C Cromme

Molecular mechanisms of cartilage remodelling in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease that is characterized primarily by progressive breakdown of articular cartilage. The loss of proteoglycans, the mineralization of the extracellular matrix (ECM) and the hypertrophic differentiation of the chondrocytes constitute hallmarks of the disease. The pathogenesis of OA includes several pathways, which in single are very well investigated and partly understood, but in their complex interplay remain mainly unclear. This review summarises recent data on the underlying mechanisms, specifically with respect to cell-matrix interactions and cartilage mineralization. It points out why these findings are of importance for future OA research and for the development of novel therapeutic strategies to treat OA.

Manipulation of pro-inflammatory cytokine production by the bacterial cell-penetrating effector protein YopM is independent of its interaction with host cell kinases RSK1 and PRK2.

The effector protein Yersinia outer protein M (YopM) of Yersinia enterocolitica has previously been identified and characterized as the first bacterial cell-penetrating protein (CPP). We found that recombinant YopM (rYopM) enters different eukaryotic cell types and downregulates the expression of several pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α]) after autonomous translocation. After infection with Y. enterocolitica or transfection of host cells, YopM interacts with isoforms of the two kinases ribosomal S6 protein kinase (RSK) and protein kinase C-related kinase (PRK). This interaction caused sustained RSK activation due to interference with dephosphorylation. Here we demonstrate by co-immunoprecipitation that rYopM interacts with RSK and PRK following cell-penetration. We show that autonomously translocated rYopM forms a trimeric complex with different RSK and PRK isoforms. Furthermore, we constructed a series of truncated versions of rYopM to map the domain required for the formation of the complex. The C-terminus of rYopM was identified to be essential for the interaction with RSK1, whereas any deletion in rYopM’s leucin-rich repeat domains abrogated PRK2 binding. Moreover, we found that the interaction of cell-penetrating rYopM with RSK led to enhanced autophosphorylation of this kinase at serine 380. Finally, we investigated whether downstream signaling of the trimeric rYopM-RSK/PRK complex modulates the expression of pro-inflammatory TNF-α. Here, we could exclude that interaction with RSK1 and PRK2 is essential for the anti-inflammatory effects of rYopM.

FHL2 regulates the resolution of tissue damage in chronic inflammatory arthritis

Objective We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease. Methods Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2−/− mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot. Results FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)β-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2−/− cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation. Conclusions These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.

Anti-inflammatory effects of YopM in rheumatoid arthritis fibroblast-like synoviocytes

Introduction Fibroblast-like synoviocytes (FLS) are key players in the pathogenesis of rheumatoid arthritis (RA). During the onset of the disease, they are transformed into an aggressive phenotype that sustains the joint-destructive inflammatory environment of RA. This transformation is most likely due to the combination of cytokines, growth factors and cellular interaction, which leads to the stable activated phenotype. YopM is the outer protein M of Yersinia species. It has cell penetrating properties and can translocate into the cell by its two N-terminal α-helices. Interestingly, YopM is capable of downregulating inflammatory response in host tissues infected with Yersinia. Based on these findings – self-delivering ability and anti-inflammatory properties – we investigated the effect of isolated recombinant YopM on RA-FLS. Material and methods We analysed the uptake of YopM into RA-FLS, using Cy3-coupled YopM and laser scanning microscopy. To analyse the effect of YopM on the inflammation in RA we used RA-FLS of four patients and investigated the production of MMPs and IL-6 using ELISA. Cells were preincubated with TNF-α for 24 h and then simultaneously incubated with TNF-α and YopM or treated with YopM during TNF-α stimulation without preincubation. To unravel the signalling pathways involved in the effects of YopM, we investigated the activation of MAP kinases (ERK, AKT and p-38) after stimulation with TNF-α, Il-1 and EGF and NFκB signalling by Western Blot analysis. Results In vitro, YopM penetrates the membrane of RA-FLS, is transported via vesicular structures into the cytosole and accumulates near the nucleus. When we investigated the effects of YopM on the expression of pro-inflammatory cytokines, we found a reduced expression of IL-6 after stimulation with TNF-α and incubation with YopM as compared to controls. The expression levels of MMP1 and MMP3 were significantly reduced after stimulation of RA-FLS with TNF-α and treatment with YopM in comparison with untreated controls. This reduction was also found after preincubation of the cells with TNF-α, indicating that YopM is also able to reduce an established inflammation. We investigated the mechanisms of this effect and found no differences in MAP kinases phosphorylation, but decreased phosphorylation of IκBα after stimulation with TNF-α, indicating a reduced activation of the NFκB pathway. Conclusions YopM penetrates the cell membrane of RA-FLS and reduces the inflammatory response of the cells. Therefore, it might be an effective regulator of inflammation in RA and could constitute a new therapeutic agent for the treatment of the disease.

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.

FRI0033 The inhibitory effects of the yersinia outer protein m (YOPM) on osteoclastogenesis and bone destruction

Background The severe bone destruction during rheumatoid arthritis (RA) is mainly mediated by osteoclasts and thus, reducing osteoclast number is a keystep in ameliorating the outcome of the disease. Yersinia strains are secreting a broad number of effector proteins. The yersinia outer protein M (YopM) has the ability to translocate into host cells by two N-terminal alpha-helices and subsequently down-regulates inflammatory response in infected host tissues. Objectives We investigated the effect of recombinant YopM on osteoclastogenesis in vitro and bone destruction in the hTNFtg mouse model in vivo. Methods After cloning recombinant YopM into pET-24 b (+) expression vector, it was expressed in Bl21 cells and purified. The uptake of YopM into bone marrow macrophages (BMMs) and osteoclasts was analysed using Cy3-coupled YopM and laser scanning microscopy. The effect of YopM on osteoclastogenesis in an in vitro osteoclast formation assay was studied. The effects of YopM on the activation of ERK, p-38, AKT and NFkappaB signalling was determined by Western Blot analysis. With respect to a potential in vivo application of YopM, we analysed the distribution of YopM-Cy5 injected in the hindpaws of hTNFtg mice by fluorescence reflection imaging (FRI). Finally, we treated hTNFtg mice with YopM and an inactive deletion mutant of YopM. Clinical parameters were recorded and histomorphometric analyses of joint destruction were performed in hind paw sections. Results In contrast to mature osteoclasts, YopM penetrated the cell membrane of BMMs in vitro and accumulated in the cells. The incubation of BMMs with YopM resulted in a strong reduction of osteoclast precursors and mature osteoclasts. We found that YopM reduced the activation of NF-kB by reducing the phosphorylation of IkB alpha. Phosphorylation of MAP kinases and AKT, however, was not altered by YopM. YopM-Cy5 injected into the hind paws was detectable by FRI during the monitored time of 48 houres. The treatment of hTNFtg mice with YopM showed a delayed onset of paw swelling and reduced bone destruction and inflammation in YopM treated hTNFtg mice in comparison to untreated hTNFtg mice. Conclusions These results suggest that YopM has the potential to reduce inflammation and bone destruction in vivo. Therefore, the underlying mechanisms and functional properties of YopM may serve as basis for the development of novel strategies for the treatment of RA. Disclosure of Interest None Declared

Heparan sulfate side chains are essential for the modulation of IL-1 signaLs by syndecan-4 in RA synovial fibroblasts

Background and objectives Activated synovial fibroblasts (SFs) are key players during RA disease progression. They exhibit a tumour-like invasive behavior, which is most likely induced by a continuous stimulation with ECM molecules, growth-factors and inflammatory cytokines, like IL-1. Syndecan-4 (sdc4), a transmembrane heparan sulfate proteoglycan, has been implicated recently in the modulation of cytokine signaling and has been found to be upregulated in RA-SF. Specifically, sdc4 is been shown to be involved in Erk signalling upon IL-1 stimulation in chondrocytes. However, the role of scd4 side chains and the underlying mechanisms of sdc4 signaling are largely unknown. Materials and methods In order investigate the influence of heperan sulfate side chains on sdc4 signaling, different sdc4 side chain mutants were designed using overlap PCR. The authors mutated the serine residues, which constitute the heparan sulphate attachment sites to alanines, thereby preventing side chain asssembly. Laser scanning fluorescence microscopy of transiently transfected Cos-7 cells was performed to analyse the membrane localisation of the generated mutants. The multimerisation pattern of the different sdc4 mutants was analysed using crosslinking upon IL-1 stimulation and subsequent Western blot analysis. Furthermore, fibroblasts lacking sdc4, the IL-1 receptor (IL1-RI) or both were stimulated with IL-1 to unravel the interaction of the IL-1RI and scd4 in IL-1 signaling. Erk1/2 was chosen as readout. Results The authors showed that all sdc4 side chain lacking mutants exhibited normal intracellular trafficking into the cell membrane. While wild type sdc4 showed normal multimerisation, the side chain mutants exhibited an impaired sdc4 complex formation, particularly with respect to tetramers. Furthermore, the authors found by our cross-linking experiments that sdc4 undergoes complex formation upon IL-1 stimulation. Analysing downstream signaling, sdc4-/- cells showed less IL-1 induced Erk1/2 phosphorylation, while IL-1 induced Erk1/2 phosphorylation was virtually abolished in cells without IL-1RI. The loss of both proteins completely prevented IL-1 dependent Erk1/2 phosphorylation. When comparing the effects of IL-1α and IL-1β on Erk1/2 phosphorylation, the authors found that IL-1β led to faster and more pronounced increase in phosphorylation levels compared to IL-1α. Conclusion In conclusion, heparan sulfate side chains are essential for multimerisation of sdc4 and IL-1 stimulation promotes sdc4 complex formation. Additionally, sdc4 seems to be involved in IL-1 signaling and has an additive or even regulatory effect on IL-1 signaling. Therefore, sdc4 complex formation in RA-SF might be an important step in signal transduction during RA disease progression.

Protective role of syndecan-4 in experimental colitis

Background Syndecan-4 (sdc4) is a transmembrane heparan sulfate proteoglycan that in addition to functioning as an adhesion a molecule can bind cytokines and impact on inflammation. Modulation of inflammatory signals by sdc4 may occur either through mere binding of cytokines, in which case sdc4 acts as decoy receptor or through initiation of sdc-dependent signalling following sdc4 complex formation. While arthritic cartilage damage is decreased in sdc4-deficient mice most likely due to reduced sdc4 signaling, osteopontin- mediated liver damage has been shown to be increased in these mice due to the lack of scd4 decoy receptor function. Based on this dual and in part opposing effects of sdc4, the aim of this study was to investigate if the loss of sdc4 changes the natural course of murine experimental colitis. Methods Experimental Colitis was induced in sdc4-/- mice and in C57BL/6 WT mice by DSS. The course of colitis was monitored by weight loss as well as assessment of colon length and blinded histological scoring of colonic changes at the end of the experiment. In addition, sdc4-/- and C57BL/6 WT mice were orally gavaged with 5×108 colony-forming units (CFU) of Citrobacter rodentium. Fecal excretion of C rodentium and changes of body weight were monitored. At day 21 postinfection, inflammatory changes of the colon were evaluated histologically. Results Beginning from day 5 after start of DSS-administration, sdc4-/- mice lost dramatically more body weight compared to WT animals (day 8: 24.8%±1.9 vs 9.2%±3.1; p=0.008). In accordance with the increased loss of body weight, the colon length of sdc4-/- mice was significantly shortened (63.3 mm±2.4 vs 74.8±2.3; p=0.01) and the histological damage according to the Dieleman-Score was markedly aggravated (16AU±3.7 vs 3.4AU±0.2; p=0.016). At day 19 postinfection, the fecal excretion of C rodentium in sdc4-/- was prolonged compared to WT animals (2.5×105±6.8×103 vs 9.6×103±6.5×102; p=0.01). Histological damage of colonic mucosa, reflected by lengthening of crypts, was increased in Sdc4-/- mice (14.3AU±1.3 vs 10.2AU±0.9; p=0.03). Conclusions The data suggest that like in inflammatory liver damage, sdc4 exerts protective effects in intestinal inflammation. Future studies are needed to explore the underlying mechanisms and to determine if these effects are due to a decoy receptor function of sdc4 or whether sdc4 complex formation and signalling is involved.

The Yersinia outer protein M inhibits osteoclastogenesis in vitro and reduces bone destruction in hTNFtg mice in vivo

Background One main phenomenon in rheumatoid arthritis (RA) is severe bone destruction, mainly mediated by osteoclasts in the arthritic joints. YopM is the outer protein M of Yersinia species. It has cell penetrating properties and can translocate into the cell by its two N-terminal α-helices. Interestingly, YopM is capable of downregulating inflammatory response in host tissues infected with Yersinia. Based on this dual function – the self-delivering ability and the anti-inflammatory properties – The authors investigated the effect of recombinant YopM on invitro osteoclast differentiation and bone destruction in the human tumour necrosis factor transgenic (hTNFtg) mouse model of RA. Methods Recombinant YopM was cloned into pET-24 b (+) expression vector, expressed in Bl21 cells and purified. The uptake of YopM into bone marrow macrophages (BMMs) and osteoclasts was analysed using Cy3-coupled YopM and laser scanning microscopy. The authors then studied the effect of YopM on osteoclastogenesis in an invitro osteoclast formation assay. To unravel the signalling pathways involved in the effects of YopM, the authors investigated the activation of extracellular-signal-regulated kinase, p-38, AKT and nuclear factor-κB (NF-κB) signalling by Western Blot analysis. With respect to a potential invivo application of YopM, the authors injected YopM-Cy5 in the hindpaws of hTNFtg mice and monitored its distribution by fluorescence reflection imaging (FRI). Finally, the authors treated hTNFtg mice with YopM and an inactive deletion mutant of YopM and recorded clinical parameters. In addition, histomorphometric analyses of joint destruction were performed in hindpaw sections. Results YopM penetrated the cell membrane of BMMs, but not osteoclasts invitro and accumulated in the cells. Most interestingly, incubation of BMMs with YopM resulted in a strong reduction of both, osteoclast precursors and mature osteoclasts. Studying the signaling pathways affected by YopM, the authors found that YopM reduced the TNF α induced activation of NF-κB by reducing the phosphorylation of IκB α. TNF α mediated phosphorylation of mitogen activated protein kinases and AKT, however, was not altered by YopM. As shown in FRI, YopM-Cy5 injected into the hindpaws was detectable during the monitored time of 48 h. YopM treated hTNFtg mice showed a delayed onset of paw swelling and histomorphometric analyses of the hindpaw sections revealed reduced bone destruction and inflammation in YopM treated hTNFtg mice in comparison to untreated hTNFtg mice. Conclusion These results suggest that YopM has the potential to reduce inflammation and bone destruction invivo. Therefore, the underlying mechanisms and functional properties of YopM may serve as basis for the development of novel strategies for the treatment of RA.

Antibodies against syndecan-4 reduce cartilage destruction and the progression after onset in RA-like disease of hTNF transgenic mice

Background Several studies have implicated syndecan-4 in cell-matrix-adhesion, cell-migration, differentiation and proliferation, but its specific function in inflammatory pathologies remains unclear. Here, the authors used the human tumour necrosis factor α (TNFα) transgenic (hTNFtg) mouse, to analyse the expression and function of syndecan-4 in chronic-destructive-arthritis and answer the question whether inhibition of syndecan-4 by specific antibodies may prevent cartilage-estruction and/or improve the phenotype after onset of the disease in this animal model of human rheumatoid arthritis. Methods Expression of syndecan-4 was investigated by immunohistochemistry in the hind-paws of 8-/12-week-old hTNFtg mice and wild type controls. In addition, synovial fibroblasts were isolated and analysed for syndecan-4-expression by reverse transcriptase PCR. For functional analyses, the authors generated blocking-antibodies against syndecan-4. To investigate their effect on TNFα mediated-destructive-arthritis, hTNFtg mice were injected with the antibodies or with IgG-control twice weekly for 4-week prophylacticly (age 4–8 weeks) and for disease treatment (age 8–12 weeks) into their hind paws. Evaluation of disease severity included clinical parameters (weight, arthritis-score, grip-strength) as well as histomorphometric analysis of toluidine-blue-stained paraffin sections. Results As seen in immunohistochemistry, there was a strong expression of syndecan-4 in the synovial membranes of hTNFtg mice, whereas only negligible staining for syndecan-4 was found in synovial tissues of wild type animals. In vitro, synovial fibroblasts isolated from hTNFtg mice showed more than 30-fold higher expression of syndecan-4 than wild type controls. Administration of the anti-syndecan-4 antibodies but not of IgG-control in pretreated 8-week-old hTNFtg mice clearly ameliorated the clinical signs of arthritis and protected the treated joints from cartilage damage. At histomorphometric analysis, this was evident for all analysed parameters but seen most prominently for area of distained cartilage. Significantly reduced cartilage damage in the anti-syndecan-4 treated hTNFtg mice was accompanied by a strinking reduction in the expression of matrix metalloproteinase 3 (MMP3). The treatment with anti-syndecan-4 in 12-week-old hTNFtg mice after onset of the arthritic disease prevented the mice from massive joint-destruction, and improved the severe cartilage-damage. The treatment also showed a clear reduction of inflammation in the paws compared to the untreated. Conclusions This findings indicate that syndecan-4 is involved prominently in fibroblast-mediated cartilage-damage in hTNFtg mice by regulating the expression of disease-relevant MMPs. More importantly, My data suggest that inhibition of syndecan-4 can not only prevent cartilage damage, but also reduces the severity after onset of the disease.