were characterized by synovitis, lymphoid aggregates and osteoproliferation. Besides arthritis, all mice spontaneously developed spondylitis as evidenced by a crinkled tail, a hunchback and histological pathophysiology. Conclusions Membrane-bound TNF is relatively overexpressed by alternatively polarized macrophages in SpA synovitis and leads to an axial and peripheral SpA phenotype in transgenic mice.

Background: Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. Materials and Methods: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFNc, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/ or RT-qPCR. Results: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MWIL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. Conclusion: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

Background Rheumatoid arthritis (RA) is characterized by inflammation and hyperplasia of the synovial membrane which ultimately results in erosion of cartilage and bone. Fibroblast-like synoviocytes (FLS) play ac ritical role in this destructive process by producing inflammatory cytokines. Interleukin 6 (IL-6) is a key cytokine in the pathobiology of RA and biological therapies targeting the IL-6 receptor have shown clinical benefit. The Fmslike tyrosine kinase 3 (Flt3) is a membrane bound tyrosine kinase receptor which has a crucial role in hematopoiesis, regulating cellular differentiation, proliferation and apoptosis. Both Flt3L and its receptor are strongly expressed in RA and it has been shown that this axis exerts both pro-inflammatory and tissue destructive properties once in the joint cavity. Therefore we examined the effect(s) of a specific Flt3 inhibitor on IL-6 production by RA synovial biopsies and FLS. Methods

Background With TNF-blockers availability for spondyloarthritis (SpA), evaluation of new drugs requires quick “go/no go” signals in small scale, short term proof-of-concept (PoC) trials. Biomarkers complementing clinical evaluations may help reducing length and size of these PoCs. We aimed to identify and validate serum biomarkers to predict clinical response at group level in these trials. Methods Matrix metalloproteinase-3 (MMP-3), Pentraxin-3 (PTX3), high sensitive C-reactive protein (hs-CRP), calprotectin, Interleukin-6 (IL-6), Vascular Endothelial Growth Factor (VEGF), and alpha-2-macroglobulin (alpha-2-MG) were selected as biomarkers [1-3]. Serum levels were determined by ELISA in healthy controls (n=20) and at week 0 and 2 in SpA patients treated with infliximab (5 mg/kg; week 0, 2, and 6) (n=18) or placebo (n=19). Patient and physician global assessment of disease activity and BASDAI were evaluated at week 0 and 12. Results Baseline serum levels of PTX-3, hs-CRP, calprotectin and VEGF (all p

Results In comparison with RA, synovial infiltration with C-kit positive mast cells was strongly and specifically increased in SpA, independently of the subtype (non-psoriatic versus psoriatic) and disease duration. Staining of mast cell granules with tryptase and toluidine blue as well as analysis of synovial fluid levels of histamine and tryptase did not indicate increased degranulation in SpA synovitis. Mast cells expressed significantly more IL-17 in SpA than RA synovitis and constituted the major IL-17 expressing cell population in SpA synovitis. Targeting mast cells with the c-kit inhibitor imatinib mesylate in ex vivo tissue cultures led to a significant decrease in the production of IL-17 as well as other pro-inflammatory cytokines. To confirm that mast cell infiltration is not a merely secondary, inflammationdependent feature of SpA, we assessed the impact of TNF blockade on mast cell infiltration in vivo. Analysis of paired synovial biopsies before and after treatment revealed that neither the number of mast cells nor the expression of IL-17 by mast cells was affected by effective TNF blockade in SpA.

Background Different inflammatory joint diseases have distinct patterns of bone damage with pronounced erosions in rheumatoid arthritis (RA), a combination of bone destruction and formation in psoriatic arthritis (PsA), and new bone formation in spondyloarthritis (SpA). Although the underlying mechanisms remain elusive, blocking of DKK-1 reverses the bone-destructive pattern to a boneforming pattern in experimental arthritis.

Background In rheumatoid arthritis (RA) synovial tissue (ST) angiogenesis can be observed already in the earliest phase of disease. The chemokine CXCL12, which is induced via the non-canonical nuclear factor-kappaB (NF-kB) pathway, plays an important role in angiogenesis, lymphocyte transendothelial migration, and the homing of endothelial progenitor cells. Therefore, the non-canonical pathway, with its key mediator NF-kB inducing kinase (NIK), may play an important role in pathological angiogenesis and the perpetuation of synovial inflammation in RA.

Background The Autoimmune Regulator (AIRE) is a transcription factor that is involved in the negative selection of self-reactive thymocytes in the thymus and is pivotal in the establishment of central tolerance. Recently, AIRE protein has also been detected in peripheral lymphoid organs, predominantly in dendritic cells (DC). In these peripheral sites, AIRE was found to regulate the expression of a group of tissue-specific antigens, suggesting that peripheral AIRE may play a complementary role in tolerance induction. It is currently unknown whether AIRE may play a role in inflamed tissues associated with ectopic lymphoid neogenesis, such as rheumatoid arthritis (RA) synovial tissue (ST).

Background Pathological angiogenesis can be observed in rheumatoid arthritis (RA) synovial tissue (ST) already in the earliest phase of disease and may be critical in the switch from acute to chronic inflammation. The chemokine CXCL12 is regulated by non-canonical NF- Bs ignaling and stimulates angiogenesis in endothelial cells (EC). Therefore, the non-canonical NF- Bp athway, with its key mediator NF- Bi nducing kinase (NIK), may play an important role in pathological angiogenesis leading to the perpetuation of synovial inflammation in RA.

Background and objectives CD55 (decay-accelerating factor) is a complement-regulatory protein, which is highly expressed by fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leucocytes. We recently showed that lack of either CD55 or CD97 ameliorates disease in murine collagen-induced and K/BxN serum transfer models of arthritis.1 Little is known regarding the regulation of CD55 expression in FLS. We therefore investigated the effect of toll-like receptors ligation and pro-inflammatory cytokines on CD55 expression. Materials and methods Synovial fibroblasts, obtained from biopsy samples of arthritis patients, were cultured and stimulated with cytokines (TNF, IFNγ, IL-1β, IL-6, IFNα) or TLR ligands (LTA, poly (I:C), LPS, imiquimod, CpG). Expression of CD55 was measured by flow cytometry using domain-specific monoclonal antibodies and recombinant CD97-loaded fluorescent beads. Chloroquine was used to inhibit TLR3 activity. Upregulation and functionality of dsRNA sensors in response to poly (I:C) or 59-triphosphate RNA was analysed by PCR. Apoptosis was measured by PI/annexinV staining and was blocked with the pan-caspase inhibitor Q-VD-OPH. Results Cultured synovial fibroblasts of patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis, and spondylarthritis express equal amount of CD55. Stimulation of RA-FLS with IL-1β (p=0.02) and poly (I:C) (p=0.001) induced a significant upregulation of CD55. Engagement of TLR3 by the dsRNA analog poly (I:C) was confirmed using chloroquine, an inhibitor of endosomal acidification that impairs TLR3 signaling. Synovial fibroblasts also expressed the cytoplasmic dsRNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Stimulation of these receptors with either poly (I:C) or 59-triphosphate RNA induced CD55 expression, but, in case of MDA5, also induced significant cell death (p Conclusions We identify dsRNA as a potent inducer of CD55 upregulation on synovial fibroblasts. Our findings suggest that CD55 induction by viral dsRNA or dsRNA may facilitate the accumulation of CD97-expressing inflammatory immune cells in the synovial tissue.