京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18160号 / 医博第3880号 / 新制||医||1003(附属図書館) / 31018 / 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 生田 宏一, 教授 宮地 良樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Haut ist das größte Organ des Menschen und bildet die Barriere gegenüber Einwirkungen aus der Umwelt. Die Störung der Hautbarriere durch exogene und endogene Reize führt zu einer Entzündungsreaktion in der Haut. In der Folge können Hauterkrankungen wie die irritative oder Atopische Dermatitis entstehen. Der Tumor Nekrose Faktor-α (TNF-α) ist ein pleiotrop wirksames Zytokin, das eine zentrale Rolle bei entzündlichen Prozessen spielt. Ziel der vorgelegten Promotionsarbeit war zu untersuchen, ob und wie TNF-α zu Entzündungsgeschehen, ausgelöst durch exogene und endogene Faktoren, beiträgt. Die Bedeutung von TNF-α wurde in TNF-ko Mäusen in verschiedenen Hautmodellen untersucht. Für das Irritationsmodell wurden chemische und physikalische Reize verwendet. TSLP (Thymic stromal lymphopoietin) wurde durch die verschiedenen Stimuli signifikant induziert. Diese Induktion war unabhängig von der endogenen TNF-α Produktion, gezeigt durch den Einsatz von TNF- ko Mäusen . Da endogenes TNF-α für die Hautirritation keine notwendige Bedingung darstellte, wurde die Bedeutung von TNF-α bei der atopischen Dermatitis (AD) untersucht. TNF-α defiziente Mäuse zeigen verstärkt Ekzeme im Vergleich zu Wildtyp Mäusen. Die Behandlung von TNF-ko Mäusen mit einem TSLP Antikörper führte zu einer Verminderung des Ekzems. Mastzellen wurden vermehrt in läsionaler Haut gefunden und korrelierten mit dem Schweregrad des atopischen Ekzems sowie der TSLP-Expression. / The skin is the largest organ of an individuum and builds the barrier for a host against the environment. Skin barrier disruption by exogenous or endogenous stimuli can lead to skin inflammation. As a consequence, irritant or atopic eczema, frequent skin diseases, may evolve. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine which plays a central role in inflammatory processes. The main aim of this thesis was to clarify whether and how endogenous TNF-α is contributing to skin inflammation driven by exogenous and endogenous triggers. The role of endogenous TNF-α was studied using TNF knockout (-/-) mice. In an irritation model, chemical and physical stimuli were applied on to mouse skin. Thymic stromal lymphopoietin (TSLP) was significantly induced by the used irritants. This TSLP induction was independent from endogenous TNF-α proven by using TNF-/- mice. Next the role of TNF-α in atopic dermatitis (AD) promoting an allergic skin inflammation was investigated. TNF-/- mice developed more severe AD compared to the wildtype mice and TSLP was significantly increased and correlated with the severity of the eczema. To prove the pathophysiological role of TSLP for AD progression, TNF-/- mice were pretreated with an TSLP antibody. Indeed, these mice developed less AD symptoms compared to the control mice. Mast cells (MCs) were also significantly increased in lesional skin in the AD model and moreover, correlated with AD severity, but also with TSLP expression.

Die Haut ist das größte Organ des Menschen und stellt die primäre Barriere gegen Umwelteinflüsse und Pathogene dar. Eine Dysbalance der Hautbarriere birgt die Gefahr einer nachfolgenden Entzündungsreaktion. Bleibt diese bestehen, können sich Hautkrankheiten wie zum Beispiel die atopische Dermatitis (AD) entwickeln. Verschiedene Zytokine wie Thymic Stromal Lymphopoietin (TSLP) werden bei entzündlichen Hauterkrankungen eine bedeutende Rolle zugeschrieben. Die Bedeutung von TSLP wurde zunächst anhand mehrerer Knockout-Mausstämme untersucht. Hier zeigte sich, dass TSLPR-KO und TNF-TSLPR-DKO Mäuse im Gegensatz zu TNF-KO Mäusen keine bzw. weniger Zeichen einer Entzündungsreaktion in der Haut entwickeln. Die Rolle äußerer Einflüsse auf die TSLP-Produktion wurde anhand eines Irritationsmodells ebenfalls in Mäusen untersucht. Dabei führte die Hautirritation, ausgelöst durch Abtragen der oberen Hautschichten mittels eines Tesa-Abriss, zu einem signifikanten Anstieg von TSLP in der Haut?. Mit Hilfe von Agonisten und Inhibitoren konnte gezeigt werden, dass dieser Irritations-vermittelte TSLP-Anstieg über Interleukin-1 (IL-1) und Protease Activated Receptor 2 (PAR-2) vermittelt wird. In diesem Zusammenhang wurde auch gezeigt, dass die Aktivierung von IL-1- sowie PAR-2-abhängigen Signalwegen zu einer gesteigerten Aktivität des TSLP-Promotors führte. Die Untersuchung der Wirkung verschiedener Mastzellmediatoren auf die TSLP-Expression ergab, dass Tryptase, über die Aktivierung von PAR-2, der wichtigste Mediator für den Anstieg von TSLP nach der Degranulation von Mastzellen ist. Dieses Ergebnis wurde mittels verschiedener in vitro, in vivo und ex vivo Experimentalansätze belegt. So konnte in einem c48/80-abhängigen Degranulationsmodell in Mäusen gezeigt werden, dss PAR-2- sowie Mastzell-KO Mäuse im Vergleich zu Wildtypen nach Injektion von c48/80 signifikant weniger TSLP exprimierten. / The skin is the first line of defense against environmental or microbial pathogens. A deviation of the skin barrier homeostasis by any kind of insult can result in an inflammatory response. The inflammatory response in turn can promote the development of an eczemaincluding atopic eczema. Thymic stromal lymphopoietin (TSLP) due to its pleiotropic nature play an important role in inflammatory disorders. The major aim of this thesis was to better understand the underlying mechanisms of TSLP production in the context of skin irritation and mast cell (MC) mediators and their contribution in the development of atopic dermatitis (AD). The role of TSLP was studied using TSLPR-/- mice. The data show that TSLPR-/- and TNF-/-/TSLPR-/- mice were protected from AD development, by contrast TNF-/- mice exhibited severe AD. The role of exogenous triggers was studied using tape stripping mediated skin irritation mouse models. Skin irritation resulted in significant enhanced TSLP production. TSLP induction was identified to depend on interleukin (IL)-1 and protease activated receptor (PAR)-2 pathways proven by using exogenous activators or inhibitors of these pathways. Moreover, PAR-2 and IL-1 concomitantly promoted NF-κB binding to the human TSLP promoter which in turn resulted in an increased TSLP promoter activity. Additionally, the role of mast cell mediators in the context of TSLP induction was investigated. Tryptase turned out to be the trigger responsible for the enhanced TSLP response by activating the PAR-2 pathway. This finding was proven by employing in vitro, ex vivo and in vivo approaches. In detail PAR-2-/- and MC-/- mice were used in a compound 48/80 (C48/80) dependent MCs degranulation model. PAR-2-/- and MC-/- mice produced significantly less TSLP in comparison to control mice.

Une fois activées, les cellules dendritiques (DCs) migrent dans les organes lymphoïdes ou elles exercent leur rôle de cellules présentatrices d’antigène professionnelles. Elles sont capables d’activer et d’induire la différenciation des lymphocytes T naïfs en différentes sous-populations de lymphocytes T auxiliaires. L’ajustement de la réponse lymphocytaire T au type d’inflammation est assuré par les DCs à deux niveaux. Premièrement, grâce à leur plasticité, les DCs adaptent leur comportement en fonction de la combinaison de signaux issus du microenvironnement inflammatoire. Deuxièmement, il existe différentes sous-populations de cellules dendritiques ayant de différentes spécialisations fonctionnelles.Mon travail de thèse s’est concentré sur l’étude de la diversité des réponses des sous-populations de cellules dendritiques humaines suite à la stimulation par la cytokine lymphopoïetine stromale thymique (TSLP). Cette cytokine est secrétée par les cellules épithéliales et la peau au cours de l’inflammation. La TSLP active principalement les DCs myéloïdes, induisant celles-ci à secréter les chimiokines inflammatoires CCL17 et CCL22. Les DCs activées par la TSLP (TSLP-DCs) induisent une réponse inflammatoire de type Th2, et sont impliquées dans le développement de l’allergie. La comparaison systématique de la réponse à la TSLP par les sous-populations de DCs du sang, BDCA1+ et BDCA3+, nous a permis de montrer que ces deux sous-populations sont activées par la TSLP. Toutefois, nos résultats montrent que la TSLP, en synergie avec le TGF-β, induit la différenciation des DCs BDCA-1+ en cellules de Langerhans, mais pas celle des DCs BDCA-3+. De plus, la TSLP induit la migration cellulaire et la sécrétion de chimiokines seulement chez la sous-population de DCs BDCA-1+. Des analyses complémentaires des mécanismes impliqués dans la migration des DCs BDCA-1+ en réponse à la TSLP révèlent que, d’une part la TSLP est indispensable à l’induction de la migration et d’autre part, qu’un récepteur de chimiokines, sensible à la Toxine Pertussique serait impliqué. Au final, nos résultats révèlent (i) de nouvelles capacités des DCs en tant que cellules précurseurs, (ii) de différentes propriétés fonctionnelles des sous-populations de DCs en réponse à la stimulation par la TSLP, (iii) et démontrent la complexité des mécanismes impliqués dans la migration des DCs induite par la TSLP. / Once activated, Dendritic Cells (DCs) migrate to the lymphoid organs and exert their role as professional antigen presenting cells. They are able to induce the activation and differentiation of naïve T cells into different types of T helper cells. The T cell response must be suited to the type of inflammation. This is ensured by DCs at two levels. First DCs are functionally plastic. This means that their behavior is subdued to the integrated signals coming from the inflammatory microenvironment. Secondly, the DC population is diverse. Indeed, different DC subsets have different functional specializations. My thesis was focused on the differential response of human DC subsets to Thymic stromal lymphopoietin (TSLP). This cytokine is secreted by inflamed skin and epithelia, and strongly activates myeloid DCs. The TSLP-activated DCs secrete the inflammatory chemokines CCL17 and CCL22, prime an inflammatory Th2 response, and have been involved in the pathogenesis of allergic inflammation. By systematically comparing the response of human blood BDCA-1+ and BDCA-3+ DCs to TSLP stimulation we found that both of these DC subsets get activated by TSLP. However TSLP synergizes with TGF-β to induce the differentiation of blood BDCA-1+ and not BDCA-3+ DCs into Langerhans Cells. Moreover, TSLP induces cell migration and chemokine secretion only on the blood BDCA-1+ subset. Further analysis of the mechanisms implicated in TSLP-induced DC migration revealed that TSLP is required to induce DC migration, but this effect is dependent on the expression of a PTX-sensitive chemokine receptor. Overall our results reveal new precursor capacities of blood DC subsets, different functional properties of blood DC subsets stimulated by TSLP and highlight intricate mechanisms underlying TSLP-induced DC migration.

Le système immunitaire humain est majoritairement commandé par les cellules dendritiques et les lymphocytes T auxiliaires. Lorsque les cellules dendritiques détectent un pathogène, elles vont instruire les lymphocytes T auxiliaires afin qu’ils adoptent le phénotype approprié à la menace rencontrée. Les lymphocytes T auxiliaires peuvent être divisés en plusieurs sous-populations, caractérisées par la production de cytokines spécifiques. Chaque sous-population de lymphocyte T auxiliaire possède des fonctions propres et est impliquée dans l’élimination de pathogènes distincts. Si les réponses des lymphocytes T auxiliaires ne sont pas finement régulées, ils peuvent devenir pathogéniques, et dans ce cas, considérés comme cibles potentielles pour des thérapies. Dans ce contexte, j’ai concentré mon travail de doctorat sur l’étude de la diversité des sous- populations de lymphocytes T auxiliaires et de leur régulation. Premièrement, j’ai démontré que les cellules dendritiques activées par la TSLP sont capables d’induire la polarisation de lymphocytes T folliculaires. Ensuite, j’ai participé à la construction d’un modèle mathématique capable de prédire la réponse lymphocytaire T auxiliaire en fonction de signaux dérivés des cellules dendritiques. Ce modèle nous a permis d’identifier un rôle spécifique pour l’IL-12p70, dépendant du contexte IL-1, dans l’induction d’IL-17F sans IL-17A. Enfin, j’ai monitoré huit populations de lymphocytes T auxiliaires et folliculaires dans le sang périphérique de patients atteints de dermatite atopique traités par Dupilumab, une immunothérapie ciblant la sous-unité alpha du récepteur de l’IL-4 et j’ai pu montré que la diminution du pourcentage de lymphocytes Th17 correlait avec l’amélioration du score clinique EASI. Globalement, mon travail sur la diversité de phénotypes Th apporte une ressource mécanistique importante, avec une potentielle application en immunothérapie. / Human immunity is essentially driven by dendritic cells and T helper cells. When dendritic cells detect a pathogen, they will instruct T helper cells to adopt the adapted phenotype for the specific threat encountered. T helper cells are subdivided in multiple subsets, characterized by particular sets of cytokines. Each T helper subset has specific functions and is involved in the clearance of distinct pathogens. If T helper responses are not precisely regulated, they can become pathogenic, in this case T helper pathways can be considered as potential targets for therapy. In this context, I focused my PhD work on studying T helper cell subset diversity and regulation. First, I demonstrated the ability of TSLP-activated dendritic cell to induce T follicular helper cell polarization. Then I participated in building a mathematical model capable of predicting T helper cell response to dendritic-cell derived signals. This model allowed us to identify the specific role of IL-12p70, in an IL-1 context, to induce IL-17F without IL-17A. Finally, I monitered eight T helper and T follicular helper cell populations in peripheral blood from atopic dermatitis patients treated with Dupilumab, an immunotherapy targeting the IL-4 receptor alpha subunit, and was able to show a correlation between decrease of Th17 cell percentage and improvement of EASI clinical score. Overall, my work on Th phenotype diversity provides key mechanistic insight with potential application in immunotherapy.

INTRODUÇÃO: A inflamação das vias aéreas na asma envolve respostas imunes inatas. Os receptores do tipo Toll (Toll-like receptors, TLRs) e a citocina linfopoetina do estroma tímico (thymic stromal lymphopoietin, TSLP) estão envolvidos na inflamação brônquica da asma, mas a expressão destas proteínas em vias aéreas grandes e pequenas de asmáticos ainda não foi investigada. Os objetivos deste estudo foram analisar a expressão protéica de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de asmáticos, comparar sua expressão entre asmáticos tabagistas e não tabagistas e investigar se a expressão dos TLRs está associada à infecção por Chlamydophila pneumoniae e Mycoplasma pneumoniae. MÉTODOS: Foram analisadas por método imuno-histoquímico e análise de imagens as expressões de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de 24 indivíduos falecidos por asma (13 não tabagistas e 11 tabagistas) e 9 controles não asmáticos. A análise das proteínas foi realizada em quatro regiões das vias aéreas: camadas epitelial, interna, muscular e externa. A presença de C. pneumoniae e M. pneumoniae no tecido pulmonar foi investigada por meio de reação em cadeia da polimerase em tempo real. RESULTADOS: Os indivíduos asmáticos apresentaram maior expressão de TLR2 nas camadas epitelial e externa de vias aéreas grandes e pequenas, e maior TLR2 na camada muscular de vias aéreas pequenas. Asmáticos tabagistas tiveram menor expressão de TLR2 nas camadas interna e externa de vias aéreas pequenas do que asmáticos não tabagistas. Indivíduos asmáticos tiveram maior expressão de TSLP na camada epitelial e externa de vias aéreas grandes, aumento de TLR3 na camada externa de vias aéreas grandes e aumento de TLR4 na camada externa de vias aéreas pequenas. O DNA de C. pneumoniae e M. pneumoniae não foi detectado em nenhum indivíduo asmático ou controle. CONCLUSÕES: Os receptores da imunidade inata TLR2, 3 e 4 e a citocina TSLP estão aumentados nas vias aéreas de pacientes falecidos por asma, e a expressão dos TLRs não está associada à presença de Chlamydophila pneumoniae e Mycoplasma pneumoniae nos pulmões. O tabagismo em asmáticos parece reduzir a expressão de TLR2 em vias aéreas pequenas. Estes resultados sugerem que os TLRs 2, 3 e 4 e a TSLP podem contribuir com a inflamação brônquica presente em exacerbações graves de asma e que as bactérias C. pneumoniae e M. pneumoniae não estão envolvidas em óbitos por asma / INTRODUCTION: Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and the cytokine thymic stromal lymphopoietin (TSLP) are involved in bronchial inflammation in asthma, but the expression of these proteins in large and small airways of asthmatics has not been investigated. The aims of this study were to analyze the protein expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics, to compare their expression in smoking and nonsmoking asthmatics and to investigate if TLR expression in associated with infection by Chlamydophila pneumoniae and Mycoplasma pneumoniae. METHODS: Using immunohistochemistry and image analysis, we investigated the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of 24 fatal asthma patients (13 nonsmokers and 11 smokers) and 9 nonasthmatic controls. The protein expression was analyzed in four regions of the airways: epithelial, internal, airway smooth muscle and outer layers. C. pneumoniae and M. pneumoniae presence in lung tissue was analyzed by real-time polymerase chain reaction. RESULTS: Fatal asthma patients had increased expression of TLR2 in the epithelial and outer layers of large and small airways, and also higher TLR2 in the muscle layer of small airways. Smoking asthmatics had lower TLR2 in the inner and outer layers of small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of large airways. Asthmatics also had greater TLR3 in the outer layer of large airways and greater TLR4 in the outer layer of small airways. C. pneumoniae and M. pneumoniae DNA was not detected in asthmatics or controls. CONCLUSIONS: Innate immunity receptors TLR2, 3 and 4 and innate cytokine TSLP are increased in the airways of fatal asthma patients, and TLRs expression is not associated with the presence of Mycoplasma pneumoniae and Chlamydophila pneumoniae in the lungs. Smoking may reduce TLR2 expression in the small airways of asthmatics. These results suggest that TLR2, 3, 4 and TSLP may contribute to the bronchial inflammation seen in severe exacerbations of asthma and that M. pneumoniae and C. pneumoniae are not involved in fatal asthma exacerbations

INTRODUÇÃO: A inflamação das vias aéreas na asma envolve respostas imunes inatas. Os receptores do tipo Toll (Toll-like receptors, TLRs) e a citocina linfopoetina do estroma tímico (thymic stromal lymphopoietin, TSLP) estão envolvidos na inflamação brônquica da asma, mas a expressão destas proteínas em vias aéreas grandes e pequenas de asmáticos ainda não foi investigada. Os objetivos deste estudo foram analisar a expressão protéica de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de asmáticos, comparar sua expressão entre asmáticos tabagistas e não tabagistas e investigar se a expressão dos TLRs está associada à infecção por Chlamydophila pneumoniae e Mycoplasma pneumoniae. MÉTODOS: Foram analisadas por método imuno-histoquímico e análise de imagens as expressões de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de 24 indivíduos falecidos por asma (13 não tabagistas e 11 tabagistas) e 9 controles não asmáticos. A análise das proteínas foi realizada em quatro regiões das vias aéreas: camadas epitelial, interna, muscular e externa. A presença de C. pneumoniae e M. pneumoniae no tecido pulmonar foi investigada por meio de reação em cadeia da polimerase em tempo real. RESULTADOS: Os indivíduos asmáticos apresentaram maior expressão de TLR2 nas camadas epitelial e externa de vias aéreas grandes e pequenas, e maior TLR2 na camada muscular de vias aéreas pequenas. Asmáticos tabagistas tiveram menor expressão de TLR2 nas camadas interna e externa de vias aéreas pequenas do que asmáticos não tabagistas. Indivíduos asmáticos tiveram maior expressão de TSLP na camada epitelial e externa de vias aéreas grandes, aumento de TLR3 na camada externa de vias aéreas grandes e aumento de TLR4 na camada externa de vias aéreas pequenas. O DNA de C. pneumoniae e M. pneumoniae não foi detectado em nenhum indivíduo asmático ou controle. CONCLUSÕES: Os receptores da imunidade inata TLR2, 3 e 4 e a citocina TSLP estão aumentados nas vias aéreas de pacientes falecidos por asma, e a expressão dos TLRs não está associada à presença de Chlamydophila pneumoniae e Mycoplasma pneumoniae nos pulmões. O tabagismo em asmáticos parece reduzir a expressão de TLR2 em vias aéreas pequenas. Estes resultados sugerem que os TLRs 2, 3 e 4 e a TSLP podem contribuir com a inflamação brônquica presente em exacerbações graves de asma e que as bactérias C. pneumoniae e M. pneumoniae não estão envolvidas em óbitos por asma / INTRODUCTION: Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and the cytokine thymic stromal lymphopoietin (TSLP) are involved in bronchial inflammation in asthma, but the expression of these proteins in large and small airways of asthmatics has not been investigated. The aims of this study were to analyze the protein expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics, to compare their expression in smoking and nonsmoking asthmatics and to investigate if TLR expression in associated with infection by Chlamydophila pneumoniae and Mycoplasma pneumoniae. METHODS: Using immunohistochemistry and image analysis, we investigated the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of 24 fatal asthma patients (13 nonsmokers and 11 smokers) and 9 nonasthmatic controls. The protein expression was analyzed in four regions of the airways: epithelial, internal, airway smooth muscle and outer layers. C. pneumoniae and M. pneumoniae presence in lung tissue was analyzed by real-time polymerase chain reaction. RESULTS: Fatal asthma patients had increased expression of TLR2 in the epithelial and outer layers of large and small airways, and also higher TLR2 in the muscle layer of small airways. Smoking asthmatics had lower TLR2 in the inner and outer layers of small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of large airways. Asthmatics also had greater TLR3 in the outer layer of large airways and greater TLR4 in the outer layer of small airways. C. pneumoniae and M. pneumoniae DNA was not detected in asthmatics or controls. CONCLUSIONS: Innate immunity receptors TLR2, 3 and 4 and innate cytokine TSLP are increased in the airways of fatal asthma patients, and TLRs expression is not associated with the presence of Mycoplasma pneumoniae and Chlamydophila pneumoniae in the lungs. Smoking may reduce TLR2 expression in the small airways of asthmatics. These results suggest that TLR2, 3, 4 and TSLP may contribute to the bronchial inflammation seen in severe exacerbations of asthma and that M. pneumoniae and C. pneumoniae are not involved in fatal asthma exacerbations