Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 139-173. Publié aussi en version électronique.

Aucun résumé disponible. / No summary available.

L’interaction épithélium/mésenchyme est cruciale pour de nombreux processus physiopathologiques. Lors de ma thèse, je me suis intéressée aux signaux mésenchymateux régulant les cellules épithéliales en utilisant comme modèle la peau, qui est composée de 2 compartiments : l’épiderme (épithélium) et le derme (mésenchyme). Les intégrines sont impliquées ces interactions. CD98hc est une protéine transmembranaire à double fonction qui chaperonne des transporteurs d’acides aminés et régule la signalisation des ß intégrines. Elle est exprimée dans les cellules prolifératives telle les cellules épithéliales. Dans la peau, CD98hc est exprimée l’épiderme mais également dans les fibroblastes, cellules post-mitotiques. Mon hypothèse a été que CD98hc participe aux régulations des interactions derme/épiderme. Grâce à un modèle de KO conditionnel de CD98hc dans les fibroblastes dermiques, j’ai mis en évidence que CD98hc permet le maintient des propriétés mécaniques et biologiques du derme, et, de ce fait, régule l’épiderme en conditions d’homéostasie, de perturbation de la barrière et lors de la formation de cancer. De plus, le rôle de CD98hc dans cette interaction apparait comme étant lié à l’âge. En conclusion, mes travaux de thèse montrent le rôle central de l’expression dermique de CD98hc dans le maintien de l’homéostasie cutanée au cours du vieillissement ainsi que lors de la tumorigenèse. / The epithelial/mesenchymal interaction is crucial for many physiopathological processes. During my PhD, I focused on mesenchymal signals that regulate epithelial cells behavior using the skin as model. The skin is composed of 2 main compartments: the epidermis (epithelium) and the dermis (mesenchyme). While this crosstalk involves integrins, its regulations are poorly understood. The transmembrane protein CD98hc interacts with amino acid transporter and regulates integrin signaling. CD98hc which is expressed at the cell membrane of proliferative cells, specifically epithelial cells, is required for tissue homeostasis. We found that besides its expression in keratinocytes, CD98hc is also expressed in post-mitotic dermal fibroblast. Hence, I hypothesized that CD98hc is involved in epidermis/dermis crosstalk. Using a conditional KO mouse model that harbor a CD98hc deletion in dermal fibroblast, I have shown that dermal CD98hc is required to maintain mechanical and biochemical properties of the dermis. Moreover, I have shown that those CD98hc-dependent dermal properties are implicated in the regulation of the epidermal cell behavior during homeostasis, cutaneous barrier disruption and tumorigenesis. Moreover, the role of CD98hc in those processes seems to be age-related. To conclude, during my PhD, I have revealed a major role of CD98hc in the maintenance of skin homeostasis during aging and tumorigenesis.

L'utilisation de la dispase pour détacher les feuillets épidermiques est une étape pouvant être améliorée pour faciliter leurs transferts sur les plaies des grands brûlés. La culture sur colle de fibrine élimine cette étape. Les analyses histologiques et immunohistochimiques n'ont révélé aucune différence importante entre ces deux types de feuillets épidermiques. Le gel de fibrine est donc un substrat adéquat pour la production de feuillets épidermiques greffables. / Les équivalents cutanés complets complèteraient le traitement des grands brûlés. Toutefois, la production de feuillets dermiques est limitée par leur temps de production. Étant donné que le RGTA accélère la réparation de plusieurs tissus in vivo, l'effet du RGTA sur la production de feuillets dermiques fut testé. Des examens physiques et histologiques ont montré que le RGTA26 rendait les feuillets fragiles. Les ELISAs et les zymogrammes ont montré une modulation de la production collagénique et des MMPs. L'utilisation du RGTA pour produire les feuillets dermiques n'apparaît pas justifié.

La lumière solaire constitue le principal facteur de risque des cancers de peau non mélanocytaires (NMSC). L'effet génotoxique de la lumière solaire est dû aux dommages dans l'ADN induits par les rayonnements ultraviolets (UV). Les rayons UVB longs (290-315 nm) sont les principaux responsables de l'initiation et de la promotion des NMSC qui prennent naissance dans les kératinocytes épidermiques. En effet, l’absorption directe des photons d’UVB par l’ADN conduit à la génération de deux principaux types de dommages, les dimères cyclobutyliques de pyrimidines (CPD) et les photoproduits de pyrimidine (6-4) pyrimidone (6-4PP). Les CPD sont les plus abondants et sont hautement mutagènes. Ils sont responsables des mutations de transitions C → T au niveau des sites dipyrimidiniques, les mutations signatures observées dans les cancers de peau. Les cellules possèdent différents mécanismes pour éviter la conversion des CPD en mutations, à savoir, l’arrêt du cycle cellulaire, la réparation des dommages dans l'ADN par le système de réparation par excision de nucléotides (NER) et la mort cellulaire par apoptose. L’importance de la NER dans la prévention des cancers de peau est bien démontrée par le fait qu’une déficience en protéines de la NER, comme chez les patients atteints de Xeroderma Pigmentosum (XP), entraîne une incidence jusqu’à 2000 fois plus élevée de cancers de peau. De nombreux facteurs influencent la NER et une meilleure compréhension de ces derniers pourrait conduire au développement de nouvelles stratégies de prévention contre les cancers de peau. La peau est un assemblage complexe de cellules et de matrice dans lequel la communication entre les composants épidermiques et dermiques est essentielle pour de nombreux mécanismes cutanés. En utilisant des peaux reconstruites dérivées uniquement de fibroblastes et de kératinocytes primaires humains, nous avons analysé l’impact des composants dermiques sur l’efficacité de réparation des CPD épidermiques. Nous avons montré que l’élimination des CPD dans les kératinocytes est positivement influencée par la présence d'un derme et nous avons déterminé que cet effet du derme sur les kératinocytes proviendrait de molécules sécrétées. En étudiant le sécrétome, nous avons découvert que la cytokine CXCL5 (ou ENA78 - Epithelial neutrophil-activating peptide 78) possède un patron d'expression unique : elle est pratiquement absente du milieu de culture des peaux reconstruites, comparativement au milieu de culture de fibroblastes et de kératinocytes seuls. En modulant les niveaux de CXCL5 dans les milieux de culture de kératinocytes, nous avons montré que CXCL5 était un inhibiteur de la réparation des CPD. Cette première étude décrit l'impact des molécules sécrétées par le derme sur la réparation iii des CPD épidermiques et met en lumière un nouveau rôle de CXCL5 dans la réparation des dommages induits par les rayons UV. L’environnement immédiat des kératinocytes n’est pas le seul facteur qui peut influencer la réparation des CPD, le régime d’irradiation a également un impact sur cette efficacité d’élimination des lésions. Jusqu’à présent, l'efficacité de la NER a été largement étudiée après une seule exposition aiguë aux rayons UV. Cependant, l'utilisation d’une irradiation unique n'est pas représentative de l'exposition solaire humaine, qui est plutôt constituée d’une multitude d'irradiations répétées. Dans ce travail, nous avons donc exposé des cellules épidermiques à un régime d’irradiation chronique composé de faibles doses d’UVB (CLUV) afin de déterminer l’impact de cette irradiation sur la réparation NER. Nous avons montré que le traitement CLUV entraîne l’accumulation de CPD résiduels, qui ne sont pas réparés mais plutôt tolérés et dilués lors de la réplication de l’ADN. Nous avons également constaté que le prétraitement CLUV réduisait la capacité d'élimination des nouveaux dommages sans induire de sensibilité accrue à la mort cellulaire. Enfin, en utilisant nos données expérimentales, nous avons élaboré un modèle théorique pour prédire l’induction, la dilution et la réparation des CPD épidermiques lors d’une irradiation chronique aux rayons UVB. Nos résultats montrant que les kératinocytes accumulent des dommages dans l'ADN après des irradiations chroniques, constituent un facteur important à prendre en compte, car l'accumulation de CPD non réparés pourrait entraîner une augmentation des mutations dans les kératinocytes. Dans l’ensemble, ces travaux soulignent l’importance d’utiliser des modèles plus complexes, visant une meilleure représentation physiologique, pour mieux comprendre les réponses de la peau à l’exposition solaire. / Skin exposure to solar light is the main risk factor for non-melanoma skin cancers (NMSC). The genotoxic effect of sunlight is attributed to DNA damage induced by ultraviolet (UV) radiations. Long UVB wavelengths (290-315 nm) are the main responsible for NMSC initiation and promotion that occur in epidermal keratinocytes. Indeed, the direct absorption of UVB photons by DNA leads to the generation of the two main types of UV-induced DNA damage, i.e. cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts (6-4PP). CPD are the most abundant and are highly mutagenic. They are responsible for the C → T transition mutations at dipyrimidine sites, the signature mutation found in sun-related skin cancers. Skin cells use different mechanisms to avoid the conversion of UVB-induced CPD into skin cancer driver mutations, i.e. cell cycle arrest, DNA damage removal by nucleotide excision repair (NER) pathway and cell death by apoptosis. The importance of NER for skin cancer prevention is well demonstrated by the fact that a deficiency in NER proteins, such as in Xeroderma Pigmentosum (XP) patients, leads to an increase of up to 2,000-fold in skin cancer occurrence. Many factors influence NER and a better understanding of those factors might lead to new prevention strategies against skin cancer. Skin is a complex assembly of cells and matrix in which a crosstalk between epidermal and dermal components is essential for many cutaneous mechanisms. Using self-assembled tissue-engineered skin equivalents derived from human primary fibroblasts and keratinocytes, we have analyzed the impact of dermal components on epidermal CPD repair efficiency. We showed that CPD repair in keratinocytes is positively influenced by the presence of the dermis and we brought evidence that this dermal effect comes from secreted molecules. We then investigated the secretome and found that the cytokine CXCL5 (also known as ENA78 - Epithelial neutrophil-activating peptide 78) has a unique expression pattern, i.e. is virtually absent in the culture medium of reconstructed skin, when compared to the media from fibroblasts and keratinocytes alone. By modulating CXCL5 levels in keratinocytes culture medium, we have shown that CXCL5 is an inhibitor of CPD repair. This work outlines the impact of the secreted dermal components on epidermal UV-induced DNA damage repair and shed light on a novel role of CXCL5 in CPD repair. The immediate environment of the keratinocytes is not the only factor that can influence the CPD repair, the irradiation protocol also has an impact on this damage removal. Until now, v NER efficiency has been extensively studied after a single acute UVB exposure. However, the use of single UVB irradiation is not representative of the human solar exposure, which is rather a multitude of repeated irradiations than a single acute one. In this work, we thus exposed keratinocytes to a chronic low-dose of UVB (CLUV) protocol to determine the impact of this irradiation procedure on CPD removal. We showed that the CLUV treatment leads to the accumulation of residuals CPD. Those residual CPD are not repaired but rather tolerated and diluted through DNA replication. We also found that a CLUV pre-treatment reduces CPD removal rate of newly generated damage without inducing a higher sensitivity to UV-induced cell death. Finally, using our experimental data, we derived a theoretical model to predict CPD induction, dilution and repair that occur in keratinocytes when chronically irradiated with UVB. These results showing that keratinocytes accumulate DNA damage after chronic irradiations is an important factor to consider since the accumulation of unrepaired CPD might lead to an increase of skin cancer driver mutations formation. Taking together, this work outlines the importance of more relevant and physiological models to study the skin response to solar exposure.

La sclérodermie est une maladie auto-immune du tissu conjonctif caractérisée par une fibrose de la peau et des organes internes. L'objectif principal du projet était d'utiliser un modèle de derme reconstruit par auto-assemblage pour vérifier la capacité intrinsèque des fibroblastes sclérodermiques à sécréter et organiser une matrice extracellulaire in vitro. L'utilisation de fibroblastes de peau lésée ou non, de patients atteints de sclérodermie diffuse depuis moins d'un an ou depuis plus de dix ans nous a permis d'établir une nouvelle hypothèse. Au début de la maladie, les fibroblastes nécessiteraient la présence d'un ou de facteurs extrinsèques pour induire une fibrose. Avec le temps et lorsque la maladie s'aggrave, les fibroblastes deviendraient indépendants de ces stimuli externes. De plus, le TGF-βl pourrait être un des facteurs importants pour induire des lésions fibrotiques. Finalement, ce même TGF-β1 pourrait être responsable des changements phénotypiques stables des fibroblastes au stade précoce de la maladie.

Le développement des nanotechnologies, tout en promettant des progrès remarquables, notamment dans le domaine biomédical, soulève de nombreuses questions : en particulier quel est comportement de ces nano-objets en milieu biologique ? La silice a une place particulière parmi ces matériaux puisqu'elle est utilisée dans des produits cosmétiques, posant ainsi la question de l'exposition cutanée, et étudiée pour développer des vecteurs de libération intracellulaire. Cette étude a pour but d'apporter des éléments de réponses sur le comportement de différentes nanoparticules de silice en milieu biologique, à travers le suivi des phénomènes suivants : agrégation et dissolution en milieu biologique, internalisation dans des cellules en culture 2D ou 3D, toxicité et devenir intracellulaire des particules. Pour se placer dans le contexte d'une exposition cutanée, l'étude a été menée sur des cellules humaines du derme, en exposition directe ou immobilisées dans un matériau modèle du derme à travers lequel les nanoparticules diffusent. On constate ainsi que la cinétique de dissolution des particules en milieu biologique est fonction de leur taille, l'internalisation est influencée par le diamètre et la composition des particules, les effets toxiques sont liés au nombre de particules plutôt qu'à la concentration massique, et enfin la dissolution intracellulaire peut être modulée par la taille et l'insertion de fonctions disulfures et permettre la libération de molécules encapsulées dans les particules. Il apparaît donc que les caractéristiques des nanoparticules (taille, charge de surface et composition) influencent leur comportement en milieu biologique et en présence de cellules

Introdução: A imunização de coelhos saudáveis com colágeno do tipo V (COLV) reproduz as principais manifestações da esclerose sistêmica (ES), incluindo fibrose, vasculopatia e produção de autoanticorpos específicos da doença. Estudos preliminares mostraram que, tanto na derme de animais imunizados com COLV (COLV-IM), como na derme de pacientes com ES, observa-se deposição aumentada de COLV anômalo, mas não se sabe qual a relevância clínica deste achado. O remodelamento da matriz extracelular é precoce nos animais COLV-IM, ocorrendo já no sétimo dia após a imunização, o que sugere que o COLV esteja relacionado com a injúria endotelial, evento primário envolvido na patogênese da ES. Desta forma, os objetivos do presente estudo foram avaliar a expressão de COLV na derme de controles saudáveis e de pacientes com ES e sua correlação com espessamento cutâneo, atividade e duração da doença; assim como pesquisar uma possível associação entre a deposição deste colágeno na derme com a expressão de marcadores de apoptose e de ativação endotelial em pacientes e no modelo animal induzido pela imunização com COLV. Pacientes e Métodos: Biópsias de pele de pacientes com ES (N=18, 6 com doença precoce e 12 com doença tardia) e de controles (N=10) pareados por idade e sexo, assim como biópsias de pele de coelhos imunizados com COLV + adjuvante de Freund (COV-IM, N=6) ou adjuvante de Freund (N=6) foram avaliadas. Nos pacientes com ES, o espessamento cutâneo foi avaliado por meio do escore de Rodnan Modificado (MRSS) e a atividade da doença foi calculada pelo índice de atividade de Valentini. Para realizar a quantificação por histomorfometria, o COLV na derme foi identificado por imunofluorescência. Caspase-3, endotelina-1, CTGF, TGF e VEGF nas células endoteliais dérmicas foram marcados por imunohistoquímica. Nos pacientes e nos controles, o COLV proveniente da cultura de fibroblastos dérmicos foi quantificado por PCR RT em tempo real e caracterizado por eletroforese, imunoblotting e reconstrução tridimensional, por meio da microscopia confocal. Resultados: O depósito de COLV foi maior na derme de pacientes com doença precoce, quando comparados aos controles e aos pacientes com doença tardia. A atividade e a duração da ES estiveram associadas com o depósito de COLV. A expressão de RNA mensageiro das cadeias COLV1 COLV2 foi aumentada em relação aos controles e a reconstrução tridimensional confirmou a presença de fibras anômalas de COLV. Observou-se maior expressão de caspase-3, endotelina-1, CTGF, TGF e VEGF nas células endoteliais dos pacientes com ES, quando comparados aos controles. Houve correlação positiva entre o depósito de COLV e a expressão de caspase-3, endotelina-1 e CTGF. Ao comparar-se os coelhos COLV-IM com os controles, observou-se aumento significativo da expressão de COLV aos 7 dias e de endotelina-1 aos 210 dias após a imunização. Embora de maneira não significativa, verificou-se maior expressão de caspase-3, CTGF e VEGF nos animais COLV-IM e, quando os animais foram comparados ao longo do tempo, percebeu-se maior expressão de COLV no sétimo dia após a imunização na derme dos animais COLV-IM, diminuindo no trigésimo dia e voltando a subir aos 75 dias e aos 210 dias. A caspase-3 e a endotelina-1 comportaram-se de maneira semelhante. Conclusão: Estes resultados sugerem que o COLV possa agir como um possível gatilho envolvido na patogênese da ES, agindo como um indutor de ativação e de apoptose endotelial, que por sua vez poderia resultar em maior expressão de COLV, perpetuando o processo de remodelamento observado na pele dos pacientes com ES / Introduction: The immunization of healthy rabbits with type V collagen (COLV) reproduces the main characteristics of systemic sclerosis (SSc), such as fibrosis, vasculopathy and specific auto-antibodies. Preliminary studies demonstrated that both COLV-immunized rabbits (COLV-IM) and SSc patients exhibit increased expression of abnormal COLV in the dermis, but the clinical relevance of this finding is unknown. The remodeling of the extracellular matrix is an early event in COLV-IM rabbits that can be detected by the seventh day after immunization; this observation suggests that COLV is involved in endothelial injury, one of the first manifestations of the disease. Thus, the objectives of this study were to evaluate COLV expression in the dermis of healthy controls and SSc patients and to determine the correlation of this expression with skin thickness, disease activity and duration and search for a possible association between this collagen with apoptosis and activation of endothelial cells markers in patients and in animal model induced by immunization with COLV. Patients and Methods: Skin biopsies from 18 patients (6 early-stage and 12 late-stage) and 10 healthy controls as well as skin biopsies from rabbits immunized with COLV (COLV-IM) and Freund adjuvant (N=6) or Freund adjuvant alone (N=6) were evaluated. Skin thickening assessment was performed with the Modified Rodnan Skin Score (MRSS), and activity was calculated using the Valentini Disease Activity Index. To perform quantification by histomorphometry, COLV was identified by immunofluorescence, and caspase-3, endothelin-1, CTGF, TGF and VEGF in dermal endothelial cells were labeled by immunohistochemistry. In SSc patients and healthy controls, COLV from dermal fibroblast culture was quantified by real-time RT-PCR and characterized by electrophoresis, immunoblotting and tridimensional reconstruction by confocal microscopy. Results: COLV deposition was increased in the dermis of the patients with early disease compared with the healthy controls and the patients with late disease. SSc activity and disease duration were associated with dermal COLV deposition. COLV1 and COLV2 mRNA expression levels were higher in SSc, and a tridimensional reconstruction of SSc dermal heterotypic fibers confirmed the presence of abnormal COLV. The dermal endothelial cell expression of caspase-3, endothelin-1, CTGF, TGF and VEGF was higher in the SSc patients than in the controls. There was a positive correlation between COLV deposition and caspase-3, endothelin-1 and CTGF expression. When the COLV-IM rabbits were compared with the controls, there was a significant increase in the expression of COLV 7 days after the immunization and a significant increase in the expression of endothelin-1 210 days after the immunization. The expression of caspase-3, CTGF and VEGF was higher in the COLV-IM animals than in the control rabbits, although not significantly, and when the rabbits were compared over time, the expression of COLV was higher in the dermis of the COLV-IM animals 7 days after immunization, decreasing at 30 days and increasing again at 75 and 210 days. Caspase-3 and endothelin-1 exhibited similar behavior. Conclusion: these results suggest that COLV can be a possible trigger involved in the pathogenesis of SSc, acting as an inducer of endothelial apoptosis and activation that could result in higher expression of COLV, perpetuating the remodeling process observed in SSc skin

Introdução: A imunização de coelhos saudáveis com colágeno do tipo V (COLV) reproduz as principais manifestações da esclerose sistêmica (ES), incluindo fibrose, vasculopatia e produção de autoanticorpos específicos da doença. Estudos preliminares mostraram que, tanto na derme de animais imunizados com COLV (COLV-IM), como na derme de pacientes com ES, observa-se deposição aumentada de COLV anômalo, mas não se sabe qual a relevância clínica deste achado. O remodelamento da matriz extracelular é precoce nos animais COLV-IM, ocorrendo já no sétimo dia após a imunização, o que sugere que o COLV esteja relacionado com a injúria endotelial, evento primário envolvido na patogênese da ES. Desta forma, os objetivos do presente estudo foram avaliar a expressão de COLV na derme de controles saudáveis e de pacientes com ES e sua correlação com espessamento cutâneo, atividade e duração da doença; assim como pesquisar uma possível associação entre a deposição deste colágeno na derme com a expressão de marcadores de apoptose e de ativação endotelial em pacientes e no modelo animal induzido pela imunização com COLV. Pacientes e Métodos: Biópsias de pele de pacientes com ES (N=18, 6 com doença precoce e 12 com doença tardia) e de controles (N=10) pareados por idade e sexo, assim como biópsias de pele de coelhos imunizados com COLV + adjuvante de Freund (COV-IM, N=6) ou adjuvante de Freund (N=6) foram avaliadas. Nos pacientes com ES, o espessamento cutâneo foi avaliado por meio do escore de Rodnan Modificado (MRSS) e a atividade da doença foi calculada pelo índice de atividade de Valentini. Para realizar a quantificação por histomorfometria, o COLV na derme foi identificado por imunofluorescência. Caspase-3, endotelina-1, CTGF, TGF e VEGF nas células endoteliais dérmicas foram marcados por imunohistoquímica. Nos pacientes e nos controles, o COLV proveniente da cultura de fibroblastos dérmicos foi quantificado por PCR RT em tempo real e caracterizado por eletroforese, imunoblotting e reconstrução tridimensional, por meio da microscopia confocal. Resultados: O depósito de COLV foi maior na derme de pacientes com doença precoce, quando comparados aos controles e aos pacientes com doença tardia. A atividade e a duração da ES estiveram associadas com o depósito de COLV. A expressão de RNA mensageiro das cadeias COLV1 COLV2 foi aumentada em relação aos controles e a reconstrução tridimensional confirmou a presença de fibras anômalas de COLV. Observou-se maior expressão de caspase-3, endotelina-1, CTGF, TGF e VEGF nas células endoteliais dos pacientes com ES, quando comparados aos controles. Houve correlação positiva entre o depósito de COLV e a expressão de caspase-3, endotelina-1 e CTGF. Ao comparar-se os coelhos COLV-IM com os controles, observou-se aumento significativo da expressão de COLV aos 7 dias e de endotelina-1 aos 210 dias após a imunização. Embora de maneira não significativa, verificou-se maior expressão de caspase-3, CTGF e VEGF nos animais COLV-IM e, quando os animais foram comparados ao longo do tempo, percebeu-se maior expressão de COLV no sétimo dia após a imunização na derme dos animais COLV-IM, diminuindo no trigésimo dia e voltando a subir aos 75 dias e aos 210 dias. A caspase-3 e a endotelina-1 comportaram-se de maneira semelhante. Conclusão: Estes resultados sugerem que o COLV possa agir como um possível gatilho envolvido na patogênese da ES, agindo como um indutor de ativação e de apoptose endotelial, que por sua vez poderia resultar em maior expressão de COLV, perpetuando o processo de remodelamento observado na pele dos pacientes com ES / Introduction: The immunization of healthy rabbits with type V collagen (COLV) reproduces the main characteristics of systemic sclerosis (SSc), such as fibrosis, vasculopathy and specific auto-antibodies. Preliminary studies demonstrated that both COLV-immunized rabbits (COLV-IM) and SSc patients exhibit increased expression of abnormal COLV in the dermis, but the clinical relevance of this finding is unknown. The remodeling of the extracellular matrix is an early event in COLV-IM rabbits that can be detected by the seventh day after immunization; this observation suggests that COLV is involved in endothelial injury, one of the first manifestations of the disease. Thus, the objectives of this study were to evaluate COLV expression in the dermis of healthy controls and SSc patients and to determine the correlation of this expression with skin thickness, disease activity and duration and search for a possible association between this collagen with apoptosis and activation of endothelial cells markers in patients and in animal model induced by immunization with COLV. Patients and Methods: Skin biopsies from 18 patients (6 early-stage and 12 late-stage) and 10 healthy controls as well as skin biopsies from rabbits immunized with COLV (COLV-IM) and Freund adjuvant (N=6) or Freund adjuvant alone (N=6) were evaluated. Skin thickening assessment was performed with the Modified Rodnan Skin Score (MRSS), and activity was calculated using the Valentini Disease Activity Index. To perform quantification by histomorphometry, COLV was identified by immunofluorescence, and caspase-3, endothelin-1, CTGF, TGF and VEGF in dermal endothelial cells were labeled by immunohistochemistry. In SSc patients and healthy controls, COLV from dermal fibroblast culture was quantified by real-time RT-PCR and characterized by electrophoresis, immunoblotting and tridimensional reconstruction by confocal microscopy. Results: COLV deposition was increased in the dermis of the patients with early disease compared with the healthy controls and the patients with late disease. SSc activity and disease duration were associated with dermal COLV deposition. COLV1 and COLV2 mRNA expression levels were higher in SSc, and a tridimensional reconstruction of SSc dermal heterotypic fibers confirmed the presence of abnormal COLV. The dermal endothelial cell expression of caspase-3, endothelin-1, CTGF, TGF and VEGF was higher in the SSc patients than in the controls. There was a positive correlation between COLV deposition and caspase-3, endothelin-1 and CTGF expression. When the COLV-IM rabbits were compared with the controls, there was a significant increase in the expression of COLV 7 days after the immunization and a significant increase in the expression of endothelin-1 210 days after the immunization. The expression of caspase-3, CTGF and VEGF was higher in the COLV-IM animals than in the control rabbits, although not significantly, and when the rabbits were compared over time, the expression of COLV was higher in the dermis of the COLV-IM animals 7 days after immunization, decreasing at 30 days and increasing again at 75 and 210 days. Caspase-3 and endothelin-1 exhibited similar behavior. Conclusion: these results suggest that COLV can be a possible trigger involved in the pathogenesis of SSc, acting as an inducer of endothelial apoptosis and activation that could result in higher expression of COLV, perpetuating the remodeling process observed in SSc skin

The concept of immunosurveillance suggests that the innate and adaptative immune system eliminate developing tumors. However, tumor development is associated with important modifications of the stroma which, by multiple mechanisms, restrain the immune response notably by affecting dendritic cells (DC) recruitment and functions. My thesis project aims at deciphering how the tumor environment alters the mechanisms and pathways of DC recruitment and impairs their functions. First, we determine if the site of tumor transplantation affects tumor immunogenicity. We show that tumors transplanted in the dermis (i.d.), an environment containing multiple DC subsets, induce a protective anti-tumoral immune response and tumor rejection. By contrast, the same tumor implanted in the subcutaneous tissue (s.c), mainly containing monocytes, is not rejected. Rejection of i.d. tumor is associated with a rapid (within 2 days) recruitment of DC within the tumor and rapid migration of DC towards tumor draining lymph nodes (dLN) where they present the tumor antigens (TAA) to CD4 and CD8 T lymphocyte. These events also occur for s.c. tumors but with a delayed kinetic. Thus the kinetic of DC mobilisation is decisive for tumor immunogenicity. Analysis of the DC subpopulations (TIDC) infiltrating the i.d. or s.c. tumors at 4 days (D4) and 8 days (D8) post-tumor transplantation, revealed that the different DC subpopulations are present at similar frequencies. Based on these findings, we proposed that i.d. tumor are rapidly infiltrated by dermal DC (DDC), whereas in s.c. tumor, the absence of inflammatory signals would limit DDC recruitment. In this latter case, DC would mainly come from local differentiation of blood-born precursors of DC (pre-cDC). Local differentiation of pre-cDC within the immunosuppressor tumor environment may affect their differentiation program and functions. We found that pre-cDC infiltrate i.d. and s.c. tumors starting at D4 and their frequency increases at D8. To determine the DC origin in tumors, we use CD11c-DTR-GFP mice in which CD11c+ cells express a fusion protein constituted by the diphtheria toxin receptor (DTR) and GFP. To track DDC trafficking within tumors, we injected anti-MHCII antibody before tumor implantation and analysed MHCII+ CD11c+ DDC infiltration in tumor by biphotonic microscopy. At D3 post-tumor transplantation, the DDC infiltration was higher in i.d. than s.c. tumors. To analyse the impact of this early DDC recruitment on anti-tumor immunity, we inhibit early recruitment of DC by injection of pertussis toxin (PTX), a chemokine receptor protein G inhibitor, during the three first days of tumor development. For i.d. tumors, PTX treatment induced 60% reduction of DC recruitment starting at D4. In s.c. tumor, while this effect was observable at D3 (60% reduction) and increased to 80% at D4. These results suggest that early recruitment of DDC to i.d. tumors may be be chemokine independent. PTX treatment, which inhibits DDC migration from tumors to dLN inhibits the early TAA presentation to CD4 and CD8 T cells but did not impaired i.d. tumor rejection. Collectively, these results suggest that a first wave of DDC may infiltrate i.d. tumor. The initial wave of DDC may rapidly activate the adaptive immune system and induce protective anti-tumoral immune response. For s.c. tumor, this first wave in delayed or limited. Tumor neoangioenesis would permit an input of pre-cDC which would differentiate locally into cDC1 and cDC2. To consolidate this model we are developing new protocols to efficiently inhibit early recruitment of DDC in i.d. tumor. Moreover, to determine the DC origin and pathways of DC recruitment in tumors, we exploit several experimental approaches to directly analyse DDC migration toward i.d. and s.c. tumor. / Le concept d’immunosurveillance postule que le système immunitaire inné et adaptatif élimine les tumeurs naissantes. Cependant, le développement de tumeurs est associé à des modifications importantes du stroma qui, par des mécanismes multiples, inhibent la réponse immunitaire en affectant notamment le recrutement et la fonction des cellules dendritiques (DC). Mon projet de thèse vise à préciser comment l’environnement tumoral affecte les mécanismes et les voies de recrutement des DC dans les tumeurs et leurs fonctions. Tout d’abord, nous avons déterminé si la composition en DC du tissu d’implantation affecte l’immunogénicité tumorale. Nous avons montré que les tumeurs implantées dans le derme (i.d.), un environnement riche en DC dermales (DDC), induisent une réponse anti-tumorale protectrice. En revanche, une même tumeur transplantée dans le tissu sous-cutané (s.c.), contenant principalement des monocytes, n’est pas rejetée. Le rejet des tumeurs i.d. est associé à un recrutement précoce et rapide des DC dans la tumeur (dès 2 jours post-injection) et une migration, dans les ganglions drainants (dLN), de DC qui présentent les antigènes tumoraux (TAA) aux lymphocytes T (LT) CD4+ et CD8+. Dans les tumeurs s.c. ces événements sont présents mais retardés. Ceci indique que la cinétique de mobilisation des DC est déterminante pour l’immunogénicité tumorale.