David Moulin

Association between adiponectin and cartilage degradation in human osteoarthritis

OBJECTIVE Conflicting findings raise questions about the role of adiponectin in osteoarthritis (OA). The current study aimed to investigate in OA patients the association between the production of adiponectin and the grade of cartilage destruction, and to provide functional evidence for a potential role of adiponectin in OA. DESIGN The expression of adiponectin was examined by immunohistochemistry in cartilage obtained from healthy individuals (n = 2; ages 56 and 41 years; 1 male and 1 female) and OA patients (n = 11; ages 64-79 years; 2 male and 9 female). The association between its production in chondrocytes and the grade of cartilage destruction was established on full-depth cartilage biopsies. The functional activity of adiponectin in OA cartilage was determined from the relation between the expression of adiponectin, its receptor, cartilage-specific components and factors involved in matrix degradation, and from the chondrocyte response to the full-length or the globular form of adiponectin. RESULTS Adiponectin was not detected in healthy cartilage. Conversely, the adipokine was up-regulated in damaged tissue, but no strong association with the grade of cartilage destruction was found. We showed a positive correlation between adiponectin and mPGES or MMP-13 while AdipoR1 was related to the expression of type 2 collagen, aggrecan and Sox9. The full-length form of adiponectin but not the globular isoform, stimulated the production of PGE2 and MMP-13 activity in cultured human chondrocytes. CONCLUSIONS The elevated level of adiponectin found in chondrocytes from OA patients might contribute to matrix remodelling during OA, the full-length isoform being the single active form.

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.

Cytokines profiling by multiplex analysis in experimental arthritis: which pathophysiological relevance for articular versus systemic mediators?

IntroductionWe have taken advantage of the large screening capacity of a multiplex immunoassay to better define the respective contribution of articular versus systemic cytokines in experimental arthritis.MethodsWe performed a follow up (from 7 hours to 14 days) multiplex analysis of 24 cytokines in synovial fluid and sera of rats developing Antigen-Induced Arthritis (AIA) and confronted their protein level changes with molecular, biochemical, histological and clinical events occurring in the course of the disease.ResultsThe time-scheduled findings in arthritic joints correlated with time-dependent changes of cytokine amounts in joint effusions but not with their blood levels. From seven hours after sensitization, high levels of chemokines (MCP-1, MIP1α, GRO/KC, RANTES, eotaxin) were found in synovial fluid of arthritic knees whereas perivascular infiltration occurred in the synovium; local release of inflammatory cytokines (IFNγ, IL-1β, IL-6) preceded the spreading of inflammation and resulted in progressive degradation of cartilage and bone. Finally a local overexpression of several cytokines/adipocytokines poorly described in arthritis (IL-13, IL-18, leptin) was observed.ConclusionsDistinct panels of cytokines were found in arthritic fluid during AIA, and the expected effect of mediators correlated well with changes occurring in joint tissues. Moreover, multiplex analysis could be helpful to identify new pathogenic mediators and to elucidate the mechanisms supporting the efficacy of putative targeted therapies.

Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation

UNLABELLED Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5-20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOF mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGFβ1 and patient-to-patient differential expression. Novel targets of TGFβ1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. BIOLOGICAL SIGNIFICANCE The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFβ1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it.

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

OBJECTIVE To 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). METHODS Male 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. RESULTS Micro-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. CONCLUSION Pioglitazone 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.

TREM-1 inhibition restores impaired autophagy activity and reduces colitis in mice.

Background and Aims Triggering receptor expressed on myeloid cells-1 [TREM-1] is known to amplify inflammation in several diseases. Autophagy and endoplasmic reticulum [ER] stress, which activate the unfolded protein response [UPR], are closely linked and defects in these pathways contribute to the pathogenesis of inflammatory bowel disease [IBD]. Both autophagy and UPR are deeply involved in host-microbiota interactions for the clearance of intracellular pathogens, thus contributing to dysbiosis. We investigated whether inhibition of TREM-1 would prevent aberrant inflammation by modulating autophagy and ER stress and preventing dysbiosis. Methods An experimental mouse model of colitis was established by dextran sulphate sodium treatment. TREM-1 was inhibited, either pharmacologically by LR12 peptide or genetically with Trem-1 knock-out [KO] mice. Colon tissues and faecal pellets of control and colitic mice were used. Levels of macroautophagy, chaperone-mediated autophagy [CMA], and UPR proteins were evaluated by western blotting. The composition of the intestinal microbiota was assessed by MiSeq sequencing in both LR12-treated and KO animals. Results We confirmed that inhibition of TREM-1 attenuates the severity of colitis clinically, endoscopically and histologically. We observed an increase in macroautophagy [ATG1/ULK-1, ATG13, ATG5, ATG16L1, and MAP1LC3-I/II] and in CMA [HSPA8 and HSP90AA1], whereas there was a decrease in the UPR [PERK, IRE-1α, and ATF-6α] protein expression levels in TREM-1 inhibited colitic mice. TREM-1 inhibition prevented dysbiosis. Conclusions TREM-1 may represent a novel drug target for the treatment of IBD, by modulating autophagy activity and ER stress.

Management of patients with inflammatory bowel disease and spondyloarthritis

ABSTRACT Introduction: More than half of the patients with inflammatory bowel disease (IBD) experience at least one extra-intestinal manifestation (EIM). The most common EIM in patients with IBD is spondyloarthritis (SpA). Microscopic intestinal inflammation is documented in almost 50% of the patients with SpA. Areas covered: We give an overview of the classification, the epidemiology and the diagnosis of IBD and SpA. The treatment goals, the pharmacologic management options and the available treatment guidelines in IBD patients with SpA are discussed. Expert commentary: The coexistence of IBD and SpA generates challenges and opportunities for both the gastroenterologist and the rheumatologist. The potential of drugs with a gut-specific mode of action in the treatment of IBD-related arthritis warrants further exploration.

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.

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.

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