hTNFtg Mice Research Articles

Acod1-mediated inhibition of aerobic glycolysis suppresses osteoclast differentiation and attenuates bone erosion in arthritis

ObjectivesMetabolic changes are crucially involved in osteoclast development and may contribute to bone degradation in rheumatoid arthritis (RA). The enzyme aconitate decarboxylase 1 (Acod1) is known to link the cellular...

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts.

Extracellular sulfatase-2 (Sulf-2) influences receptor-ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

Single-cell multimodal analysis identifies common regulatory programs in synovial fibroblasts of rheumatoid arthritis patients and modeled TNF-driven arthritis

BackgroundSynovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined.MethodsWe applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas.ResultsBy comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1− SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks.ConclusionsWe revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.

Analysis of combined deficiency of interleukin-1 and -6 versus single deficiencies in TNF-mediated arthritis and systemic bone loss.

AimsInsufficient treatment response in rheumatoid arthritis (RA) patients requires novel treatment strategies to halt disease progression. The potential benefit of combination of cytokine-inhibitors in RA is still unclear and needs further investigation. To explore the impact of combined deficiency of two major cytokines, namely interleukin (IL)-1 and IL-6, in this study double deficient mice for IL-1αβ and IL-6 were investigated in different tumour necrosis factor (TNF)-driven inflammatory bone disorders, namely peripheral arthritis and sacroiliitis, as well as systemic bone loss.MethodsDisease course, histopathological features of arthritis, and micro-CT (µCT) bone analysis of local and systemic bone loss were assessed in 15-week-old IL1-/-IL6-/- hTNFtg in comparison to IL1-/- hTNFtg, IL6-/- hTNFtg, and hTNFtg mice. µCT bone analysis of single deficient and wild-type mice was also performed.ResultsCombined deficiency of IL-1/IL-6 markedly ameliorated TNF-mediated arthritis and bilateral sacroiliitis, but without additive benefits compared to single IL-1 deficiency. This finding confirms the important role of IL-1 and the marginal role of IL-6 in TNF-driven pathways of local joint damage, but questions the efficacy of potential combinatorial therapies of IL-1 and IL-6 in treatment of RA. In contrast, combined deficiency of IL-1/IL-6 led to an additive protective effect on TNF-driven systemic bone loss compared to single IL-1 and IL-6 deficiency. This finding clearly indicates a common contribution of both IL-1 and IL-6 in TNF-driven systemic bone loss, and points to a discrepancy of cytokine dependency in local and systemic TNF-driven mechanisms of inflammatory arthritis.ConclusionCombinatorial treatments in RA might provide different benefits to inflammatory local arthritis and systemic comorbidities.Cite this article: Bone Joint Res 2022;11(7):484–493.

Deletion of activin A in mesenchymal but not myeloid cells ameliorates disease severity in experimental arthritis

ObjectiveThe aim of this study was to assess the extent and the mechanism by which activin A contributes to progressive joint destruction in experimental arthritis and which activin A-expressing cell...

Efficacy of novel bispecific antibody targeting TNF-α/CXCL10 in the treatment of experimental arthritis

This study was aimed at generating and investigating the efficacy of a novel monoclonal bispecific antibody (BsAb) for the combined inhibition of tumor necrosis factor-α (TNF-α) and CXCL10 as a treatment option for rheumatoid arthritis (RA). A novel BsAb targeting TNF-α and CXCL10 was generated by conjugating a single-chain variable fragment (scFv) of the anti-CXCL10 monoclonal antibody to the Fc region of adalimumab (ADA). The effects of the BsAb on the inflammatory response in the in vitro and in vivo development of arthritis and joint destruction were evaluated in human TNF transgenic (hTNF-Tg) mice, and K/BxN serum transfer arthritis models. The BsAb inhibited CXCL10-mediated CD8+ T cell migration. The binding affinity of the BsAb to TNF-α was comparable to that of ADA and suppressed TNF-α induced cell death and inhibited TNF-α induced ICAM-1 and VCAM-1 in RA fibroblast-like synoviocytes (FLSs). The BsAb decreased the expression of TNFSF11 and the production of IL-6 in RA-FLS cells stimulated with TNF-α and CXCL10. Treatment with the BsAb attenuated the development of arthritis in hTNF-Tg mice and suppressed LPS-induced bone erosion. In the K/BxN serum transfer model, BsAb effectively attenuated ankle swelling, synovial inflammation, cartilage damage, and bone destruction, reducing the activation of osteoclasts. The additional neutralization of TNF-α and CXCL10 from treatment with the novel BsAb was more effective than TNF-α inhibition alone in the in vitro and in vivo models of RA. Thus, the BsAb, targeting both TNF-α and CXCL10, may provide a new therapeutic opportunity for RA patients who fail to respond to the blockade of a single cytokine.

Concomitant Treatment with Etanercept and Tacrolimus Synergistically Attenuates Arthritis Progression via Inhibition of Matrix Metalloproteinase-3 Production and Osteoclastogenesis in Human TNF-α Transgenic Mice.

In the present study, we investigated the effects and mechanisms of action of a combined treatment with etanercept, a soluble tumor necrosis factor receptor (p75) Fc fusion protein, and tacrolimus, a calcineurin inhibitor on the progression of arthritis in human tumor necrosis factor-α (TNF-α) transgenic (hTNF-Tg) mice. Single-drug treatments with etanercept and tacrolimus attenuated the clinical signs but not the radiographic changes associated with the development of arthritis in mice. On the contrary, combined treatment significantly suppressed the radiographic progression and also improved the clinical signs. The combined treatment exhibited synergistic effects of the two drugs in reducing the serum matrix metalloproteinase-3 level and the number of peripheral CD11bhigh osteoclast precursor cells. Moreover, tacrolimus inhibited the cytokine-induced osteoclast differentiation in synergy with etanercept in an in vitro assay. Interestingly, tacrolimus did not inhibit the production of antidrug antibodies (ADAs) against etanercept in the hTNF-Tg mice. This result implies that the synergistic effects of etanercept and tacrolimus are not due to secondary effects derived from the suppression of ADA production by tacrolimus but are due to their primary effects. These findings suggest that concomitant treatment with etanercept and tacrolimus may be one of preferable treatment options to control disease activities for patients with rheumatoid arthritis, especially for those with bone resorption.

Binding Immunoglobulin Protein (BIP) Inhibits TNF-α-Induced Osteoclast Differentiation and Systemic Bone Loss in an Erosive Arthritis Model.

ObjectiveThe association between inflammation and dysregulated bone remodeling is apparent in rheumatoid arthritis and is recapitulated in the human tumor necrosis factor transgenic (hTNFtg) mouse model. We investigated whether extracellular binding immunoglobulin protein (BiP) would protect the hTNFtg mouse from both inflammatory arthritis as well as extensive systemic bone loss and whether BiP had direct antiosteoclast properties in vitro.Methods hTNFtg mice received a single intraperitoneal administration of BiP at onset of arthritis. Clinical disease parameters were measured weekly. Bone analysis was performed by microcomputed tomography and histomorphometry. Mouse bone marrow macrophage and human peripheral blood monocyte precursors were used to study the direct effect of BiP on osteoclast differentiation and function in vitro. Monocyte and osteoclast signaling was analyzed by Western blotting, flow cytometry, and imaging flow cytometry.ResultsBiP‐treated mice showed reduced inflammation and cartilage destruction, and histomorphometric analysis revealed a decrease in osteoclast number with protection from systemic bone loss. Abrogation of osteoclast function was also observed in an ex vivo murine calvarial model. BiP inhibited differentiation of osteoclast precursors and prevented bone resorption by mature osteoclasts in vitro. BiP also induced downregulation of CD115/c‐Fms and Receptor Activator of NF‐κB (RANK) messenger RNA and protein, causing reduced phosphorylation of the p38 mitogen–activated protein kinases, extracellular signal–regulated kinases 1/2 and p38, with suppression of essential osteoclast transcription factors, c‐Fos and NFATc1. BiP directly inhibited TNF‐α– or Receptor Activator of NF–κB Ligand (RANKL)–induced NF‐κB nuclear translocation in THP‐1 monocytic cells and preosteoclasts by the canonical and noncanonical pathways.ConclusionBiP combines an anti‐inflammatory function with antiosteoclast activity, which establishes it as a potential novel therapeutic for inflammatory disorders associated with bone loss.

An integrative transcriptome analysis framework for drug efficacy and similarity reveals drug-specific signatures of anti-TNF treatment in a mouse model of inflammatory polyarthritis.

Anti-TNF agents have been in the first line of treatment of various inflammatory diseases such as Rheumatoid Arthritis and Crohn’s Disease, with a number of different biologics being currently in use. A detailed analysis of their effect at transcriptome level has nevertheless been lacking. We herein present a concise analysis of an extended transcriptomics profiling of four different anti-TNF biologics upon treatment of the established hTNFTg (Tg197) mouse model of spontaneous inflammatory polyarthritis. We implement a series of computational analyses that include clustering of differentially expressed genes, functional analysis and random forest classification. Taking advantage of our detailed sample structure, we devise metrics of treatment efficiency that take into account changes in gene expression compared to both the healthy and the diseased state. Our results suggest considerable variability in the capacity of different biologics to modulate gene expression that can be attributed to treatment-specific functional pathways and differential preferences to restore over- or under-expressed genes. Early intervention appears to manage inflammation in a more efficient way but is accompanied by increased effects on a number of genes that are seemingly unrelated to the disease. Administration at an early stage is also lacking in capacity to restore healthy expression levels of under-expressed genes. We record quantifiable differences among anti-TNF biologics in their efficiency to modulate over-expressed genes related to immune and inflammatory pathways. More importantly, we find a subset of the tested substances to have quantitative advantages in addressing deregulation of under-expressed genes involved in pathways related to known RA comorbidities. Our study shows the potential of transcriptomic analyses to identify comprehensive and distinct treatment-specific gene signatures combining disease-related and unrelated genes and proposes a generalized framework for the assessment of drug efficacy, the search of biosimilars and the evaluation of the efficacy of TNF small molecule inhibitors.

Multimodal [18 F]FDG PET/CT Is a Direct Readout for Inflammatory Bone Repair: A Longitudinal Study in TNFα Transgenic Mice.

ABSTRACTIn rheumatoid arthritis (RA), chronic joint inflammation leading to bone and cartilage damage is the major cause of functional impairment. Whereas reduction of synovitis and blockade of joint damage can be successfully achieved by disease modifying antirheumatic therapies, bone repair upon therapeutic interventions has only been rarely reported. The aim of this study was to use fluorodeoxyglucose ([18F]FDG) and [18F]fluoride µPET/CT imaging to monitor systemic inflammatory and destructive bone remodeling processes as well as potential bone repair in an established mouse model of chronic inflammatory, erosive polyarthritis. Therefore, human tumor necrosis factor transgenic (hTNFtg) mice were treated with infliximab, an anti‐TNF antibody, for 4 weeks. Before and after treatment period, mice received either [18F]FDG, for detecting inflammatory processes, or [18F]fluoride, for monitoring bone remodeling processes, for PET scans followed by CT scans. Standardized uptake values (SUVmean) were analyzed in various joints and histopathological signs of arthritis, joint damage, and repair were assessed. Longitudinal PET/CT scans revealed a significant decrease in [18F]FDG SUVs in affected joints demonstrating complete remission of inflammatory processes due to TNF blockade. In contrast, [18F]fluoride SUVs could not discriminate between different severities of bone damage in hTNFtg mice. Repeated in vivo CT images proved a structural reversal of preexisting bone erosions after anti‐TNF therapy. Accordingly, histological analysis showed complete resolution of synovial inflammation and healing of bone at sites of former bone erosion. We conclude that in vivo multimodal [18F]FDG µPET/CT imaging allows to quantify and monitor inflammation‐mediated bone damage and reveals not only reversal of synovitis but also bone repair upon TNF blockade in experimental arthritis. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Non-classical monocytes as mediators of tissue destruction in arthritis

ObjectivesBone destruction in rheumatoid arthritis is mediated by osteoclasts (OC), which are derived from precursor cells of the myeloid lineage. The role of the two monocyte subsets, classical monocytes (expressing...

The Role of Macrophages in the Response to TNF Inhibition in Experimental Arthritis.

The reduction of synovial tissue macrophages is a reliable biomarker for clinical improvement in patients with rheumatoid arthritis (RA), and macrophages are reduced in synovial tissue shortly after initiation of TNF inhibitors. The mechanism for this initial response is unclear. These studies were performed to identify the mechanisms responsible for the initial reduction of macrophages following TNF inhibition, positing that efflux to draining lymph nodes was involved. RA synovial tissue and synovial fluid macrophages expressed CCR7, which was increased in control macrophages following incubation with TNF-α. Human TNF transgenic (hTNF-Tg) mice were treated with infliximab after development of arthritis. Ankles were harvested and examined by histology, immunohistochemistry, quantitative RT-PCR, ELISA, and flow cytometry. hTNF-Tg mice treated with infliximab demonstrated significant clinical and histologic improvement 3 d after the initiation of therapy, at which time Ly6C+ macrophages were significantly reduced in the ankles. However, no evidence was identified to support a role of macrophage efflux to draining lymph nodes following treatment with infliximab. In contrast, apoptosis of Ly6C+ macrophages in the ankles and popliteal lymph nodes, decreased migration of monocytes into the ankles, and a reduction of CCL2 were identified following the initiation of infliximab. These observations demonstrate that Ly6C+ macrophage apoptosis and decreased ingress of circulating monocytes into the joint are responsible for the initial reduction of macrophages following infliximab treatment in hTNF-Tg mice.

Impaired periodontium and temporomandibular joints in tumour necrosis factor-α transgenic mice.

Tumour necrosis factor (TNF)-α is a pathological factor causing the characteristic symptoms of periodontal disease and rheumatoid arthritis. In this study, we describe the phenotypes of human TNF-α transgenic mice (hTNFtg) with respect to their periodontium and the temporomandibular joint (TMJ). Periodontal structures, TMJ and skull shape of hTNFtg mice and wild-type (WT) littermates were assessed by microcomputed tomography, automated segmentation, geometric morphometrics and histologic ground sections. We show that hTNFtg mice have an eroded lamina dura and reduced periodontal ligament space compared to (WT) littermates. Transgenic mice further exhibit severe destruction of the TMJ. Geometric morphometrics revealed that hTNFtg mice have a more laterally positioned TMJ with a concomitantly enlarged zygomatic process. Mandibular and maxillary teeth occluded properly. Our findings suggest that chronic inflammation in hTNFtg mice causes destructive changes of the periodontium and the TMJ.

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.

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction.

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.

Cartilage damage and bone erosion are more prominent determinants of functional impairment in longstanding experimental arthritis than synovial inflammation.

ABSTRACTChronic inflammation of articular joints causing bone and cartilage destruction consequently leads to functional impairment or loss of mobility in affected joints from individuals affected by rheumatoid arthritis (RA). Even successful treatment with complete resolution of synovial inflammatory processes does not lead to full reversal of joint functionality, pointing to the crucial contribution of irreversibly damaged structural components, such as bone and cartilage, to restricted joint mobility. In this context, we investigated the impact of the distinct components, including synovial inflammation, bone erosion or cartilage damage, as well as the effect of blocking tumor necrosis factor (TNF) on functional impairment in human-TNF transgenic (hTNFtg) mice, a chronic inflammatory erosive animal model of RA. We determined CatWalk-assisted gait profiles as objective quantitative measurements of functional impairment. We first determined body-weight-independent gait parameters, including maximum intensity, print length, print width and print area in wild-type mice. We observed early changes in those gait parameters in hTNFtg mice at week 5 – the first clinical signs of arthritis. Moreover, we found further gait changes during chronic disease development, indicating progressive functional impairment in hTNFtg mice. By investigating the association of gait parameters with inflammation-mediated joint pathologies at different time points of the disease course, we found a relationship between gait parameters and the extent of cartilage damage and bone erosions, but not with the extent of synovitis in this chronic model. Next, we observed a significant improvement of functional impairment upon blocking TNF, even at progressed stages of disease. However, blocking TNF did not restore full functionality owing to remaining subclinical inflammation and structural microdamage. In conclusion, CatWalk gait analysis provides a useful tool for quantitative assessment of functional impairment in inflammatory destructive arthritis. Our findings indicate that cartilage damage and bone erosion, but not synovial inflammation, are the most important determinants for progressive functional impairment in this chronic erosive arthritis model.

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.

Loss of SH3 domain-binding protein 2 function suppresses bone destruction in tumor necrosis factor-driven and collagen-induced arthritis in mice.

SH3 domain-binding protein 2 (SH3BP2) is a signaling adapter protein that regulates the immune and skeletal systems. The present study was undertaken to investigate the role of SH3BP2 in arthritis using 2 experimental mouse models, i.e., human tumor necrosis factor α-transgenic (hTNF-Tg) mice and mice with collagen-induced arthritis (CIA). First, Sh3bp2(-/-) and wild-type (Sh3bp2(+/+) ) mice were crossed with hTNF-Tg mice. Inflammation and bone loss were examined by clinical inspection and histologic and micro-computed tomography analysis, and osteoclastogenesis was evaluated using primary bone marrow-derived macrophage colony-stimulating factor-dependent macrophages (BMMs). Second, CIA was induced in Sh3bp2(-/-) and Sh3bp2(+/+) mice, and the incidence and severity of arthritis were evaluated. Anti-mouse type II collagen (CII) antibody levels were measured by enzyme-linked immunosorbent assay, and lymph node cell responses to CII were determined. SH3BP2 deficiency did not alter the severity of joint swelling but did suppress bone erosion in the hTNF-Tg mouse model. Bone loss at the talus and tibia was prevented in Sh3bp2(-/-) /hTNF-Tg mice compared to Sh3bp2(+/+) /hTNF-Tg mice. RANKL- and TNFα-induced osteoclastogenesis was suppressed in Sh3bp2(-/-) mouse BMM cultures. NF-ATc1 nuclear localization in response to TNFα was decreased in Sh3bp2(-/-) mouse BMMs compared to Sh3bp2(+/+) mouse BMMs. In the CIA model, SH3BP2 deficiency suppressed the incidence of arthritis and this was associated with decreased anti-CII antibody production, while antigen-specific T cell responses in lymph nodes were not significantly different between Sh3bp2(+/+) and Sh3bp2(-/-) mice. SH3BP2 deficiency prevents loss of bone via impaired osteoclastogenesis in the hTNF-Tg mouse model and suppresses the induction of arthritis via decreased autoantibody production in the CIA model. Therefore, SH3BP2 could potentially be a therapeutic target in rheumatoid arthritis.

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.

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.