Yves-Marie Pers

Interleukin‐6 Receptor Blockade Enhances CD39+ Regulatory T Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

The rationale for blocking interleukin‐6 (IL‐6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti–IL‐6 receptor (IL‐6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti–IL‐6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA.

p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

IntroductionRecent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).MethodsWe used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.Resultsp16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.ConclusionsWe disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives

Osteoarthritis (OA) is the most common form of degenerative arthritis, mainly characterized by the degradation of articular cartilage and associated with subchondral bone lesions. Novel therapeutic approaches for OA include cell-based therapies that have become thriving areas of research and development. In this context, mesenchymal stem or stromal cells (MSCs) have gained much interest based on their trophic and immunomodulatory properties that can help tissue repair/regeneration. The present review article discusses the interest of using MSCs in cell-therapy approaches with a focus on the mechanisms by which MSCs might exhibit a therapeutic potential in OA. Special attention is given to the anti-inflammatory function of MSCs and on miRNA modulation in OA for possible future innovative strategies. The paper also presents the current data on the undergoing MSCs-based clinical trials in OA.

A3.10 IL-6 Receptor Blockade Enhances CD39+ Regulatory T-Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

Background and Objectives Studies have demonstrated the clinical efficacy of tocilizumab, a humanised anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and mainly focuses on their frequency. Our objective was to elucidate anti-IL-6R mode of action on Tregs in RA patients treated with tocilizumab and in a RA model. Methods Mice with collagen-induced arthritis (CIA) were treated at day 0 by MR16–1 (a rat anti-mouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co. LTD, Japan) and the evolution of CD4+ FoxP3+ Tregs during arthritis course was assessed at key time points (day 8–18–28 and 42 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN), thymus and spleen by flow cytometry. Numerical analysis of Th17 and Th1 cells was also performed by flow cytometry. Twenty patients with severe and active RA were recruited and treated with 8 mg/kg of tocilizumab monthly. Peripheral blood was recovered at day 0, as well as 1 and 3 month, and Th17and Tregs were analysed by flow cytometry. Results Clinical and histological evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. Th17 frequency was unchanged, but Tregs frequency was enhanced in the LN of MR16–1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4+CD8−FoxP3+. Tregs phenotype was also modified in treated mice, with an increased frequency of CD39+ Tregs (LN and spleen), suggesting an enhanced ATP hydrolysis immunosuppressive activity of Tregs. In RA patients, Th17 frequencies were not modified by tocilizumab therapy and did not differ between responders and non-responders. Interestingly, CD39+ Treg cell among CD4+ cells frequencies were significantly higher in responders than in non-responders after 3 months of tocilizumab therapy. Conclusions Tregs, but not Th17, are modified by anti-IL-6R treatment in both CIA and RA. These results support a beneficial effect in RA of treatments responsible for CD39+ Tregs enhancement and emphasise the relevance of the monitoring cell populations after cytokine blockade used to treat arthritis.

Non–TNF-Targeted Biologic vs a Second Anti-TNF Drug to Treat Rheumatoid Arthritis in Patients With Insufficient Response to a First Anti-TNF Drug: A Randomized Clinical Trial

ImportancenOne-third of patients with rheumatoid arthritis show inadequate response to tumor necrosis factor α (TNF-α) inhibitors; little guidance on choosing the next treatment exists.nnnObjectivenTo compare the efficacy of a non-TNF-targeted biologic (non-TNF) vs a second anti-TNF drug for patients with insufficient response to a TNF inhibitor.nnnDesign, Setting, and ParticipantsnA total of 300 patients (conducted between 2009-2012) with rheumatoid arthritis, with persistent disease activity (disease activity score in 28 joints-erythrocyte sedimentation rate [DAS28-ESR] u2009≥u20093.2 [range, 0-9.3]) and an insufficient response to anti-TNF therapy were included in a 52-week multicenter, pragmatic, open-label randomized clinical trial. The final follow-up date was in August 2013.nnnInterventionsnPatients were randomly assigned (1:1) to receive a non-TNF-targeted biologic agent or an anti-TNF that differed from their previous treatment. The choice of the biologic prescribed within each randomized group was left to the treating clinician.nnnMain Outcomes and MeasuresnThe primary outcome was the proportion of patients with good or moderate response according to the European League Against Rheumatism (EULAR) scale at week 24. Secondary outcomes included the EULAR response at weeks 12 and 52; at weeks 12, 24, and 52; DAS28ESR, low disease activity (DAS28 ≤3.2), remission (DAS28 ≤2.6); serious adverse events; and serious infections.nnnResultsnOf the 300 randomized patients (243 [83.2%] women; mean [SD] age, 57.1 [12.2] years; baseline DAS28-ESR, 5.1 [1.1]), 269 (89.7%) completed the study. At week 24, 101 of 146 patients (69%) in the non-TNF group and 76 (52%) in the second anti-TNF group achieved a good or moderate EULAR response (OR, 2.06; 95% CI, 1.27-3.37; Pu2009=u2009.004, with imputation of missing data; absolute difference, 17.2%; 95% CI, 6.2% to 28.2%). The DAS28-ESR was lower in the non-TNF group than in the second anti-TNF group (mean difference adjusted for baseline differences, -0.43; 95% CI, -0.72 to -0.14; Pu2009=u2009.004). At weeks 24 and 52, more patients in the non-TNF group vs the second anti-TNF group showed low disease activity (45% vs 28% at week 24; OR, 2.09; 95% CI, 1.27 to 3.43; Pu2009=u2009.004 and 41% vs 23% at week 52; OR, 2.26; 95% CI, 1.33 to 3.86; Pu2009=u2009.003).nnnConclusions and RelevancenAmong patients with rheumatoid arthritis previously treated with anti-TNF drugs but with inadequate primary response, a non-TNF biologic agent was more effective in achieving a good or moderate disease activity response at 24 weeks than was the second anti-TNF medication.nnnTrial Registrationnclinicaltrials.gov Identifier: NCT01000441.

The micrornas as biomarkers in knee osteoarthritis

Among the new biomarkers associated with rheumatoid arthritis (RA), we describe the microRNA (miRNAs). miRNAs are small RNAs of 21–23 nucleotides able to inhibit gene expression. The objectives of this work are to identify the original miRNAs as biomarkers in two different chronic bone and joint diseases, osteoarthritis (OA) and RA.nnSerum samples and fresh …

Reply: To PMID 24504799.

arthritis. Arthritis Rheumatol 2014;66:273–83. 2. Samson M, Audia S, Janikashvili N, Ciudad M, Trad M, Fraszczak J, et al. Inhibition of interleukin-6 function corrects Th17/Treg cell imbalance in patients with rheumatoid arthritis. Arthritis Rheum 2012;64:2499–503. 3. Van Gestel AM, Prevoo ML, van ’t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL. Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis: comparison with the preliminary American College of Rheumatology and the World Health Organization/ International League Against Rheumatism criteria. Arthritis Rheum 1996;39:34–40. 4. Pesce B, Soto L, Sabugo F, Wurmann P, Cuchacovich M, Lopez MN, et al. Effect of interleukin-6 receptor blockade on the balance between regulatory T cells and T helper type 17 cells in rheumatoid arthritis patients. Clin Exp Immunol 2013;171:237–42. 5. Komatsu N, Okamoto K, Sawa S, Nakashima T, Oh-hora M, Kodama T, et al. Pathogenic conversion of Foxp3 T cells into TH17 cells in autoimmune arthritis. Nat Med 2014;20:62–8.

BIK is a novel pro-apoptotic target gene for miR-125b in human monocytes

IntroductionThe high numbers of activated cells in rheumatoidarthritic (RA) joints critically contribute to the persis-tence of chronic disease and have been associated withtheir resistance to undergo apoptosis [1]. Activity ofpro- and anti-apoptotic Bcl-2 family members is con-strained by transcriptional and/or post-transcriptionalcontrols. As part of the post-transcriptional gene regula-tory network machinery, micro(mi)RNAs represent animportant class of endogenous, short, non-coding RNAthat decreases gene expression by pairing to target tran-scripts and inducing gene silencing. Among miRNAsthat have been assigned oncogenic and/or tumor sup-pressor-like functions, miR-125b is of particular interest.Among the dozen of genes that are identified so far asmiR-125b targets, three are directly involved in apopto-sis, all encoding for pro-apoptotic proteins: Bmf, BAK1and p53 [2-4].AimTo provide novel insight into the integrated genetic reg-ulatory network specifying cell fate, we have exploredother pro-apoptotic members of the Bcl-2 family in thecontext of miR-125b and analyzed their respectiveexpression levels in RA.MethodsmiRNA databases were usedto detect potential miR-125b responsive elements within the 3’-UTR mRNA ofpro- and anti-apoptotic genes. The human monocyticcell line THP-1 was co-transfected with the reporter sys-tem containing the BIK 3’-UTR down-stream of theluciferase together with either a synthetic miR-125bprecursor or antagonist. Inhibition of the luciferaseactivity was compared with cells transfected with themock construct. BIK expression and apoptosis weremonitored at mRNA and protein levels after transfectionof THP-1 monocytes with pre- or antago-miR-125b.Finally, miR-125b and BIK were quantified by RT-qPCRin blood samples from RA and healthy donors.ResultsWe identify miR-125b, whose expression was highly cor-related with patients with RA, as a post-transcriptionalregulator of the pro-apoptotic BH3-like protein BIK.Moreover, enforced expression of miR-125b in humanmonocytes promoted apoptosis by targeting BIK in adose-dependent manner, and broadly impacted apopto-sis-related genes as assessed by transcriptomic analysis.ConclusionUnderstanding the mechanisms that alter the balance ofanti- and pro-apoptotic proteins and identifying keymolecules able to re-establish tissue homeostasis willopen novel fundamental and therapeutic potentials.

Cardiac Complications Attributed to Chloroquine and Hydroxychloroquine: A Systematic Review of the Literature

IntroductionChloroquine and hydroxychloroquine are widely used in the long-term treatment of connective tissue disease and usually considered safe. However, chloroquine- or hydroxychloroquine-related cardiac disorder is a rare but severe adverse event, which can lead to death. This systematic review investigates cardiac complications attributed to chloroquine and hydroxychloroquine.MethodsPubMED, EMBASE, and Cochrane database searches were conducted using keywords derived from MeSH terms. Reports published prior to 31 July, 2017 were eligible for inclusion, without restriction to study design. Searches were also conducted on reference lists of included studies.ResultsEighty-six articles were identified, reporting individual cases or short series, providing information on 127 patients (65.4% female). A majority of patients were treated with chloroquine (58.3%), with the remaining treated with hydroxychloroquine (39.4%), or both in succession. Most patients had been treated for a long time (median 7xa0years, minimum 3xa0days; maximum 35xa0years) and with a high cumulative dose (median 1235xa0g for hydroxychloroquine and 803xa0g for chloroquine). Conduction disorders were the main side effect reported, affecting 85% of patients. Other non-specific adverse cardiac events included ventricular hypertrophy (22%), hypokinesia (9.4%), heart failure (26.8%), pulmonary arterial hypertension (3.9%), and valvular dysfunction (7.1%). For 78 patients reported to have been withdrawn from treatment, some recovered normal heart function (44.9%), while for others progression was unfavorable, resulting in irreversible damage (12.9%) or death (30.8%).LimitationsThe risk of cardiac complications attributed to chloroquine/hydroxychloroquine was not quantified because of the lack of randomized controlled trials and observational studies investigating the association.ConclusionsClinicians should be warned that chloroquine- or hydroxychloroquine-related cardiac manifestations, even conduction disorders without repercussion, may be initial manifestations of toxicity, and are potentially irreversible. Therefore, treatment withdrawal is required when cardiac manifestations are present.

04.03 Tgfβ-induced protein (tgfβi) is dysregulated in osteoarthritis

Backgrounds and objectives Osteoarthritis (OA) is the most common rheumatic disease affecting all joint tissues and Transforming Growth Factor β (TGFβ) pathway dysregulation in bone marrow mesenchymal stem cells (MSCs) has been proposed to be involved in OA physiopathology. Based on secretome analysis of MSCs, we identified several TGFβ family members and focused our attention on Transforming Growth Factor β-Induced (TGFβI), a poorly studied extracellular matrix (ECM) component. Materials and methods Human bone marrow MSCs were isolated from OA patients (OA-MSCs) and healthy donors (H-MSCs) while chondrocytes were isolated from OA patients (OA-CH). Chondrogenic differentiation of MSCs was induced in micropellet for 21 days. TGFβI expression was analysed by RT-qPCR and ELISA. Murine articular chondrocytes were isolated from 3 days old C57/BL6 mice and OA-like murine chondrocytes were obtained by addition of 1u2009ng/mL IL1β for 24u2009hour. Transfection of siRNA directed against TGFβI (siTGFβI) was performed using oligofectamine. The collagenase-induced murine model of OA was used for immunohistochemical analysis of cartilage and RNA extraction with TRIzol and acid phenol. Results In humans, TGFβI mRNA expression was 2.5 fold lower in OA-MSCs than in H-MSCs and 10 fold lower in OA-MSCs than in OA-CH. We also found out that TGFβI was highly induced (by a 10 fold factor) during the first three days of chondrogenic differentiation but decreased to reach levels similar to those found in MSCs. In the mouse, immunohistochemical analysis revealed high expression of TGFβI in healthy cartilage as compared to OA cartilage. In murine MSCs, expression of TGFβI is 3.8 fold lower than in chondrocytes. In OA-like chondrocytes, characterised by reduced expression of anabolic markers and increased levels of catabolic markers, TGFβI mRNA levels were significantly lower than in healthy chondrocytes. Rapid decline of TGFβI expression was also observed in OA-like dedifferentiated chondrocytes. Finally, transfection of siTGFβI in murine chondrocytes resulted in alteration of their metabolic activity. Conclusion Altogether, our results indicate that expression of TGFβI is higher in articular cartilage than in MSCs, and loss of TGFβI is associated with OA phenotype. TGFβI might be a key regulator of joint homeostasis involved in chondrogenesis and ECM integrity.