Athanasios Stratis

Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation

Psoriasis is a common skin disease, the pathogenesis of which has not yet been resolved. In mice, epidermis-specific deletion of inhibitor of NF-kappaB (IkappaB) kinase 2 (IKK2) results in a skin phenotype that mimics human psoriasis in several aspects. Like psoriasis, this skin disease shows pronounced improvement when mice are treated with a TNF-neutralizing agent. We have found previously that this phenotype does not depend on the presence of alphabeta T lymphocytes. In order to evaluate contributions of other immune cell populations to the skin disease, we selectively eliminated macrophages and granulocytes from the skin of mice with epidermis-specific deletion of IKK2 (K14-Cre-IKK2fl/fl mice). Elimination of skin macrophages by subcutaneous injection of clodronate liposomes was accompanied by inhibition of granulocyte migration into the skin and resulted in a dramatic attenuation of psoriasis-like skin changes. The hyperproliferative, inflammatory skin disease in K14-Cre-IKK2fl/fl mice was a direct consequence of the presence of macrophages in the skin, as targeted deletion of CD18, which prevented accumulation of granulocytes but not macrophages, did not lead to major changes in the phenotype. Targeted deletion of the receptor for IFN-gamma revealed that the pathogenesis of the skin disease does not depend on classical IFN-gamma-mediated macrophage activation. Our results demonstrate that in mice epidermal keratinocytes can initiate a hyperproliferative, inflammatory, IFN-gamma-independent, psoriasis-like skin disease whose development requires essential contributions from skin macrophages but not from granulocytes or alphabeta T lymphocytes.

Myostatin is a direct regulator of osteoclast differentiation and its inhibition reduces inflammatory joint destruction in mice

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.

Early structural changes in cartilage and bone are required for the attachment and invasion of inflamed synovial tissue during destructive inflammatory arthritis

Objective To elucidate the mechanisms involved in cartilage damage in an experimental model of rheumatoid arthritis (RA) by specifically addressing the time course of extracellular matrix degradation and the contribution of cell–matrix interactions for initiation and perpetuation of this process. Methods The human tumour necrosis factor (TNF) transgenic (hTNFtg) mouse model of RA was used to analyse the time course of pannus attachment to the cartilage and cartilage destruction, respectively, and crossed hTNFtg mice with interleukin (IL)-1−/− animals were used to investigate the role of IL-1 on these TNF-induced mechanisms in vivo. In addition, an in vitro attachment assay using synovial fibroblasts (SFs) from hTNFtg mice and freshly isolated articular cartilage was used to determine the role of proteoglycan loss in attachment of SFs and the role of the transmembrane heparan sulfate proteoglycan syndecan-4. Results In vivo analyses of hTNFtg mice showed that proteoglycan loss induced by IL-1 precedes and constitutes an important prerequisite for these processes as, in hTNFtg mice, IL-1 deficiency protected from the loss of cartilage proteoglycans and almost completely prevented the attachment and subsequent invasion of inflamed synovial tissue into cartilage. In vitro studies confirmed that loss of cartilage proteoglycans is required for attachment of SFs and that syndecan-4 is prominently involved in SF attachment and activation. Conclusions The results of this study suggest that the loss of cartilage proteoglycans is an early event in the course of destructive arthritis that facilitates the attachment of the inflamed synovial membrane and also initiates matrix degradation and inflammation through cell–matrix interactions.

Translational 18F-FDG PET/CT Imaging to Monitor Lesion Activity in Intestinal Inflammation

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.

The Role of the WNT inhibitor sclerostin in rheumatoid arthritis

Joints affected by rheumatoid arthritis (RA) display signs of bone resorption and usually lack signs of repair which contributes to rapid and progressive loss of joint structures. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway, a key regulator of bone turnover. Since it has been shown that sclerostin is expressed under inflammatory conditions, we analysed the …

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation

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-&agr;–driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9–/– mice with 2 independent TNF-&agr;–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.

A4.22 Syndecan-4 controls interleukin (IL)-1 receptor trafficking and IL-1 signalling in chronic destructive arthritis

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.

Loss of the Wnt inhibitor sclerostin promotes pannus formation and accelerates joint destruction in the hTNFtg mouse model of rheumatoid arthritis

Background In rheumatoid arthritis (RA), progressive joint destruction is a hallmark of disease and results from both increased bone resorption and the lack of repair mechanisms. tumour necrosis factor α (TNFα) contributes to both aspects of pathologic joint remodelling by increasing the number of bone-resorbing osteoclasts and decreasing the number of bone-forming osteoblasts. Sclerostin is a potent inhibitor of osteoblast development by antagonising the Wnt/β-catenin signalling pathway. Based on recent data that have shown increased expression of sclerostin under inflammatory conditions, the authors studied its expression in human RA and in human TNF transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. Moreover, the authors analysed the effects of sclerostin deficiency on the development and severity of the arthritis in these mice. Methods Expression of sclerostin was determined by immunohistochemistry, western blot and reverse transcriptase PCR. To assess the functional role of sclerostin in vivo, sclerostin knockout (SOST−/−) mice were crossed with hTNFtg mice. In addition to determining the clinical severity of disease in SOST−/−/hTNFtg and hTNFtg mice, histological changes including bone erosion, cartilage destruction and inflammation were evaluated by histomorphometric analyses. The number of osteoclasts was quantified using tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed to analyse the expression of molecules of the Wnt pathway. Results Immunohistochemistry and western blot analyses revealed a strong overexpression of sclerostin in synovial tissue of RA compared to osteoarthritis patients. Likewise, ankle joints of hTNFtg mice showed high levels of sclerostin, especially in the infiltrating pannus, whereas only negligible staining was observed in wild-type animals. In vitro, expression of sclerostin was only found in osteoblasts and osteoclasts, but could be induced in RA synovial fibroblasts by TNFα. Surprisingly, the lack of sclerostin not only increased the clinical severity of arthritis in hTNFtg mice but most dramatically accelerated joint damage in this mouse model of RA. SOST−/−/hTNFtg mice displayed significant higher bone erosion, synovial hyperplasia and osteoclast numbers compared to hTNFtg mice. Moreover, immunohistochemistry revealed higher levels of dickkopf 1 (DKK-1) and Wnt-5a in joints of SOST-deficient hTNFtg mice. Conclusions The authors hypothesise that under inflammatory conditions, higher levels of DKK-1 and Wnt-5a in joints of sclerostin-deficient arthritic mice counteract the beneficial effect of sclerostin deficiency by increasing osteoclast development through enhanced blockade of the Wnt-3a pathway by DKK-1 as well as by promoting synovial hyperplasia through Wnt-5a-mediated synoviocyte activation. These results may have an impact on the use of sclerostin inhibitors in inflammatory joint diseases.

Syndecan-4 Modulates Epithelial Gut Barrier Function and Epithelial Regeneration in Experimental Colitis

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.

01.03 Ttp/s100a9 deficient mice promote a tnf-dependent psoriatic arthritis phenotype triggered by the bacterial environment

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.