Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Background: The transmembrane heparan sulfate proteoglycan Syndecan-4 (Sdc4) plays an important role in the regulation of various inflammatory disorders. However, the involvement of Sdc4 in intestinal inflammation remains unknown. Therefore, we assessed the impact of Sdc4 deficiency on experimental colitis and epithelial wound healing in vitro and in vivo. Methods: Dextran sulfate sodium (DSS)-induced colitis was monitored in wild type and Sdc4-deficient (Sdc4-/-) mice by assessment of body weight, histology, inflammatory cellular infiltration, and colon length. Syndecan-4 expression was measured by immunohistochemistry, Western blot, and quantitative real-time PCR. Epithelial permeability was evaluated by Evans blue measurements, Western blot, and immunohistological analysis of tight junction protein expression. Impact of Sdc4 on epithelial wound healing was determined by scratch assay in vitro and by colonoscopy following mechanical wounding in vivo. Results: In Sdc4(-/-) mice, colitis-like symptoms including severe weight loss, shortened colon length, histological damage, and invasion of macrophages and granulocytes were markedly aggravated compared with wild type (WT) animals. Moreover, colonic epithelial permeability in Sdc4(-/-) mice was enhanced, while tight junction protein expression decreased. Furthermore, Sdc4(-/-) colonic epithelial cells had lower cell proliferation and migration rates which presented in vivo as a prolonged intestinal wound healing phenotype. Strikingly, in WT animals, Sdc4 expression was reduced during colitis and was elevated during recovery. Conclusions: The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

Background The ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is crucial in inflammatory diseases, like rheumatoid arthritis. Depending on the tissue, it can either protect or promote an inflammatory process. By its binding of molecules, such as cytokines and growth factors, it can initiate signalling pathways and it has been implicated in cell-matrix adhesion, cell migration, differentiation as well as proliferation. However, the involvement of Sdc4 in intestinal inflammation is unknown so far. Our group revealed a protective function of Sdc4 in experimental intestinal inflammation. Material and methods We monitored the course of DSS-induced colitis in Scd4-/- and C57BL/6 WT mice and analysed the changes in body weight, colon length, histology and inflammatory cellular infiltrate. We also evaluated Sdc4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR. Colon-permeability was examined in vivo by using the Evans Blue method and measuring the clearance for Citrobacter rodentium in vivo. Wound healing effects of Scd4 were analysed in vitro by scratch assay analysis with human epithelial colon cell line (T-84) and in vivo by mechanically induced wounds in colonoscopies of Scd4-/- compared to WT mice. Results The expression of Scd4 is decreased upon the course of colitis and increased during remission. The course of colitis was markedly aggravated in Scd4-/- mice, reflected by dramatically loss of body weight, increased mortality rates and histological damage, emphasised by increased invasion of macrophages and granulocytes into the colon. Also colonic epithelial permeability of DSS-treated Scd4-/- mice was enhanced associated with an altered expression of tight junction proteins. Furthermore, Sdc4 deficiency resulted in a prolonged intestinal wound healing in vitro and in vivo due to reduced proliferation rates in vitro. Conclusions Our data indicates that Scd4 is crucial in experimental intestinal inflammation. It exerts protective effects by maintaining epithelial barrier integrity and regeneration. Further studies are needed to explore the mechanisms of Sdc4-signalling in colitis.

Background Psoriatic-Arthritis (PsA) is a type of inflammatory chronic arthritis with a seronegative spondyloarthropathy and associated psoriasis. The Danger-Associated Molecular Pattern molecules (DAMPs) S100A8 and S100A9 are both antimicrobial proteins with chemotactic activity and are the most abundant DAMPs expressed during many inflammatory disorders. The expression of the S100A8/S100A9-complex is highly elevated in psoriasis and psoriatic arthritis. However, the mechanisms that regulate S100A8/S100A9-complex-activities are poorly understood, which has led us to examine the role of S100A8 and S100A9 under chronic inflammatory conditions. Material and methods We crossed S100A9-deficient mice with TTP (tristetraprolin)-deficient mice into a systemic inflammatory model featuring high levels of TNF with an arthritic joint destruction phenotype. Disease progression in TTP-/- x S100A9-/- mice was analysed by immunostaining, immunohistochemistry and the adapted PASI-score. To neutralise TNF in TTP-/- x S100A9-/- mice we used an aTNF-inhibiting monoclonal antibody already in clinical use for therapy of arthritis and psoriasis. To measure altered protein levels we used Western blot analysis. Primary keratinocytes were isolated of the skin from newborn mice and infected with E.coli isolated from the faeces of mice. Results TTP/S100A9 deficiency led to highly elevated levels of the S100A9 complex partner S100A8 in the epidermis and to a severe psoriatic phenotype of TTP-/- x S100A9-/- mice. Furthermore the mice showed an accelerated course of arthritis compared to TTP-/- mice, including increased articular cartilage loss and bone destruction. Inhibition of TNF by application of anti-TNF clearly reduced the psoriatic phenotype of TTP-/- x S100A9-/- mice. Additionally, the reduction of the environmental bacterial levels led to a milder phenotype and decelerated pathogenesis. The in vitro infection of isolated keratinocytes with isolated E.coli resulted in a high expression of S100A8. Conclusions The data reveal that the S100A8/S100A9-complex acts not only as a systemic danger signal molecule, but is also TNF dependent and is essential for the regulation of inflammation. The loss of S100A9 led to a disregulated inflammatory response and this to a severe psoriasis with enhanced cartilage and bone destruction. Furthermore, an exogenic bacterial factor, such as E. coli, is also demonstrated to be important in the activation of the disease.

Background and objectives Psoriatic-Arthritis (PsA) is a type of inflammatory chronic arthritis with a seronegative spondyloarthropathy and associated psoriasis. The S100 calcium-binding molecules S100A8 and S100A9, known as damage-associated molecular pattern molecules (DAMP), are highly increased during many inflammatory disorders and their expression correlates with the severity of disease. S100A8 and S100A9 are expressed in low levels in normal epidermis, but are highly expressed in psoriasis. Interestingly a high expression of epidermal S100A8/S100A9 is also an early marker found in patients suffering from systemic onset of juvenile idiopathic arthritis, which has led us to investigate the role of DAMPs under chronic inflammatory conditions. Material and methods To analyse the role S100A8 and S100A9 have during inflammation, we crossed S100A9-deficient mice withTTP (tristetraprolin)-deficient mice as a systemic inflammatory model featuring high levels of TNF. Disease progression in TTP-/- x S100A9-/- mice was analysed by immunostaining, immunhistochemistry and the adapted PASI-score. RNA was extracted from snap-frozen mouse tissue for Real-time quantitative PCR analysis. To neutralise TNF in TTP-/- x S100A9-/- mice we used an aTNF-inhibitor monoclonal antibody already in clinical use for therapy of arthritis and psoriasis. To measure altered protein levels we used Western blot analysis. Results The loss of S100A9 in TTP-deficient mice leads to highly elevated amounts of S100A8 in the epidermis and furthermore to a severe psoriasis-like phenotype at postnatal day 8. The expression of other effector molecules in the TTP-/-/S100A9-/- mice know to be involved in the pathogenesis of psoriasis, incuding IL-17, IL-23 and IL-22 is markedly increased compared to TTP-/- mice. Treatment withanti-TNF preventedthe mice from developing the psoriatic phenotype, indicating that the psoriasis-like skin disease of TTP-/-/S100A9-/- mice is tumour necrosis factor (TNF) dependent. Conclusions Our results demonstrate that under inflammatory conditions, S100A9 is essential for the regulation of inflammation, suggesting that S100A9 released from epidermal cells may act not only as a systemic danger signal that is involved in the initiation of inflammatory disorders like psoriasis and arthritis, but may also have a homeostatic anti-inflammatory function in the skin.

Background and objectives The ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is able to act in different ways. By binding a variety of molecules, such as cytokines, it can function either as a decoy receptor or it can initiate signalling pathways. It has been also implicated in cell-matrix adhesion, cell migration, differentiation, proliferation and plays an important role during inflammation in rheumatoid arthritis. Our group showed that the loss of Sdc4 has a protective effect in cartilage damage, but in osteopontin-mediated liver damage, in which syndecan-deficiency increased the pathology. Because of these dual effects, we investigate the role of Sdc4 in murine experimental colitis. Material and methods We performed DSS-induced colitis in Scd4-/- and C57BL/6 WT mice at different time points. First, we analysed the Scd4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR, followed by measuring the course of colitis by weight loss, colon length, histological scoring of colonic modifications and IF of inflammatory marker cells. Colon-permeability was examined in vitro by using the Evans Blue method. Wound healing effects of Scd4 were analysed in vitro by performing scratch assay analyses withhuman epithelial colon cell line (T-84) and in vivo by colonoscopy of Scd4-/- compared to WT mice. Results The expression of Scd4 was downregulated in the course of colitis and was elevated during the recovery phase. DSS-treated Scd4-/- mice lost dramatically more body weight compared to WT mice and the histological damage according to the Dieleman-Score was markedly elevated in the Scd4-/- mice. Also, we found increased invasion of macrophages and granulocytes into the colon. In DSS-treated Scd4-/- mice colon-permeability was higher compared to WT mice. The knockdown of Scd4 as well as treatment of T-84 cells with the anti-Scd4 antibody led to a delayed cell migration. Furthermore, colonoscopy experiments showed a decelerated wound healing effect in the Scd4-/- mice. Conclusions Our data indicate that Scd4 is a major molecule in the homeostasis of the colon epithelium. It exerts protective effects in intestinal inflammation with Scd4 deficiency leading to a higher permeability of the colon as well as a delayed cell migration and diminished wound healing.

Sclerostin, an inhibitor of the Wnt/β-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities.

Background and objectives Wnt-inhibitor sclerostin has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Loss of sclerostin expression results in high bone mass and bone strength in patients with sclerosteosis and sclerostin knockout mice. Therefore, antibody-mediated inhibition of sclerostin is currently evaluated for the treatment of osteoporosis in humans. Since it has been shown that sclerostin is upregulated by TNFα, we studied its impacton inflammatory arthritis using RA mouse models such as the human TNF transgenic (hTNFtg) model, the G6PI-induced arthritis model and the K/BxN serum transfer-induced arthritis model. Materials and methods Sclerostin knockout (sost-/-) mice were crossed with hTNFtg mice to generate sost-/ -/hTNFtg. Mice with serum transfer-induced arthritis were generated by injection of arthritogenic serum collected from K/BxN mice in sost-/- and wild type (WT) mice. Arthritis was induced in DEREG mice by immunisation with recombinant glucose-6-phosphate isomerase (G6PI). To switch to the non-remitting G6PI-induced arthritis, Foxp3+ regulatory T-cells were depleted by diphtheria toxin. hTNFtg and G6PI-induced arthritis mice were treated with a neutralising antibody against human and murine sclerostin. Clinical disease severity, bone erosion, cartilage destruction and pannus formation were evaluated by histomorphometric, x-ray and micro-CT analysis. Sclerostin expression and p38 activation was assessed by immunohistochemistry, western-blot-analysisor RT-PCR. Knockdown of LRP5 and LRP6 was performed by transfection of fibroblast-like synoviocytes (FLS) with siRNA. Results This study showed for the first time that TNFα induces sclerostin expressionin RA-FLS. Surprisingly, the lack of sclerostin and its antibody-mediated inhibition led to deterioration of RA-like disease in hTNFtgmice with enhanced pannus formation and joint destruction. Suggesting a specific role for sclerostin in TNFα signalling, inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent G6PI-induced arthritis, but ameliorated disease severity in K/BxN serum transfer-induced arthritis,in which TNFα plays only a minor role. FLS from sost-/- /hTNFtg mice displayed increased TNFα-mediated p38 activation, a key step in arthritis development. In turn, sclerostin effectively blocked TNFα-induced but not IL-1-induced activation of p38 with participation of the canonical Wnt receptor LRP6. Conclusion Collectively, these data demonstrated that sclerostin appears to have a protective function in TNF-mediated chronic inflammation.

Background and objectives Syndecan-4 (sdc4) is a transmembrane heparan sulfate (HS) proteoglycan. We have shown previously that sdc4 blockade protects from osteoarthritis like changes in mice. Here, sdc4 has been implicated in the modulation of IL-1 mediated Erk signalling and regulation of MMP-3 expression in fibroblasts by modulating IL-1 receptor trafficking. Hence, we hypothesised that sdc4 blockade reduces cytokine signalling in RA synovial fibroblasts. Materials and methods Evaluating the impact of sdc4 on the RA phenotype in vivo, hTNFα transgenic (hTNFtg), sdc4 knockout (sdc4-/-) and hTNFtg/sdc4-/-as well as hTNFtg mice treated with sdc4 blocking antibody were histological analysed. To quantify the histomorphometrical changes toluidin-blue stained sections were analysed for cartilage destaining and erosion. Furthermore, immunohistological stainings for MMP-3 and the IL-1 receptor (IL-1RI) were performed. MMP-3 production was analysed via ELISA after TNFα or IL-1 stimulation and the influence on the Erk signalling pathway was evaluated by Western blot analysis in cells lacking sdc4, IL-1RI or both proteins. Additionally, the expression and presentation of TNF and IL-1RI was assessed via quantitative RT-PCR and FACS analysis. Influence of IL-1 stimulation on sdc4 complex formation was characterised via crosslinking and subsequent Western blot detection. Results The loss of sdc4 or its antibody-mediated inhibition reduced the severity of chronic destructive arthritis in mice by impairing IL-1 responsiveness of resident fibroblast-like cells. RT-PCR revealed, that neither the expression of IL-1RI nor TNF receptor was altered in cells lacking sdc4 compared to wild type fibroblasts, whereas FACS analysis and histological stainings showed that the inhibition of sdc4 largely abolished the IL-1R presentation on fibroblasts in vitro and in vivo. We demonstrated that IL-1 directly binds to sdc4 and in an IL-1R independent manner leads to its dimerisation. Strikingly, IL-1 induced dimerisation of sdc4 and its loss diminished caveolin vesicle-mediated trafficking of IL-1RI. Hence, Western Blot analysis of Erk phosphorylation showed reduced IL-1 induced Erk1/2 phosphorylation in sdc4-/- compared to wild type fibroblasts. The absence of IL1RI prevented IL-1 mediated Erk1/2 phosphorylation completely. Focusing at MMP-3 levels as read-out for the activation of the Erk-pathway, we found lower MMP-3 production upon IL-1 stimulation in sdc4-/- compared to wild type controls, while IL-1RI-/- cells did not respond at all. MMP-3 production was not altered upon TNFα stimulation. Conclusion We could show that the loss plus the antibody-mediated sdc4 blockade reduced IL-1RI presentation and thereby IL-1 signalling in firboblasts, which constitutes a novel function of sdc4 and might be of great value for RA treatment.

Myostatin (also known as growth and differentiation factor 8) is a secreted member of the transforming growth factor-β (TGF-β) family that is mainly expressed in skeletal muscle, which is also its primary target tissue. Deletion of the myostatin gene (Mstn) in mice leads to muscle hypertrophy, and animal studies support the concept that myostatin is a negative regulator of muscle growth and regeneration. However, myostatin deficiency also increases bone formation, mainly through loading-associated effects on bone. Here we report a previously unknown direct role for myostatin in osteoclastogenesis and in the progressive loss of articular bone in rheumatoid arthritis (RA). We demonstrate that myostatin is highly expressed in the synovial tissues of RA subjects and of human tumor necrosis factor (TNF)-α transgenic (hTNFtg) mice, a model for human RA. Myostatin strongly accelerates receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast formation in vitro through transcription factor SMAD2-dependent regulation of nuclear factor of activated T-cells (NFATC1). Myostatin deficiency or antibody-mediated inhibition leads to an amelioration of arthritis severity in hTNFtg mice, chiefly reflected by less bone destruction. Consistent with these effects in hTNFtg mice, the lack of myostatin leads to increased grip strength and less bone erosion in the K/BxN serum-induced arthritis model in mice. The results strongly suggest that myostatin is a potent therapeutic target for interfering with osteoclast formation and joint destruction in RA.

Hintergrund: Syndecan (Sdc)4 ist ein transmembranöses Protein mit Rezeptorfunktion und moduliert Wundheilung und Entzündung in der Haut. Ziel der Studie war den Einfluss von Sdc4 auf die intestinale epitheliale Wundheilung in vitro und in vivo sowie auf den Verlauf einer experimentellen Kolitis zu evaluieren.

Notably, DC T5 was absolutely critical for flagellin-induced IL-22 production, which is thought to play a key role in protecting the gut in infection, while both IEC and Hep T5 were important for robust systemic production of CXCL1 and IL-6.However, IEC T5 was the predominant determinant of intestinal and clinical phenotypes previously associated with loss of T5.Specifically, IEC-specific T5-deficient mice exhibited low-grade intestinal inflammation and metabolic syndrome, which were associated with altered microbiota composition, increased levels of fecal flagellin and LPS, decreased microbiota-epithelial distance, and reduced ability to quickly clear flagellated pathobionts.In contrast, mice lacking T5 in DC appeared similar to WT mice by these measures.While phenotype of mice lacking T5 in Hep remains under study, we note that loss of IEC T5 was not sufficient to recapitulate dietinduced liver disease previously associated with complete loss of T5 (steatosis), suggesting a role for Hep T5 in this disease model.CONCLUSION: IEC T5 is critical for keeping the microbiota in-check to minimize risk for developing diseases associated with intestinal inflammation.DC T5 may be important for protection against enteric pathogens while Hep T5 may protect against microbiota-mediated liver disease.

Background and Objectives The transmembrane heparan sulphate proteoglycan syndecan-4 (Scd4) has been implicated in cell-matrix adhesion, cell migration, differentiation, proliferation and plays an important role during inflammation in rheumatoid arthritis. Scd4 is a mediator and modulator of inflammatory signals, upon its binding of cytokines Scd4 acts either as a decoy receptor or through the initiation of Scd-dependend signalling, followed by the formation of a Scd4 complex. Cartilage damage is decreased in sdc4-deficient mice, but osteopontin-mediated liver damage is increased. Because of these dual effects we investigate the impact of sdc4 in murine experimental colitis. Materials and Methods We performed DSS-induced colitis in Scd4-/- and C57BL/6 WT mice. We used weight loss, colon length and histological scoring of colonic modifications to measure the course of colitis. Scd4-/- and WT mice were orally gavaged with 5 × 108 colony-forming units (CFU) of invasive bacterium Citrobacter rhodentium (C. rhodentium). The changes of body weight and faecal excretion of C. rhodentium were monitored for 21 days followed by evaluation of histological changes after infection. The permeability of the colon was examined in vitro by infection of colon samples from Scd4-/- and C57BL/6 WT mice with C. rhodentium. The migration behaviour of endothelial human cells (T-84) and scd4-siRNA T-84 knockdown cells was analysed by scratch assay. Results DSS-treated Scd4-/- mice lost dramatically more body weight compared to the WT mice and the histological damage according to the Dieleman-Score was markedly increased. At day 19 of post infection the clearance of C. rhodentium in Scd4-/- mice was markedly prolonged. In vitro infection of colon samples from Scd4-/- mice with C. rhodentium revealed a higher permeability for the bacterium compared to WT colon samples. The knockdown of Scd4 in human endothelial T-84 cells leads to delayed cell migration. Conclusions Like in inflammatory liver damage, Scd4 appears to play an important role in colitis and exerts protective effects in intestinal inflammation. The Scd4 deficiency leads to a higher permeability of the colon to C. rhodentium and a delayed cell migration. Further analysis are needed to explore the mechanisms of Sdc4-signalling in colitis.

Background In rheumatoid arthritis (RA) pathogenesis, activated synovial fibroblasts (SFs) play an important role, because of their tumor-like and agressive behavior. This phenotype is promoted by a steady stimulation with growth-factors and inflammatory cytokines, such as IL-1. Syndecan-4 (sdc4), a transmembrane heparan sulfate proteoglycan, has been found to be upregulated in RA-SF. Sdc4 can act as membrane receptor for a large array of ligands and modulate cytokine signaling by binding of molecules, such as TGFbeta, to its side chains. Very recently, sdc4 was discovered to modulate Erk signaling in chondroytes in response to IL-1 stimulation. Objectives Since the exact mechanisms of sdc4 signaling according to the role of sdc4 side chains are still unknown, we investigated sdc4 signal transduction with sdc4 side chain mutants as well as knock-out fibroblasts. Methods In order to unravel the role of sdc4 side chains for sdc4 signal transduction, we designed different sdc4 side chain mutants via overlap PCR. The serine residues, which constitute the side chain attachment sides, where replaced by alanine residues, thereby abolishing side chain formation. To analyze membrane localization of the designed mutants, laser scanning fluorescence microscopy of transiently transfected Cos-7 cells was performed. To investigate the multimerization pattern of the different sdc4 mutants crosslinking was performed upon IL-1 stimulation and subsequent Western blot analysis. Moreover, fibroblasts lacking sdc4, the IL-1 receptor (IL1-RI) or both receptors were stimulated with IL-1 to elucidate the interaction between IL-1RI and scd4 in IL-1 signaling. The phosphorylation pattern of Erk1/2 was chosen as readout. Results Every sdc4 side chain lacking mutant revealed normal intracellular trafficking into the cell membrane. Although wild type sdc4 showed normal multimerization, an impaired complex formation could be observed for the side chain mutants, particularly with regard to tetramers. Additionally, our crosslinking experiments displayed that sdc4 undergoes dimerization due to IL-1 stimulation. Analyzing sdc4 signal transduction in sdc4-/- fibroblasts revealed a diminished IL-1 induced Erk1/2 phosphorylation, while IL-1 induced Erk1/2 phosphorylation was nearly completely abolished in cells lacking the IL-1RI. Fibroblasts lacking both, IL-1RI and sdc-4, did not respond to IL-1 stimulation with an increased Erk1/2 phosphorylation. Comparing the impact of IL-1α and IL-1β on Erk1/2 phosphorylation, IL-1β was found to lead to a faster and more pronounced elevation of Erk1/2 phosphorylation in comparison to IL-1α. Conclusions In conclusion, we have shown that heparan sulfate side chains are essential for sdc4 multimerization and IL-1 stimulation induces sdc4 dimerization. Furthermore, sdc4 plays a role during IL-1 signaling and can be assumed to play an additive or even regulatory role in IL-1 signaling. Consequently, sdc4 complex formation might be a keystone in signal transduction during RA disease progression in RA-SF. Disclosure of Interest None Declared

In patients with inflammatory bowel disease (IBD) and in murine IBD models, mucosal disease activity is routinely assessed by endoscopy and histologic evaluation. This information is valuable for monitoring treatment response, with mucosal healing being a major treatment goal. The aim of this study was to evaluate the translational potential of noninvasive 18F-FDG PET/CT for the assessment of mucosal damage in murine dextran sodium sulfate (DSS) colitis and human IBD. Methods: After induction of DSS colitis, 18F-FDG uptake was serially assessed from colonic volumes of interest defined on PET/CT scans and intraindividually correlated to histologic findings and to infiltrating cell types. In addition, 18F-FDG PET/CT scans of 25 Crohn disease patients were analyzed, and colonic 18F-FDG uptake was correlated to endoscopically assessed damage. Results: At days 4 and 7 after DSS induction, colonic 18F-FDG uptake was significantly increased, with a distinct peak in the medial colon. 18F-FDG uptake strongly correlated with histologic epithelial damage. Additionally, 18F-FDG uptake increased in the bone marrow in the course of the disease, correlating with an increase in intestinal 18F-FDG uptake. Histology and fluorescence-activated cell sorting analysis of the bone marrow of DSS mice revealed an increased number of immature neutrophils, whereas mucosal polymerase chain reaction suggested a correlation of 18F-FDG uptake to T cell infiltration. In accordance with the results of 18F-FDG PET/CT in DSS colitis, an increased 18F-FDG uptake was found in 87% of deep mucosal ulcerations in IBD patients, whereas mild endoscopic lesions were detected only by 18F-FDG PET/CT in about 50% of patients assessed. Conclusion:18F-FDG PET/CT is a noninvasive method for evaluation of both experimental colitis and Crohn disease patients and thereby offers promising translational potential.