京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18886号 / 医博第3997号 / 新制||医||1009(附属図書館) / 31837 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 戸口田 淳也, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La polyarthrite rhumatoïde (PR) est une maladie auto-immune qui mène à une inflammation chronique et à une destruction progressive des articulations. Les effecteurs principaux de cette pathologie sont les synoviocytes de type fibroblastique (FLS). Ces derniers utilisent les invadosomes, des structures riches en actine et en métalloprotéases, afin de dégrader la matrice extracellulaire (ECM). Ce phénotype pro-destructif résulte d’une activation des FLS par différents facteurs de croissance, dont le PDGF et le TGF-β. Les récepteurs à activité tyrosine kinase, dont le PDGFR, sont impliqués dans la pathogenèse de plusieurs maladies, incluant le cancer et la PR. Une activation de ces récepteurs peut mener, entre autres, à la survie, à la différenciation et à la prolifération des cellules. L’étude présentée dans ce mémoire montre que parmi les RTK les plus communs, le PDGFR est spécifiquement phosphorylé chez les cellules synoviales de patients atteints de PR, contrairement aux cellules de patients non arthritiques ou atteints d’arthrose. De plus, l’activation du PDGFR résulte en une augmentation de la formation d’invadosomes par les FLS. Nous avons aussi démontré que la formation d’invadosomes par le PDGFR nécessite l’activation de la voie de signalisation PI3K/Akt faisant intervenir les isoformes α et δ de la PI3K. De plus, l'inhibition de l’activation du PDGFR ou la neutralisation du PDGF endogène inhibe la formation des invadosomes et la dégradation de l'ECM par les synoviocytes, ce qui suggère la présence d'une boucle d'activation autocrine impliquant le PDGF. Parmi les isoformes du PDGF, nous avons démontré que le PDGF-B est exprimé de façon significativement plus élevée dans les synoviocytes provenant de patients atteints de PR. Nos données indiquent également une association entre le PDGF et le TGF-β dans la formation des invadosomes. Cette dernière implique la production autocrine de ligands du PDGFR induite par le TGFβ via la signalisation TβR1/Smad et PI3K/Akt. L’inhibition des isoformes de PI3K de classe I indique que le PI3Kα est impliquée de façon sélective dans l'expression de PDGF-B. Ces résultats démontrent que le PDGFR est un RTK nécessaire au phénotype destructeur des cellules synoviales d’arthrite. Ils fournissent aussi des preuves d'une association entre le TGF-β et le PDGFR dans la formation d’invadosomes chez les synoviocytes de patients atteints de la PR. / Abstract : Rheumatoid arthritis (RA) is an autoimmune disease that leads to chronic inflammation and progressive joint destruction. The main effectors of this pathology are fibroblast-like synoviocytes (FLS). They use invadosomes, actin-rich structures that concentrate metalloproteinases to degrade the extracellular matrix (ECM). This pro-destructive phenotype is due to the activation of FLS by various growth factors, including PDGF and TGF-β. Receptor tyrosine kinases, including PDGFR, are involved in the pathogenesis of several diseases, including cancer and RA. Activation of these receptors may lead to cell survival, differentiation and proliferation. The study presented in this thesis shows that among the most common RTKs, PDGFR is specifically phosphorylated in synovial cells of RA patients, unlike cells of non-arthritic or osteoarthritic patients. In addition, activation of PDGFR results in an increase in invadosome formation by FLS. We also shown that formation of invadosome by PDGFR requires the activation of the signaling pathway PI3K/Akt, that specifically involves the α and δ isoforms of PI3K. In addition, inhibition of PDGFR activation or neutralization of endogenous PDGF inhibits the formation of invadosomes and the degradation of the ECM by synoviocytes, suggesting the presence of an autocrine activation loop involving PDGF. Among the PDGF isoforms, we demonstrate that PDGF-B expression is significantly higher in synoviocyte cell lines from RA patients. Our data also indicates an association between PDGF and TGF-β for invadosome formation that involves autocrine production of PDGF-B induced by TGF-β through the Smad/T β R1 and PI3K/Akt pathways. Inhibition of class I PI3K isoforms indicates that PI3K α is selectively involved in the expression of PDGF-B. These results demonstrate that PDGFR is an RTK necessary for the pro-destructive phenotype of RAFLS. They also provide evidence of an association between TGF-β and PDGFR in invadosome formation by synovial cells from RA patients.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13195号 / 論医博第2159号 / 新制||医||1030(附属図書館) / (主査)教授 松田 秀一, 教授 三森 経世, 教授 妻木 範行 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

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.

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.