La polyarthrite rhumatoïde (PR) est la maladie auto-immune la plus fréquente d’une prévalence de 1%. Les cellules résidentes de la cavité synoviale, les fibroblast-like synoviocytes (FLS), sont des acteurs majeurs de la PR. Leur activation par des récepteurs de l’immunité innée participe à l’acquisition d’un phénotype agressif menant à la destruction ostéo-articulaire. Dans cette étude, nous avons évalué le rôle régulateur de miARN sur les voies de signalisation des Toll-like receptors (TLR). L’activation de TLR2 et de TLR4 dans les FLS induit la diminution de l’expression de plusieurs miARN, dont miR-19a et b (miR-19), alors que TLR2 est surexprimé. Nous avons pu ainsi montrer que miR-19 régule Tlr2 et que la transfection de mir-19 dans les FLS activés induit une diminution de l’expression de TLR2 et de la synthèse d’IL-6 et de MMP-3. Mir-19 appartient au cluster miR-17~92, dont l’expression est abaissée dans les FLS. Il code pour 6 miARN dont miR-20a. miR-20a est également sous-régulé après activation de TLR2 et TLR4 dans les FLS et les THP-1. Nous avons montré que miR-20a régule directement l’expression d’Ask1, impliquée et surexprimée après activation de TLR4. La transfection de miR-20a in vitro nous a permis de montrer que miR-20a contrôle l’expression d’ASK1 et induit une inhibition de la synthèse de cytokines majeures de la PR dans les FLS et les THP-1. Des résultats équivalents ont été obtenus ex vivo chez la souris. Ces travaux ont permis d’identifier dans les FLS rhumatoïdes des miARN anti-inflammatoires dont la baisse d’expression permet une augmentation de l’expression de TLR2 et d’ASK1. Ces miARN pourraient donc constituer de nouvelles cibles thérapeutiques. / Rheumatoid arthritis (RA) is the most frequently autoimmune disease with a prevalence of 1%. Resident cells of joints, the fibroblast-like synoviocytes (FLS), act as key players in RA. Their activation through Pattern-recognition receptors leads to an aggressive phenotype, leading in the osteo-articular destruction of the joints. In this study, we aimed to discuss the link between Toll-like receptors (TLR) and miRNA pathway. We established the down-regulation of a few miRNA when FLS were activated through TLR2 and TLR4, including miR-19a and miR-19b (miR-19). We showed that miR-19 regulates directly Tlr2 and that transfection of miR-19 mimics leads to a decrease of IL-6 and MMP-3 synthesis in FLS. miR-19 belongs to the cluster miR-17~92, which is also down-regulated in activated FLS. This primary transcript encodes for 6 miRNA, including miR-20a, which is also down regulated upon TLR2 and TLR4 activation in FLS and further in THP-1, a monocyte cell-line. Then, we validated the predicted regulation of miR-20a on Ask1, an important kinase involved in TLR4 pathway. The transfection of miR-20a mimics in vitro represses ASK1 expression and inhibits several major cytokines in RA both in FLS and THP-1. Further, we confirmed these results on ex vivo experiments on peritoneal macrophages. These works allowed us to identify new anti-inflammatory miRNA that are downregulated and allow overexpression of TLR2 and ASK1 in RA FLS. These results open new experiments on in vivo models. All together, these data give new insights for identify new therapeutics in RA.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

Mémoire en recherche-création / L'arthrose (OA) est la maladie musculosquelettique la plus fréquente au monde et peut affecter toutes les articulations. L’arthrose est caractérisée par la dégradation progressive du cartilage, l’inflammation de la synoviale et le remodelage de l’os sous-chondral. Ces changements sont dus à une augmentation d’expression des médiateurs pro-inflammatoires tels que la cyclooxygénase 2 (COX-2) et de facteurs cataboliques, notamment les métalloprotéinases 1 et 13 (MMP-1 et 13). Les métabolites de la 12-lipoxygénase jouent un rôle important dans de nombreux processus physiologiques et pathologiques. À la suite des réactions d'oxygénation / réduction de la 12 LOX, un facteur eicosanoïde particulier est produit: le 12-HydroxyEicosaTétraÉnoique (12-HETE), dont le rôle dans la pathogenèse de l’OA n’est pas caractérisé. L’objectif de ce travail est de définir le rôle de la 12-HETE dans la régulation des réponses inflammatoires et cataboliques dans les tissus articulaires, chondrocytes et synoviocytes, humains. Nous avons démontré que la 12-HETE n’affecte pas la prolifération (test MTT) des chondrocytes et des synoviocytes et n’a aucun effet sur leur migration (test de rayure). Un traitement avec la 12-HETE augmente l’induction de l’expression de la COX-2 dans les deux types cellulaires. La 12-HETE n’avait aucun effet sur l’expression de la MMP-1 et la MMP-13. La 12-HETE induit ses effets à l’aide d’un récepteur couplé à la protéine G : la GRP31. Nous avons décrit l’arthrose sur des coupes histologiques humaines, puis nous avons observé que l’expression de GPR31 était similaire dans le cartilage dégradé et le cartilage non dégradé. Finalement, nous avons montré que les chondrocytes et les synoviocytes expriment la GPR31 et son niveau est diminué en présence de l’interleukine-1 béta (IL-1β). En conclusion, nous avons démontré que la 12-HETE a des effets divers sur les réponses inflammatoires et cataboliques dans les tissus articulaires : elle augmente l’expression de la cyclooxygénase 2 (COX- 2) et n’a pas d’effet sur l’expression de la MMP-1 et la MMP-13. / Osteoarthritis (OA) is the world's most common musculoskeletal disease and can affect all joints. Osteoarthritis is characterized by progressive degradation of cartilage, inflammation of the synovium and remodelling of the subchondral bone. These changes are due to increased expression of pro-inflammatory mediators such as cyclooxygenase 2 (COX-2) and catabolic factors including metalloproteinases 1 and 13 (MMP-1 and 13). 12-lipoxygenase metabolites play an important role in many physiological and pathological processes. Following the oxygenation/reduction reactions of 12 LOX, a particular eicosanoid factor is produced: 12-HydroxyEicosaTetraenoic (12-HETE), whose role in the pathogenesis of AO is uncharacterized. This work aims to define the role of 12-HydroxyEicosaTetraenoic (12-HETE) in regulating inflammatory and catabolic responses in human joint tissues, chondrocytes and synoviocytes. We have demonstrated that 12-HETE does not affect the proliferation (MTT assay) of chondrocytes and synoviocytes and has no effect on their migration (scratch assay). Treatment with 12-HETE increased the induction of COX-2 expression in both cell types. 12-HETE had no effects on MMP-1 and MMP-13 expression. 12-HETE induces its effects using a G protein-coupled receptor: GRP31. We described osteoarthritis on human histological sections and then observed that GPR31 expression was the same in degraded and non-degraded cartilage. Finally, we showed that chondrocytes and synoviocytes express GPR31 and its level is decreased in the presence of Interleukin-1 beta (IL-1β). In conclusion, we demonstrated that 12-HETE has different effects on inflammatory and catabolic responses in joint tissues by increasing COX-2 expression and has no effect on MMP-1 and MMP-13 expression.

La polyarthrite rhumatoïde (PR) est la maladie auto-immune la plus fréquente d'une prévalence de 1%. Les cellules résidentes de la cavité synoviale, les fibroblast-like synoviocytes (FLS), sont des acteurs majeurs de la PR. Leur activation par des récepteurs de l'immunité innée participe à l'acquisition d'un phénotype agressif menant à la destruction ostéo-articulaire. Dans cette étude, nous avons évalué le rôle régulateur de miARN sur les voies de signalisation des Toll-like receptors (TLR). L'activation de TLR2 et de TLR4 dans les FLS induit la diminution de l'expression de plusieurs miARN, dont miR-19a et b (miR-19), alors que TLR2 est surexprimé. Nous avons pu ainsi montrer que miR-19 régule Tlr2 et que la transfection de mir-19 dans les FLS activés induit une diminution de l'expression de TLR2 et de la synthèse d'IL-6 et de MMP-3. Mir-19 appartient au cluster miR-17~92, dont l'expression est abaissée dans les FLS. Il code pour 6 miARN dont miR-20a. miR-20a est également sous-régulé après activation de TLR2 et TLR4 dans les FLS et les THP-1. Nous avons montré que miR-20a régule directement l'expression d'Ask1, impliquée et surexprimée après activation de TLR4. La transfection de miR-20a in vitro nous a permis de montrer que miR-20a contrôle l'expression d'ASK1 et induit une inhibition de la synthèse de cytokines majeures de la PR dans les FLS et les THP-1. Des résultats équivalents ont été obtenus ex vivo chez la souris. Ces travaux ont permis d'identifier dans les FLS rhumatoïdes des miARN anti-inflammatoires dont la baisse d'expression permet une augmentation de l'expression de TLR2 et d'ASK1. Ces miARN pourraient donc constituer de nouvelles cibles thérapeutiques.

In rheumatoid arthritis (RA), the expression of many pro-destructive/pro-inflammatory proteins depends on the transcription factor AP-1. Therefore, our aim was to analyze the presence and functional relevance of mutations in the coding regions of the AP-1 subunits of the fos and jun family in peripheral blood (PB) and synovial membranes (SM) of RA and osteoarthritis patients (OA, disease control), as well as normal controls (NC). Using the non-isotopic RNAse cleavage assay, one known polymorphism (T252C: silent; rs1046117; present in RA, OA, and NC) and three novel germline mutations of the cfos gene were detected: (i) C361G/A367G: Gln121Glu/Ile123Val, denoted as “fos121/123”; present only in one OA sample; (ii) G374A: Arg125Lys, “fos125”; and (iii) C217A/G374A: Leu73Met/Arg125Lys, “fos73/125”, the latter two exclusively present in RA. In addition, three novel somatic cjun mutations (604–606ΔCAG: ΔGln202, “jun202”; C706T: Pro236Ser, “jun236”; G750A: silent) were found exclusively in the RA SM. Tansgenic expression of fos125 and fos73/125 mutants in NIH-3T3 cells induced an activation of reporter constructs containing either the MMP-1 (matrix metalloproteinase) promoter (3- and 4-fold, respectively) or a pentameric AP-1 site (approximately 5-fold). Combined expression of these two cfos mutants with cjun wildtype or mutants (jun202, jun236) further enhanced reporter expression of the pentameric AP-1 construct. Finally, genotyping for the novel functionally relevant germline mutations in 298 RA, 288 OA, and 484 NC samples revealed no association with RA. Thus, functional cfos/cjun mutants may contribute to local joint inflammation/destruction in selected patients with RA by altering the transactivation capacity of AP-1 complexes.

Les maladies ostéoarticulaires représentent environ 10% de l'ensemble des pathologiessurvenant en France chaque année. Les difficultés pour identifier les causes de ces maladiesproviennent pour une part d'un manque de compréhension du mécanisme de lubrificationd'une articulation synoviale saine. Dans ce contexte, un premier objectif de ce travail a été d'analyser la structurediscontinue du liquide synovial à partir de prélèvements animaux sains et de la reproduire àpartir de composants biomoléculaires commerciaux afin de comprendre le mécanisme delubrification dans le cas sain. Le deuxième objectif de cette thèse a été d'analyser l'évolution de la structure et despropriétés lubrifiantes du liquide synovial dans le stade précoce de pathologies noninflammatoiresou inflammatoires à partir de prélèvements pathologiques humains. Afind'étudier plus finement l'évolution de la lubrification pathologique cette thèse a visé àdévelopper aussi des modèles de lubrifiants obtenus à partir de cultures cellulaires desynoviocytes humains en combinant l'action de facteurs inflammatoire (cytokines).L'ensemble des résultats montre l'importance de la structure supramoléculaire duliquide synovial dans l'obtention de bonnes propriétés lubrifiantes ; cette relation devraitconstituer d'une part un paramètre clé dans le diagnostic précoce des pathologies articulaireset d'autre part une voie de développement de liquides thérapeutiques à base de lubrifiantsnanostructurés

Die rheumatoide Arthritis ist eine chronisch-entzündliche Bindegewebserkrankung mit symmetrischem Befall der Gelenke. Die genaue Ätiologie ist bisher unbekannt. Aktivierte synoviale Fibroblasten sollen durch gesteigerte Adhäsion und Produktion von proinflammatorischen Zytokinen und Matrix-lysierenden Proteasen maßgeblich an der Gelenkdestruktion beteiligt sein. Ziel dieser Arbeit war es, ein neues in-vivo-Knorpeldestruktions-Modell zu etablieren, in welchem unter immunkompetenten Bedingungen, die Invasion und Destruktion von Gelenkknorpel durch die Fibroblasten-Zelllinie LS48 über einen längeren Zeitraum simuliert werden kann. Die am Institut für klinische Immunologie der Medizinischen Fakultät der Universität Leipzig etablierte Zelllinie LS48 wurde in die ipsilateralen Kniegelenke von BALB/c-Mäusen injiziert. Die dadurch induzierte Gewebsdestruktion wurde über zehn Wochen in zweiwöchigem Abstand histopathologisch beurteilt und klassifiziert. Als vergleichende Fibroblasten-Zelllinie wurden nicht-invasive NIH/3T3-Zellen eingesetzt. An Hand der Score-Parameter Zellinvasion, Pannusformation und Knorpeldestruktion wurde eine mäßige bis schwer-wiegende Gewebsdestruktion durch die LS48-Zellen bereits ab der zweiten Untersuchungswoche lichtmikroskopisch nachgewiesen, ohne dass dabei pathologische Effekte in den kontralateralen Kniegelenken aufgetreten sind. Polarisationsmikroskopisch wurden für den Parameter Knorpeldestruktion vergleichbare Ergebnisse erzielt. Damit wurde gezeigt, dass das Modell BALB/c LS48 ein erfolgversprechendes Instrument darstellt, das zur Testung neuer therapeutischer Strategien gegen die Gelenkdestruktion verwendet werden kann. Inwieweit die Auseinandersetzung der LS48-Zellen mit dem spezifischen Immunsystem der BALB/c-Maus Auswirkungen auf den Verlauf der Gewebsdestruktion hat, sollte in weiterführenden Experimenten untersucht werden.

Die rheumatoide Arthritis ist eine chronisch-entzündliche Bindegewebserkrankung mit symmetrischem Befall der Gelenke. Die genaue Ätiologie ist bisher unbekannt. Aktivierte synoviale Fibroblasten sollen durch gesteigerte Adhäsion und Produktion von proinflammatorischen Zytokinen und Matrix-lysierenden Proteasen maßgeblich an der Gelenkdestruktion beteiligt sein. Ziel dieser Arbeit war es, ein neues in-vivo-Knorpeldestruktions-Modell zu etablieren, in welchem unter immunkompetenten Bedingungen, die Invasion und Destruktion von Gelenkknorpel durch die Fibroblasten-Zelllinie LS48 über einen längeren Zeitraum simuliert werden kann. Die am Institut für klinische Immunologie der Medizinischen Fakultät der Universität Leipzig etablierte Zelllinie LS48 wurde in die ipsilateralen Kniegelenke von BALB/c-Mäusen injiziert. Die dadurch induzierte Gewebsdestruktion wurde über zehn Wochen in zweiwöchigem Abstand histopathologisch beurteilt und klassifiziert. Als vergleichende Fibroblasten-Zelllinie wurden nicht-invasive NIH/3T3-Zellen eingesetzt. An Hand der Score-Parameter Zellinvasion, Pannusformation und Knorpeldestruktion wurde eine mäßige bis schwer-wiegende Gewebsdestruktion durch die LS48-Zellen bereits ab der zweiten Untersuchungswoche lichtmikroskopisch nachgewiesen, ohne dass dabei pathologische Effekte in den kontralateralen Kniegelenken aufgetreten sind. Polarisationsmikroskopisch wurden für den Parameter Knorpeldestruktion vergleichbare Ergebnisse erzielt. Damit wurde gezeigt, dass das Modell BALB/c LS48 ein erfolgversprechendes Instrument darstellt, das zur Testung neuer therapeutischer Strategien gegen die Gelenkdestruktion verwendet werden kann. Inwieweit die Auseinandersetzung der LS48-Zellen mit dem spezifischen Immunsystem der BALB/c-Maus Auswirkungen auf den Verlauf der Gewebsdestruktion hat, sollte in weiterführenden Experimenten untersucht werden.:Bibliographische Zusammenfassung II Inhaltsverzeichnis III Abkürzungsverzeichnis IV 1 Einleitung 1 1.1 Rheumatische Erkrankungen 1 1.2 Die rheumatoide Arthritis 2 1.2.1 Immunologische Grundlagen der rheumatoiden Arthritis 2 1.2.1.1 Hypothese der Fibroblasten-Abhängigkeit 3 1.2.1.2 Hypothese der T-Zell-Abhängigkeit 4 1.3 Allgemeine Anatomie und Histologie des Kniegelenks 6 1.4 Die Histopathologie der rheumatoiden Arthritis 9 1.4.1 Verschiedene Synovialmembrantypen bei rheumatoider Arthritis 10 1.5 Tiermodelle zur Untersuchung der rheumatoiden Arthritis 11 1.5.1 Das Tiermodell der Fibroblasten-induzierten Gelenkdestruktion in der BALB/c-Maus 12 1.6 Histopathologische Score-Systeme der rheumatoiden Arthritis in Tiermodellen 13 1.7 Ziel 13 1.7.1 Fragestellungen 14 2 Material und Methoden 16 2.1 Zelllinien und Versuchstiere 16 2.1.1 Die Fibroblasten-Zelllinie NIH/3T3 16 2.1.2 Die Fibroblasten-Zelllinie LS48 16 2.1.3 Die BALB/c-Maus 17 2.2 Tierversuchsplan 18 2.3 Zellkultur 19 2.3.1 Geräte und Verbrauchsmaterialien 19 2.3.2 Reagenzien 20 2.3.3 Durchführung 21 2.4 Isolation der murinen Kniegelenke 22 2.5 Histologische Aufarbeitung 23 2.5.1 Geräte und Verbrauchsmaterialien 23 2.5.2 Reagenzien 24 2.5.3 Entkalkung, Entwässerung, Einbettung und Schneiden der Präparate 26 2.5.4 Azanfärbung nach Heidenhain (Kernechtrubin-Anillinblau-Orange G-Färbung) 27 2.6 Klassifikation mit dem Durchlichtmikroskop für das Modell der Fibroblasten-induzierten Knorpeldestruktion (Balb/c-LS48) 29 2.7 Klassifikation mit dem Polarisationsmikroskop für das Modell der Fibroblasten-induzierten Knorpeldestruktion (Balb/c-LS48) 29 2.8 Statistik 30 3 Ergebnisse 31 3.1 Score-Erhebung mit dem Lichtmikroskop für das Modell der Fibroblasten-induzierten Knorpeldestruktion (BALB/c-LS48) 31 3.1.1 Bewertungsmodus für den Score-Parameter Zellinvasion 32 3.1.2 Bewertungsmodus für den Score-Parameter Pannusformation 35 3.1.3 Bewertungsmodus für den Score-Parameter Knorpeldestruktion 38 3.2 Datenanalyse der lichtmikroskopisch untersuchten Parameter Zellinvasion, Pannusformation und Knorpeldestruktion für das Modell der Fibroblasten-induzierten Knorpeldestruktion (BALB/c-LS48) 41 3.2.1 Zellinvasion 41 3.2.2 Pannusformation 45 3.2.3 Knorpeldestruktion 48 3.2.4 Gesamtscore 51 3.3 Score-Erhebung mit dem Polarisationsmikroskop für das Modell der Fibroblasten-induzierten Knorpeldestruktion (BALB/c-LS48) 56 3.3.1 Bewertungsmodus für den Score-Parameter Knorpeldestruktion 56 3.4 Datenanalyse des polarisationsmikroskopisch untersuchten Parameters Knorpel-destruktion für das Modell der Fibroblasten-induzierten Knorpeldestruktion (BALB/c-LS48) 59 3.5 Statistischer Vergleich der licht- und polarisationsmikroskopischen Analysemethoden für den Parameter Knorpeldestruktion 62 3.6 Statistischer Vergleich der medialen und lateralen histologischen Sagittalschnitte der Kniegelenke 63 4 Diskussion 64 4.1 Die Bedeutung der histopathologischen Score-Parameter für das Modell der Fibroblasten-induzierten Gelenkdestruktion in der BALB/c-Maus 65 4.1.1 Der Score-Parameter Zellinvasion 65 4.1.1.1 Die pathophysiologische Bedeutung des Score-Parameters Zellinvasion 67 4.1.1.2 Interpretation der lichtmikroskopischen Befunde des Score-Parameters Zellinvasion für die Zelllinie LS48 im ipsilateralen Kniegelenk im Verlauf von zehn Wochen 69 4.1.2 Der Score-Parameter Pannusformation 70 4.1.2.1 Die pathophysiologische Bedeutung des Score-Parameters Pannusformation 71 4.1.2.2 Interpretation der lichtmikroskopischen Befunde des Score-Parameters Pannusformation für die Zelllinie LS48 im ipsilateralen Kniegelenk im Verlauf von zehn Wochen 73 4.1.3 Der Score-Parameter Knorpeldestruktion 74 4.1.3.1 Die pathophysiologische Bedeutung des Score-Parameters Knorpeldestruktion 75 4.1.3.2 Interpretation der lichtmikroskopischen Befunde des Score-Parameters Knorpeldestruktion für die Zelllinie LS48 im ipsilateralen Kniegelenk im Verlauf von zehn Wochen 76 4.1.4 Interpretation des lichtmikroskopisch erhobenen Gesamtscores für das Knorpeldestruktionsmodell (BALB/c-LS48) im ipsilateralen Kniegelenk im Verlauf von zehn Wochen 78 4.1.4.1 Verlaufsvergleich zu anderen Tiermodellen 81 4.2 Die histopathologischen Auswirkungen der Zelllinien LS48 und NIH/3T3 im ipsilateralen Kniegelenk der BALB/c-Maus im Vergleich 83 4.3 Vergleich der medialen und lateralen Sagittalebenen der histologischen Präparate der Kniegelenke 85 4.4 Beurteilung histopathologischer Veränderungen in den kontralateralen Kniegelenken im Verlauf von zehn Wochen 86 4.5 Vergleich der licht- und polarisationsmikroskopischen Untersuchungsergebnisse 87 4.6 Schlussfolgerungen und Ausblick 89 Zusammenfassung 94 Literaturverzeichnis 99 Abbildungs- und Tabellenverzeichnis 116 Eigenständigkeitserklärung VIII Danksagung IX