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Background The transmembrane-TNF transgenic mouse, TgA86, has been shown to develop spontaneously peripheral arthritis with signs of axial involvement. To assess similarity to human spondyloarthritis, we performed detailed characterization of the axial, peripheral, and comorbid pathologies of this model. Methods TgA86 bone pathologies were assessed at different ages using CT imaging of the spine, tail vertebrae, and hind limbs and characterized in detail by histopathological and immunohistochemical analysis. Cardiac function was examined by echocardiography and electrocardiography and bone structural parameters by μCT analysis. The response of TgA86 mice to either early or late anti-TNF treatment was evaluated clinically, histopathologically, and by μCT analysis. Results TgA86 mice developed with 100% penetrance spontaneous axial and peripheral pathology which progressed with time and manifested as reduced body weight and body length, kyphosis, tail bendings, as well as swollen and distorted hind joints. Whole-body CT analysis at advanced ages revealed bone erosions of sacral and caudal vertebrae as well as of sacroiliac joints and hind limbs and, also, new ectopic bone formation and eventually vertebral fusion. The pathology of these mice highly resembled that of SpA patients, as it evolved through an early inflammatory phase, evident as enthesitis and synovitis in the affected joints, characterized by mesenchymal cell accumulation, and neutrophilic infiltration. Subsequently, regression of inflammation was accompanied by ectopic bone formation, leading to ankylosis. In addition, both systemic bone loss and comorbid heart valve pathology were evident. Importantly, early anti-TNF treatment, similar to clinical treatment protocols, significantly reduced the inflammatory phase of both the axial and peripheral pathology of TgA86 mice. Conclusions The TgA86 mice develop a spontaneous peripheral and axial biphasic pathology accompanied by comorbid heart valvular dysfunction and osteoporosis, overall reproducing the progression of pathognomonic features of human spondyloarthritis. Therefore, the TgA86 mouse represents a valuable model for deciphering the role of transmembrane TNF in the pathogenic mechanisms of spondyloarthritis and for assessing the efficacy of human therapeutics targeting different phases of the disease.

Background New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. Methods We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. Results Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. Conclusion Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment.

Synovial fibroblasts (SFs) are key pathogenic drivers in rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models, and TNF blockade proved efficacious for a high percentage of patients with RA albeit coinducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF-transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely amisulpride, which reduced SFs' inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that amisulpride function was neither through its known targets dopamine receptors D2 and D3 and serotonin receptor 7 nor through TNF-TNF receptor I binding inhibition. Through a click chemistry approach, potentially novel targets of amisulpride were identified, which were further validated to repress hTNFtg SFs' inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, amisulpride could prove beneficial to patients experiencing RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its antidepressive activity, serving further as a \"lead\" compound for the development of novel therapeutics against fibroblast activation.

ObjectivesPatients with rheumatoid arthritis and spondyloarthritisshow higher mortality rates, mainly caused by cardiac comorbidities. The TghuTNF (Tg197) arthritis model develops tumour necrosis factor (TNF)-driven and mesenchymalsynovial fibroblast (SF)-dependent polyarthritis. Here, we investigate whether this model develops, similarly to human patients, comorbid heart pathology and explore cellular and molecular mechanisms linking arthritis to cardiac comorbidities.MethodsHistopathological analysis and echocardiographic evaluation of cardiac function were performed in the Tg197 model. Valve interstitial cells (VICs) were targeted by mice carrying the ColVI-Cretransgene. Tg197 ColVI-Cre Tnfr1 fl/fl and Tg197 ColVI-Cre Tnfr1 cneo/cneo mutant mice were used to explore the role of mesenchymal TNF signalling in the development of heart valve disease. Pathogenic VICs and SFs were further analysed by comparative RNA-sequencing analysis.ResultsTg197 mice develop left-sided heart valve disease, characterised by valvular fibrosis with minimal signs of inflammation. Thickened valve areas consist almost entirely of hyperproliferative ColVI-expressing mesenchymal VICs. Development of pathology results in valve stenosis and left ventricular dysfunction, accompanied by arrhythmic episodes and, occasionally, valvular regurgitation. TNF dependency of the pathology was indicated by disease modulation following pharmacological inhibition or mesenchymal-specific genetic ablation or activation of TNF/TNFR1 signalling. Tg197-derived VICs exhibited an activated phenotype ex vivo, reminiscent of the activated pathogenic phenotype of Tg197-derived SFs. Significant functional similarities between SFs and VICs were revealed by RNA-seq analysis, demonstrating common cellular mechanisms underlying TNF-mediated arthritides and cardiac comorbidities.ConclusionsComorbidheart valve disease and chronic polyarthritis are efficiently modelled in the Tg197 arthritis model and share common TNF/TNFR1-mediated, mesenchymal cell-specific aetiopathogenic mechanisms.

Targeting receptor activator of nuclear factor-κB ligand (RANKL) with the monoclonal antibody Denosumab decreases osteoclast-mediated bone resorption and is approved for the treatment of postmenopausal osteoporosis. Since RANKL is also implicated in mammary gland homeostasis and breast tumorigenesis, Denosumab is being currently pursued as a candidate for drug repurposing in oncology, including breast cancer, while its efficacy remains controversial. In this study, by developing a humanized transgenic mouse model of human RANKL overexpression, we demonstrated that RANKL mediated hormone-induced mammary carcinogenesis, while its prophylactic inhibition by Denosumab prevented tumorigenesis. Our humanized transgenic mice provide a unique genetic tool for investigating the involvement of human RANKL in breast cancer pathogenesis and can serve as a preclinical platform for anticancer therapies. Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.

BackgroundSpondyloarthritis (SpA) describes a group of diseases characterised by a paradoxical simultaneous bone destruction and formation manifested as axial and peripheral pathologies. TNF is a key pathogenic factor with the strongest evidence of its leading role arising from the therapeutic effect of its inhibition in SpA patients. The TgA86 is a mouse model that overexpresses TNF and exhibits signs of spontaneous SpA. Our objective was to characterise the TgA86 pathology, identify its similarity to human disease and ultimately explore its potential to be used as the basis of a preclinical platform for the evaluation of SpA therapeutics and diagnostics.Materials and methodsTgA86 transgenic mice express deregulated mouse transmembrane TNF and develop with 100% incidence chronic inflammatory arthritis and axial pathology clinically manifested by a characteristic tail bending. We used clinical observations, histopathological analysis, x-ray and mu CT radiographic read-outs to establish a standardised pipeline for the evaluation of the TgA86 pathology.ResultsHistopathological, x-ray and mu CT analysis of the vertebral column revealed the presence of pathological findings closely resembling those observed in human SpA patients. More specifically, disease characteristics included soft tissue swelling, bone erosion, increased bone formation and replacement of the vertebra bar-bell shape with a rectangular shape. Moreover, TgA86 mice exhibited a concomitant development of aortic valve disease, an extraarticular co-morbidity observed in a significant percentage of SpA patients. This data support the similarity of the TgA86 pathology to that of the human disease. We further used a standardised set of the above-mentioned read-outs to validate the response of this model to human therapeutics. TgA86 animals treated with the commercially available human therapeutic Etanercept, exhibited significant amelioration of both peripheral and axial pathologies in a reproducible and dose-dependent manner thus establishing the translational value of this model in the evaluation of human therapeutics.ConclusionBiomedcode has standardised and validated a novel TgA86-based highly reproducible and sensitive efficacy preclinical platform for the evaluation of therapeutics targeting SpA. This platform can be further used to validate and evaluate the therapeutic effect of the blockade of additional pathological pathways associated with SpA including IL-17, Wnt signalling and others.

BackgroundChronic inflammatory diseases, such as Rheumatoid arthritis (RA) and Inflammatory Bowel Disease (IBD) are often co-manifested with additional comorbidities. Studies performed in the established TNFΔARE/+ mouse model of TNF dependent RA and Crohn’s-like IBD, have demonstrated that stromal cells in the joint and the gut are primary and sufficient targets of TNF leading to the development of these two pathologies. Valvular heart disease (VHD) is one of the most common comorbid conditions in RA patients, that frequently leads to heart failure and premature death. We have recently observed that VHD is also displayed in the TNFΔARE/+ mouse model and our current aim has been to identify whether TNF signalling in stromal cells of the heart play also a role in regulating the VHD pathology similarly to what has been shown in the joint-gut axis.To this end we have used ColVI-Cre mice (specific for mesenchymal cells) and dissected genetically the role of TNFR2 in the stroma of the TNFΔARE/+ mouse model by assessing its function in RA and the VHD comorbidity.Materials and methodsMesenchymal cells specific deletion of TNFR2 was achieved by crossing TNFΔARE/+ to ColVICre/p75f/f. We further used histopathological analysis and cell isolation, characterisation, signalling analysis and RNAseq expression profiling analysis to decipher the pathologic mechanisms.ResultsInterestingly, we found that stroma-specific TNFR2 deletion leads to amelioration of RA and VHD pathologies. These results point to a common role of stroma-expressing TNFR2 in RA and VHD. Furthermore, we identified that isolated stromal cells from joint (SFs) and aortic heart valve (VICs) lacking TNFR2, failed to acquire pathogenic ‘activated phenotypes’ and display increased expression of the anti-inflammatory IL1RA which correlated with increased AKT signalling. Comparative RNAseq experiments performed in SFs and VICs lacking TNFR2 showed that the majority of deregulated pathways in TNFΔARE/+ are reversed to normal levels.ConclusionTaken together, our data demonstrate that the RA and VHD comorbidities developing in the TNFΔARE/+ mouse model, share common cellular mechanisms converging at the TNF/TNFR2 dependent activation of their mesenchymal stroma.

BackgroundSpondyloarthritis (SpA) describes a group of diseases characterised by a paradoxical simultaneous bone destruction and formation manifested as axial and peripheral pathologies. TNF is a key pathogenic factor with the strongest evidence of its leading role arising from the therapeutic effect of its inhibition in SpA patients. The TgA86 is a mouse model that overexpresses TNF and exhibits signs of spontaneous SpA. Our objective was to characterise the TgA86 pathology, identify its similarity to human disease and ultimately explore its potential to be used as the basis of a preclinical platform for the evaluation of SpA therapeutics and diagnostics.Materials and methodsTgA86 transgenic mice express deregulated mouse transmembrane TNF and develop with 100% incidence chronic inflammatory arthritis and axial pathology clinically manifested by a characteristic tail bending. We used clinical observations, histopathological analysis, x-ray and μCT radiographic read-outs to establish a standardised pipeline for the evaluation of the TgA86 pathology.ResultsHistopathological, x-ray and μCT analysis of the vertebral column revealed the presence of pathological findings closely resembling those observed in human SpA patients. More specifically, disease characteristics included soft tissue swelling, bone erosion, increased bone formation and replacement of the vertebra bar-bell shape with a rectangular shape. Moreover, TgA86 mice exhibited a concomitant development of aortic valve disease, an extraarticular co-morbidity observed in a significant percentage of SpA patients. This data support the similarity of the TgA86 pathology to that of the human disease. We further used a standardised set of the above-mentioned read-outs to validate the response of this model to human therapeutics. TgA86 animals treated with the commercially available human therapeutic Etanercept, exhibited significant amelioration of both peripheral and axial pathologies in a reproducible and dose-dependent manner thus establishing the translational value of this model in the evaluation of human therapeutics.ConclusionBiomedcode has standardised and validated a novel TgA86-based highly reproducible and sensitive efficacy preclinical platform for the evaluation of therapeutics targeting SpA. This platform can be further used to validate and evaluate the therapeutic effect of the blockade of additional pathological pathways associated with SpA including IL-17, Wnt signalling and others.

BackgroundChronic inflammatory diseases, such as Rheumatoid arthritis (RA) and Inflammatory Bowel Disease (IBD) are often co-manifested with additional comorbidities. Studies performed in the established TNF Delta ARE/+ mouse model of TNF dependent RA and Crohn's-like IBD, have demonstrated that stromal cells in the joint and the gut are primary and sufficient targets of TNF leading to the development of these two pathologies. Valvular heart disease (VHD) is one of the most common comorbid conditions in RA patients, that frequently leads to heart failure and premature death. We have recently observed that VHD is also displayed in the TNF Delta ARE/+ mouse model and our current aim has been to identify whether TNF signalling in stromal cells of the heart play also a role in regulating the VHD pathology similarly to what has been shown in the joint-gut axis.To this end we have used ColVI-Cre mice (specific for mesenchymal cells) and dissected genetically the role of TNFR2 in the stroma of the TNF Delta ARE/+ mouse model by assessing its function in RA and the VHD comorbidity.Materials and methodsMesenchymal cells specific deletion of TNFR2 was achieved by crossing TNF Delta ARE/+ to ColVICre/p75f/f. We further used histopathological analysis and cell isolation, characterisation, signalling analysis and RNAseq expression profiling analysis to decipher the pathologic mechanisms.ResultsInterestingly, we found that stroma-specific TNFR2 deletion leads to amelioration of RA and VHD pathologies. These results point to a common role of stroma-expressing TNFR2 in RA and VHD. Furthermore, we identified that isolated stromal cells from joint (SFs) and aortic heart valve (VICs) lacking TNFR2, failed to acquire pathogenic 'activated phenotypes' and display increased expression of the anti-inflammatory IL1RA which correlated with increased AKT signalling. Comparative RNAseq experiments performed in SFs and VICs lacking TNFR2 showed that the majority of deregulated pathways in TNF Delta ARE/+ are reversed to normal levels.ConclusionTaken together, our data demonstrate that the RA and VHD comorbidities developing in the TNF Delta ARE/+ mouse model, share common cellular mechanisms converging at the TNF/TNFR2 dependent activation of their mesenchymal stroma.

Background Chronic inflammatory diseases, such as Rheumatoid arthritis (RA) and Inflammatory Bowel Disease (IBD) are often co-manifested with additional comorbidities. Studies performed in the established TNFΔARE/+ mouse model of TNF dependent RA and Crohn's-like IBD, have demonstrated that stromal cells in the joint and the gut are primary and sufficient targets of TNF leading to the development of these two pathologies. Valvular heart disease (VHD) is one of the most common comorbid conditions in RA patients, that frequently leads to heart failure and premature death. We have recently observed that VHD is also displayed in the TNFΔARE/+ mouse model and our current aim has been to identify whether TNF signalling in stromal cells of the heart play also a role in regulating the VHD pathology similarly to what has been shown in the joint-gut axis. To this end we have used ColVI-Cre mice (specific for mesenchymal cells) and dissected genetically the role of TNFR2 in the stroma of the TNFΔARE/+ mouse model by assessing its function in RA and the VHD comorbidity. Materials and methods Mesenchymal cells specific deletion of TNFR2 was achieved by crossing TNFΔARE/+ to ColVICre/p75f/f . We further used histopathological analysis and cell isolation, characterisation, signalling analysis and RNAseq expression profiling analysis to decipher the pathologic mechanisms. Results Interestingly, we found that stroma-specific TNFR2 deletion leads to amelioration of RA and VHD pathologies. These results point to a common role of stroma-expressing TNFR2 in RA and VHD. Furthermore, we identified that isolated stromal cells from joint (SFs) and aortic heart valve (VICs) lacking TNFR2, failed to acquire pathogenic 'activated phenotypes' and display increased expression of the anti-inflammatory IL1RA which correlated with increased AKT signalling. Comparative RNAseq experiments performed in SFs and VICs lacking TNFR2 showed that the majority of deregulated pathways in TNFΔARE/+ are reversed to normal levels. Conclusion Taken together, our data demonstrate that the RA and VHD comorbidities developing in the TNFΔARE/+ mouse model, share common cellular mechanisms converging at the TNF/TNFR2 dependent activation of their mesenchymal stroma.