Chronic 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.

Introduction Bile acids play an important role in cholesterol metabolism and act as intestinal detergents for digestion and absorption of fats and fat-soluble vitamins. Disruption of bile flow causes cholestatic liver diseases. Derivatives of cholic acid (CA) such as nor-ursodeoxycholic acid (norUDCA) are promising therapeutic agents in the treatment of cholangiopathies. Previous studies also demonstrated anti-inflammatory and anti-fibrotic properties of norUDCA in experimental sclerosing cholestasis. Objective To investigate the anti-inflammatory potential of CA and its derivatives ursodeoxycholic acid (UDCA) and nor-UDCA in in Collagen-induced arthritis (CIA), an animal model for inflammatory, erosive arthritis. Methods Mice were prophylactically treated with CA, UDCA or nor-UDCA enriched diet pellets (5 mg/kg diet) or standard diet pellets (Placebo) ad libitum starting 1 week before the first immunisation with collagen. Animals were weekly assessed for clinical signs of arthritis, body weight and food consumption during the experimental period. After 10 weeks of treatment hind paws, liver, sera and lymph nodes were isolated for further analysis. Sera were investigated for anti-collagen antibodies, cytokine responses and liver parameters such as alkaline phosphatase (AP) and alanine transaminase (ALT). Paraffin-sections of hind paws were examined for histoptahological changes in synovial inflammation, subchondral bone erosion, cartilage damage and osteophyte formation. Cell populations within synovial pannus were identified by immunhistochemical stainings and were determined using HistoQuest software (from TissueGnostics). Results Uptake of CA, UDCA and norUDCA was confirmed by serum analysis. Prophylactic treatment of CIA mice with UDCA and norUDCA could not significantly prevent disease incidence. In contrast, treatment with CA led to a marked increase in disease incidence and severity compared to Placebo treated animals. Whereas UDCA and norUDCA showed a similar course of clinical signs of arthritis. Histological analysis of hind paws revealed an increase in synovial inflammation, bone erosion and cartilage damage in the CA treated animals compared to the Placebo group. No changes in joint pathology were observed in the UDCA or norUDCA treated group. Despite differences in absolute numbers of infiltrating cells, immunhistochemical staining for cell specific markers demonstrated no markedly changes in the distribution of neutrophils, macrophages, T- and B-lymphocytes within inflammatory synovial tissue. Similar amounts of total anti-collagen IgG and its subtypes IgG2 and IgG2c were found in all treatment groups. Moreover, FACS analysis of lymph node cells revealed similar amounts of T-cells, B-cells, dendritic cells and macrophages. In contrast, serum analyses revealed a trend toward increased IL-6 levels suggesting that CA exacerbates IL-6 levels driving disease onset and severity in CIA model. Conclusions Bile acid derivatives UDCA and norUDCA could not modulate collagen-induced arthritis. In contrast, chol...

a reduction or increase in the activation of canonical WNT signalling pathway, respectively. Fluctuating levels of Frzb did not influence the Wnt/CamKII signalling pathway. The semi-quantitative OArSI score showed a significant increase in cartilage erosion in DMM-operated Frzb -/-mice compared to wild-type. Conclusions Our data show that, in addition to the higher susceptibility to OA in acute induced models, Frzb -/-mice are more prone to OA in a full translational model of the disease characterised by slowly progressive joint damage. Overexpression of Frzb stimulates chondrogenesis by its inhibitory role on Wnt/beta-catenin signalling. Objective To investigate the individual impact of synovial inflammation, subchondral bone erosion or cartilage damage on functional impairment in an animal model of Rheumatoid Arthritis (RA). Methods We analysed gait profiles in human tumour necrosis factor transgene (hTNFtg) animals, using the video-based Catwalk gait analysis system (from Noldus, Netherlands). In this system, mice run along an illuminated glass plate. A digital camera measures light emissions resulting from the contact of paws on the glass plate. We evaluated gait profiles at different time points of disease (6, 10, 15 week of age) in hTNFtg animals. Wildtype littermates served as controls. Bodyweight and clinical signs of arthritis including paw swelling and grip strength were also evaluated. To investigate whether gait changes are pain related, we treated hTNFtg animals with diclofenac (50 mg/kg, i.p.) at week 10 and week 15 after birth and analysed gait profiles before, as well as 1 h and 3 h after treatment. To investigate reversibility in impaired gait profils, we treated hTNFtg mice with anti-TNF (Infliximab 10 mg/kg body weight, 2× per week) for 5 weeks starting 6 and 10 weeks after birth. To analyse inflammatory joint destruction, we quantitatively assessed the extend of synovial inflammation, subchondral bone erosion and cartilage damage on hematoxylin and eosine (H&E), tartrate-resistant acid phosphatase (TRAP) and toluidine-blue stained paw sections. We performed correlation studies between gait parameters and the histopathological components as well as clinical signs. Results We identified several gait parameters among them weight bearing, stride length and contact area of the paw to be significantly decreased in hTNFtg animals compared to sex-and age-matched wildtype animals. Moreover, we found a marked reduction in maximum intensity, an indicator for weight bearing, in week 10 and 15 compared to week 6 old hTNFtg mice. Similar effects were seen in print width, print area, print length, max contact max intensity and max contact area at different stages of disease. Interestingly, analgesic treatment with diclofenac (50 mg/kg, i.p.), resulted in a better improvement of weight-bearing parameters in 10 week old hTNFtg mice than in 15 week old hTNFtg animals indicating an pain independent, irreversible functional impairment in progressed disease. To further investigate to which extend synovial inflamm...

Objective To investigate skeletal myopathy in a chronic infl ammatory, erosive animal model for rheumatoid arthritis, the human tumour necrosis factor transgenic (hTNFtg) mice. Methods To evaluate whether hTNFtg mice are suffering from skeletal muscle atrophy, the authors isolated Triceps surae muscles from hTNFtg animals from various time-points of age starting 4 weeks until 16 weeks after birth. Muscle weight and body weight were assessed from these animals. Muscle tissue, muscle weight and bodyweight from age and sex-matched wildtype animals served as controls. To investigate whether tumour necrosis factor (TNF) blockade protects hTNFtg animals from muscle atrophy, 5 female hTNFtg animals were treated with anti-TNF ab (Infl iximab, 10mg/kg, 3x per week, ip). Untreated hTNFtg animals served as controls. To identify proteolysis pathways and pro-infl ammatory cytokine expression involved in muscle atrophy, the authors performed quantitative real-time PCR for Cathepsin L, B, S, H, D, MMP-9 and Interleukin (IL)-1 and IL-6 from mRNA isolated from muscle tissues of hTNFtg and wildtype animals. To further investigate infi ltration of infl ammatory cells, muscle tissue sections are stained for macrophages, neutrophils, T cells and B cells and convential hematoxylin/eosin. Results The authors demonstrate that hTNFtg mice show signifi cantly less triceps surae muscle weight compared to sex-and age matched wildtype animals. Reductions in muscle weight became already manifest at the early age of 4 weeks and were continuously reduced until week 16. Due to decreased muscle weight, bodyweight was also signifi cantly decreased in hTNFtg animals compared to their wildtype littermates. The authors found a signifi cantly increased mRNA expression levels of Cathepsin L, a lysosomal endopeptidase responsible for muscle protein degradation, in muscles from hTNFtg compared to their wildtype littermates. In contrast, other proteases such as cathepsin B, S, H, D did not reach signifi cantly increased expression levels between these 2 genotypes. Moreover, proinfl ammatory cytokines such as IL1 and IL6 are also signifi cantly upregulated in muscles from hTNFtg mice. Conclusion Despite spontaneous development of chronic infl amed, erosive arthritis, chronic overexpression of TNF leads to skeletal muscle atrophy due to increased tissue-degrading cathepsin L in hTNFtg animals.

Objectives To investigate the impact of systemic inflammation on skeletal muscles in human tumor necrosis factor transgenic (hTNFtg) animals. Methods We isolated triceps surae, as well as rectus abdominis muscles from hTNFtg animals at different time-points of disease starting at week 4 after birth. Muscle weight and body weight were assessed from these animals. Age and sex-matched wildtype (wt) animals served as controls. Moreover, hTNFtg animals were treated with anti-TNF ab (Infliximab, 10mg/kg, 3x per week, i.p.) and we performed quantitative real-time PCR for Cathepsin L, B, S, H, D, LC3-B, MMP-9 and Interleukin (IL)-1 and IL-6 from mRNA isolated from muscle tissues of hTNFtg and wt animals. Muscle tissue sections were also stained for macrophages, neutrophils, T cells and B cells. Results We could demonstrate that hTNFtg mice significantly lost muscle weight when compared to sex- and age matched wt animals. Reductions in muscle weight became already manifest at early stages of the disease, at week 4, and continuously progressed until week 16. Due to decreased muscle weight, bodyweight was also significantly lower in hTNFtg animals compared to their wt littermates. We also found significantly increased mRNA expression levels of Cathepsin L, a lysosomal endopeptidase responsible for muscle protein degradation, in muscles from hTNFtg compared to their wt littermates. In contrast, other proteases such as cathepsin B, S, H, D did not significantly increased differ between these 2 genotypes. In addition, we also found LC3B, an enzyme for autophagy-lysosome-mediated proteolysis, to be upregulated in hTNFtg mice compared to wt animals. Moreover, proinflammatory cytokines such as IL-1 and IL-6 were also significantly upregulated in muscles from hTNFtg mice. Interestingly, we observed an increased presence of macrophages and granulocytes in the muscle vascular system but no accumulation of inflammatory cells into the muscle tissue. To evaluate whether the observed changes in muscular tissue are a result of local inflammation or of systemic nature we also investigated the rectus abdominis muscle which is not located between inflamed articular joints. We found elevated levels of Cathepsin L and LC3B in this muscle, indicating that hTNFtg mice suffer from a systemic muscle proteolysis due to systemic inflammation. Conclusions Despite spontaneous development of chronic inflamed, erosive arthritis, chronic overexpression of TNF leads to skeletal muscle atrophy due to increased tissue-degrading cathepsin L in hTNFtg animals. Disclosure of Interest None Declared