Meriem Koufany

Oxidative stress-induced expression of HSP70 contributes to the inhibitory effect of 15d-PGJ2 on inducible prostaglandin pathway in chondrocytes

The inhibitory effect of 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) on proinflammatory gene expression has been extensively documented and frequently ascribed to its ability to prevent NF-κB pathway activation. We and others have previously demonstrated that it was frequently independent of the peroxisome proliferator activated receptor (PPAR)γ activation. Here, we provide evidence that induction of intracellular heat shock protein (HSP)70 by oxidative stress is an additional regulatory loop supporting the anti-inflammatory effect of 15d-PGJ2 in chondrocytes. Using real-time quantitative PCR and Western blotting, we showed that 15d-PGJ2 stimulated HSP70, but not HSP27 expression while increasing oxidative stress as measured by spectrofluorimetry and confocal spectral imaging. Using N-acetylcysteine (NAC) as an antioxidant, we demonstrated further that oxidative stress was thoroughly responsible for the increased expression of HSP70. Finally, using an HSP70 antisense strategy, we showed that the inhibitory effect of 15d-PGJ2 on IL-1-induced activation of the NF-κB pathway, COX-2 and mPGES-1 expression, and PGE2 synthesis was partly supported by HSP70. These data provide a new anti-inflammatory mechanism to support the PPARγ-independent effect of 15d-PGJ2 in chondrocyte and suggest a possible feedback regulatory loop between oxidative stress and inflammation via intracellular HSP70 up-regulation. This cross talk is consistent with 15d-PGJ2 as a putative negative regulator of the inflammatory reaction.

The peroxisome proliferator-activated receptor γ agonist pioglitazone preserves bone microarchitecture in experimental arthritis by reducing the interleukin-17-dependent osteoclastogenic pathway.

OBJECTIVEnTo investigate the effect of pioglitazone on inflammation-induced bone loss and changes in bone microarchitecture in rats with adjuvant-induced arthritis (AIA), focusing on the contribution of interleukin-17 (IL-17) and the balance of RANKL and osteoprotegerin (OPG).nnnMETHODSnMale Lewis rats sensitized with Freunds complete adjuvant were treated orally for 21 days with 30 mg/kg/day of pioglitazone or vehicle. Arthritis severity was evaluated by clinical and histologic examination. Bone mineral density (BMD) was assessed by dual x-ray absorptiometry. The therapeutic effect of pioglitazone on changes of the bone architecture was determined by micro-computed tomography (micro-CT). Levels of RANKL, OPG, and IL-17 were determined by serum immunoassay and by synovial tissue immunohistochemistry. Messenger RNA for IL-17 and retinoic acid receptor-related orphan nuclear receptor γt (RORγt) was evaluated by quantitative reverse transcription-polymerase chain reaction and IL-17 promoter activity by gene-reporter assay.nnnRESULTSnMicro-CT analysis revealed that pioglitazone treatment reduced arthritis severity and bone erosion scores and increased BMD in comparison to vehicle treatment. Cortical bone thickness was preserved, although the major beneficial effect of pioglitazone was on indices of the trabeculae, especially trabecular separation. Pioglitazone reduced the ratio of RANKL to OPG, in both the serum and the inflamed synovium. Circulating levels of IL-17 were significantly reduced by pioglitazone treatment, as were the percentages of IL-17-positive cells, mainly polymorphonuclear cells, in the inflamed synovium. Induction of IL-17 was strictly dependent on the binding of RORγt to IL-17 promoter, and lentiviral overexpression of peroxisome proliferator-activated receptor γ (PPARγ) reduced the expression of RORγt.nnnCONCLUSIONnPioglitazone decreased the level of inflammatory bone destruction and protected the bone microarchitecture in rats with AIA by controlling the circulating and local expression of IL-17, with a subsequent decrease in the RANKL-to-OPG ratio. Along with the inhibition of RORγt expression after PPARγ overexpression, these findings provide evidence of the major contribution of reduced IL-17/RANKL-dependent osteoclastogenesis.

MicroRNA-29b Contributes to Collagens Imbalance in Human Osteoarthritic and Dedifferentiated Articular Chondrocytes

Objective Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA- (miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype. Methods After preliminary microarrays screening, miR-29b levels were measured by RT- quantitative PCR in in vitro models of chondrocyte phenotype changes (IL-1β challenge or serial subculturing) and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identified in silico in 3′-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining) during chondrogenesis. Results MiR-29b level increased significantly in IL-1β-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3′-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased. Conclusions By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.

Eplerenone treatment alleviates the development of joint lesions in a new rat model of spontaneous metabolic-associated osteoarthritis

Increasing epidemiological and clinical studies suggest that metabolic syndrome (MetS) plays a role in the incidence and progression of osteoarthritis (OA).1 2 However, in absence of an appropriate MetS-associated OA experimental model,3 the MetS contribution to the joint phenotype in OA remains difficult to investigate and the evaluation of potential disease-modifying OA drugs (DMOADs) is complicated. Noteworthy, in contrast to their lean SHHF+/+(spontaneously hypertensive heart failure) controls, obese SHHFcp/cp rats, a well-characterised model of MetS,4 develop drastic metabolic, cardiovascular and renal alterations that are substantially improved through an early chronic mineralocorticoid receptor antagonism (MRA) treatment.5 Thus, by comparing young (1.5u2009months) and aged (12.5u2009months) lean SHHF+/+ and obese SHHFcp/cp rats, we sought to evaluate for the first time the potential (1) contribution of MetS to joint alterations and (2) therapeutic benefits derived from chronic MRA treatment by eplerenone (figure 1A).nnnnFigure 1 nPreventive 11-month treatment with mineralocorticoid receptor antagonist eplerenone alleviated the metabolic syndrome (MetS)-associated joint lesions in SHHF model. (A) Experimental design of the study. Lean spontaneously hypertensive heart failure (SHHF+/+) and obese SHHFcp/cp rats were divided randomly into treatment groups. Untreated groups (n=4 for SHHF+/+ and n=4 for SHHFcp/cp) were given …

Association between rheumatoid arthritis and systemic mastocytosis: a case report and literature review

Classically, mast cells (MC) are considered as important actors of the innate immune response playing a pivotal role in IgE-mediated allergic and antiparasite responses. In the last two decades, many experimental evidences demonstrated that these hematopoietic-derived cells present in both connective and mucosal tissues are also key modulators of the adaptive immune response and could contribute to autoimmune disease notably in rheumatoid arthritis (RA). Recently, Bader-Meunier et al. reported a series of 31 patients suffering from inflammatory joint diseases associated with mastocytosis, suggesting that mastocytosis was associated with a higher prevalence in spondyloarthritis. We discuss here the possible link between chronic inflammatory arthritis and mastocytosis through the report of a clinical case describing a patient developing RA after a long history of mastocytosis. Of great interest, antihistamine treatment alone was sufficient to treat RA in this patient.

Fenofibrate vs pioglitazone: Comparative study of the anti-arthritic potencies of PPAR-alpha and PPAR-gamma agonists in rat adjuvant-induced arthritis.

BACKGROUNDnRheumatoid arthritis is characterized by synovial hyperplasia, inflammatory infiltration, cartilage destruction and juxta-articular as well as generalized bone demineralization. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily which behave as ligand-activated transcription factors in response to endogenous fatty acids and eicosanoids or isotype selective synthetic compounds as fibrates and thiazolidinediones. Beyond their key role in lipid metabolism, increased evidence has shown a role of the three isotypes in inflammatory modulation. We and others demonstrated previously that PPAR-gamma agonists reduced the severity of experimental polyarthritis and the overall inflammatory-induced bone loss.nnnOBJECTIVEnTo compare the anti-arthritic potencies of a PPAR-alpha agonist (fenofibrate, a lipid lowering drug) and a PPAR-gamma agonist (pioglitazone, formerly used as an antidiabetic drug) in rat adjuvant-induced arthritis.nnnMETHODSnMale Lewis rats were sensitized by an intra-dermal injection of 1 mg complete Freunds adjuvant at the basis of the tail and were treated orally for 21 days with fenofibrate 100 mg/kg/day (FENO) or pioglitazone 30 mg/kg/day (PIO), or with vehicle only. Arthritis severity was evaluated by clinical observations (oedema, clinical score, body weight). Global and femoral bone mineral density (BMD), femoral bone mineral content (BMC) were measured by dual-energy X-ray absorptiometry (DEXA) before sensitization and at day 20. Synovial mRNA levels of IL-1beta and IL-6 were determined by real-time RT-PCR.nnnRESULTSnAdministration of fenofibrate (100mg/kg/d) and pioglitazone (30 mg/kg/d) significantly reduced hindpaw oedema and arthritis score. Treatment with fenofibrate exerted a better effect on clinical scoring. DEXA analysis revealed that pioglitazone and fenofibrate treatment to a greater extent, reduced inflammatory-bone loss and increased BMD versus vehicle-treated rats. Finally, we demonstrated that both agonists decreased synovial expression of IL-1beta and IL-6.nnnCONCLUSIONnPioglitazone and fenofibrate decreased arthritis severity in adjuvant-induced arthritis. Both agonists partially protected animals from inflammatory induced-bone loss.

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.

Response to: ‘Spontaneous hypertensive rat exhibits bone and meniscus phenotypes of osteoarthritis: is it an appropriate control for MetS-associated OA?’ by Chan and Wen

We thank Dr Chan and Dr Wen for their interest in our report1 and their resulting eLetter.2 We fully agree that, among the different components of metabolic syndrome (MetS), hypertension has very recently been brought out as a critical feature in the development of osteoarthritis (OA) in humans.3 4 In these reports using either data from the Framingham OA study3 or the Osteoarthritis Initiative study,4 it has been emphasised that high blood pressure (diastolic or systolic, respectively) was associated with increased incidence of radiographic knee OA.nnIn order to further experimentally investigate the actual role of hypertension in OA onset and development, Chan and colleagues describe in a yet unpublished study the development of OA features in the spontaneous hypertensive rat (SHR) model, a widely characterised model of systemic hypertension.2 5 Although the spontaneous hypertensive heart failure (SHHF) rat strain we employed is deriving from the SHR strain6 and suffers high …

FRI0017 Pharmacological Blockade of CCR3 Reduces Collagen-Induced Arthritis Severity

Background Chemokines are key pathophysiological mediators in the early phase of arthritis. Eotaxin-1 (Paquet and al., 2012) and its receptor, CCR-3 (CC Chemokine Receptor 3) (Haas and al., 2005), were previously shown to be highly expressed in the course of experimental arthritis. In human, CCR-3 positive monocytes are more abundant in the synovial fluid of arthritic patients than in healthy ones (Katschke and al., 2001). Moreover, RANTES, another chemokine with pathophysiological relevance to arthritis, is also a ligand of CCR-3, further suggesting that CCR-3 could be a potential therapeutic target for joint diseases. Objectives The main obective of this project was to study consequences of CCR-3 inactivation by a pharmacological antagonist in collagen-induced arthritis. Methods CIA was induced in 20 DBA/1J mice by intradermal injection of 100mg of bovine type II collagen in CFA at the basis of the tail, with a booster injection of 50mg by day 21. From day 15, mice were administered daily with antagonist of CCR-3 at a concentration of 10 mg/kg/day (i.p). Mice were regularly weighed and evaluated for the severity of arthritis with an arthritic score and by measuring hindpaw oedema by plethysmography. Mice were sacrificed at day 41, and histological analysis was performed on ankle joints after HES and safranin-O fast green staining. At that time, serum levels of IL-6 and IL-17F were measured by the multiplex technology. Results The clinical parameters showed that mice treated with a specific antagonist of CCR-3 develop less severe arthritis (respective clinical score 3.89 ± 1.25 vs 8.57 ± 1.63). Histological analyzes indicated that antagonist reduced the intensity of inflammatory process and limits cartilage degradation in arthritic joints. Blood level of IL-6 and IL-17F cytokines were reduced in mice treated with antagonist of CCR-3 compared to untreated arthritic mice. Conclusions Thus, our study shows that CCR-3 has an important role in arthritis development and designates it as a potential therapeutic target. References Haas C.S., Martinez R.J., Attia N., Kenneth H., Campbell P.L., Koch A.E., Chemokine receptor expression in rat adjuvant-induced arthritis. Arthritis and Rheumatism, 2005, 52, 3718–1730. Katschke K.J., Jr., Rottman J.B., Ruth J.H., Qin S., Wu L., LaRosa G., Ponath P., Park C.C., Pope R.M., Koch A.E. Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis. Arthritis and Rheumatism, 2001, 44: 1022–1032. Paquet J., Goebel, J-C, Delaunay C., Pinzano A., Grossin L., Cournil-Henrionnet C., Gillet P., Netter P., Jouzeau J-Y., Moulin D. Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators? Arthritis Research and Therapy, 2012, 14: 60–75 Acknowledgement The authors thank fondation Arthritis for granting this study. Disclosure of Interest None declared

OP0174 Ppar Gamma Deficient Mice Develop Spontaneous Polyarthritis

Background Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor superfamily of transcription factors. PPARγ is involved in the regulation of adipocyte differentiation, glucose homeostasis and inflammation. Others and ourselves have previously demonstrated that PPARγ agonists could be successfully used in the treatment of experimental arthritis, representing an attractive potential approach for rheumatoid arthritis (RA) therapy. Indeed, we recently demonstrated that treatment with synthetic agonist of PPARγ isotype (pioglitazone) in addition to decrease arthritis severity, specifically reduced inflammatory-bone destruction and protected bone micro-architecture in experimental arthritis by controlling circulating and local expression of Interleukin-(IL-)17 with a subsequent decrease of the (RANKL/OPG) ratio [1]. Furthermore, recent genetic studies have demonstrated an association between a known coding SNP in the PPARG gene and psoriatic [2] or rheumatoid arthritis [3]. In recent clinical studies, a PPARγ agonist, used alone or in combination with NSAIDs or methotrexate appears to represent a promising therapeutic strategy for rheumatoid or psoriatic arthritis patients [4]. Objectives This study aims to characterize the articular phenotype of PPARγ deficient mice. Methods Fully viable PPARγ-null mice were generated through specific and total epiblastic gene deletion using Sox2Cre/PPARγL2/L2 mice. Histologic examination of ankle and knee joints after specific staining of cartilage and synovium tissues was performed. Synovial mast cells were specifically stained with toludine blue. Finally, serum levels of the pro-inflammatory cytokines IL-17 and TNFα, the anti-inflammatory/immunomodulatory cytokine IL-10 and the chemokine KC were determined using multiplex cytokine ELISA. Results We have observed that adult PPARγ–/– mice show evidence of arthritis in the ankle and knee with 100% incidence. We have determined the onset of the disease around 3 weeks of age. Joints share many histological features with RA, such as synovial lining hyperplasia, synovial hypervascularization, infiltrating immune cells, cartilage erosion and bone remodeling (Figure 1: Safranin O- Fast Green (A) and Hematoxylin-Eosin (B) staining of knee joint sections of wild type and PPARγ–/– littermates showing evidence of severe arthritis in PPARγ deficient mice: cartilage (black arrow) and bone erosion (red arrow) (A), synovial lining hyperplasia (yellow arrow), synovial hypervascularization (blue arrow), infiltrating immune cells (green arrow) (B). We explored concomitantly the circulating levels of several cytokines in these mice. We found notably elevated levels of the pro-inflammatory cytokines IL-17 and TNFα, the chemokine KC and reduced levels of the anti-inflamatory/immunomodulatory cytokine IL-10. Of major interest, we observed a great increased number (more than 50 fold) of mast cells in the synovium of PPARγ–/– mice, as compared to heterozygous or wild type littermates. Conclusions Taken together these data suggest that PPARγ may be a key regulator of arthritis development in mice. We described here a new model of spontaneous polyarthritis in mice. References Arthritis Rheum. 2013 Dec;65(12):3084-95. Ann Rheum Dis. 2012 Feb;71(2):313-4. Rheumatol Int. 2013 Apr 30. J Am Heart Assoc. 2013 Nov 19;2(6):e000441. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3291