Denise Beckmann

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.

Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells

Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.

Structural cartilage damage attracts circulating rheumatoid arthritis synovial fibroblasts into affected joints

BackgroundRheumatoid arthritis synovial fibroblasts (RASFs) are known to travel via the bloodstream from sites of cartilage destruction to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces. In this study, we examined the role of interleukin (IL)-1-induced cartilage changes and their chemotactic effect on RASF transmigratory capacity.MethodsTo investigate synovial fibroblast (SF) transmigration through endothelial layers, we used a modified Boyden chamber with an endothelioma cell layer (bEnd.5) as a barrier and IL-1-treated murine cartilage explants as a chemotactic stimulus for SFs from human tumor necrosis factor–transgenic (hTNFtg) mice. We injected recombinant IL-1 or collagenase into knee joints of wild-type mice, followed by tail vein injection of fluorescence-labeled hTNFtg SFs. The distribution and intensity of transmigrating hTNFtg SFs were measured by fluorescence reflectance imaging with X-ray coregistration. Toluidine blue staining was performed to evaluate the amount of cartilage destruction.ResultsHistomorphometric analyses and in vivo imaging revealed a high degree of cartilage proteoglycan loss after intra-articular IL-1 and collagenase injection, accompanied by an enhanced in vivo extravasation of hTNFtg SFs into the respective knee joints, suggesting that structural cartilage damage contributes significantly to the attraction of hTNFtg SFs into these joints. In vitro results showed that degraded cartilage was directly responsible for the enhanced transmigratory capacity because stimulation with IL-1-treated cartilage, but not with IL-1 or cartilage alone, was required to increase hTNFtg SF migration.ConclusionsThe present data indicate that structural cartilage damage facilitates the migration of arthritic SF into affected joints. The prevention of early inflammatory cartilage damage may therefore help prevent the progression of rheumatoid arthritis and its spread to previously unaffected joints.

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis

IntroductionInflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA.MethodsExpression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses.ResultsRA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro.ConclusionsThese data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

02.36 Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis

Background In rheumatoid arthritis (RA) the attachment of synovial fibroblasts to articular cartilage is an important prerequisite in the process of cartilage degradation. The actin-associated protein Lasp-1 is involved in processes of actin organisation and polymerization and focal adhesion turnover, respectively. Therefore, we investigated its role in regulating cell-cell contacts and ECM interactions of synovial fibroblasts in RA. Methods Lasp-1 expression was analysed in human RA tissue and in arthritic mouse models such as the hTNFtg and G6PI mouse model by using WB and immunohistochemistry. Lasp-1 expression and its subcellular distribution was investigated in isolated synovial fibroblasts by immunofluorescence in all genotypes. Furthermore, the formation of cell-cell and -ECM contacts was investigated in an electrical cell/substrate impedance sensing assay (ECIS) as well as by immunofluorescence. Additionally, we performed an in vitro three-dimensional organ culture system to identify genotype-specific differences of cellular distribution and formation of the artificial synovial lining layer. Results An increased Lasp-1 expression was observed in synovial tissue from RA patients as well as in RASF. Isolated SF from hTNFtg mice also produced higher levels of Lasp-1 compared to wild type (wt) SF. Interestingly, this was completely reproducible in the G6PI model. Lasp-1 deficient mice that had been crossed into the hTNFtg background exhibited less cartilage degradation (−4% vs. hTNFtg) and less attachment of synovial tissue to the cartilage (−30% vs. hTNFtg) compared to hTNFtg mice. Functional analyses indicated that Lasp-1-/- hTNFtg SF form closer (+20% vs hTNFtg SF) and more stable cell-cell contacts in comparison to hTNFtg SF. Furthermore, we detected Lasp-1 to be located at structures of adherens junction complexes. The loss of Lasp-1 led to alterations of these structures. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusions Lasp-1 regulates the migratory behaviour of synovial fibroblasts and their invasion into cartilage matrix in rheumatoid arthritis by controlling the dynamics of cell-cell contacts.

FRI0018 Targeted inhibition of janus kinases abates IFN-GAMMA-INDUCED invasive behavior of fibroblast-like synoviocytes

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA pools were used to suppress Janus Kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA. Disclosure of Interest None declared

A7.05 Baricitinib abrogates IFNγ-induced focal adhesion kinase (FAK) activation in fibroblast-like synoviocytes

While evidence implicates both the adaptive and innate immune system in rheumatoid arthritis (RA) pathogenesis, accumulating data indicate that the synovial tissue itself actively participates in the destructive inflammatory process. Specifically, resident fibroblast-like synoviocytes (FLS), together with macrophages, re-organise to form an aggressive cell mass, called pannus, which destroys the articular cartilage and the subchondral bone. The exact molecular mechanisms of synovial pannus formation, FLS expansion and invasion into adjacent tissues are not yet known. Our data strongly suggest that the T-cell derived cytokine IFNγ is involved in FLS-mediated joint destruction. Migration and invasion assays revealed increased migratory activity for IFNγ-stimulated FLS, when compared to unstimulated FLS. Further, biochemical studies showed that IFNγ promotes the migratory and invasive activity of FLS via Janus kinase 2 (JAK2) and the focal adhesion kinase (FAK), a kinase known to integrate focal adhesion turnover and thus, regulates cell migration. In detail, IFNγ stimulation of FLS distinctly resulted in the phosphorylation of FAK-Y925, a phospho-site that has recently been demonstrated to be required for FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated the IFNγ-induced activation of FAK. Correspondingly, baricitinib, a JAK inhibitor that is currently successfully probed in RA clinical trials, abrogated IFNγ-stimulated activation of FAK. In conclusion, our studies contribute insight into the synovial response to IFNγ and reveal JAK2 and FAK as potential targets for synoviocyte-mediated joint destruction in arthritis, especially in RA.

A10.15 LASP-1 modifies ECM-synovial fibroblast interactions in a mouse model of ra

Background and objectives The LIM-and-SH3-domain-protein-1 (Lasp-1) is an actin-associated protein and is localised at focal adhesion sites where it is involved in organisation of actin polymerization and focal adhesion turnover prozesses. We investigated its role in regulating synovial fibroblast (SF) interaction with components of the extracellular matrix (ECM) and in establishing cell-cell contacts during RA. Materials and methods Lasp-1 expression was analysed in tissue from RA patients and in the hind paws of arthritic hTNFtg mice by Western blot, immunofluorescence and immunohistochemistry. Furthermore, Lasp-1-/- mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Migration characteristics of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were analysed in a modified scratch assay and by live cell imaging. Cell-matrix interactions and cell-cell contacts of isolated SF from all different genotypes were investigated using fibronectin coating in an electrical cell/substrate impedance sensing assay (ECIS). Additionally, we used an in vitro three-dimensional organ culture system for functional analyes. Results Lasp-1 expression levels were increased in human RA tissue and hTNFtg mice in comparison to healthy controls. Evaluation of Lasp-1-/-/hTNFtg mice revealed milder arthritis score compared to hTNFtg mice,which was confirmed by immunohistochemistry. Results of the scratch assays demonstrated a significantly reduced migration rate of Lasp-1-/- SF (-43,7% vs wt SF) and Lasp-1-/-/hTNFtg SF (-69,11% vs hTNFtg SF). Furthermore, live cell imaging studies demonstrated striking differences in the migration velocity and in migration edge formation of Lasp-1-/-/hTNFtg SF compared to hTNFtg SF. ECIS analysis demonstrated an increased cell-cell contact formation in Lasp1-/- compared to wt SF (+22% versus wt SF) and prolonged cell-cell interaction remodelling of Lasp-1-/-/hTNFtg SF in comparison to hTNFtg SF. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusion Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis by controlling the dynamics of cell-matrix and cell-cell contacts.

A6.7 The junctional adhesion molecule JAM-3 regulates the motility of fibroblast-like synoviocytes

Background and objectives Rheumatoid arthritis (RA) is a disease of a continuous activation and expansion of the synovium involving migration and dissemination of fibroblast-like synoviocytes (FLS). Several lines of evidence indicate that adhesion molecules play a crucial role in this process. We have focused on the junctional adhesion molecule 3 (JAM-3), known to be involved in adhesion, polarity and migration of other cells. So far it has been demonstrated that blocking of JAM-3 in a serum transfer model of RA ameliorates the disease. However, the question of whether JAM-3 is involved in fibroblast motility or primarily regulates the influx of inflammatory cells remains unknown. Therefore, our objective was to clarify the involvement of JAM-3 in the establishment of cell-cell and cell-matrix contacts as well as in the migration of FLS. Materials and methods As an arthritis model we use human TNFalpha transgenic (hTNFtg) mice that due to high levels of human TNFalpha develop a RA-like disease. FLS form these animals as well as form JAM-3 floxed mice CreERT2 (tamoxifen inducible cre line) and control mice were isolated by collagenase digestion of the hind paws. Cells were subjected to electrical cell-substrate impedance sensing (ECIS) surface staining (FACS), immunofluorescence, in vitro cell migration assays and standard molecular biology methods. Results JAM-3 was found on both wild type and hTNFtg synoviocytes. Blocking or silencing of JAM-3 significantly increased cell spreading in an modified scratch assay but decreased migration in an in vitro transmigration assay, indicating that JAM-3 is involved in the establishment of cell-cell contacts between FLS. In line with this notion ECIS experiments revealed altered adhesion properties of these cells. Explaining these findings, we found that JAM-3 co-localises with the tight junction molecule zo-1 at cell contact sites, which are connected to actin cell filaments. The ultrastructure of these structures and an influence of Jam-3 on the cytoskeleton dynamics is under investigation. Conclusions JAM-3 modulates migration of FLS in vitro, which may contribute to the beneficial effects of JAM-3 inhibition as seen in the serum transfer model of RA in vivo. We suggest that Jam-3 is an adhesion molecule, responsible for controlling motility by organising the cytoskeleton in FLS.