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Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc−/−Ihh−/− double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc

Transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine with normal microscopic morphology after birth and before weaning. Intestinal enlargement by CD97 depends on signaling but does not require binding of its ligand, CD55., Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein–coupled receptor CD97 shows an expression gradient along the crypt–villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.

Adhesion G protein–coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97–CD55-mediated cell adhesion to tissue sites.

Backgroundand objectives Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. The induction and maintenance of T cell tolerance to tissue antigens is essential to prevent autoimmunity. Combinations of central and peripheral mechanisms act in parallel to inactivate, eliminate or control 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. Traditional approaches to treatment of autoimmune diseases through immunosuppression have focused on direct inhibition of T cells. In the present study, the authors examined the targeted inhibition of APCs as a mean to downregulate /prevent autoimmune disease in a mouse model for rheumatoid arthritis. Materials and methods Collagen-induced arthritis (CIA) was induced in mice lacking Flt3L (Flt3L −/− ) and WTlittermates (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 (chronic phase) phenotypical and functional analysis of spleen and lymph nodes was performed: T and B cell markers, FoxP3 expression, activation markers, co-stimulatory markers and cytokine production. Results In steady-state, Flt3L−/− mice show reduced celularity in both spleen (p=0.007) and lymph nodes (p=0.01) and reduced T and B cell numbers compared with WT. In CIA abrogation of Flt3 signaling led to decreased disease incidence and severity. As shown for steady-state conditions, in CIA Flt3L-/- mice showed reduced spleen and lymph node cellularity (p + CD25 + T cells compared with WT (p=0.03). Flt3L−/− CD4 + T cells also produce significantly less IL-17 (p=0.016) and tumour necrosis factor α (p=0.010), and CD8 + T cells less interferon (IFNγ) (p=0.029) compared to WT. Conclusions Mice lacking Flt3L are protected from collagen-induced arthritis. The authors observed that Flt3L deletion influences the magnitude (cell numbers) and quality (CD25 expression) of T cell responses. Stimulation of lymphocytes by different types of DC, DC at different stages of maturity and producing and responding to different growth factors might contribute for this change in T cell numbers and/or effector functions in Flt3L−/− mice. Targeting this signalling pathway might be considered as a good therapeutic strategy in RA.

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.

Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS.

Background & Aims Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation. Methods Hedgehog activity was modulated genetically in both cell type–specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence. Results Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells. Conclusions We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria., Graphical abstract

Introduction CD55, a glycosylphosphatidylinositol-anchored, complement-regulating protein (decay-accelerating factor), is expressed by fibroblast-like synoviocytes (FLS) with high local abundance in the intimal lining layer. We here explored the basis and consequences of this uncommon presence. Methods Synovial tissue, primary FLS cultures, and three-dimensional FLS micromasses were analyzed. CD55 expression was assessed by quantitative polymerase chain reaction (PCR), in situ hybridization, flow cytometry, and immunohistochemistry. Reticular fibers were visualized by Gomori staining and colocalization of CD55 with extracellular matrix (ECM) proteins by confocal microscopy. Membrane-bound CD55 was released from synovial tissue with phospholipase C. Functional consequences of CD55 expression were studied in the K/BxN serum transfer model of arthritis using mice that in addition to CD55 also lack FcγRIIB (CD32), increasing susceptibility for immune complex-mediated pathology. Results Abundant CD55 expression seen in FLS of the intimal lining layer was associated with linearly oriented reticular fibers and was resistant to phospholipase C treatment. Expression of CD55 colocalized with collagen type I and III as well as with complement C3. A comparable distribution of CD55 was established in three-dimensional micromasses after ≥3 weeks of culture together with the ECM. CD55 deficiency did not enhance K/BxN serum-induced arthritis, but further exaggerated disease activity in Fcgr2b−/− mice. Conclusions CD55 is produced by FLS and deposited on the local collagen fiber meshwork, where it protects the synovial tissue against immune complex-mediated arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0518-4) contains supplementary material, which is available to authorized users.

BACKGROUND: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS.METHODS: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry.RESULTS: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55.CONCLUSIONS: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast like synoviocytes (FLS) m rheumatoid arthritis a prototypical immune mediated inflammatory disease Broad inhibition of Ras and related proteins has shown protective effects In animal models of arthritis but each of the Ras family homologues (ie H K and N Ras) makes distinct contributions to cellular activation We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis Each Ras protein was expressed in synovial tissue and cultured FLS Each homolog was also activated following FLS stimulation with tumor necrosis factor alpha or interleukin (IL) 1 beta Constitutively active mutants of each Ras protein enhanced IL 1 beta induced FLS matrix metalloproteinase 3 production while only active H Ras enhanced IL 8 production Gene silencing demonstrated that each Ras protein contributed to IL-1 beta dependent IL 6 production while H Ras and N Ras supported IL 1 beta dependent matrix metalloproteinase 3 and IL 8 production, respectively The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GT Pases in vivo suppresses global inflammation and joint destruction in arthritis Consistent with this simultaneous silencing of H Ras K Ras and N Ras expression significantly reduces inflammation and Joint destruction in murine collagen induced arthritis while specific targeting of N Ras alone is less effective in providing clinical benefits (Am J Pathol 2010 177 3010-3024, DOI 10 2353/ajpath 2010 091053)

CD55 (decay accelerating factor) is best known for its role in the negative regulation of the complement system. Indeed, lack of this molecule leads to disease aggravation in many inflammatory disease models. However, CD55 is abundantly present on fibroblast-like …

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

Background and Objectives CD55 (decay-accelerating factor) is a complement-regulating protein, expressed at unusually large amounts by fibroblast-like synoviocytes (FLS) in the intimal lining in rheumatoid arthritis (RA). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor, broadly expressed by immune cells invading the inflamed synovium. We previously reported a protective effect of lack of CD55 in experimental models of RA (Hoek et al , Arthritis Rheum 2010; 62(4): 1036–42). Current data cannot explain the high expression of CD55 by FLS. Therefore, we explored in detail the pattern of CD55 expression in RA synovial tissue and 3-D organ cultures. Materials and Methods Synovial tissue was obtained by miniarthroscopy and analysed by immunohistochemistry staining to visualise CD55 and collagen type III, a constituent of extracellular matrix. Reticular fibres were visualised with Gomori silver staining. Expression of CD55 mRNA in synovial tissue was detected using antisense locked nucleic acid (LNA) oligomers. CD55 expression on 2-D-cultured RA-FLS and on blood cells of healthy individuals was detected by flow cytometry and related to mRNA levels measured by qPCR. 3-D micromasses of RA-FLS were generated in matrigel and analysed by immunohistochemistry for CD55 and reticular fibres. Results CD55 was highly expressed in the synovial lining of RA tissue, both at the mRNA and the protein level. Notably, CD55 showed an extracellular staining pattern, which coincided with Gomori silver staining of reticular fibres on sequential sections. CD55 expression on 2D-cultured FLS was less abundant and comparable to PBMCs. In 3D-micromasses of RA-FLS, CD55 was upregulated after 3–4 weeks of culture and showed an extracellular distribution that resembled reticular fibres. Conclusions CD55 mRNA and protein is abundantly expressed by FLS in the intimal lining of RA synovial tissue. We provide evidence that FLS-derived CD55 is deposited in extracellular matrix structures such as reticular fibres, where it may contribute to the synovial stromal address code that facilitates the recruitment of immune cells.

Background and objectives Rheumatoid arthritis is characterised by hyperplasia of the synovial tissue due to local proliferation of stromal and recruitment of inflammatory immune cells. Accumulation of synovial fibroblasts likely is due to an imbalance between cell proliferation, survival and death. Virus RNA receptors can initiate pro-apoptotic signalling in a variety of cell types. Based on our recent observation that fibroblast-like synoviocytes (FLS) are equipped with functional endosomal (TLR3) and cytosolic (MDA5 and RIGI) sensors for double-stranded (ds)RNA, the authors have studied the consequences of stimulation with dsRNA on the survival of FLS. Materials and methods FLS from arthritis patients were stimulated with extracellular poly(I:C) or poly(I:C)/3pRNA complexed with PEI for intracellular uptake. Activation of TLR3, MDA5 and RIG-I was confirmed by semi-quantitative PCR of response genes. Cell death of FLS after triggering of dsRNA sensors was detected by FACS analysis (annexin V/PI staining) and MTT assay. mRNA level of 46 genes involved in apoptosis was tested with a multiplex ligation-dependent probe amplification (MLPA) assay. Changes in protein expression were confirmed by Western blot analysis. Results At concentrations >0.01 µg/ml, stimulation of MDA5 by intracellular delivery of poly(I:C) caused massive cell death in up to 75% of cultured FLS. In contrast, transfection of 3pRNA triggering RIG-I had a much smaller effect on cell viability, and extracellular administration of the TLR3 ligand poly(I:C) induced death only at the high concentration of 100 µg/ml. Type I IFNs did not kill FLS, indicating that cell death was not secondary to IFN receptor signaling. MLPA and biochemical analysis demonstrated a strong induction of the BH3-only pro-apoptotic proteins Puma and Noxa upon dsRNA triggering but was not restricted to MDA5 signaling, suggesting the existence of additional regulatory mechanisms. Treatment of cells with the pan-caspase inhibitor QVD reduced cell death by about 50%, indicating that MDA5-induced cell death in FLS may involve also caspase-independent mechanisms. Conclusions Triggering of the endosomal dsRNA sensor MDA5 induces expression of pro-apoptotic Puma and Noxa and causes death in cultured FLS. Ligation of MDA5 might be a therapeutic target to overcome apoptosis resistance of FLS in rheumatoid arthritis.

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.

Introduction Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. Methods ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. Results We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Conclusions Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.