京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22144号 / 医博第4535号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 生田 宏一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rheumatoid arthritis (RA) is a chronic inflammatory disease with an unknown aetiology characterized by joint destruction. Both genetic and environmental factors contribute to the disease development with HLA-DRB1* alleles and smoking identified as most important. The disease is characterized by the presence of autoantibodies, originally by rheumatoid factor (RF) and more recently by anti citrullinated protein/peptide antibodies (ACPA) and antibodies against carbamylated peptides (CarP). These autoantibodies are present, not only after the onset of disease, but also prior to the onset of symptoms. The development of RA is a gradual process lasting several years before the onset of any joint symptom, but when and if there is a temporal difference in the development both between and within the different antibody systems is currently unknown. B-cells produce the antibodies, and a subset of B-cells, i.e., B-regulatory (Breg) cells, produces interleukin-10, and thus have the ability to down-regulate pro-inflammatory cytokines. Whether the Breg cells are involved in the pathogenesis of RA is, as yet, unknown. The aim of this thesis was to increase knowledge of the pathophysiological processes in the development of RA through identification of factors involved. The analyses involved detection of autoantibodies to post-translationally modified peptides/proteins in addition to RF isotypes, cell surface markers on immune cells in asymptomatic individuals, who have an increased risk of developing RA. In a co-analysis of the registers of patients with RA attending the Department of Rheumatology, with the registers from population based screening programmes within the Biobank of Northern Sweden, blood samples collected from individuals prior to the onset of symptoms were identified, as were those from population control subjects. A cohort of pre-symptomatic individuals also donated samples at the time of receiving a diagnosis of RA. First-degree relatives (FDR) of patients with RA were also identified and included for analyses. The levels of ten different ACPAs, i.e., (fibrinogen (Fib) α563-583(573), Fibα580-600(591), Fibβ62-81a(72), Fibβ62-81b(74), Fibβ36-52, a-enolase (CEP-1), triple helical collagen type II (citC1III), filaggrin (Fil307-324), vimentin (Vim) 2-17, and Vim60-75) were measured using the ImmunoCAP ISAC system (Phadia/ThermoFischer, Uppsala, Sweden) in blood samples from individuals before the onset of symptoms and when diagnosed with RA in comparison with those in population based controls. In a subset of samples, the levels of anti-CarP antibodies were measured using ELISA coated with anti-CarP-FCS, as well as analysis of RF of IgM, IgG and IgA isotype using the EliA assay (Phadia, Uppsala, Sweden). Breg cells were analysed both with and without stimulation ex vivo along with other cell types using flow cytometry in samples from patients with RA, their first degree relatives (FDR) and healthy controls. In paper I it was shown that levels of ACPA were initially restricted to a few antibodies but disseminated over time to involve additional different antibodies. The levels of antibodies to CEP-1, Fibß36-52, and filaggrin were significantly increased. In paper II, anti-CarP antibodies were positive in 5-13% of the individuals negative for the various ACPA studied. The presence of anti-CarP antibodies was significantly related to radiological destruction of joints at baseline, at follow-up after 24 months and to the radiological progress between baseline and 24months. In paper III, the relationships between the frequencies of RF isotypes, the ten different ACPA, anti-CCP2 and anti-CarP antibodies before the onset of any symptoms and the presence of certain combinations of antibodies were associated with a very high risk of developing RA. In paper IV Breg cells from patients with RA are functionally impaired and FDR showed a similar pattern by responding less to stimulation ex vivo than cells from healthy controls. In conclusion, individuals who subsequently develop RA have an increased number and amount of ACPAs, anti-CarP antibodies and RF of IgM, IgG and IgA isotype, several years before symptom onset. Most of the different antibodies analysed remain associated with disease development after adjustments for each separate antibody. In FDRs, Breg cells were functionally altered in that they produce less IL-10 and consequently contribute to a more inflammation-prone status, as in their relatives with RA. These findings contribute to information about the development of RA as well as a given individual’s risk(s) of developing RA and its progression.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24837号 / 医博第5005号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤, 能永, 教授 生田, 宏一, 教授 上野, 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23773号 / 医博第4819号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 杉田 昌彦, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A artrite reumatÃide à uma doenÃa auto-imune crÃnica e inflamatÃria que evolui com graus de destruiÃÃo articular e alteraÃÃes extra-articulares podendo levar a incapacidade funcional. AlÃm da avaliaÃÃo clÃnica o diagnÃstico baseia-se na determinaÃÃo do fator reumatÃide (FR) sendo que este à tambÃm positivo em indivÃduos saudÃveis como tambÃm em outras doenÃas auto-imunes e infecciosas. Os anticorpos antipeptÃdeos citrulinados cÃclicos (anti-CCP) tem sido usados no diagnÃstico da artrite reumatÃide sendo superiores ao fator reumatÃide (FR) no diagnÃstico da artrite reumatÃide (AR) recente. O estudo teve como objetivo demonstrar a presenÃa de anticorpos anticitrulina em pacientes com o diagnÃstico de artrite reumatÃide comparando com parÃmetros clÃnicos, laboratoriais e com a avaliaÃÃo da qualidade de vida desses pacientes. Para avaliar a qualidade de vida foi aplicado o questionÃrio âHealth Assessment Questionnaire.â Foi realizado um teste por imunoturbidimetria, para a detecÃÃo de FR (Roche, Indianopolis, EUA) e ELISA para o anti-CCP (Inova , San Diego, EUA) em 69 pacientes apresentando, ao menos, 4 dos critÃrios do ColÃgio Americano de Reumatologia para a classificaÃÃo de AR e em 20 controles saudÃveis. A anÃlise estatÃstica utilizou o teste exato de Fisher e teste de Spearmann com significÃncia alcanÃada com P<0.05. Os pacientes com AR tinham entre 18-75 (mÃdia = 43.9 anos), 66 (95.7%) eram mulheres, os controles com idades variando entre 20-60 anos. O inÃcio da AR variou de 4 a 384 meses (mÃdia = 74.0 e mediana = 48.0). FR foi positivo em 48 (69.6%) pacientes e 1 (0,5%) controle. O anti-CCP foi positivo em 36 (52.2%) pacientes e em 2 (10%) controles. Foi observada uma correlaÃÃo significante dos testes FR e anti-CCP com P< 0.0001 e este estudo sugere que o anti-CCP nÃo foi superior ao FR no diagnÃstico da AR estabelecida / Rheumatoid arthritis is an inflammatory, chronic and auto-immune disease that develops in degrees of articular destruction and extra-articular changes being able to lead to functional disability. Besides clinical assessment the diagnosis is based on the determination of the rheumatoid factor being this one also positive in healthy people as well as in other infectious and auto-immune diseases. Anticyclic citrullinated peptide (anti-CCP) antibodies have been used in diagnosis of rheumaoid arthritis (RA) and seen to be superior to rheumatoid factor (RF) in early onset RA diagnosis. The target of the study is to demonstrate the presence of anticitrulline antibodies in patients with rheumatoid arthritis diagnostic comparing with laboratory, clinical parameters and with the assessment of the quality of life of these patients. The âHealth Assessment Questionnaireâ has been used to assess the quality of life. We performed an immunoturbidimetry test for detection of RF (Roche,Indianopolis, USA) and an ELISA for anti-CCP antibodies (Inova, San Diego, USA) in 69 patients presenting, at least, 4 of the American College of Rheumatology criteria for classification of RA and in 20 healthy controls. For statistical analysis we used thr Fisher exact test and the Spearmann test Significance was reached wuth P<0.05. RA patients were aged between 18-75 years (mean = 43.9 years), 66 (95.7%) of then were female, controls age ranged between 20-60 years.The period of RA onset varied from 4 to 384 months (mean = 74.0 and median = 48.0). RF was positive in 48 (69.6%) patients and in 1 (0,5%) control. The anti-CCP was positive in 36 (52.2%) patients and in 2 (10%) controls. A significant correlation of RF and anti-CCP tests was observed with P< 0,0001 and this study suggests that anti-CCP was not superior to RF in diagnosis of established RA

La polyarthrite rhumatoïde est une pathologie auto-immune qui affecte près de 1% de la population mondiale et se caractérise par une inflammation articulaire, des altérations cartilagineuses et osseuses notamment associées à des douleurs chroniques articulaires, souvent résistantes aux thérapeutiques actuelles. Que ce soit à un stade préclinique, où l’on parle d’arthralgie, ou à un stade établi de la pathologie, ces douleurs constituent un réel handicap pour les patients atteints avec plus de 60% d’entre eux insatisfaits de sa prise en charge. La présence d’une synovite étant nécessaire au diagnostic de la PR, aucune stratégie thérapeutique n’est mise en place à un stade préclinique. En outre, à un stade établi, la stratégie actuelle vise en premier lieu à diminuer l’activité de la pathologie sans prise en charge de la douleur en tant que telle. Parmi les acteurs de la synovite dans la PR, un rôle essentiel est attribué au facteur de croissance des nerfs ou Nerve Growth Factor dans la mise en place et le maintien des symptômes douloureux. Les anti-NGF sont connus comme des molécules antalgiques prometteuses. Néanmoins, de par son action pléiotropique, cibler cette neurotrophine conduit à des effets indésirables potentiellement importants. Dans la première partie de ce travail, nous avons cherché à mieux caractériser l’implication spécifique des voies de signalisation intracellulaires au récepteur tyrosine kinase de type A (TrkA) de haute affinité au NGF, dans un contexte de douleur articulaire inflammatoire (arthrite) mais aussi de douleurs somatique et viscérale. Un modèle de knock-out total pour le récepteur TrkA n’étant pas viable, nous avons réalisé une étude multimodale chez des souris knock-in TrkA/C, exprimant un récepteur chimérique composé de la partie extracellulaire native du récepteur TrkA et des parties transmembranaire et intracellulaire fonctionnelles du récepteur à la neurotrophine 3 : le récepteur TrkC. Ce dernier n’étant que peu ou pas impliqué dans la douleur inflammatoire. Ainsi, le NGF pourra se lier normalement au récepteur TrkA/C mais activera les voies de signalisation intracellulaires du récepteur TrkC. Les résultats de nos études ont mis en évidence qu’une absence d’activation de certaines voies en aval de TrkA (i.e. c-Jun et p38 MAPK) au niveau des DRGs chez les souris TrkA/C, impacte significativement la mise en place des symptômes douloureux, en particulier l’hypersensibilité mécanique, que ce soit dans un contexte de douleur articulaire, somatique ou viscérale, sans affecter l’hyperalgie thermique au chaud. Ces résultats résultent d’une part de la diminution de la néo-innervation CGRP+ mais aussi de changements transcriptionnels de certains neurotransmetteurs et mécanotransducteurs dont le canal ionique sensible aux protons : ASIC3. De plus, un lien entre NGF/TrkA et le remodelage osseux, en particulier, l’activation ostéoclastique, a été démontré mettant en avant un rôle doublement bénéfique de l’inhibition de certaines voies associées à TrkA, à la fois dans certains symptômes douloureux et l’érosion osseuse retrouvée dans la PR. Dans un deuxième temps, nous nous sommes intéressés aux mécanismes impliqués dans l’arthralgie induite par l’injection d’autoanticorps anti-peptides citrullinées (ACPA). La majorité des patients PR sont positifs pour les ACPA qui peuvent être produits des mois voire des années, avant son diagnostic et semblent être directement associés au développement des symptômes douloureux. Cette arthralgie constitue l’un des premiers signes d’une PR émergente et peu persister, même suite à la prise en charge thérapeutique des patients PR. Tout d’abord, nous avons confirmé que les sous-types monoclonaux IgG1 ACPA diffèrent par leurs propriétés pronociceptives et érosives de l’os, certainement liées à leurs différentes réactivités vis-à-vis des épitopes citrullinés. (...) / Rheumatoid arthritis is an autoimmune disease that affects nearly 1% people worldwide and is characterized by joint inflammation, cartilage and bone damages, associated with chronic joint pain, often resistant to current therapies. Whether at a preclinical stage, where we talk about arthralgia, or at an established stage of the pathology, pain constitutes a real burden for the patients with more than 60% rating their pain management has unsatisfactory. The presence of synovitis is necessary for the diagnosis of established RA, therefore, no real therapeutic strategy is used at a preclinical stage. In addition, at an established stage, the current strategy aimed primarily at reducing the activity of the pathology without an actual management of the pain as such. Among the actors of synovitis in RA, Nerve Growth Factor plays a critical role in the establishment and maintenance of painful symptoms. Anti-NGF are known as promising analgesic drugs. Nevertheless, due to pleiotropic effects of NGF, targeting this neurotrophin leads to significant adverse effects. In the first part of this work, we sought to better characterize the specific involvement of intracellular signaling pathways of the high affinity tyrosine kinase A (TrkA) receptor of NGF in a context of inflammatory joint pain (arthritis), but also of somatic and visceral pain. Since a total knockout of TrkA receptor in mice is not viable, we performed a multimodal study in TrkA/C knock-in mice, expressing a chimeric receptor composed of the native extracellular part of TrkA receptor and, the transmembrane and intracellular functional parts of the neurotrophin 3 receptor: TrkC receptor, which is not really involved in inflammatory pain. Thus, NGF can bind normally to the TrkA/C receptor but activates the intracellular signaling pathways downstream of TrkC receptor. Our results have shown that a lack of activation of certain TrkA pathways (i.e. c-Jun and p38 MAPK) in the DRGs of TrkA/C mice, has a significant impact on the development of painful symptoms, especially mechanical hypersensitivity in a context of articular, somatic, or visceral pain, without affecting heat thermal hyperalgesia. These effects result, on one hand, from the decrease of CGRP+ nerve sprouting and in another hand, from the transcriptional changes of some neurotransmitters and mechanotransducers including the proton-sensitive ion channel: ASIC3. In addition, our studies highlight a direct link between NGF/TrkA and bone remodeling, in particular, osteoclastic activity, suggesting a beneficial role of the inhibition of some specific TrkA-associated pathways, in both mechanical hypersensitivity and bone erosion found in RA.In a second part of our work, we investigated the mechanisms involved in arthralgia induced by the injection of autoantibodies against citrullinated peptides (ACPA). The majority of RA patients is positive for ACPA that can be produced months to years before RA diagnosis and appear to be directly associated with the development of pain. Arthralgia is one of the first signs of an emerging RA and can persist even following RA treatment. First, we confirmed that monoclonal ACPA IgG1 subtypes differ in their pronociceptive and bone erosive properties certainly link to their reactivity patterns against citrullinated epitopes on different targets especially those engaging osteoclast activity. Thus, the combination of B02/B09 ACPA clones induced pain like behaviour without any inflammation, but is associated with an alteration of bone homeostasis in injected mice. We suggest that as a result of ACPA-induced osteoclast activation, certain factors (e.g. protons and/or lipids) are released, which sensitize ASIC3, ultimately leading to pain.