Monocytes and macrophages are mediator cells of cartilage and bone erosion in the synovia of rheumatoid arthritis (RA) patients due to secretion of the inflammatory cytokine Interleukin-1β (IL-1β). Calcium, phosphate and fetuin are liberated from the affected bone matrix, and the formation of calciproteinparticles (CPPs) is likely. IL-1β production in monocytes in vitro is stimulated by high concentrations of extracellular calcium. Additionally, the rise of extracellular calcium concentrations leads to increased macropinocytosis in mononuclear phagocytes. Flow cytometry analyses in this study show that peripheral blood monocytes from patients with RA perform more calcium stimulated macropinocytosis of the fluorescent dye calcein than monocytes from healthy donors. Stimulation of monocytes with calcium and preformed CPPs leads to more IL-1β production, quantified using ELISA, by monocytes from RA patients. Experiments with macrophages show similar results. Furthermore calcium-stimulated macropinocytosis and IL-1β secretion are significantly positively correlated. However, there was no connection of in vitro findings and the severity of RA in patients.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Rheumatoide Arthritis 1 1.1.1 Epidemiologie und Klinik der rheumatoiden Arthritis 1 1.1.2 Ätiopathogenese der rheumatoiden Arthritis 1 1.2 Monozyten und Makrophagen 3 1.2.1 Inflammasomaktivierung und Interleukin-1β-Sekretion in Monozyten und Makrophagen 4 1.2.2 Makropinozytose in Monozyten und Makrophagen 6 1.2.3 Beitrag der Monozyten und Makrophagen zur rheumatoiden Arthritis 7 1.3 Kalzium – lokale Dysregulation trotz systemischer Regulation 9 1.3.1 Entstehung von Kalziumproteinpartikeln 10 1.3.2 Kurzportrait des G-Protein-gekoppelten Kalziumrezeptors CaSR 11 2 Fragestellungen 13 3 Forschungsdesign, Material und Methoden 15 3.1 Forschungsdesign 15 3.2 Materialien 15 3.2.1 Laborgeräte 16 3.2.2 Verbrauchsmaterialien 17 3.2.3 Materialien und Chemikalien 17 3.2.4 Medien, Lösungen und Puffer 19 3.2.5 Stimulanzien und Inhibitoren 20 3.2.6 Fluoreszenzfarbstoffe 20 3.2.7 Software 20 3.3 Methoden 21 3.3.1 Separation von PBMCs mittels Ficolldichtegradientenzentrifugation 21 3.3.2 Separation von Monozyten mittels negativer Magnetseparation 22 3.3.3 Differenzierung von Monozyten zu Makrophagen in Zellkulturbeuteln 23 3.3.4 Makropinozytose von Monozyten und Makrophagen in der Durchflusszytometrie 23 3.3.4.1 Makropinozytose von Calcein in Monozyten 24 3.3.4.2 Makropinozytose fluoreszenzgefärbter Kalziumproteinpartikel in Monozyten und Makrophagen 25 3.3.4.3 Inhibition der Makropinozytose in Monozyten 26 3.3.4.4 Auswertung der am Durchflusszytometer generierten Rohdaten mit FlowJo 26 3.3.5 Makropinozytose von Monozyten in der Fluoreszenzmikroskopie 28 3.3.6 Bestimmung der Interleukin-1β-Produktion von Monozyten und Makrophagen mittels ELISA 29 3.3.7 Erhebung des DAS28 33 3.3.8 Bestimmung von Laborparametern 33 3.4 Statistische Auswertung 33 4 Ergebnisse 35 4.1 Charakterisierung der Kohorten 35 4.2 Vorversuche zur Auswahl eines geeigneten Fluoreszenzfarbstoffes für die Detektion der Makropinozytose 37 4.3 Stimulation von Monozyten mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion 39 4.3.1 Kalziumstimulierte Calceinaufnahme von Monozyten 39 4.3.2 Kalziumstimulierte Interleukin-1β-Produktion von Monozyten 44 4.4 Stimulation von Monozyten mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion unter Zugabe von Kalziumproteinpartikeln 47 4.4.1 Kalziumstimulierte Aufnahme fluoreszierender Kalziumproteinpartikel 47 4.4.2 Kalziumstimulierte Interleukin-1β-Produktion in Monozyten unter Zugabe von Kalziumproteinpartikeln 51 4.5 Stimulation von Makrophagen mit Kalzium zur Makropinozytose und Interleukin-1β-Produktion 53 4.5.1 Kalziumstimulierte Makropinozytose von fluoreszierenden Kalziumproteinpartikeln in Makrophagen 53 4.5.2 Kalziumstimulierte Interleukin-1β-Produktion mit und ohne Zugabe von Kalziumproteinpartikeln in Makrophagen 54 4.5.3. Visualisierung von Monozyten und Makrophagen nach 16 Stunden Inkubation 57 4.6 Korrelation zwischen kalziumstimulierter Makropinozytose und Interleukin-1β-Sekretion 59 5 Diskussion 61 5.1 Kalziumstimulierte Makropinozytoseaktivität von Monozyten und Makrophagen 61 5.2 Kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen 64 5.2.1 Auswirkung der Phosphatkonzentration im Zellkulturmedium auf die kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen 65 5.2.2 Kalziumstimulierte Interleukin-1β-Sekretion von Monozyten und Makrophagen von RA-Patienten und Kontrollprobanden 66 5.3 Zusammenhang von kalziumstimulierter Makropinozytose und Interleukin-1β-Sekretion in Monozyten und Makrophagen von RA-Patienten und Kontrollprobanden 70 5.4 Ausblick und offene Fragen 71 6 Zusammenfassung der Arbeit 73 8 Erklärung über die eigenständige Abfassung der Arbeit 88 9 Danksagung 89

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L’arthrose et l’arthrite rhumatoïde sont deux pathologies articulaires caractérisées par la dégradation du cartilage articulaire, subséquente à la production de divers médiateurs inflammatoires. Le traitement de ces pathologies se limite généralement à soulager le patient des épisodes douloureux et inflammatoires et à améliorer sa qualité de vie. Dans le cas de l’arthrose, peu de traitements permettent d’enrayer significativement l’évolution de la dégradation du cartilage et donc de la maladie. Par contre, l’arthrite rhumatoïde peut être efficacement ralentie grâce à l’administration de certaines molécules. Néanmoins, ces traitements n’ont généralement montré qu’une efficacité à court-terme, requérant une administration fréquente. L’objectif de ce travail repose donc sur l’élaboration de nouvelles options thérapeutiques permettant de réduire la fréquence d’administration ainsi que les effets indésirables des traitements actuels. La délivrance de molécules en intra-articulaire associée à une libération prolongée offre l’avantage d’exposer les sites directement impliqués dans l’évolution de la maladie à une ou plusieurs molécules efficaces contre l’inflammation et la douleur, et aidant à la régénération du cartilage, durant plusieurs semaines, voire des mois.<p>Des microparticules de PLGA chargées en clonidine ou en bétaméthasone ont donc été optimisées afin d’obtenir des efficacités d’encapsulation appréciables (clonidine HCl :EE ≈ 20% ;dipropionate de bétaméthasone :EE ≈ 70%), une taille adaptée à l’administration intra-articulaire (12 – 38 µm) et une libération de la molécule s’échelonnant sur 5 à 8 semaines. La libération prolongée de la clonidine implique des mécanismes de diffusion de la molécule ainsi que de dégradation/érosion du polymère. Au vu de l’absence de réaction inflammatoire, les microparticules développées sont correctement tolérées par les chondrocytes, synoviocytes, PBMC et neutrophiles, principales cellules impliquées dans les mécanismes inflammatoires de l’arthrose et de l’arthrite rhumatoïde. L’évaluation de l’efficacité anti-inflammatoire des microparticules vides et chargées en clonidine ou en bétaméthasone via l’étude de l’expression et de la sécrétion de différents médiateurs de l’inflammation a permis d’aboutir à plusieurs conclusions :(i) les microparticules vides sont associées à un effet anti-inflammatoire, (ii) les microparticules chargées en clonidine n’ont pas montré d’activité anti-inflammatoire propre pouvant être attribuée à la clonidine, et (iii) les microparticules de bétaméthasone ont confirmé l’effet anti-inflammatoire de la bétaméthasone. Enfin, l’étude de la toxicité des principes actifs et microparticules vides ou chargées a montré une toxicité significative de la clonidine sur les synoviocytes. Néanmoins, l’encapsulation des principes actifs dans les microparticules de PLGA a permis d’éliminer cette toxicité, protégeant donc efficacement les cellules articulaires.<p>Les microparticules développées permettent alors d’envisager l’encapsulation d’autres molécules anti-inflammatoires ou une combinaison de molécules ayant des effets complémentaires (anti-inflammatoire et antidouleur). L’utilisation de la clonidine dans ces indications devra être réévaluée en étudiant de façon approfondie son efficacité dans la douleur. / Both osteoarthritis and rheumatoid arthritis are articular diseases characterized by the degeneration of the joint cartilage, resulting from the production of various inflammatory mediators. The current treatment of these diseases is restricted to alleviate the painful and inflammatory episodes of the patients and to improve its quality of life. In osteoarthritic patients, few treatments allow to significantly stop the evolution of the degradation of the cartilage and, consequently, the disease. In rheumatoid arthritis, the evolution can be slowed down following the administration of some drugs. Nevertheless, these treatments are often associated to a short-term efficacy. The objective of this work is to develop new therapeutic options that allow to reduce the frequency of administration and the side effects of the current treatments. The intraarticular delivery combined to controlled-release presents the advantage to expose the sites directly involved in the evolution of the disease to one or more molecules effective to relieve the pain, inflammation and to help the regeneration of the cartilage.<p>Clonidine or betamethasone-loaded PLGA microparticles were optimized to reach suitable encapsulation efficiencies (clonidine HCl: EE ≈ 20%; betamethasone dipropionate: EE ≈ 70%), an appropriate size for an intraarticular delivery (12 – 38 µm) and a controlled-release of the molecule over 5 to 8 weeks. The release of clonidine implies mechanisms of diffusion and degradation/erosion of the polymer. Given the absence of an inflammatory reaction, the developed microparticles were properly tolerated by the chondrocytes, synoviocytes, PMBC and neutrophils, which are the main cells involved in the inflammatory reaction of osteoarthritis and rheumatoid arthritis. The assessment of the anti-inflammatory efficacy of the drug-free and drug-loaded microparticles through the evaluation of the expression and the secretion of various inflammatory mediators allowed to draw several conclusions: (i) drug-free microparticles were associated to an anti-inflammatory effect, (ii) clonidine-loaded microparticles did not show any anti-inflammatory activity that could be attributed to clonidine, and (iii) betamethasone- loaded microparticles confirmed the anti-inflammatory effect of betamethasone. Finally, the evaluation of the toxicity of the drugs and microparticles showed a significant toxicity of clonidine against synoviocytes. Nevertheless, the encapsulation of the drugs in PLGA microparticles induced the suppression of this toxicity, protecting in this way the articular cells. <p>Entrapping other anti-inflammatory molecules or a combination of molecules with complementary effects (anti-inflammatory and anti-nociceptive drugs) in the PLGA microparticles developed has to be considered. Moreover, the use of clonidine in these indications has to be reassessed by a thorough study of its anti-nociceptive potential.<p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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Le costimulateur inductible (ICOS) est un récepteur costimulatoire des cellules T crucial pour l’immunité humorale. Chez l’humain, une déficience de ICOS est associée à des infections récurrentes dû à des défauts de différentiation des lymphocytes T auxiliaires folliculaires (Tfh) et un manque d’anticorps protecteurs. Paradoxalement, certains patients déficients en ICOS développent des signes d’autoimmunité causés par des autoanticorps. Ces observations sont en accord avec des résultats suggérant que ICOS joue un double rôle dans la promotion des fonctions de cellules T effectrices et de cellules T régulatrices. L’arthrite rhumatoïde (RA) est une maladie autoimmune caractérisée par l’inflammation articulaire et la destruction progressive des os. Dans la première étude présentée au Chapitre 2, nous avons démontré que ICOS est requis pour l’initiation et la maintenance de l’arthrite induite par le collagène (CIA), un modèle murin de la RA. Nous avons démontré que des événements clés de l’initiation de la CIA incluant la production d’anticorps contre le collagène et la prolifération de cellules T inflammatoires sont dépendants de la voie de signalisation ICOS-PI3K. Aussi, nous avons trouvé que la signalisation par ICOS-PI3K promouvoit le maintien de la CIA. De plus, nous avons établi que l’inhibition de la glycolyse réduit la sévérité de la CIA, ce qui suggère un chevauchement entre la signalisation ICOS-PI3K et le métabolisme du glucose dans la pathogenèse de l’arthrite autoimmune. Dans la deuxième étude présentée au Chapitre 3, nous avons utilisé des souris dont les cellules T régulatrices sont déficientes en ICOS (ICOS FC) pour évaluer l’impact de ICOS dans les cellules Tfr. Nous avons trouvé que les souris ICOS FC possèdent un taux réduit de cellules Tfr, mais aucune diminution des cellules T régulatrices (Treg) précurseurs. De plus, les souris ICOS FC ont un taux élevé de cellules B du centre germinatif (GC) non-spécifiques ainsi qu’une production accrue d’anticorps anti-nucléaires. Nous avons aussi observé une perturbation des réponses anti-virales et de la production d’anticorps spécifiques dans les souris ICOS FC ce qui suggère des rôles non-régulateurs pour les cellules Tfr. Nous avons effectué une analyse de transcriptome de cellule unique avec des Tregs et nous avons observé une accumulation de précurseurs de Tfr dans les souris ICOS FC, ce qui suggère un défaut de la transition Treg à Tfr. Nos données suggèrent que ICOS participe à la différentiation des cellules Tfr en régulant KLF2 et NFAT2, ce qui contribue à l’établissement de traits folliculaires. En résumé, nous avons démontré que ICOS promouvoit le développement de l’arthrite autoimmune en soutenant les fonctions des cellules T inflammatoires par la voie de signalisation ICOS-PI3K. De plus, nos résultats prouvent que ICOS peut prévenir la production d’autoanticorps en supportant la différentiation des cellules Tfr. Notre travail contribue à l’avancement des connaissances en ce qui concerne le rôle double de ICOS dans l’immunité cellulaire et humorale et fournit des paramètres importants à considérer lors de la recherche de nouvelles cibles thérapeutiques. / The inducible costimulator (ICOS) is a T cell costimulatory receptor crucial for humoral immunity. In humans, ICOS deficiency is associated with recurrent infections due to defects in T follicular helper (Tfh) differentiation and lack of protective antibodies. Paradoxically, some ICOS-deficient patients were found to exhibit signs of antibody-mediated autoimmunity. These observations are congruent with findings suggesting that ICOS plays a dual role in promoting T effector and T regulatory cell functions. Due to this ambivalence, the role of ICOS in autoimmunity remains unresolved. Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and progressive bone destruction. The number of ICOS+ T cells in the synovial tissues of RA patients was found to be elevated, suggesting a potential involvement of ICOS signaling in the pathogenesis of RA. In the first study presented in Chapter 2, we showed that ICOS is required for the initiation and maintenance of collagen-induced arthritis (CIA), a murine model of RA. We found that key CIA initiation events such as anti-collagen antibody production and inflammatory T cell proliferation were dependent on ICOS-PI3K signaling. Further, we found that ICOS-PI3K signaling promotes maintenance of established CIA. Additionally, we show that glycolysis inhibition ameliorates CIA, thus suggesting potential overlaps between ICOS-PI3K signaling and glucose metabolism in the pathophysiology of autoimmune arthritis. The initiation of autoimmunity depends on the action of autoimmune effector cells, but also on a failure of regulatory cells. Amongst the latter, T follicular regulatory (Tfr) cells are thought to prevent autoantibody production. In the second study presented in Chapter 3, we used regulatory T cell-specific ICOS knockout (ICOS FC) mice to investigate the impact of ICOS signaling in Tfr cells. We found that ICOS FC mice display a significant reduction in Tfr cell numbers, but no depletion of their T regulatory (Treg) precursors. Further, ICOS FC mice exhibited a rise of extraneous germinal center (GC) B cells numbers and increased production of anti-nuclear antibodies. We also observed disruptions of anti-viral responses and antigen-specific antibody production in ICOS FC mice, suggesting non-regulatory roles for Tfr cells. We performed single-cell transcriptome analysis of regulatory T cells and observed an accumulation of Tfr precursors in ICOS FC mice suggestive of a Treg-to-Tfr transition defect. Mechanistically, we found that ICOS participates in Tfr differentiation by regulating KLF2 and NFAT2, thereby contributing to the establishment of follicular T cell traits. In sum, we demonstrate that ICOS promotes the development of autoimmune arthritis by fostering inflammatory T cell responses in a PI3K-dependent manner. In addition, our work shows that ICOS can prevent autoantibody production by supporting Tfr differentiation. Thus, we contribute insights into the dual role of ICOS in the cellular and humoral arms of autoimmunity, providing important parameters to be considered when searching for novel therapeutic targets.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with high levels of tumour necrosis factor-alpha (TNF-a) in synovial fluid and synovial tissue (Saxne et al., 1989). Thalidomide is a proven inhibitor of the biological synthesis of TNF-a (Sampaio et al., 1991) and is believed to rely on this action for its suppression of the wasting of tissue which accompanies RA. Oral administration of thalidomide has proven to be effective in RA, but unacceptable side effects are easily provoked (Gutierrez-Rodriguez, 1984). Administration of thalidomide via the dermal route can down-regulate TNF-a production in and around the affected joint, and this without raising the systemic blood level to a problematical level. Based on thalidomide's physicochemical properties, it is unlikely that it can be delivered percutaneously at a dose required for RA. Therefore, we have embraced the idea of using N-alkyl analogues of thalidomide. The most important feature that an analogue of this compound might contribute is decreased crystallinity and increased lipophilicity. Ordinarily both these parameters should favour percutaneous delivery. The current study was primarily aimed at exploring the feasibility of percutaneous delivery of thalidomide and subsequently, three of its odd chain IV-alkyl analogues (methyl, propyl and pentyl) via physicochemical characterization and assessment of their innate abilities to diffuse through skin as an initial step towards developing a topical dosage form for the best compound. The biological activities, more specifically their potential to inhibit the production of TNF-a was determined for thalidomide and its N-alkyl analogues. In order to achieve the objectives, the study was undertaken by synthesizing and determining the physicochemical parameters of thalidomide and its N-alkyl analogues. A high level of crystallinity is expressed in the form of a high melting point and heat of fusion. This limits solubility itself, and thus also sets a limit on mass transfer across the skin. Generally, the greater a drug's innate tendency to dissolve, the more likely it is that the drug can be delivered at an appropriate rate across the skin (Ostrenga et al., 1971). Therefore, the melting points and heats of fusion were determined by differential scanning calorimetry. Aqueous solubility and the partition coefficient (relative solubility) are major determinants of a drug's dissolution, distribution and availability. N-octanollwater partition coefficients were determined at pH 6.4. Solubilities in water, a series of n-alcohols and mixed solvents were obtained, as well as the solubility parameters of the compounds in study. Secondly, in vitro permeation studies were performed from these solvents and vehicles using vertical Franz diffusion cells with human epidermal membranes. Thirdly, tumour necrosis factor-alpha (TNF-a) inhibition activities were assessed for thalidomide and its N-alkyl analogues. By adding a methyl group to the thalidomide structure, the melting point drops by over 100°C and, in this particular instance upon increasing the alkyl chain length to five -CH2- units the melting points decrease linearly. Heats of fusion decreased dramatically upon thalidomide's alkylation as well. Methylation of the thalidomide molecule enhanced the aqueous solubility 6-fold, but as the alkyl chain length is further extended from methyl to pentyl, the aqueous solubility decreased exponentially. The destabilization of the crystalline structure with increasing alkyl chain length led to an increase in lipophilicity and consequently an increase in solubility in nonpolar media. Log partition coefficients increased linearly with increasing alkyl chain length. Solubilities in a series of n-alcohols, methanol through dodecanol, were found to be in the order of pentyl > propyl > methyl > thalidomide. The N-alkyl analogues have more favourable physicochemical properties than thalidomide to be delivered percutaneously. The in vitro skin permeation data proved that the analogues can be delivered far easier than thalidomide itself. N-methyl thalidomide showed the highest steady-state flux through human skin from water, n-alcohols and combination vehicles. Thalidomide and its N-alkyl analogues were all active as TNF-a inhibitors. Finally, active as a TNF-a inhibitor, N-methyl thalidomide is the most promising candidate to be delivered percutaneously for treatment of rheumatoid arthritis, of those studied. / Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

Background: The ability to measure health and the value of improving or declining health is crucial to the evaluation of health care interventions. Many generic and disease specific health status measures exist for use in patients with rheumatoid arthritis (RA). The Overall Status in Rheumatoid Arthritis (OSRA) measure is a new and simple measure with early evidence of construct validity. Generic health profiles with attached utility weights such as the EuroQol EQ-5D and the SF-6D (calculated from the Medical Outcome Study 36-item short-form health survey) allow the quantification of a patient's health relative to perfect health and death, and can be used to estimate quality adjusted life years (QALYs). The EQ-5D is extensively used in RA, but has potential limitations. The SF-6D appears to have potential, but needs further evaluation. The aim of this thesis was to assess the validity and responsiveness of the EQ5D, SF-6D and OSRA in UK RA patients, and compare the performance and implications of the use of the EQ-5D and SF-6D.Methods and subjects: Patient data were obtained from three sources; the Steroids in Very Early Arthritis (STIVEA) (n=256) and British Rheumatoid arthritis Outcome Study Group (BROSG) (n=466) randomised controlled trials, and the British Society for Rheumatology Biologics Register (BSRBR) (n=129). The data used included lifestyle and demographic factors, disease activity (DAS28), functional disability (HAQ), X-rays to assess erosive damage, the EQ-5D and the SF-6D. The OSRA was collected only in the BROSG trial. Visual analogue scales (VAS) of pain and fatigue were collected in BROSG and STIVEA. Construct validity was tested by correlating the EQ-5D, SF-6D and OSRA with a range of outcome measures for RA. Responsiveness to change was assessed using minimum important differences (MID), effect size (ES) and standardised response means (SRM), and compared using ratios. EQ-5D profiles placing arthritis patients in utility states 'worse than death' (negative scores) were described and assessed using linear and logistic regression. The implications of using the EQ-5D and SF-6D in economic evaluation were compared by cost-effectiveness analyses of the BROSG trial. Results: The correlation of the EQ-5D and SF-6D was moderate to high (0.67). Both measures had moderate to high correlations with disease activity, physical function, joint damage and fatigue. The OSRA Activity (OSRA-A) and Damage (OSRA-D) correlated strongly with measures of related aspects of disease. The EQ-5D, SF-6D and OSRA discriminated between known differences in health status across groups defined by social deprivation and disease activity. The EQ-5D MID was 0.04 for improvement and 0.10 for deterioration. The SF-6D MID was 0.04 in both directions. The SF-6D was more responsive to improvement (EQ-5D: SF-6D ES ratio 0.78-0.88) and the EQ-5D more responsive to deterioration (ES ratio 1.14) in health. The OSRA-A was the most sensitive disease specific measure in the BROSG trial, and the OSRA-D was more responsive than the HAQ. The factors associated with being in a 'worse than death' health state were male gender, the HAQ, SF-36 mental composite scale, pain VAS, and erythrocyte sedimentation rate (a marker of inflammation). Pain was the predominant factor and was scored at the most extreme level in every worse than death profile. The cost-effectiveness analyses (BROSG trial), found net quality adjusted life years (QALYs) were greater for the EQ-5D (0.07) than the SF-6D (0.05), but had higher variance than the SF-6D. Conclusions: The EQ-5D and SF-6D appear valid and responsive to changes in health in RA, but measure subtly different aspects of health. There are issues with both measures, and cost-effectiveness conclusions of a study could differ according to which measure was used. The EQ-5D may be more likely to demonstrate that an intervention is cost effective than the SF-6D, due to its larger mean change in response to change in health status. The OSRA is valid for use in RA and its responsiveness suggests potential for inclusion in clinical trials.

La polyarthrite rhumatoïde (PR) est le plus fréquent des rhumatismes inflammatoires et représente un problème de santé publique majeur. A l'heure actuelle, les biothérapies anti-TNF sous forme de protéines recombinantes constituent une avancée considérable dans le traitement de la polyarthrite rhumatoïde (PR). Néanmoins, il convient de développer des approches thérapeutiques alternatives pour traiter les 40% de patients non-répondeurs ainsi queceux qui échappent à plusieurs années de traitement. La recherche de nouvelles cibles thérapeutiques est indispensable pour proposer des approches alternatives à ces biothérapies. Par ailleurs, les techniques de transfert de gène offrent une alternative thérapeutique possible pour pallier aux limitations des biothérapies actuelles, à condition de les adapter aux contraintes du tissu cible de la PR, les articulations. Les projets ont consisté à développer et valider dans des modèles expérimentaux d'arthrite de nouvelles stratégies anti-inflammatoires basées sur l'utilisation de l'ARN interférence comme outil thérapeutique. En effet, la possibilité d'interférer au niveau des mécanismes responsables de l'expression des protéines,la régulation de la stabilité des ARNm et de l'efficacité de la machinerie traductionnelle, présente un intérêt thérapeutique supérieur aux biothérapies actuelles basées sur l'inhibition des protéines sécrétées (anticorps ou récepteurs solubles) mais nécessite cependant de posséder un vecteur qui transduit efficacement les cellules productrices de la molécule ciblée. / Rheumatoid arthritis (RA) is the most frequent chronic inflammatory systemicautoimmune disease that remains a major medical challenge as the exact causes of the disease are not completely elucidated. The principal treatment strategies arebased on the inhibition of TNF-α, one of the major inflammatory cytokine in RA.Although risk and benefit analyses are in favour of the use of monoclonal antibodiesagainst TNF-α, the most currently used biotherapy, they are not devoid from multipleside effects. The search for new therapeutic targets is essential to proposealternative approaches to non responders to such biotherapies. The possibility to interfere in the mechanisms responsible for regulating mRNA stability andeffectiveness of the translational machinery also present a therapeutic benefitsuperior to current biologic therapies based on inhibition secreted proteins(antibodies or soluble receptors). Such approach however requires developingvectors that efficiently transduced the specific cell type producing the targeted gene.Projects of my PhD fellowship have included both the development of gene therapyvehicles for RNAi-based intervention in experimental mouse models of arthritis andevaluation of novel candidate genes for alternative anti-inflammatory therapy in RA.

La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique qui touche les articulations synoviales. La PR se caractérise par une expansion pseudo-tumorale des cellules synoviales de types fibroblastiques (FLS) qui envahissent et détruisent les articulations. Le Facteur de Necrose Tumorale (TNF) alpha joue un rôle primordial dans cette pathologie et le blocage de son action constitue une thérapie efficace contre la PR. Il existe néanmoins des patients non-répondeurs aux anti-TNFs ce qui suggére la participation d'autres cytokines dans la PR. La Lymphotoxine (LT) alpha est le plus proche homologue du TNFalpha, mais son rôle dans la PR reste peu étudié et le potentiel thérapeutique de son blocage pour traiter la maladie doit encore être établi. La déplétion des FLS hyperprolifératives constitue une autre stratégie pour le traitement de la PR. L'utilisation des membres de la Famille du TNF, TRAIL et Fas ligand (FasL), a été proposée pour induire la mort cellulaire pa r apoptose des FLS dans les articulations synoviales. Toutefois, ces cytokines sont pleiotropiques et peuvent causer des effets secondaires. Nous avons caractérisé les effets de la LTalpha, de TRAIL et FasL dans les FLS pour mieux comprendre ses rôles dans la PR. Nos résultas montrent que les niveaux sériques de la LTalpha sont augmentés dans la PR comparés aux patients sains ou atteints d'arthrose. Toute comme le TNFalpha, la LTalpha induit la prolifération et l'activation des FLS. Ainsi, l'inhibition simultanée de la LTalpha et du TNFalpha pourrait fournir un avantage thérapeutique dans le cadre des traitements contre la PR. Nous avons par ailleurs démontré que TRAIL est un facteur protecteur en début de la PR mais a un rôle promoteur au cours de la maladie. Le double rôle de TRAIL est corrélé à l'expression de TRAIL récepteur 1. Enfin, nous avons montré que FasL joue un rôle non-apoptotique dans les FLS, en modulant leur prolifération. Pour cette raison, une thérapie basée sur TRAIL ou FasL demande une sensibilisation à l'apoptose des FLS. / Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting synovial joints. A hallmark of RA is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), which invade and destroy the joint. Blocking of Tumor Necrosis Factor (TNF) alpha is effective to treat RA. However, some patients are nonresponsive to anti-TNF therapies, suggesting the participation of other cytokines in RA. Lymphotoxin (LT) alpha is the closest homologous to TNFalpha, but little is known about its role in RA and therapeutic potential of blocking this cytokine to treat RA. Another strategy to treat RA is the depletion of hyperproliferative FLS. The TNF family members TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) have been proposed for targeting FLS in arthritic joints. However, these cytokines are pleiotropic and can thus cause unwanted effects. We aimed to characterize the effects of LTalpha, TRAIL and FasL in RA FLS and better understand their role in the pathog enesis of RA. Our results show that serum levels of LTalpha are increased in RA compared to osteoarthritis and healthy controls and LTalpha induces proliferation and activation of RA FLS to the same extent that TNFalpha. Thus, simultaneous blocking of LTalpha and TNFalpha appears to be of benefit for RA patients. Additionally, we demonstrated that TRAIL could be a protective factor in the initial phase of RA but subsequently has a disease-promoting role. The dual role of TRAIL is correlated to TRAIL receptor 1 expression of RA FLS. Moreover, we showed that FasL induces non-apoptotic effects in RA FLS, such as proliferation. Therefore, a TRAIL or FasL based therapeutic strategy in RA requires sensitization for apoptosis of FLS.

Heparan sulfate (HS), an unbranched sulfated carbohydrate chain, and the HS-degrading enzyme heparanase play important roles in physiological and pathological processes during all stages of life, from early embryogenesis to ageing. Accumulated information shows that HS and heparanase are involved in inflammatory processes and associated diseases, e.g. rheumatoid arthritis (RA) and Alzheimer’s disease. In this thesis I have investigated the role of HS and heparanase (Hpa) in inflammatory-related pathologies. In the first project, Hpa overexpressing mice (Hpa-tg) were induced with a murine model of RA. We found a pro-inflammatory role of Hpa through enhancing the activity of T-cells and innate immune cells, which contributed to an augmented severity of clinical symptoms in the Hpa-tg mice. In my second project, we revealed co-current interaction of heparin with both ApoA1 and SAA of HDL isolated from plasma of inflamed mouse. Mass spectrometry analysis indicated close proximity of ApoA1 and SAA on the HDL surface, providing a molecular and structural mechanism for the simultaneous binding of heparin to apoA1 and SAA. In my third project, we investigated the role of Hpa in AA amyloid formation and resolution in mice in a model of AA-amyloidosis. We found a similar degree of amyloid formation in Hpa-KO mice compared to the wildtype control mice, but the resolution process was faster in Hpa-KO mice. The rapid clearance of deposited SAA in Hpa-KO mice was associated with upregulated expression of matrix metalloproteases. The results suggest an associated function of ECM proteases with heparanase in the process of AA amyloid resolution. In my fourth project, we found that overexpression of heparanase impaired inflammation associated beta amyloid (Aβ) clearance in the brain of an Alzheimer’s disease mouse model. Examination of the cytokine profile of brain lysates revealed an overall lower inflammatory reaction in the double transgenic (tgHpa*Swe) mice compared to single APP-tg (tg-Swe) mice in response to LPS-induced inflammation.