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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23814号 / 医科博第135号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 松村 由美, 教授 森本 尚樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Epithelial cells are the first line of defense against invading pathogens and external threats in the environment. Keratinocytes, often not perceived of as immune cells, release cytokines in response to infection or injury to signal danger to neighboring cells and recruit effector leukocytes to prevent further damage to the host. IL-1 and IL-36 cytokines are a group of closely related proteins that share similarities in structure and function and have been shown to play key roles in inflammatory responses of epithelial tissues. While IL-1, consisting of IL-1α and IL-1β, have been widely studied and recognized as pinnacle cytokines in a variety of inflammatory responses, relatively little is understood about IL-36 cytokines since their discovery more than 15 years ago, and how they differ from their better-known IL-1 relatives. IL-36 cytokines, consisting of IL-36α, IL-36β, and IL-36γ, signal through the same receptor, IL-36R, which is expressed most abundantly on epithelial cells. IL-36 proteins garnered attention when it was discovered that a missense mutation in the gene encoding the naturally occurring receptor antagonist, IL-36Ra, was associated with the deadly form of psoriasis, generalized pustular psoriasis (GPP). This disease is characterized by episodic flares of keratinocyte hyperproliferation leading to red scaly lesions all over the body, excessive neutrophil recruitment to the epidermis resulting in pustule formation, and severe fever. Our data presented here demonstrate that IL-36α, but not IL-36β or IL-36γ is critical for the psoriatic phenotype, including epidermal thickening and neutrophil recruitment, generated during a murine model of psoriasis induced by the drug Imiquimod. Furthermore, IL-36α was found to induce IL-1α expression and vice versa through a signaling feedback loop which perpetuated disease. These data provide insight into mechanisms whereby IL-36 signaling can lead to excessive inflammatory effects in patients with pre-existing regulation deficiencies, which can lead to acute flares of disease. Beyond their association with disease, IL-1 has been shown to contribute to anti-bacterial and anti-viral responses of the immune system by upregulating inflammatory signals and chemoattractants. Herpes Simplex Virus-1 (HSV-1) is a human pathogen that has developed several strategies to manipulate elements of the immune system to avoid detection by the host. One such mechanism is the prevention of activation and release of IL-1β from infected cells thereby blocking its pro-inflammatory responses. Our data show that keratinocytes infected with HSV-1 actively release IL-1α to alert danger to neighboring cells to circumvent this blockage of IL-1β signaling. This release of IL-1α initiates recruitment of leukocytes to early HSV-1 microinfection sites resulting in increased protection against disease, as evident by the increased mortality rate of mice deficient in the IL-1 receptor, IL-1R1. This study, for the first time in vivo, demonstrates the ability of IL-1α to act as an alarmin to initiate an immune response to combat infection. The role of IL-36 cytokines during viral infections has been less defined than that of IL-1. Several studies have shown the upregulation of IL-36 expression during viral infections in epithelial tissues, such as HSV-1 and Influenza, yet a direct link has not been established between these proteins and anti-viral responses. Our research presented within this thesis show that IL-36β, but not IL-36α nor IL-36γ, provides protection against the lethal outcome of cutaneous HSV-1 infection, as demonstrated by IL-36β knockout mice dying earlier and more often than wild type mice. Surprisingly, while previous reports have found IL-36 cytokines to be capable of activating the adaptive immune system, our results found no significant differences in development of HSV-1 specific antibodies or CD8+ T cell development between wild type and IL-36β knockout mice. Furthermore, we found no significant differences in viral copy numbers at infection sites between the two groups. Although our data show that IL-36β clearly plays a critical role in controlling the outcome of HSV-1 infection, further studies are necessary to define the mechanisms behind this protection. The final section of this thesis focuses on the endogenous nature of IL-36 cytokines, specifically IL-36γ, and their potential processing. IL-36 cytokines were originally believed to be synthesized as full-length fully active proteins; however, large concentrations of the recombinant proteins were required to elicit cellular responses in vitro. Since then, studies have shown that IL-36 cytokines gained up to 1000-fold increases in reactivity following processing at very specific N-terminal locations of each individual cytokine, however this processing has never been shown to occur in vivo. These studies were recently expanded when neutrophil proteases were found to be responsible for processing of these proteins in vitro. Data presented here show, for the first time, that IL-36γ may be endogenously processed by neutrophils in wounded murine skin in vivo, yet, the amino acid processing site appears to be different from that predicted. Although further studies are required to fully characterize the nature of this processing, these data provide valuable insight into the natural mechanisms involved in the potential activation of these cytokines. Taken together, the research presented within this thesis sheds light on the mechanisms whereby IL-1 and IL-36 cytokines enhance immunological defenses against potential threats, and yet, can contribute to disease if unregulated. Furthermore, these studies demonstrate the evolutionary advantage of producing multiple cytokines that appear to have redundant roles within the body, yet can provide multiple levels of protection to the host. This knowledge contributes to our overall understanding of these proteins and their contribution to immunological systems within the body. / Microbiology and Immunology

No / Agents which increase intracellular cyclic adenosine monophosphate (cAMP) may have an antagonistic effect on pro-inflammatory molecule production so that inhibitors of the cAMP degrading phosphodiesterases have been identified as promising drugs in chronic inflammatory disorders. Although many such inhibitors have been developed, their introduction in the clinic has been hampered by their narrow therapeutic window with side effects such as nausea and emesis occurring at sub-therapeutic levels. The latest generation of inhibitors selective for phosphodiesterase 4 (PDE4), such as apremilast and roflumilast, seems to have an improved therapeutic index. While roflumilast has been approved for the treatment of exacerbated chronic obstructive pulmonary disease (COPD), apremilast shows promising activity in dermatological and rheumatological conditions. Studies in psoriasis and psoriatic arthritis have demonstrated clinical activity of apremilast. Efficacy in psoriasis is probably equivalent to methotrexate but less than that of monoclonal antibody inhibitors of tumour necrosis factor (TNFi). Similarly, in psoriatic arthritis efficacy is less than that of TNF inhibitors. PDE4 inhibitors hold the promise to broaden the portfolio of anti-inflammatory therapeutic approaches in a range of chronic inflammatory diseases which may include granulomatous skin diseases, some subtypes of chronic eczema and probably cutaneous lupus erythematosus. In this review, the authors highlight the mode of action of PDE4 inhibitors on skin and joint inflammatory responses and discuss their future role in clinical practice. Current developments in the field including the development of topical applications and the development of PDE4 inhibitors which specifically target the subform PDE4B will be discussed.

No

Yes / The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence. / Faculty of Life Sciences, University of Bradford. MRC, Grant/Award Number: MR/M01942X/1; British Skin Foundation, Grant/Award Number: BSF 5035.

Le psoriasis est une maladie cutanée grave pour laquelle il n’existe encore aucun traitement curatif. Des modèles de peau psoriasique in vitro sont alors nécessaires afin de tester de nouveaux traitements. Cette étude visait, en premier lieu, à optimiser la production de substituts cutanés psoriasiques par la méthode d’auto-assemblage, pour ensuite les utiliser afin d’évaluer l’efficacité d’une nouvelle molécule potentiellement antipsoriasique, la tBEU. Dans un premier temps, des substituts sains et psoriasiques ont été produits par la méthode d’auto-assemblage partiellement modifiée en utilisant des plaques 6 puits et 12 puits. Ces expériences ont permis de démontrer que les plaques 6 puits étaient plus efficaces pour la production de substituts reproductibles et représentatifs d’une peau psoriasique in vivo. Ensuite, ces substituts ont été traités avec la tBEU, démontrant que cette molécule diminue la prolifération des kératinocytes et améliore leur différenciation. Ainsi, la tBEU pourrait éventuellement être utilisée comme un traitement efficace du psoriasis. / Psoriasis is a serious cutaneous disease for which there is currently no cure. In vitro psoriatic skin models could become new tools to assess the potency of new drugs during the development of new therapies. First, this study aimed to optimize the production of psoriatic skin substitutes by the self-assembly method, and then use them to assess the effectiveness of a new potentially antipsoriatic molecule, tBEU. Initially, healthy and psoriatic substitutes have been produced by the self-assembly method partially modified, using 6-well and 12-well plates. These experiments demonstrated that the 6-well plates were more effective for the production of reproducible substitutes and representative of psoriatic skin in vivo than the 12-well plates. Then, these substitutes were treated with tBEU, showing that this molecule decreases significantly keratinocytes proliferation and improves their differentiation. Thus, tBEU may eventually be used as an effective topical treatment for psoriasis.

Le psoriasis se caractérise par la présence d’infiltrats leucocytaires inflammatoires au niveau du derme et de l’épiderme cutané humain, menant à des désordres cutanés considérables : hyperprolifération des cellules épidermiques basales, différenciation altérée des kératinocytes et sécrétion accrue de médiateurs pro-inflammatoires dans le microenvironnement local. Cette dysfonction immuno-épithéliale menant au développement de lésions blanchâtres, agit tel un cercle vicieux incontrôlable, stimulant réciproquement les kératinocytes et les cellules immunitaires. Dans cette optique, mes travaux de doctorat ont visé à : (1) générer un microenvironnement immunocompétent par l’intégration de cellules T pré-activées dans un modèle de peau saine, non-lésionnelle et lésionnelle, produit selon la méthode d’auto-assemblage afin (2) d’étudier le rôle de cette composante importante sur les caractéristiques-clés associées au psoriasis. De surcroit, nous avions pour autre objectif (3) d’analyser l’impact de macrophages dérivés de monocytes dans un modèle tridimensionnel de peau saine afin (4) de déterminer l’influence de ces cellules sur le remodelage matriciel et la différenciation épidermique à l’état d’équilibre pour, finalement, (5) analyser l’empreinte du microenvironnement sur leur polarisation in vitro. Nos données mettent en lumière le caractère déterminant de la composante immunitaire, lorsqu’associée aux cellules cutanées pathologiques, dans le développement des lésions psoriasiques, puisque la présence de cellules T, dans un modèle de peau saine, n’induit pas l’ensemble des caractéristiques histopathologiques associées à cette dermatose. D’autre part, ces modèles de peau immunocompétente répondent à un traitement de choix pour le psoriasis, le méthotrexate, qui inhibe la prolifération cellulaire et atténue l’inflammation. Ces nouveaux modèles organotypiques serviront d’outils précliniques performants pour le criblage d’actifs visant à traiter certaines pathologies d’origine auto-immune inflammatoire chronique, dans lesquelles le système immunitaire joue un rôle crucial, à l’exemple du psoriasis. Par ailleurs, l’intégration de macrophages dérivés de monocytes dans un modèle de peau normale, affecte l’état de différenciation des kératinocytes épidermiques tout en réduisant l’état inflammatoire basal. Ces macrophages induisent, en outre, un remodelage matriciel tissulaire important modifiant de ce fait le microenvironnement avoisinant. Finalement, ce nouveau modèle tridimensionnel nous a permis, d’une part, de mimer davantage la structure cutanée physiologique et, d’autre part, de mieux comprendre l’influence de ces cellules sur le phénotype cutané à l’état d’équilibre. Bien que d’autres études soient nécessaires avant l’utilisation de ces modèles en phase préclinique, ces travaux constituent une avancée majeure dans le développement de modèles produits par génie tissulaire, plus complexes et plus pertinents d’un point de vue physiologique. / Psoriasis is characterized by the presence of inflammatory leukocyte infiltrates in the human skin dermis and epidermis, leading to important skin disorders: hyperproliferation of basal epidermal cells, altered differentiation of keratinocytes, and increased secretion of proinflammatory mediators in the local microenvironment. This immuno-epithelial dysfunction leading to the development of whitish lesions acts as an uncontrollable vicious circle, stimulating the keratinocytes and the immune cells. With this in mind, my thesis work aimed to: (1) generate an immunocompetent microenvironment by integrating pre-activated T cells into a healthy, non-lesional and lesional skin model, produced using the self-assembly method (2) to study the role of this essential component on key features associated with psoriasis. In addition, our other objective was (3) to analyze the impact of monocyte-derived macrophages in a three-dimensional model of healthy skin (4) to determine the influence of these cells on tissue remodeling and epidermal differentiation at steady state, (5) in order to finally analyze the microenvironment effect on their in vitro polarization. Our data highlight the essential feature of the immune component, when associated with pathological skin cells, in the development of psoriatic lesions, since the presence of T cells, in a healthy skin model, does not induce all of the histopathological key features, associated with the dermatosis. On the other hand, these models of immunocompetent skin respond to a treatment of choice for psoriasis, methotrexate, which inhibits cell proliferation and reduces inflammation. These new organotypic models will serve as powerful preclinical tools for the screening of active agents to treat some chronic inflammatory pathologies of autoimmune origin, in which the immune system plays a crucial role, like psoriasis. Moreover, the integration of monocytederived macrophages into a normal skin model affects the differentiation state of epidermal keratinocytes while reducing the basal inflammatory state. These macrophages induce, in addition, a significant matrix remodeling thereby modifying the surrounding microenvironment. Finally, this new three-dimensional model allowed us, on the one hand, to further mimic the physiological cutaneous structure and, on the other hand, to better understand the influence of these cells on the skin phenotype at steady state. Although further studies are needed prior to the use of these preclinical models, this work represents a major breakthrough in the development of more complex tissue-engineered models that are physiologically relevant.

Le psoriasis est une maladie cutanée inflammatoire multifactorielle chronique qui affecte 2 à 3 % de la population mondiale. Le mécanisme d'entrée en jeu de la pathologie est encore inconnu. De même, aucun traitement curatif n'existe sur le marché pharmaceutique menant à un rétablissement complet du phénotype des lésions psoriasiques. Certaines voies signalétiques ont été montrées comme étant dérégulées au niveau des kératinocytes de l'épiderme suite à l'infiltration de cellules immunitaires et de cytokines, mais certaines sont encore peu étudiées. L'objectif de ce projet a été d'étudier les trois principales voies de la famille des MAPKS dans la pathologie du psoriasis, soit ERK1/2, JNK et p38, en les inhibant, afin d'en observer les effets soit sur le modèle de peaux psoriasiques 3D reconstruites par génie tissulaire ou sur un modèle de monocouche. Il a été intéressant d'étudier le mécanisme d'action des inhibiteurs aux niveaux cytosolique et nucléaire, au regard du niveau d'expression de différentes protéines et de différents facteurs de transcription. Celles-ci ont été identifiées comme étant surexprimées chez les patients psoriasiques. Des substituts cutanés sains et psoriasiques ont été préparés selon la méthode d'auto-assemblage en présence de cytokines et de différents traitements visant les voies signalétiques ERK 1/2, JNK et p38. Des monocouches ont été préparées dans le but d'obtenir des extraits nucléaires permettant de déterminer le niveau d'activation de certains facteurs de transcription par retard sur gel (EMSA). Des analyses histologiques et protéiques ont été effectuées sur des substituts lésionnels et sains. Les résultats démontrent que le traitement des substituts avec les différents inhibiteurs tend vers un rétablissement partiel du phénotype des lésions psoriasiques vers un phénotype sain suite à une diminution de l'épaisseur de l'épiderme chez les substituts cutanés psoriasiques. La régulation de l'expression de différents marqueurs de prolifération et de différenciation est également observée. Les inhibiteurs introduits en monocouche montrent un rétablissement de l'expression de certains facteurs de transcription potentiellement impliqués dans le psoriasis. En conclusion, une meilleure compréhension des voies signalétiques affectées dans le psoriasis permettra le développement efficace et plus ciblé des traitements envisagés pour soigner cette pathologie. / Psoriasis is a chronic multifactorial inflammatory skin disease that affects 2-3% of the world's population. The mechanism of the onset of the disease is still unknown. Also, no curative treatment exists on the pharmaceutical market that leads to a complete recovery of the psoriatic lesion phenotype. Some signaling pathways have been shown to be deregulated in epidermal keratinocytes following infiltration of immune cells and cytokines, but many are still poorly studied. The objective of this project was to study the three main pathways of the MAPKS family in psoriasis pathology, ERK1/2, JNK and p38, by inhibiting them, to observe their effects on the 3D psoriatic skin model reconstructed by tissue engineering and on a monolayer model. It was interesting to study the mechanism of action of the inhibitors at the cytosolic and nuclear levels, with regard to the expression level of different proteins and transcription factors. These pathways were identified as being overexpressed in psoriatic patients. Healthy and psoriatic skin substitutes were prepared using the self-assembly method in the presence of cytokines and different treatments targeting the s6 ribosomal kinase (RSK) of the ERK 1/2 pathway kinase, JNK and p38. Monolayers were prepared to obtain nuclear extracts to determine the level of activation of certain transcription factors by gel electrophoretic mobility shift assay (EMSA). Histologic and protein analyses were performed for the study of lesional and healthy substitutes. The results show that treatment of the substitutes with the different inhibitors leads to a tendency of partial recovery of the psoriatic lesion phenotype towards a healthy phenotype following a decrease in epidermal thickness in psoriatic skin substitutes. Variations in the expression of different proliferation and differentiation markers were also observed. Inhibitors introduced in monolayers show a restoration of the expression of some transcription factors potentially involved in psoriasis. In conclusion, a better understanding of the signaling pathways affected in psoriasis will allow for the efficient and more targeted development of treatments envisaged to treat this pathology.

Die Psoriasis ist mit einer Prävalenz von 1-3% eine der häufigsten chronisch-entzündlichen Erkrankungen. Histopathologisch ist sie gekennzeichnet durch epidermale Hyperkeratose und Akanthose, gesteigerte Angiogenese sowie ein gemischtzelliges leukozytäres Infiltrat. Aus aktueller Sicht wird sie als eine komplexe primär T-Zell vermittelte Autoimmunerkrankung mit genetischer Prädisposition verstanden. Psoriasis kann mit kardiovaskulären, metabolischen und psychiatrischen Erkrankungen assoziiert sein (Komorbidität). Hier wurde erstmals die Wirkung des inositoylierten Plättchen-aktivierenden Faktors (Ino-C2-PAF), eines synthetischen Alkylphospholipids mit geringer Toxizität, in experimentellen Modellen chronisch-entzündlicher (Haut)-Erkrankungen untersucht. Die Wirkung beruht auf Zellmembran-Interaktionen, wodurch der Cholesterol- und Phospholipidmetabolismus verändert und zelluläre Signalkaskaden der Proliferation, Apoptose und Motilität beeinträchtigt werden. Ino-C2- PAF reguliert außerdem entzündungsrelevante Proteine herab. In vitro lag der Schwerpunkt auf der Wirkung von Ino-C2-PAF auf Endothelzellen und Leukozyten. Ino-C2-PAF hemmte die Proliferation humaner Endothelzellen moderat, steigerte allerdings die Apoptose TNFα-stimulierter Endothelzellen sehr deutlich. Diese Wirkung war begleitet von einer Reduktion der durch TNFα stimulierbaren endothelialen Adhäsionsmoleküle VCAM-1, ICAM-1 und E-Selektin sowie der lymphozytären Adhäsionsmoleküle CD49d, CD11a, CD62L und CLA. Funktionell führte dies zu einer signifikanten Abnahme dynamischer Interaktionen (Rollen und feste Adhäsion) von Leukozyten und aktivierten Endothelzellen in Flusskammerexperimenten. Die anti-entzündliche Wirkung von Ino-C2-PAF wurde in zwei komplementären Modellen in vivo bestätigt. Sowohl in K5.hTFGß-transgenen als auch in JunB/c-Jun-defizienten Mäusen bewirkte Ino-C2-PAF eine signifikante Besserung des psoriasiformen Phänotyps sowohl auf makroskopischer als auch auf histopathologischer Ebene. Die Ergebnisse dieser Arbeit legen nahe, dass Ino-C2-PAF oder verwandte Substanzen zur Behandlung entzündlicher Erkrankungen wie der Psoriasis eingesetzt werden könnten.