La spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent avec une prévalence de 0,43% en France, fortement associée à HLA-B27. À l’heure actuelle, il n’existe aucun traitement curatif et les mécanismes physiopathologiques impliqués restent méconnus. Afin de mieux comprendre les mécanismes immunologiques impliqués dans le développement de la SpA, nous avons étudié deux populations cellulaires clé, les cellules dendritiques (DC) et les lymphocytes T (LT) CD4+, chez le rat transgénique pour le HLA-B27 et la β2 microglobuline humaine (rat B27) qui développe spontanément tous les symptômes de la SpA. Il a été démontré que l’accumulation de lymphocytes T helper producteurs d’interleukine 17 (IL-17) pathogénique (lymphocyte Th17), et plusieurs défauts fonctionnels des cellules dendritiques (DCs) sont corrélés avec le développement de la SpA chez les rats B27.Nous nous sommes tout d’abord intéressés aux lymphocytes T régulateurs (Treg), dont le rôle est d’empêcher l’établissement d’une réponse immune pathogène pour l’hôte, chez le rat B27. Nous avons découvert que les Treg de rats B27 présentent un phénotype pro-inflammatoire (surexpression d’IL-17 et sous-expression d’IL-10 anti-inflammatoire), lié à la surexpression de la molécule ICOS. De plus, la sévérité des signes cliniques chez les rats B27 n’exprimant pas ICOS (rats B27 ICOS KO) est diminuée comparé aux animaux HLA-B27 sauvages. Cette protection partielle est corrélée à une réduction de la proportion de lymphocytes Th17. Ces résultats mettent en lumière le rôle majeur d’ICOS dans la physiopathologie de la SpA du rat.La deuxième partie de ce travail s’est concentrée sur les conséquences de la sous-expression d’IL-27 par les DC de rats B27, cytokine connue pour inhiber le développement des Th17. Nous avons observé que l’addition d’IL-27 exogène permet de diminuer la production d’IL-17 et d’augmenter la synthèse d’IL-10 anti-inflammatoire par les LT différenciés (T effecteurs et Treg) et les LT naïfs de rats B27 différenciés in vitro. De façon intéressante, l’IL-27 réduit également la synthèse d’IL-17 par les LT CD4+ circulants de patients atteints de SpA.Ces travaux démontrent pour la première fois le rôle clé de l’IL-27 et d’ICOS dans le contrôle de l’inflammation chez le rat B27 et suggèrent fortement que ces deux molécules sont de nouvelles cibles thérapeutiques prometteuses dans la SpA. / Spondyloarthritis (SpA) is a frequent chronic rheumatic inflammatory disorder with a prevalence of 0.43% in France and closely associated to HLA-B27. To date, there is no curative treatment and pathophysiological mechanisms involved in this pathology remain elusive. To better understand these mechanisms, we studied two crucial cell populations, dendritic cells (DC) and CD4+ T cells in rats transgenic for HLA-B27 and human β2 microglobulin (B27 rats) which spontaneously develop a phenotype closely resembling human spndyloarthritis. Previous studies demonstrated that accumulation of pathogenic IL-17 producing T cells (Th17 cells) and several function defects of DCs are correlated with SpA development in B27 rats.First, we focused on regulatory T cells, whose role is to prevent the establishment of pathogenic immune responses. We discovered that Treg from B27 rats have a pro-inflammatory phenotype. They overexpress IL-17 and underexpress anti-inflammatory IL-10, linked to ICOS overexpression. Furthermore, B27 rats knock-out for ICOS (B27 ICOS KO rats) have reduced severity of clinical symptoms compared to B27 ICOS WT rats. This protective effect is correlated with a reduced proportion of Th17 cells. These results highlight the crucial role of ICOS in rat SpA physiopathology.In the second part of this work we studied the consequences of IL-27 underexpression by B27 DC, a cytokine known to inhibit Th17 development. Addition of exogenous IL-27 reduces IL-17 and increases IL-10 productions by differentiated T cells (Teff and Treg) and by naive T cells polarized in vitro. Interestingly, IL-27 also reduces IL-17 production by circulating CD4+ T cells isolated from blood of SpA patients.This work demonstrate for the first time the key role of IL-27 and ICOS in the control of inflammation in B27 rats and highly suggest that these molecules may be new promising therapeutic targets in SpA.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18684号 / 医博第3956号 / 新制||医||1007(附属図書館) / 31617 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊達 洋至, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Au cours des deux dernières décennies, la stéatose hépatique non alcoolique (NAFLD) a été une maladie épidémique croissante, non seulement dans les pays occidentaux mais également dans le monde entier en raison de l’augmentation continue des modes de vie sédentaires, de l’obésité, et de la résistance à l’insuline. La prévalence mondiale de la NALFD est actuellement estimée à 25% dans la population générale adulte. NALFD est composé d’un éventail d’affections hépatiques s’étendant du foie gras non-alcoolique (NAFL), stéatohépatite non-alcoolique (NASH), fibrose avancée et cirrhose qui peut progresser au carcinome hépatocellulaire (HCC). L’inflammation induite par NASH peut moduler l’activation des cellules stellaires hépatiques (CSH) et donc influencer la progression de la fibrose hépatique. Le rôle de l’inflammation de type 3, qui est caractérisée par la production des cytokines IL-17A et IL-22, dans la fibrose de type NAFLD demeure incompris. Dans cette thèse, nous avons évalué le rôle d’IL-22 et d’IL-17A dans la fibrose liée à la NAFLD. Des biopsies cliniques de foie NAFLD humain et un modèle murin in vivo de NAFLD ont été utilisés et des expériences in vitro ont été effectuées. Nous avons démontré que l’expression hépatique d’IL-22 est plus élevée chez les femmes et chez les femelles avec NAFLD versus les hommes et les mâles. Nous avons identifié les neutrophiles et les cellules T, y compris les cellules T Th17, Th22 et γδ, en tant que principaux producteurs d’IL-22 chez les sujets féminins et les souris atteintes de NAFLD. De plus, nous avons démontré que l’absence de la signalisation endogène du récepteur IL-22 (modèle IL-22RA1 knockout) chez les souris femelles avec NAFLD, aggravait les lésions hépatiques, l’inflammation et la fibrose, comparé aux mâles. Cet effet hépatoprotecteur dépend des mécanismes anti-apoptotiques médiés par la signalisation du récepteur IL-22 qui favorisent la survie des hépatocytes et réduisent au minimum les dommages au foie. Nous avons également montré que l’expression hépatique d’IL-22BP est régulé à la hausse chez les souris femelles avec NAFLD comparé aux mâles. Dans ces femelles, le ratio d’ARN messager hépatique de l’IL-22 envers celui de l’IL-22BP est corrélé positivement avec les gènes en aval de cible d’IL-22 (gènes anti-apoptotiques et antioxydants). Par ailleurs, nous avons prouvé que les neutrophiles intrahépatiques produisent l’IL-17A in situ dans notre modèle NAFLD et ceci correspondait fortement avec la progression de la fibrose de foie et les dommages hépatiques. Nous avons fourni des preuves préliminaires que l’IL-17A peut induire des pièges extracellulaires de neutrophiles (NET) in vitro, et la signature de NETs est impliquée dans la progression de la fibrose hépatique dans notre NAFLD. Pris ensemble, Ces résultats démontrent qu’identifié un nouveau rôle de l’inflammation de type 3 dans la fibrose liée au NAFLD, où l’action de l’IL-22 est dépendante du sexe et possède des 4 fonctions hépatoprotectrices contre la fibrose du foie chez les femelles, alors que l’IL-17A agit en tant que cytokine profibrogénique et favorise la fibrose de foie. / Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic, not only in western countries but also worldwide due to the continuous rise in sedentary lifestyles, obesity, and insulin resistance over the last two decades. The global prevalence of NALFD is currently estimated to be 25% in the general adult population. NAFLD is comprised of a spectrum of liver disease ranging from non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), advanced fibrosis, and finally cirrhosis that can progress to hepatocellular carcinoma (HCC). NASH-induced inflammation can modulate hepatic stellate cells (HSCs) activation and hence influence hepatic fibrosis progression. The role of type 3 inflammation, which is characterized by the production of the cytokines IL-17A and IL-22, in NAFLD-related fibrosis remain not clear. In this thesis, we evaluated the role of IL-22 and IL-17A in NAFLD-related fibrosis using clinical liver biopsies from a NAFLD human cohort, an in vivo NAFLD mouse model and in vitro experiments. We report that hepatic IL-22 expression had sexually dimorphic differences in both humans and mice with NAFLD where it was elevated in females versus males. We identified intrahepatic neutrophils in female subjects with NAFLD as well as T cells, including Th17, Th22, γδ T cells, in female mice with NAFLD as major producers of IL-22. In addition, we demonstrated that lack of endogenous IL-22 receptor signaling (IL-22RA1 knockout model), exacerbated liver injury, inflammation, and fibrosis in female but not male mice with NAFLD. This hepatoprotective effect was dependent on IL-22 receptor signaling-induced anti-apoptotic signals that promote hepatocyte survival and minimize liver damage. We also demonstrated that hepatic IL-22BP expression was upregulated in female mice with NAFLD compared to males, and the hepatic IL22/IL-22BP mRNA ratio positively correlated with IL-22 downstream target genes (anti-apoptotic and antioxidant genes) in those females. Moreover, we showed that intrahepatic neutrophils produce IL-17A in situ in our NAFLD model and this was strongly correlated with progression of liver fibrosis and liver injury. We provided preliminary evidence that IL-17A can induce neutrophil extracellular traps (NETs) in vitro, and that NETs are implicated in liver fibrosis progression in our NAFLD model. Taken together, we identified a novel role for type 3 inflammation in NAFLD-related fibrosis, where IL-22 act in sex-dependent manner and provided hepatoprotective functions against liver fibrosis in females, while IL-17A act as profibrogenic cytokine and promotes liver fibrosis through enhancing NETs.

HIV infection has been shown to predispose patients to accelerated development of heart disease. One mechanism for this pathology may involve endothelial activation either by HIV itself or by its secreted proteins, gp120 (a viral envelope protein) and tat (a protein that upregulates transcription of viral genes). We have studied the effects of gp120 and tat on signaling and production of inflammatory cytokines by Human Pulmonary Artery Endothelial Cells (HPAEC). HPAEC were stimulated at varying time points with combinations of gp120, tat, and monokines (IL-1β and TNFα). Cell lysate fractions were analyzed for MAP Kinase activity and NFκB activation, and culture supernatants were assayed for inflammatory cytokines (IL-6 and IL-8). The production of IL-6 and IL-8 was significantly enhanced by tat but not by gp120. Both gp120 and tat, however, induced significant morphological changes in HPAEC. The only synergy noted was between high levels of tat and TNFα acting on the production of IL-6. When HPAEC were stimulated with IL-1β and TNFα, peak phosphorylation of p38 MAP Kinase was found at 45 minutes, while NFκB was maximally activated at two hours. Both the ERK1,2 and p38 cascades of MAP Kinase were activated by tat, and an increase in NFκB phosphorylation and translocation were noted. We conclude that the HIV tat protein could be involved in inflammatory changes in endothelium leading to the accelerated development of heart disease in HIV patients.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24547号 / 薬科博第164号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 生田 宏一, 教授 橋口 隆生, 教授 木村 郁夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The urban climate of the city of Trento, adopted as a representative case study of urban weather and climate phenomena in a mid-sized city lying in a mountain valley, is investigated using different methods and on different spatial scales. First the intensity of the Urban Heat Island (UHI) is analyzed evaluating the differences between air temperatures measured at an urban automated weather station on a tower, over mean rooftop level, and at five suburban/rural weather stations, located few kilometers around the city boundaries. It is found that the extra-urban weather stations, being affected by different local-scale climatic conditions, display different temperature contrasts compared to the urban site. However the diurnal cycle of the UHI is characterized by similar behaviors at all the extra-urban weather stations: the UHI intensity is stronger at night, while during the central hours of the day an “urban cool island†is likely to occur. The diurnal maximum UHI intensity turns out to be typically of order 3°C, but under particularly favorable conditions it may be higher than 6°C. Wind speed and cloud cover are the weather factors which most affect UHI intensity, making it weaker with stronger winds and cloudier skies. The investigation of the urban climate of Trento focuses then on a smaller spatial scale, analyzing in detail the thermal field inside an urban canyon located in the city center, by means of two experimental campaigns and the use of a simple model. This simple model simulates the energy balance of the different surfaces composing the urban canyon, calculating both surface and air temperatures inside the canopy. During the two field measurements, carried out in the summer 2007 and in the winter 2008- 2009, temperature sensors were placed at various levels near the walls flanking the canyon and on a traffic light in the center of the street. It is found that the air temperature near the walls, both in summer and in winter, is strongly influenced by direct solar radiation, thus inducing a quite strong imbalance within the canyon: during sunny days an overheating of the east-facing sensors is found in the morning, while in the afternoon west-facing sensors are the warmest. On the other hand, when solar radiation is weak or absent, the temperature field inside the canyon is homogeneous. Moreover air temperature inside the canyon is generally higher than above roof level, the differences being larger during summertime, when solar radiation is stronger and can penetrate for longer inside the street. The measurements performed during the field campaigns, along with observations of wall surface temperatures taken from the literature, allow to validate the results of the urban canyon model. A good agreement between experimental measurements and numerical results is found for both surface and air temperatures, in different seasons and under different weather conditions. The urban area of Trento, being located in the Alpine Adige Valley, interacts with the atmospheric phenomena typical of these contexts, in particular thermally driven local circulation systems. Moreover the city is located at a point where various narrow tributary valleys or gullies join the Adige Valley, and, as a consequence, complex interactions of local circulation systems are present in the area of Trento. In order to study these phenomena, first the main features of local circulation systems developing in the Adige, Sarca and Lakes valleys, which directly influence the climate of the city, are investigated by means of the analysis of a dataset from surface weather stations covering the period 2004-2011. After that, high-resolution (500 m) numerical simulations with the mesoscale meteorological WRF model, coupled with the multilayer Building Environment Parameterization (BEP) scheme, are utilized to study the urban climate of Trento in the Adige Valley context. Suitable datasets of land use, urban morphology and anthropogenic heat flux have been specifically prepared for these numerical simulations. Both methods highlight the substantial differences occurring between the local circulation system developing in the Adige Valley, and that blowing in the Sarca and Lakes valleys. The former is a typical valley wind, while the latter is a combination of a lake breeze and a valley wind. The along-valley wind developing in the Adige Valley is mainly determined by the local geometry of the valley, which controls the penetration of solar radiation and the heating of the valley slopes. The lake breeze, the so-called Ora del Garda, starts to blow from the shores of Lake Garda in the morning and then propagates with its cooler air towards north in the Sarca and Lakes valleys, outbreaking into the Adige Valley north of Trento in the first part of the afternoon. In some days the lake breeze is even able to reach the central part of the urban area of Trento, thus lowering the temperature in the city in hot summer afternoons. Focusing on the urban effects, the model is able to simulate correctly the daily cycle of the UHI, with high intensities during the night and negligible values in the central part of the day. Numerical results suggest that at night the temperature sharply increases at the city boundaries, while the thermal field is quite homogeneous inside the urban area, with only slightly higher temperatures where the urban morphology is more compact. Finally it is found that the presence of the city influences considerably also the wind field, due to the high roughness of the urban area.

Thermally-driven slope winds are mesoscale atmospheric circulations, known as breezes, that take place because of the heating (cooling) of the air layer close to the ground during daytime (nighttime). Mostly known to occur on days with weak synoptic forcing and under clear sky conditions, the wind blows up valleys and slopes during the daytime, and in the opposite direction during nighttime. A better comprehension of slope winds can improve the understanding of the soil-atmosphere turbulent exchange processes and of the energy budget over complex terrain, in addition to the evaluation of the along-slope transport of dangerous species (pollutants, pesticides), as well as water vapor (relevant for the development of convection). This research project aims to improve the knowledge of thermally-driven slope winds, with particular attention to the differences between the diurnal and nocturnal regimes. This is done through a multiple-way approach. Field data analysis, analytical solutions with realistic forcing, and numerical models are all employed to fulfill the objective. At first, data from two stations located on slopes were analyzed. Measurements were taken in the surroundings of the Alpine city of Innsbruck, as part of the i-Box field campaign, covering a period of 7 years (2013 to 2020). Observation indicates a marked seasonality of the phenomena, with warm season months being more prone to the occurrence of slope winds. Moreover, the results highlighted the key role played by the local topographical characteristics in the development of pure slope wind days, with both slope angle and orientation playing a major role in the interplay between valley and slope winds. Previous results suggested the development of an improved analytical model which uses the available net radiation at the surface as the forcing for slope circulations, in the form of a truncated Fourier series expansion. The net radiation model accounts for both the seasonality (day of the year) and the local topographic characteristics (latitude, slope angle, orientation, elevation). Therefore, differences in the properties of slope winds occurring in different seasons and on slopes with different slope angles and orientations are highlighted and studied. The last chapter of the thesis investigates the structure of the eddy viscosity and diffusivity employing numerical models. These parameters govern the mass, momentum, and heat turbulent exchanges from slope winds. A simple one-dimensional model was developed to test different turbulence closures. In particular, the attention focused on the so-called K-l closure, meaning that the eddy viscosity and diffusivity parameters are bounded to the turbulence length scale l, representing the distance a turbulent eddy can travel “carrying” heat, momentum, and mass. In the current work, different parameterizations of the turbulence length scale l are tested and compared. Results show how simple K-l closures are compared with other non-constant K profiles proposed in the literature for the case of katabatic winds. Nevertheless, such simple parameterizations for the turbulence length scale l still fail to properly discriminate between the daytime and nighttime regimes of slope winds.

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