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Abstract
Objectives Alarmins S100A8/A9 regulate pathology in experimental osteoarthritis (OA). Paquinimod is an immunomodulatory compound preventing S100A9 binding to TLR-4. We investigated the effect of paquinimod on experimental OA and human OA synovium.
Materials and methods Two OA mouse models differing in level of synovial activation were treated prophylactic with paquinimod. Synovial thickening, osteophyte size and cartilage damage were measured histologically, using an arbitrary score, adapted Pritzker OARSI score or imaging software, respectively. Human OA synovia were stimulated with S100A9, with or without paquinimod.
Results Paquinimod treatment of collagenase-induced OA (CIOA) resulted in significantly reduced synovial thickening (57%), osteophyte size at the medial femur (66%) and cruciate ligaments (67%) and cartilage damage at the medial tibia (47%) and femur (75%; n=7, untreated n=6). In contrast, paquinimod did not reduce osteophyte size and reduced cartilage damage at one location only in destabilised medial meniscus, an OA model with considerably lower synovial activation compared with CIOA. In human OA synovium, paquinimod blocked proinflammatory (interleukin (IL)-6, IL-8, tumour necrosis factor-α) and catabolic (matrix metalloproteinases 1 and 3) factors induced by S100A9 (n=5).
Conclusions Prophylactic treatment of paquinimod reduces synovial activation, osteophyte formation and cartilage damage in experimental OA with high synovial activation (CIOA) and ameliorates pathological effects of S100A9 in OA synovium ex vivo.
- Osteoarthritis
- Inflammation
- Synovitis
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Introduction
Osteoarthritis (OA), one of the ten most disabling diseases in developed countries, has no effective therapy to date. Although historically considered a disease of the cartilage only, it is now clear that other tissues such as the synovium also play an important role in the pathophysiology of OA.1 Synovial activation is present in at least 50% of patients with OA2 and several proinflammatory mediators have been shown to be involved in OA pathology,3 ,4 among which alarmins S100A8 and S100A9. In recent studies, we showed that S100A8/A9 is high in serum and synovium of patients with OA and predictive for cartilage damage and osteophyte formation.5 ,6 We also found that paquinimod binds S100A9, but not S100A8, using photoaffinity labelling and surface plasmon resonance and specifically blocks the interaction of S100A9.7 S100A9−/− mice show reduced joint pathology in collagenase-induced OA (CIOA), a model with considerable synovitis. However, no effect of the absence of S100A9 was seen in the surgical, non-inflammatory destabilised medial meniscus (DMM) model.5 S100A8 and S100A9 are endogenous ligands for TLR-48 and blocking this interaction in OA chondrocytes reduces the catabolic effects of these alarmins.9 Quinoline-3-carboxamide paquinimod (ABR-215757) is effective in experimental lupus and encephalomyelitis and proven to be safe and well tolerated in patients with systemic lupus erythematosus.10 ,11 In the current study, we investigated the effect of the S100A9-blocking compound paquinimod on experimental OA with different degrees of synovial activation. Furthermore, we tested the in vitro effects of paquinimod on human OA synovial tissue.
Material and methods
Human OA synovium preparation and stimulation
Human synovium was obtained anonymously from patients with OA undergoing arthroplasty. Synovium pieces (2×2 mm) taken from each patient were mixed and three pieces per well were cultured and stimulated in quadruplo for 24 h with 1 µg/mL human S100A9, 350 µM paquinimod or both (30 min preincubated). Thereafter, synovium pieces were fixed in formalin and embedded in paraffin for histological analysis with H&E staining and with a specific mouse anti-human CD68 (clone KP1, Dako). OA synovium supernatant was collected for protein measurement on the Bio-Plex 100 Luminex system (Bio-rad). Five different patients with OA were compared.
Experimental OA models
Experimental OA was elicited in male C57BL/6J (Janvier) aged 12–14 weeks in a C57BL/6 background. Mice were housed in filter-top cages, and water and food were provided ad libitum. CIOA was performed by two times 1U collagenase (C0773, Sigma) injection into the knee-joint.12 DMM OA was induced by transection of the medial anterior meniscotibial ligament.13 Ten animals were allocated to each treatment group. Due to dislocations we used seven and six of the original 10 in the treated and untreated group. All animal experiments were approved by the local authority Animal Care and Use Committee and local ethics committee (RU-DEC 2012-232) and were performed by personnel certified by the Dutch Ministry of WVC.
Paquinimod preparation and administration
Paquinimod (ABR-215757) was synthesised by Active Biotech AB and administered in the drinking water of mice to a final concentration of 3.75 mg/kg. Normal tap water in which the paquinimod was dissolved served as control.
Histological analysis
Total knee joints were fixed in formalin, embedded in paraffin and cut in 7 µm sections. Osteophyte size was assessed in Safranin-O/Fast Green stained sections using the Leica Application Suite image analysis software. Cartilage damage was measured blinded using an arbitrary score based on the OARSI scoring method by Pritzker et al.14 Briefly, lateral and medial tibia and femur were scored for the grade of cartilage destruction (0–5) and the extent of damaged cartilage surface (0–6). These scores were multiplied and added together for five sections to form the total OA cartilage score. Synovial thickening/activation was scored on H&E-stained paraffin sections using an arbitrary score from 0 to 3 as previously described.15
Statistical analysis
Statistical differences were calculated with Student's t test as indicated using Graph Pad Prism V.5 (GraphPad Software).
Results
Paquinimod reduces synovial activation, osteophyte formation and cartilage damage in collagenase-induced OA
We treated experimental OA with high (CIOA) and low synovial activation (DMM model) with paquinimod, administered via the drinking water. Treatment started 4 days prior to induction of either model and was refreshed twice a week. Paquinimod was well tolerated and caused no apparent side effects in both models. Control-treated and paquinimod-treated animals did not differ in weight when measured either on day 11 (weight increase control vs paquinimod in CIOA 105% vs 106%, in DMM 114% vs 114%) or on day 42 (weight increase control vs paquinimod in CIOA 117% vs 117%, in DMM 117% vs 115%; data not shown), indicating that drinking behaviour was not affected.
CIOA was treated with paquinimod and on day 42 we measured synovial thickening, osteophyte formation and cartilage damage, three important hallmarks of OA pathology. Synovial thickening was significantly reduced by paquinimod treatment at the medial side of the patella-femur region (57%; figure 1A). Osteophyte size was significantly reduced at the medial femur (66%) and cruciate ligament (67%; figure 1B). A trend towards reduction was also observed in the medial collateral ligament and in total osteophyte size, but this did not reach significance. Finally, cartilage damage was significantly reduced by paquinimod treatment of CIOA at both medial tibia and femur (47% and 75%, respectively) and in the total joint score (47% reduction; figure 1C).
Treatment of collagenase-induced osteoarthritis (CIOA) with paquinimod significantly reduced synovial thickening, osteophyte formation and cartilage damage. CIOA was induced in C57BL/6J mice, an experimental OA model with high synovial activation. We treated mice 4 days prior to induction of OA with 3.75 mg/kg paquinimod and assessed OA pathology on day 42. (A) Synovial thickening, scored on an arbitrary scale from 0 to 3 on H&E-stained histological slides, was significantly reduced by paquinimod at the medial side of the femur. (B) Osteophyte size was measured with image analysis software on Safranin-O/Fast Green-stained histological slides. Paquinimod reduced osteophyte size on day 42 of CIOA at the medial femur and cruciate ligament, while a trend towards reduction was observed at the medial collateral ligament and in the total medial and lateral osteophyte size. (C) Cartilage damage (indicated by arrows) was assessed on Safranin-O/Fast Green-stained histological slides with the modified Pritzker OARSI score. Paquinimod significantly reduced cartilage damage on day 42 of CIOA at the medial tibia and femur and in the total joint. Horizontal bars represent mean. Ten animals were used per group, but due to dislocations four in the control group and three in the paquinimod group were omitted from analysis. p Values calculated with Student's t test, 95% CI for (A) 0.22 to 1.30, for (B) medial femur 2.00 to 62.50 and cruciate ligament 18.25 to 59.06, for (C) medial femur 1.56 to 81.5 and total 5.29–153.2. p Values from Student's t test. col, collateral; control, open dots; cruc, cruciate; fem, femur; lig, ligament; Med, medial; paquinimod, black dots; tib, tibia.
Paquinimod has marginal effects in DMM
Paquinimod treatment of DMM OA, in which synovial activation is scant, did not reduce osteophyte size in neither medial tibia nor femur (figure 2A) when measured at day 56. Cartilage damage was significantly reduced by paquinimod treatment only at the medial femur (64%), but not at the medial tibia where most damage occurs in DMM (figure 2B).
Paquinimod does not reduce osteophyte size and only marginally reduces cartilage damage in surgically induced experimental osteoarthritis . Destabilised medial meniscus (DMM) was surgically induced in C57BL/6J mice and treatment with 3.75 mg/kg paquinimod was started 4 days prior. Synovial activation in the DMM model is low and cartilage damage and osteophyte formation occur mostly at the medial femur and tibia. Osteophyte size and cartilage damage were assessed on day 56. (A) Paquinimod did not reduce osteophyte size on day 56 of DMM at medial tibia or femur, as measured with image analysis software on Safranin-O/Fast Green-stained histological slides. (B) Cartilage damage was assessed on Safranin-O/Fast Green-stained histological slides with the modified Pritzker OARSI score. Paquinimod significantly reduced cartilage damage at the medial femur on day 56 of DMM, however, not at the medial tibia where most damage occurs. Control, open dots; paquinimod, black dots. Horizontal bars represent mean. Eight animals were used per group. p Values from Student's t test, 95% CI for (B) medial femur 0.52 to 3.67.
Paquinimod reduces proinflammatory/catabolic effects in vitro in macrophages and OA synovial tissue
To test paquinimod in a human OA setting, we investigated the effect of the compound on synovial specimen obtained after joint replacement of patients with end-stage OA. Macroscopically, we excluded non-synovial tissue like cartilage, tendon and fat and we could confirm this on histology using H&E staining. We stained for CD68 demonstrating the presence of macrophages (data not shown). Then we incubated OA synovium with human S100A916 and/or paquinimod and measured the protein release. Addition of paquinimod alone did not have an effect on proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 nor on matrix metalloproteinases (MMP)-1, MMP-3 and MMP-9 (IL-1β and MMP-13 were not detectable; figure 3). S100A9 alone clearly upregulated IL-6, IL-8 and TNF-α protein levels in OA synovia compared with medium control (ninefold, 12-fold and 20-fold increase, respectively), and addition of paquinimod significantly inhibited, although partially, this upregulation for IL-6 and IL-8, but not TNF-α (figure 3). S100A9 significantly upregulated MMP-1 and MMP-3 (up to 2.5-fold) and this catabolic effect was completely inhibited by adding paquinimod. No effects were found on MMP-9 (figure 3).
Paquinimod reduces induction of proinflammatory and catabolic mediators by S100A9 in human osteoarthritis (OA) synovium. Synovia were collected from patients with end-stage OA undergoing arthroplasty, characterised and stimulated for 24 h with 1 µg/mL S100A9 and/or 350 µM paquinimod. Treatment of OA synovia with 350 µM paquinimod alone did not affect production of proinflammatory mediators tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 nor of catabolic matrix metalloproteinases (MMP)-1, MMP-3 and MMP-9 (IL-1β and MMP-13 were not detectable). S100A9 upregulated release of TNF-α, IL-6 and IL-8 and MMP-1 and MMP-3 by synovial specimen. Addition of paquinimod significantly inhibited upregulation of IL-6 and IL-8 (95% CI 11896 to 81780 and 3111 to 236437, respectively), while a clear trend towards inhibition was observed for TNF-α (p=0.06). Paquinimod significantly inhibited S100A9-induced upregulation of MMP-1 and MMP-3 (95% CI 36.5 to 669.9 and 8361 to 55640), but not MMP-9. Measurements were done in five donors in total, although in some cytokine or MMP levels were below detection level. p Values from Student's t test. Every dot represents one donor. Horizontal bars represent mean.
Discussion
In the current study, we show that prophylactic treatment with S100A9 inhibitor paquinimod reduces severity of pathology in CIOA that is characterised by high synovial activation. Furthermore, we show that paquinimod can reduce induction of proinflammatory and catabolic mediators by S100A9 in human OA synovium.
We showed earlier that paquinimod binds S100A9 preventing it from binding to TLR-47 but also to S100A8 which is then rapidly degraded.8 Furthermore, we found that S100A9−/− mice showed reduction of synovial thickening, osteophyte formation and cartilage damage during CIOA.5 Therefore, the beneficial effects of paquinimod on experimental OA are probably due to a specific inhibition of functional S100A9. We earlier showed that the effects of paquinimod on encephalomyelitis run via Ly6Chi monocytes,11 providing another possible mechanism for the observed effects on OA pathology. A prophylactic approach was chosen since early treatment has been shown to be effective in suppressing development of OA pathology.17
In recent years, researchers and clinicians have given more focus to the role of inflammation in OA and it is believed that synovial inflammation might be one of the parameters to classify different phenotypes of OA patients.18 We found that the effect of paquinimod was largest in an OA model with high synovial activation (CIOA), compared with low (DMM) and mainly inhibits S100A9-dependent upregulation of IL-6 and IL-8. In translation of this study to the clinic, one could imagine that targeting S100A9 by paquinimod would especially be suitable for treating patients with considerable synovial activation and high S100A8/A9 levels. Levels of S100A8/A9 in serum could be used as marker to assess synovial inflammation, as they have been described as inflammatory markers in various forms of arthritis such as rheumatoid arthritis, juvenile idiopathic arthritis and gout.19–21 Recently, we described the presence of S100A8/A9 in serum of patients with early symptomatic OA correlating with joint damage and osteophyte size.5 ,6 We now show that paquinimod is able to block production of proinflammatory and catabolic mediators induced by S100A9 in human OA synovium, in which CD68+ macrophages are present. A recent study in our lab showed that S100A9 had a stimulating effect on macrophages but not on synovial fibroblasts (personal communication). Moreover, the activation status of synovial macrophages is much higher in CIOA when compared with DMM reflected by a higher S100A8/A9 expression .This suggests that the effects of paquinimod ex vivo run via synovial macrophages. We have shown earlier that macrophages are crucial in mediating OA pathology as selective removal of macrophages prior to CIOA effectively reduces synovial activation, fibrosis, osteophyte formation and MMP-induced cartilage damage.22 ,23
Taken together, our study indicates that paquinimod may be a promising compound to suppress joint destruction, inflammation and osteophyte formation in patients with OA with high synovial activation and expressing high levels of S100A8/A9.
Acknowledgments
The authors would like to thank Birgitte Walgreen, Monique Helsen and Elly Vitters (Dept. Experimental Rheumatology, Radboud umc Nijmegen) for their excellent technical assistance.
References
Footnotes
Handling editor Tore K Kvien
Contributors All authors meet the criteria recommended by ICMJE. Conception and design of study: RFS, PMvdK, ABB, PLEMvL, WBvdB and TL. Acquisition of data: RFS, EJG, MHJvdB and TV. Analysis and interpretation of data: RFS, HE, TL, JR, TV, WBvdB, PMvdK, FAJvdL and MIK. Drafting the article: RFS, PLEMvL and ABB. Revising article critically: EJG, MHJvdB, HE, TL, TV, JR, FAJvdL, MIK, PMvdK, WBvdB, ABB and PLEMvL. Final approval of the submitted manuscript and agreement to be accountable for all aspects of the work: all authors.
Funding RFS was financially supported by a research grant from Active Biotech.
Competing interests RFS is a full time employee and owns stock in Active Biotech that develops paquinimod for the treatment of systemic sclerosis. TL is a part time employee and owns stock in Active Biotech that develops paquinimod for the treatment of systemic sclerosis.
Provenance and peer review Not commissioned; externally peer reviewed.