Thèses sur le sujet « ST2/IL-33 »

L'interleukine-33 (IL-33), une cytokine de type alarmine de la famille de l’IL-1, est essentiellement exprimée par les cellules endothéliales et les cellules épithéliales dans diverses pathologies inflammatoires chez la souris et chez l'Homme. IL-33 induit son activité biologique par l'interaction d’un récepteur hétérodimérique composé de ST2 et IL-1RAcP. Les nouvelles sources cellulaires de l'IL-33 et la régulation de son expression restent mal connues dans le foie. L'objectif de ma thèse était de mieux comprendre les mécanismes d'expression, de régulation et d’activité de l'IL-33 en tant qu’alarmine au cours des hépatites aiguës murines induites par CCl4, ConA, FasL/Jo2, D-GalN-TNFα, Poly(I:C) et MHV3 ainsi que chez les patients souffrant d’hépatite fulminante à VHB. Nous avons montré que l’IL-33 était surexprimée dans tous les modèles hépatiques étudiés chez la souris avec une expression dans les cellules endothéliales sinusoïdales du foie et les cellules endothéliales vasculaires. L'IL-33 dont l'expression est associée à l'hypervascularisation du foie a également été observée chez les patients atteints d’hépatites fulminantes à VHB. Plus surprenant, nous avons trouvé que l'IL-33 était inductible dans les hépatocytes au cours des hépatites à ConA, à Poly(I:C) et induites par le MHV3 chez la souris. Nous avons démontré que les cellules NKT et la cytokine de mort cellulaire TRAIL, régulaient l’expression de l’IL-33 dans les hépatocytes au cours de l’hépatite à ConA. De plus, la nécroptose (ou nécrose programmée) dépendante des kinases PARP-1, RIPK1 et RIPK3 contrôle l’expression de l’IL-33 dans les hépatocytes au cours de l’hépatite à ConA. Ceci aboutit au concept d’IL-33 comme marqueur de la nécroptose. Au niveau fonctionnel, l’IL-33 a un effet protecteur dans les dommages hépatiques induits par la ConA suggérant un rôle bénéfique de l’axe IL-33/ST2 dans certaines pathologies du foie. En conclusion, nous mettons en évidence que les cellules NKT, la molécule TRAIL, la molécule PARP-1 et les kinases RIPK1, -3 contrôlent l’expression de l'IL-33 dans la physiopathologie de foie. Ces résultats suggèrent un rôle important de l’axe IL-33/ST2 dans les maladies du foie
Interleukin-33 (IL-33), an alarmin cytokine of IL-1 family, is primarily expressed by endothelial cells and epithelial cells in various inflammatory pathologies in mice and human. IL-33 mediates its biological activity by interaction with specific ST2 and IL-1RAcP receptors. The novel cellular sources of IL-33 and its regulation particularly in liver remain obscure. The objective of my thesis was to better determine the expression, regulation and functions of IL-33 as an alarmin during murine acute hepatitis induced by CCl4, ConA, FasL/Jo2, D-GalN-TNF-α, Poly(I:C) and MHV3 as well as in human patients suffering from HBV fulminant hepatitis. IL-33 was over-expressed in all studied murine hepatic models with induced expression in liver sinusoidal endothelial cells and vascular endothelial cells. The IL-33 over-expression associated with hypervascularisation was also found in HBV fulminant hepatic patients. More surprisingly, we found that IL-33 was expressed in hepatocytes during ConA, Poly(I:C) and MHV3 mediated acute hepatitis in mice. We demonstrated that NKT cells and cell death inducing molecule TRAIL regulated the hepatocyte-specific IL-33 expression during ConA-hepatitis. The PARP-1-RIPK1-RIPK3 mediated necroptosis (or programmed necrosis) in ConA liver injury regulated IL-33 expression in hepatocytes. This leads to the concept of IL-33 as a marker of necroptosis. At functional level, IL-33 had a protective impact in ConA-induced liver injury supporting the current protective role of IL-33/ST2 axis in liver pathology. In conclusion, we evidence a novel NKT cells, TRAIL, PARP-1 and RIP kinases dependent regulatory mechanism for IL-33 in liver pathophysiology. These findings suggest an important role of IL-33/ST2 axis in liver diseases

Regulation of the growth and differentiation of trophoblast cells is critical for successful embryo implantation and placentation. Cytokines are key players in these processes, as well as modulating the maternal immune response to prevent rejection of the conceptus. This thesis focused on the investigation of the cytokine interleukin (IL) - 33 and its receptor, ST2. ST2 has two isoforms, a functional cell surface receptor (ST2L) and a soluble decoy receptor (sST2). Previous work in this laboratory had shown that the human placenta expresses both IL-33 and sST2 at term. The aim of this thesis was to investigate IL-33 and ST2 in early pregnancy, the time when trophoblast is at its most active, with a view to better understanding their role. IL-33 and ST2 mRNA and protein were examined in 14 first trimester placentas from 6-12 weeks of gestation. IL-33 was localized to cells in the villous stroma, whereas ST2 was present in the syncytiotrophoblast, villous cytotrophoblast and the invasive extravillous cytotrophoblast of the cell columns. Secretion of sST2, but not IL-33, by the placenta was found. Investigation of pre-implantation embryos showed the presence of ST2, but not IL-33 protein. Decidualized endometrium was investigated as a potential source of IL-33 and sST2 at the maternal-fetal interface and, although mRNA for both was present, no protein could be found. The key finding was that sST2, rather than ST2L, was the predominant isoform in the placenta. This led us to reconsider the hypothesis that IL-33/ST2 interactions in the placenta are important for successful pregnancy and raised the possibility that they may have independent roles. Using trophoblast cell lines as a model, it was shown that sST2 binds to trophoblast cells, significantly inhibits their proliferation and stimulates their invasion in vitro. This is the first report of this novel role for sST2 in pregnancy. Thus these studies have shown that sST2 may play an important role in implantation and placentation through controlling trophoblast invasion.

Background: ST2 has been identified in playing an important role in Th2-mediated inflammation and asthma. IL-33 acts as the ligand for ST2; it is a novel cytokine that induces innate Th2/type-2 responses when delivered to the lung. The hierarchy of IL-33 and type-2 cytokines and chemokines in Th2 inflammation in the lung has not been fully elucidated. Furthermore, the role of IL-33 in the adaptive response in allergic mediated airways disease is unclear. Epithelial cells (ECs) are increasingly recognised as having an immunological role in airway inflammation and asthma, in particular releasing cytokines such as IL-33. Little is known about whether ST2 is expressed on these cells and what function IL-33 responsive ECs may have in Th2 diseases. Soluble ST2 (sST2) has emerged as a biomarker correlating with disease activity in cardiovascular disease. It is not known if there is a clear association between sST2 and asthma, nor whether measurable IL-33 concentrations are present and if so, their association with disease severity. The influence of smoking and corticosteroid treatment on these parameters has also not been determined. Aim: To ascertain the levels of systemic sST2 and IL-33 in asthmatic patients. To determine cytokine, chemokine and airway dynamics of IL-33-driven innate airway inflammation. To determine the role of epithelial cells in IL-33-driven innate airway inflammation. To investigate the function of ST2/IL-33 axis in the innate and adaptive responses in allergic airways inflammation and asthma. Methods and Results: sST2 and IL-33 levels in plasma of never smokers, ex-smokers and smokers were determined by immunoassay before and after a corticosteroid trial. Corticosteroid treatment resulted in increased sST2 levels in all smoking status patient groups; there was no effect attributable to smoking. Time course and dosage interval experiments were performed in mice treated with intranasal IL-33. IL-5, IL-13, eotaxin/CCL11 and eotaxin2/CCL24 mediated eosinophilic airway inflammation (AI). Treatment of mice with both anti- CCL11 and -CCL24 partially ameliorated the AI. IL-4 gene deficient mice were protected from IL-33-induced inflammation. BALB/c mice displayed airways hyperreactivity following IL-33 treatment. Murine, human cell line and primary human ECs were assessed for ST2 expression by immunohistochemistry and fluorescence activated cell sorting (FACS). ST2 expression was clearly demonstrated in the ECs. Subsequently ECs were treated with IL-33 in an in vitro setting including in a pseudostratifed epithelium model. ECs produced a range of inflammatory and angiogenic mediators in response to IL-33. In particular IL-33 driven EC-derived VEGF promoted angiogenesis in vitro. Intranasal IL-33 induced increased endothelial cells and vascular remodelling in vivo. Experimental allergic airways inflammation (AAI) was generated in BALB/c mice which were co-treated with IL-33 or PBS at allergen sensitisation. IL-33 induced the polarisation of IL-5+IL-4- T cells in the draining lymph nodes and these mice developed more severe inflammation. AAI was induced in WT, ST2-deficient, IL-4-deficient and ST2/IL-4 deficient mice. These experiments showed IL-4 was necessary for generation of AAI which could not be overcome by ST2-pathway stimulation in an adjuvant-free model. Conclusions: The data presented further extends the current understanding of the ST2/IL-33 axis in the innate and adaptive aspects of Th2 inflammation in AAI and asthma. In particular the hierarchy of mediators and cells involved in Th2 inflammation, including at the sensitisation phase, have been explored. This identifies ST2/IL-33 as a potential target in the development of biological therapies for asthma.

Asthma is a chronic disease characterised by variable airflow obstruction, bronchial hyperresponsiveness and airways inflammation. At an immunological level Th2 inflammation and the presence of activated eosinophils and mast cells are key features of asthma. ST2, the receptor for the novel cytokine IL-33, is expressed upon Th2 lymphocytes and mast cells but its role in clinical and experimental asthma remains unclear. IL-33 has been shown to induce local and systemic eosinophilia when administered to the peritoneum of mice. In this thesis I have set out to test the hypothesis that the activation of mast cells by IL-33 acting on cell surface ST2 plays a critical role in allergic airways inflammation. I began by studying the function of ST2 on mast cells in vitro. I found that ST2 was expressed at an early stage of development, and correlated closely with the expression of the stem cell factor receptor (c-kit), a marker present on mast cells from a progenitor stage. Despite this mast cells generated form ST2 gene deleted mice proliferated and matured normally. When mast cells were activated by IL-33, acting in an ST2-dependent manner, pro-inflammatory cytokines and chemokines were released that have potential roles in asthma, specifically IL-6, IL-13, MIP-1α and MCP-1. To extend these findings I looked at the role of ST2 in allergic airways inflammation. I first optimised and validated an ovalbumin and adjuvant based ‘short’ twelve day model of murine asthma and demonstrated that ST2 gene deletion results in attenuated eosinophilic inflammation. In addition to being ST2 dependent it is possible that this adjuvant based short model is mast cell dependent, unlike longer adjuvant based models which are mast cell and ST2 independent. Therefore I went on to study an adjuvant-free model of asthma which has been demonstrated to be mast cell dependent. In this adjuvant-free model of asthma the airway inflammation was attenuated in ST2 gene deficient mice compared with wild type mice, while AHR was unaffected. There was an associated reduction in IgE production and thoracic lymph node recall Th2 cytokine responses. I then examined the effect of ST2 activation in the lungs. When IL-33 was administered directly to the airways of naïve mice it induced the features of experimental asthma. There was extensive eosinophilic inflammation within the lung tissue and airspaces. The Th2 cytokines IL-5 and IL-13, and the eosinophil chemoattractant chemokines eotaxin-1 and eotaxin-2 were detected at increased concentrations. Significant airways hyperresponsiveness was also generated. Using ST2 gene deleted mice I demonstrated that these effects were ST2 specific. Although I have shown that mast cells are activated by IL-33 in vitro, I used mast cell deficient mice to demonstrate that the eosinophilic inflammation generated by IL-33 is unaffected by the absence of mast cells. These data show that IL-33 can induce in the lungs the cardinal pathological characteristics of asthma, and that it appears to act upstream of other important mediators such as IL-13 and the eotaxins. Furthermore the IL-33 receptor ST2 is required in an adjuvant free model of asthma, which is more akin to human disease. Placing these findings in the context of recent evidence that IL-33 is released by structural cells in response to damage or injury suggests that IL-33 may play a key role in initiating the immunological features of clinical asthma. As a consequence of this position in the hierarchy of inflammation IL-33 offers a promising direct target for novel biological therapies in asthma.

A preparação da suspensão bacteriana e o intervalo de confiança de UFC nessa suspensão é um importante procedimento utilizado em laboratórios como métodos para avaliação de respostas inflamatórias e pode ser obtido por diferentes métodos, tais como diuições seriadas e pela análisa visual da turbidez através da escala de McFarland. Nós investigamos a influência do armazenamento da suspensão de Staphylococcus aureus na viabilidade de bactérias e sua influência na inflamação induzida por essa suspensão. O armazenamento da suspensão de S. aureus a 8 º C por 24 h diminuiu a viabilidade bacteriana não só em suspensões preparadas por diluições seriadas, mas também ao seguirmos a escala de McFarland 0,5. O aumento do tempo de armazenamento reduziu o número de UFC de S. aureus. Como conseqüência da viabilidade bacteriana reduzida, foi detectada redução do recrutamento de leucócitos em um modelo de peritonite bacteriana. Em modelo de artrite séptica foi detectada redução da hiperalgesia mecânica, edema e recrutamento de leucócitos. Esses resultados demonstram que o armazenamento da suspensão bacteriana afeta a viabilidade bacteriana e também a resposta inflamatória "in vivo". Uma solução possível para determinar/estimar o número de UFC é o uso da escala de McFarland, que permitirá a elaboração e utilização de uma suspensão bacteriana no mesmo dia para testes in vivo e assim, evita-se a diminuição da viabilidade bacteriana e a influência de resultados experimentais.
The preparation of bacterial suspension is an important procedure used in laboratories for inflammatory evaluation protocols and can be obtained by different methods such as CFU (colony forming unities), needs storage counting and McFarland scale turbidity (does not need storage). We investigated the influence of storage of Staphylococcus aureus suspension as bacterial viability and its influence in bacteria-induced inflammation. . The increase of time of storage reduced the S. aureus CFU. As a consequence of reduced bacterial viability, it was detected reduced leukocyte recruitment in a model of bacterial peritonitis, and reduced mechanical hyperalgesia, edema and leukocyte recruitment in septic arthritis. These results demonstrate that storage of bacterial suspension affected bacterial viability and also the inflammatory response in vivo, raising the importance of standard procedures for bacterial suspension preparation. A conceivable approach would be to determine the number of CFU at a specific McFarland"s scale degree, which will allow the preparation and use a bacterial suspension in the same day for in vivo testing and avoiding reduced bacterial viability.

Interleukin 33 (IL-33) is a dual function cytokine. It is a member of the IL-1 family and it acts as a pro-inflammatory factor (18 kilo Dalton, 18 kD) like other cytokines in IL-1 family. IL-33 is also a transcription factor (32 kD - form) which can suppress or activate gene transcription in diverse cases. A variety of cell types and tissues in the central nervous system (CNS) can release IL-33 after injury. The 18 kD IL-33 binds to the membrane receptor protein ST2 ligand, then regulates downstream gene expression, triggers cytokine synthesis, and modulates the immune system response. After traumatic brain injury (TBI) in the CNS, glial cells become key players in the nervous tissue response. Astrocytes undergo activation, proliferation, release pro-inflammatory factors and, as a consequence, a glial scar barrier around the injury is formed. Simultaneously, resting microglia are activated and able to remove debris. Lastly, oligodendrocytes together with microglia and astrocytes are activated and communicate with the immune system. In addition, as a severe kind of injury to the CNS, brain tumours share some similar characteristics of brain injury, such as hypoxia and inflammation. Therefore, IL-33 may play a role in neuroinflammation and also in brain tumours. In this project, our aim was to investigate the role of IL-33 and the IL-33/ST2 pathway in traumatic brain injury and brain tumours (e.g glioma). We found that IL-33 can influence the CNS immune resonse, and may be important in CNS pathology.

O carcinoma espinocelular (CEC) é um dos cânceres humanos mais incidentes. A despeito do entendimento da fisiopatologia do CEC, as opções terapêuticas ainda são limitadas e o(s) exato(s) mecanismo(s) envolvido(s) na progressão deste tipo de tumor ainda não foi descrito. Estudos recentes mostram a existência de uma associação direta entre a resposta imune TH1 e um melhor prognóstico em pacientes com CEC. Aumento da expressão de componentes do eixo IL-33/ST2 foi demonstrado contribuir para transformação neoplásica em diversos modelos tumorais, incluindo cânceres de estômago e de mama. Trabalho recente do nosso e de outros laboratórios indicam que IL-33 pode impedir a resposta imune TH1 . Baseado nessas observações, a hipótese testada foi que o impedimento da resposta imune pela interação IL-33/ST2 pode contribuir para iniciação e progressão do CEC. Utilizando modelo de carcinogênese química em camundongos WT e deficientes de ST2 (ST2KO), os resultados mostram que a deficiência de ST2 leva a uma notável redução da severidade das lesões 20 semanas após a carcinogênese química, sugerindo que a sinalização ST2 é necessária para o desenvolvimento tumoral neste modelo. Análises do infiltrado inflamatório presente nas lesões em camundongos WT e ST2KO revelaram redução significativa nas percentagens de macrófagos, células T CD4+ e células dendríticas, mas não em células T CD8+, células B e células natural killer (NK) no microambiente tumoral de camundongos ST2KO. Além disso, células NK esplênicas isoladas de camundongos ST2KO exibiram atividade citotóxica aumentada contra células YAC quando comparado com células de camundongos do grupo controle (WT). Os resultados indicam que a via IL-33/ST2 contribui para o desenvolvimento de carcinoma espinocelular recrutando células T CD4+, macrófagos e células dendríticas e reduzindo a citotoxicidade de células NK.
Squamous cell carcinoma (SCC) is the second most common form of skin cancer and is most commonly observed in photo-exposed areas of the body. The mechanism(s) involved in the progression of this tumor are unknown. Recent studies have shown that there is a direct association between a TH1-related immune response and a better prognosis in patients with SCC. Increased expression of the IL33/ST2 axis components has been demonstrated to contribute to neoplastic transformation in several tumor models, including gastric and breast cancer. Recent work from ours and other laboratories indicate that can IL-33 impair TH1-type immune responses. Based on these observations, we hypothesized that TH1-type immune response impairment by IL33/ST2 could contribute to the initiation and progress SCC. We found that ST2 deficiency led to a marked decreased in severity of skin lesions at 20 weeks post-DMBA, suggesting that ST2 signaling is necessary for tumor development in this model. Analysis of tumor lesions in WT and ST2KO mice revealed that lack of ST2 led to a specific and significant reduction in the frequency of macrophages, T CD4+ and dendritic cells, but not CD8+, B and NK cells. In addition, splenic NK cells isolated from DMBA-treated ST2KO mice exhibited increased cytotoxicity activity against YAC cells targets when compared with WT splenic NK cells in the same cytotoxic assay. Altogether, our findings indicate that IL-33/ST2 pathway contributes to the SCC development by recruitment T CD4+ cells, macrophages, and dendritic cells and impairing NK cytotoxicity.

Asthma is a common and complex inflammatory disease of the airways characterized by deregulated immune responses that involves activation of multiple cell types including Th2 cells, IgE producing B cells, mast cells, basophils and eosinophils as well as resident lung cells such as epithelial, smooth muscle cells and macrophages. Despite intensive research, there are still unmet needs in the treatment of asthma. Recently, a new cytokine of IL 1 family, named IL 33 emerged as a potentially important factor in the immunopathogenesis of allergy and asthma. It was recently shown in our laboratory that intranasal administration of IL 33 can induce certain physiological features that are characteristic of experimental asthma, such as eosinophilic inflammation, Th2 cytokine and antibody production as well as increased airway hyperresponsiveness. The effect of IL 33 on the activation and differentiation of allergen specific Th2 cells has been well studied. However, the contribution of IL 33 to the activation of lung resident and inflammatory innate cells remains undefined. In this project I focused on alveolar macrophages and eosinophils as both cell types were reported to express IL 33R, ST2L and are thought to play a crucial role in asthma pathogenesis. I raised the hypothesis that IL 33 released locally in the lungs may trigger symptoms resembling asthma through the activation of airway alveolar macrophages. Furthermore, I hypothesize that IL 33 may exacerbate and maintain inflammation in the lungs by the direct activation of eosinophils. In our previous study we showed that IL 33 could switch the quiescent phenotype of alveolar macrophages toward the alternatively activated phenotype (M2, AAM). In the first part of my thesis I looked at the consequences of this phenomenon for airway inflammation. Using clodronate liposomes in vivo I was able to eliminate macrophage population from the lungs and demonstrated that resident alveolar macrophages are crucial for the development of IL 33 induced eosinophilic inflammation in the airways. I then examined the contribution of IL 13, a known M2 differentiation factor, to airway inflammation. Using anti IL 13 neutralizing antibodies I showed that IL 13 is required for the IL 33 triggered differentiation of alveolar macrophages toward M2 phenotype as well as for eosinophilic inflammation. Next, I looked at how IL 33/ST2 pathway modulates the differentiation and activation of eosinophil. I demonstrated that bone marrow hematopoietic progenitors CD117+ express ST2L and that IL 33 is able to differentiate these cells toward eosinophils. By employing deficient mice or neutralizing antibodies I found that this process is ST2 and IL 5 dependent and independent of IL 13. I then extended my research interests to include mature mouse and human eosinophils. I showed that both human and mouse resting eosinophils express low levels of ST2L which can be markedly increased by IL 33. Moreover, I demonstrated that eosinophils that are recruited to the lungs during experimental allergic airway inflammation express high levels of ST2L. Furthermore, I carried out a study on effector function of eosinophils. I found that IL 33 induces IL 13, IL 6 and increases TARC, TGF production by mouse eosinophils. In addition, IL 33 exacerbated IgG induced human and mouse eosinophil degranulation, likely by enhancing FcRII expression. Having shown earlier that IL 13 is requited for the polarization of alveolar macrophages toward AAM by IL 33 in vitro and in light of the fact that IL 33 stimulated eosinophils can be a significant source of IL 13; I went on to investigate the interaction between macrophages and eosinophils. Using co cultures of ST2 / macrophages with WT eosinophils in Transwell system, I demonstrated that IL 33 but not IL 5 activated eosinophils can support macrophage polarization toward the pro inflammatory AAM phenotype, partially through the production of IL 13. Finally, given the role of IL 33/ST2L axis in eosinophil activation in vitro, I investigated the contribution of IL 33 activated eosinophils to airway inflammation in vivo. Using adoptive transfer protocol I showed that the contribution of IL 33 activated eosinophils to airway inflammation is mediated primarily by the release of cytokines from these cells which, in turn, recruits other inflammatory cells and supports the differentiation of alveolar macrophages towards AAM. These data show that IL 33/ST2 pathway regulates multiple features of alveolar macrophage and eosinophil biology that can have a significant impact on asthma pathophysiology in the airways. Studies carried out in our laboratory and elsewhere suggest that IL 33 is equally capable of activating other cell types that have been implicated in asthma pathology such as Th2, B1 cells, DCs, mast cells and basophils. Therefore, targeting IL 33/ST2 pathway may potentially offer a promising therapeutic approach to asthma and allergy.

A citocina IL-33 apresenta papel dual e está envolvida com a resolução ou progressão de inúmeras doenças, além disso, acredita-se que a via IL-33/ST2 esteja envolvida no equilíbrio entre a atividade de osteoclastos e osteoblastos. O objetivo deste estudo foi avaliar o papel do receptor ST2 no desenvolvimento e progressão de lesões periapicais experimentalmente induzidas em camundongos. Lesões periapicais foram induzidas em primeiros molares inferiores de camundongos WT e ST2 knockout (KO). Decorridos 7 e 14 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos e do volume por microtomografia computadorizada (μCT); contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); expressão gênica de marcadores osteogênicos e osteoclastogênicos por q-PCR; quantificação de neutrófilos por ensaio de mieloperoxidases. Os linfonodos foram submetidos à análise da expressão dos fatores transcricionais T-bet, GATA-3, RORc e Foxp-3 por q-PCR. Análise estatística utilizada foi One-way ANOVA, seguido de pós-teste de Bonferroni. Aos 14 dias, observou-se maior extensão da lesão periapical em animais WT que em ST2KO (p<0,05). O tamanho da lesão nos animais ST2KO permaneceu igual em função do tempo. Foi observada maior quantidade de neutrófilos na lesão do grupo WT aos 7 dias, em comparação aos animais ST2KO (p<0,05). Na expressão de T-bet, GATA-3, RORc e Foxp-3 não foram observadas diferenças estatisticamente significantes. O número de osteoclastos contados nos animais ST2KO foi maior que o observado em WT aos 7dias e aos 14 dias (p<0,05). A expressão de Runx2 foi maior no grupo lesão dos animais ST2KO quando comparado a seu respectivo controle. Os outros marcadores relacionados com a formação óssea não apresentaram diferenças estatisticamente significantes. Dentre os marcadores relacionados com a reabsorção óssea, a catepsina K e o MMP-9 apresentaram maior expressão aos 14 dias, na lesão dos animais WT quando comparada à expressão na lesão dos animais ST2KO (p<0,05). Com base nos resultados obtidos no presente estudo, pode-se concluir que na ausência do receptor ST2 as lesões periapicais são menos extensas e embora em maior quantidade, os osteoclastos são menos ativos. Nossos resultados sugerem um importante papel da via IL-33/ST2 na ativação dos osteoclastos e desenvolvimento da lesão periapical.
The IL -33 cytokine presents a dual role and is involved either in the resolution and progression of many diseases. Furthermore, it is believed that this pathway is involved between osteoclast and osteoblast activity balance. The aim of this study was to evaluate the role of ST2 receptor in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and ST2 knockout (KO) mice. After 7 and 14 days, jaw samples were subjected to various analysis: determination of periapical lesions area by histology and volume by computed microtomography (μCT); osteoclasts number by TRAP histoenzymology; osteogenic and osteoclastogenic markers expression by q-PCR; neutrophil quantification by myeloperoxidase activity. The expression of transcription factors T-bet, GATA-3, RORC and Foxp-3 in lymph nodes were analysed by q-PCR. Statistical analysis was done by One-way ANOVA and Bonferroni post-test. It was observed a greater extent in periapical lesions of WT compared to ST2KO animals at 14 days (p<0.05). There is no progression in the lesion of ST2KO mice with the time. A larger number of neutrophils in WT group was observed, compared to ST2KO mice evaluated at 7 days (p<0.05). The expression of T-bet, GATA-3, RORc and Foxp-3 were not statistically significant different among the groups. The number of osteoclasts in lesions of ST2KO animals were greater than the observed in WT, at 7 and 14 days (p<0.05). Although, other osteogenic markers showed no statistically significant difference, Runx2 expression in ST2KO was higher in lesion side compared to control side at 14 days. The markers related to bone resorption, cathepsin K and MMP-9, were significantly abrogated in the lesion side of ST2KO mice, at 14 days (p<0.05). Based on the results, it can be concluded that although larger amounts of osteoclast were counted in ST2KO, the lesion was less extensive and osteoclasts less active. It all suggests that the IL-33/ST2 pathway play an important role in osteoclasts activation and periapical lesion development.

Les maladies pulmonaires, responsables de 3,1 millions de décès de part le monde représentent un problème majeur de santé publique. En particulier, la fibrose pulmonaire et la broncho-pneumopathie chronique obstructive (BPCO) conduisent à la perte de la fonction pulmonaire. Aucun traitement efficace n’a été identifié à ce jour pour lutter contre ces maladies, la seule alternative étant la transplantation. Au cours de ma thèse, j’ai exploré les mécanismes du développement de ces maladies en utilisant différents modèles chez la souris, soit par l’instillation de nanoparticules de métaux ou de bléomycine, conduisant à l’inflammation et/ou à la fibrose pulmonaire, soit par exposition à la fumée de cigarette provoquant une inflammation. Nous avons montré le rôle de la voie de signalisation IL-33/ST2 dans les réponses inflammatoires induites par les nanoparticules ou la bléomycine et identifié de nouveaux mécanismes de régulation de l’IL-33 au sein des macrophages, différents de ceux décrits pour les cellules épithéliales. Nos résultats indiquent que l’expression intracellulaire de l’IL-33 et de son récepteur ST2, joue un rôle important dans l’inflammation, ainsi que la translocation nucléaire de l’IL-33. D’autre part, mes travaux de thèse ont permis d’identifier le rôle clef du senseur intracytosolique NLRP6 dans l’inflammation provoquée par l’exposition à la fumée de cigarette. Nos résultats indiquent que NLRP6, aux fonctions pulmonaires inexplorées, contrôle l’activation des cellules épithéliales et le recrutement des neutrophiles de façon indépendante de la formation d’un inflammasome mais dépendante de la signalisation par les récepteurs des interférons de type I et III
Pulmonary diseases are a major health problem with 3.1 million deaths in the worldwide. Among them pulmonary fibrosis and chronic obstructive pulmonary disease (COPD), which occur after repeated lung epithelium injury, are characterized by impaired lung functions. To date, no effective therapy against pulmonary fibrosis and COPD were developed, lung transplantation being the only alternative. During my thesis, I studied the mechanisms leading to disease development using different experimental models in mice in particular by metal dioxide nanoparticles or bleomycin instillation leading to inflammation and/or pulmonary fibrosis, or by cigarette smoke exposure promoting pulmonary inflammation which may lead to emphysema. We show the crucial role of IL-33/ST2 signaling pathway in response to nanoparticles or bleomycine and identify new mechanisms for IL-33 regulation in macrophages which are different from those described in epithelial cells. Our results indicate that intracellular expression of IL-33 and of its receptor ST2, together with nuclear IL-33 translocation, play an important role in inflammatory response to nanoparticles instillation. On the other hand, my thesis work allowed identifying that the cytosolic sensor NLRP6 as a key player in pulmonary inflammation developed upon mouse cigarette smoke exposure. Interestingly, our results show that the receptor NLRP6, whose pulmonary functions are still unexplored, controls epithelial cells activation leading to neutrophils recruitment in the airways, in an inflammasome-independent manner but dependently of type I and III interferon receptors signaling

In addition to their role in fighting infection, mast cells have long been implicated in the pathogenesis of allergic and autoimmune inflammatory diseases and cancers. Increasingly, however, there is recognition that these cells may also play a part in protecting against development of pathologies. Indeed, there is increasing evidence that mast cells comprise heterogeneous phenotypes that exhibit functional plasticity to allow them to play both pro- and anti-inflammatory roles during an immune response. This plasticity appears to reflect that immature mast cells are tailored by their particular microenvironment not only to trigger protective inflammatory responses but also to limit pathology by resolving inflammation and promoting wound healing and tissue repair. Mast cells can be activated by a range of stimuli including (pathogen-derived) antigen/allergen-mediated crosslinking of antibody-bound to Fc receptors, most notably FcεRI, pathogen-associated molecular patterns (PAMP) such as bacterial lipopolysaccharide (LPS) acting on TLR4, inflammatory cytokines such as IL-33 (via the IL-1R/TLR-like receptor ST2) and tissue-derived signals such as SCF (via cKIT). During infection these stimuli provoke a response optimised for pathogen clearance however in autoimmune or allergic disease such responses can initiate and exacerbate host pathology. Thus the challenge for therapeutic targeting of mast cells in inflammatory or malignant disease is to limit mast cells with pathogenic phenotypes whilst preserving those contributing to protective homeostatic and anti-pathogen responses. Thus, as a first step, it was a core aim of these studies to generate in vitro mast cell models representing the phenotypic and maturational heterogeneity of mast cells in vivo, as these are difficult to isolate and purify due to their limited numbers in tissue. Distinct murine mast cell phenotypes, namely mature serosal peritoneal-derived mast cells (PDMC), connective tissue-like mast cells (CTMC) and mucosal-like mast cells (BMMC), the latter two subtypes both derived from bone marrow progenitors, were found to differentially respond, in terms of cytokine production and degranulation, to important immunoregulatory receptors in health and disease, namely FcεRI and TLR4. Consistent with their distinct functional responses, these mast cell subtypes were also found to display differential calcium signalling profiles in response to FcεRI and TLR4 signalling, further highlighting the importance in investigating phenotypically relevant and microenvironment-specific (serosal versus mucosal) mast cells in drug discovery programmes. Recently there has been great interest in IL-33, a pro-inflammatory cytokine increasingly recognised as playing an important role in a variety of mast cell responses associated with allergic inflammatory disorders and tumour pathogenesis. Consistent with this, whilst IL-33 can stimulate mast cell cytokine production, but not degranulation, via the IL-1R/TLR-like receptor ST2, responses to this cytokine are amplified following IgE sensitization and/or exposure to SCF or serum. Such augmented responses reflect increased calcium mobilization, PLD, SphK, ERK and NF-κB signalling and mTOR activation and can be suppressed by existing therapeutics targeting the costimulatory signal, for example, Imatinib or Dasatinib for SCF/cKIT and potentially Omalizumab for IgE/FcεR1. Moreover, IL-33/ST2 signalling can modulate mast cell responses resulting from antigen-mediated crosslinking of FcεRI and LPS-TLR4 signalling. Indeed, ST2 signalling can differentially modulate LPS/TLR4 responses depending on the presence (enhances) or absence (inhibits) of IL-33, as in the latter case ST2 acts to limit LPS cytokine production, potentially by sequestering MyD88. This receptor crosstalk is likely to occur under pathological conditions, thus targeting of such cooperative signalling may allow the downregulation of hyper-inflammatory responses, whilst leaving protective and homeostatic mast cell responses intact. ES-62 is an immunomodulator produced by filarial nematodes to dampen immune responses in order to promote parasite survival and prevent tissue damage without immunocompromising the host to infection. As a serendipitous side effect of its anti-inflammatory actions, ES-62 exhibits therapeutic potential in both allergic and autoimmune inflammatory disease and thus to further explore the potential for safe, targeted downregulation of pathogenic mast cell responses, the parasite product was exploited in order to identify signals regulating mast cell activation. ES-62 was found to be able to induce hypo-responsiveness of all three mast cell phenotypes in terms of degranulation and cytokine production in response to stimulation of FcεR1-, TLR4- or IL-33/ST2, either alone or in combination. ES-62 mediated these effects, at least in part, by mechanisms involving downregulation of PKCα (and in BMMC, MyD88) expression and calcium mobilisation and, in PDMC, potentially by subverting the negative feedback interactions of ST2 on TLR4. The precise mechanism of modulation varies both with receptor usage and mast cell phenotype as ES-62 exhibits differential effects in PDMC and BMMC. Nevertheless, collectively these data support the role of calcium-, PKCα- and MyD88- as key regulatory intersection sites in the functional crosstalk amongst these important immunoregulatory receptors and importantly, suggest they are potential targets for therapeutic intervention in pathogenic mast cell responses.

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Infecções por Schistosoma mansoni e geohelmintos, na presença de IgE anti-Ascaris, modulam a intensidade das alergias. Neste cenário, as quimiocinas que alteraram o recrutamento celular, IL-33 indutora de resposta Th2, e seu receptor solúvel ST2 (ST2s) não foram investigados. Objetivou-se avaliar CXCL8/IL-8, CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1, CXCL10/IP-10 e a expressão do RNAm de IL-33 e ST2s, bem como, os níveis de IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α e IFN-γ e dos anticorpos IgE total, anti-Blomia e anti-Asc, eosinófilos no hospedeiro infectado com S. mansoni ou gehelmintos e investigar sua associação com asma/rinite e a positividade para teste cuâneo (SPT) para aeroalérgenos. Para isto, realizou-se estudo de Caso-Controle aninhado a um corte transversal. Aplicou-se o questionário ISAAC, obtiveram-se dados socioeconômicos e parasitológicos e quatro grupos (carga parasitária baixa): Alérgicos/Infectados (A-I); Alérgicos/Não Infectados (A-NI); Não Alérgicos/Infectados (NA-I); Não Alérgicos/Não Infectados (NA-NI), realizou-se o teste de reatividade cutânea para aeroalérgenos. Coletou-se sangue para cultura (Fitohemaglutinina; 24hs) e, no sobrenadante foram dosadas citocinas e quimiocinas (cytometric bead array) e nas células a expressão do RNAm (qPCR). A infecção por S. mansoni constitui proteção para reatividade do SPT (OR=0,351, IC95%=0,153 – 0,802; p=0,017) e asma/rinite (OR=0,274, IC95%= 0,135 - 0,554; p<0,0001). A IgE anti-Asc não interferiu na proteção induzida pelo S. mansoni. Em comparação ao grupo NA-NI, no grupo A-I, houve menor expressão da IL-33 e produção de CCL2/MIP-1, acompanhado de mais IL-10. No grupo NA-I, foi detectado menos IL-33 e mais CXCL10/IP10 e CXCL9/MIG e no grupo A-NI mais IL-10. Os geohelmintos constituíram proteção para asma/rinite (OR=0,337; IC95% = 0,132 – 0,829; p=0,008) com menor frequencia de crise no último ano, chiado no peito após exercício físico e tosse seca à noite. Em comparação ao grupo NA-NI, os grupos infectados com ou sem alergia apresentaram menor razão na expressçao IL-33/ST2s. Sendo assim, os dados sugerem que houve uma modulação negativa na produção do CCL2/MIP-1, pelo S. mansoni e geohelmintos, e na atuação da IL-33. Contudo, o primeiro parasita prejudicou a produção de IL-33 e segundo levou ao sequestro via ST2s. Estes achados podem explicar a atenuação das manifestações alérgicas em áreas endêmicas para esquistossomose e geohelmintíases.
Schistosoma mansoni and geohelminths infections, in the presence of anti-Ascaris IgE, module the intensity of allergies. Chemokines that altered cell recruitment, and IL-33-inducing Th2 response, and its soluble ST2 receptor (sST2) were not investigated. The aim of study was to evaluate CXCL8/IL-8, CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1, CXCL10/IP-10 and IL-33 mRNA expression and ST2s, as well as IL-2 , IL-4, IL-6, IL-10, IL-17, TNF-α and IFN-γ levels, and total IgE, anti-Blomia and anti-Asc antibodies, eosinophils in the host infected with S. mansoni or gehelmintos Its association with asthma/rhinitis, and the Skin Prick Test (SPT) for aeroallergens. Case-Control study was carried out nested to a cross-section. The ISAAC questionnaire was applied, socioeconomic and parasitological data and four groups (low parasite load): Allergic / Infected (A-I); Allergic / Non-Infected (A-NI); Non-allergic / Infected (NA-I); Non-Allergic / Non-Infected (NA-NI), the skin reactivity test for aeroallergens was performed. Culture blood (Phytohemagglutinin, 24hs) was collected and cytometric bead array (cytometric bead array) and mRNA expression (qPCR) were measured in the supernatant. S. mansoni infection provides protection for SPT reactivity (OR = 0.351, 95% CI = 0.153 - 0.802, p = 0.017) and asthma / rhinitis (OR = 0.274, 95% CI = 0.135-0.554, p <0.0001). Anti-Asc IgE did not interfere with S. mansoni induced protection. Compared to the NA-NI group, in the A-I group, there was lower expression of IL-33 and production of CCL2 / MIP-1, accompanied by more IL-10. In the NA-I group, less IL-33 and more CXCL10 / IP10 and CXCL9 / MIG and in group A-NI plus IL-10 was detected. The geohelminths constituted protection for asthma/rhinitis (OR = 0.337, 95% CI = 0.132 - 0.829, p = 0.008) with a lower frequency of crisis in the last year, chest wheezing after exercise and dry cough at night. Compared to the NA-NI group, the groups infected with or without allergy had a lower ratio in IL-33/ST2s expression. Thus, the data suggest that there was a negative modulation in the production of CCL2/MIP-1, by S. mansoni and geohelminths, and in the performance of IL-33. However, the first parasite impaired IL-33 production and second led to sequestration via ST2s. These findings may explain the attenuation of allergic manifestations in endemic areas for schistosomiasis and geohelminthiasis.

Obesity has become an increasing public health issue worldwide; it is estimated than over 65% of American adult population is overweight and 35% are actually obese. Criteria for obesity definition is traditionally linked to Body Mass Index (BMI). Although an important correlation exists between BMI and body fat deposit, the BMI criteria cannot actually predict body composition in any given individual due to variability determined by age, sex, race and ethnicity. Besides the weight of an individual, both fat localization and type represent more important estimators of cardiovascular disease. Importantly, the accumulation of visceral fat – more than subcutaneous fat – is linked to cardiovascular disease severity and mortality. Among ectopic fat depots, pericardial and epicardial fat accumulation are intriguingly important. Epicardial adipose tissue (EAT) represents a biological active tissue that secretes adipokines known to promote low-grade inflammation and diabetic vascular complications. The cause is due to the shared blood supply between EAT and myocardium, as indeed EAT provides the 50–70% of the energy used for contraction trough the release of free fatty acids (FFAs). It is intuitive that molecular/metabolic alteration of EAT will be reflected on its secretome and on cardiac metabolism. EAT thickness seems to be associated to ventricular myocardial mass and importantly to myocardial steatosis. A plethora of works highlighted the role of IL-33 and its receptor ST2 within cardiac dysfunction and inflammation associated with obesity. Once secreted in activated or damaged cells, IL-33 modulates the functions of various immune cells through ST2 binding, affecting the proliferation of T cells, macrophages, and innate lymphoid cells. Accordingly, IL-33/ST2 pathway is involved in lipid metabolic diseases and - following its role as immune sensor to infection and stress – it is linked to pro-fibrotic remodeling of myocardium and more in general in cardiovascular diseases. Our group demonstrated in coronary artery disease (CAD) a direct correlation between EAT thickness and IL-33/ST2 signalling imbalance further highlighting a role for EPAC protein involved in cAMP signal transduction In this PhD Project we aimed to further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on the molecular pathway that links adipose/cardiac-derived IL-33 to development of fibrosis or hypertrophy. We choose Zucker Fatty rats (ZF) and Zucker Diabetics Fatty rats (ZDF) to overcome the problems linked to tissue availability and to the homogeneity of human EAT samples that need to be collected during cardiac surgery. Since genetic animal models do not fully recapitulate human pathology, we developed in vitro models to mimic adipose and myocardial relationship in vitro. Indeed, we evaluated the effects of visceral adipose tissue (VAT)-derived cellular medium in the modulation of adipose secretome and how they affect myocardial gene expression. Finally, we compared obtained results with those derived from Diet induced obesity (DIO) mice, a naturally occurring model of obesity that reliably resembles human disease. Following both molecular and proteomic analysis, we demonstrated a dysregulation of IL-33/ST2 signalling in both adipose and cardiac tissue, where they affected myocardial gene expression and determined a pro-fibrotic signature. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression (such as MEF2a), but also increased sST2 and not cardioprotective ST2L form. In this context we observed a reduction of EPAC signalling, that is promoted by VAT secretome and linked to ST2 isoforms balancing regulation. Similar results were obtained in ZDF rats, as both models did not develop evident alteration of cardiac architecture, although we described the increase of the pro-fibrotic signature. To avoid the genetic bias linked to leptin mutation and ghrelin up-regulation we repeated our analysis in DIO mice that fully recapitulates human obesity and presents gradual appearance of hyperglycaemia and progression of metabolic syndrome. Thanks to the analysis of cardiac proteome, we observed an enrichment in proteins and networks involved in extra-cellular matrix remodeling and ventricular function. Interestingly, IL-33 was considered among the possible up-stream regulators of this process. In conclusion, this PhD project demonstrated a dysregulation of IL-33/ST2 signalling in obesity, that directly correlate with Epac expression in ZF rats. The alteration of these pathways in adipose tissue could influence IL-33/ST2 expression and hypertoric/fibrotic response in cardiac tissue. Importantly the effect of IL-33 signalling could be modulated by hormones (such as Ghrelin) and other stimuli. Importantly, the final effect of IL-33 signalling activation is dependent on several factor, as cell types’ origin and balancing of ST2 isoforms. Noteworthy, extreme importance has to be ascribed to the animal models used in in vivo experiments, the chronic or acute condition as such as the time of IL-33 secretion. This way, it is reasonable that to define a unique protective role of IL-33 is over-simplistic and further studies are needed to confirm and unveil mechanisms of IL-33 as a gene expression regulator in cardiac obesity.

L'interleukine-33 (IL-33) est une cytokine nucléaire de la famille des interleukines-1. L'IL-33 est exprimée par de nombreux épithéliums en contact avec l'environnement et peut être libérée sous sa forme pleine taille lors de nécrose. Cette forme active peut interagir avec le récepteur ST2 appartenant à la famille des récepteurs de l'IL-1 (IL-1R4). ST2 est présent sur de nombreuses cellules du système immunitaire aussi bien impliquées dans l'immunité innée (cellules lymphoïdes innées de type 2 ou ILC2, mastocytes, NK,. . . ) qu'adaptative (lymphocytes Th2, T CD8,. . . ). L'ensemble de ces propriétés suggèrent que l'IL-33 fonctionne comme une alarmine chargée d'alerter le système immunitaire en cas de dommage cellulaire ou tissulaire. L'axe IL-33/ST2 semble jouer un rôle prépondérant dans les pathologies inflammatoires chroniques, allergiques ou encore infectieuses. Compte tenu de l'implication des alarmines et des membres de la famille IL-1 lors de la tumorigenèse, l'IL-33 pourrait également jouer un rôle important lors de la progression tumorale. Cependant, à ce jour, aucune étude n'a encore caractérisé l'expression et le rôle potentiel de l'IL-33 endogène au cours de la tumorigenèse. Pour réaliser ce projet, nous avons développé un modèle murin invalidé pour l'Il-33 grâce à une stratégie " Gene Trap ". Dans la première partie du travail, nous avons caractérisé le profil d'expression de l'IL-33 chez la souris et nous l'avons comparé à celui décrit précédemment chez l'Homme. Nous avons montré que ce profil d'expression recouvre partiellement celui de l'Homme puisque l'IL-33 est exprimée de manière constitutive par les épithéliums. En revanche, de manière surprenante, l'IL-33 n'est pas retrouvée au niveau de l'endothélium, même si son expression peut être induite dans les cellules endothéliales en contexte inflammatoire comme par exemple lors de colite. Nous avons ensuite utilisé ce modèle murin " Il-33 Gene Trap " pour décrire le profil d'expression de l'IL-33 et étudier son rôle potentiel au cours de la tumorigenèse. Nous montrons que dans un contexte tumoral associé à une inflammation chronique, l'expression de l'IL-33 est fortement induite dans les noyaux des cellules endothéliales et des cellules mésenchymateuses du microenvironnement tumoral, ainsi que dans ceux des cellules cancéreuses. Dans la deuxième partie du travail, nous montrons à l'aide d'un modèle de carcinome du côlon induit chimiquement dans notre lignée invalidée pour l'Il-33, que la perte d'IL-33 augmente la susceptibilité des souris à développer un cancer colorectal. De façon intéressante, la restauration en IL-33 dans ces souris " Il-33 Gene Trap " inhibe le développement des tumeurs du colon. Ainsi, pour la première fois, nous impliquons l'IL-33 dans la réponse anti-tumorale. Nous avons caractérisé les acteurs cellulaires impliqués dans cette réponse anti-tumorale initiée par l'IL-33 et nous avons découvert que les éosinophiles en sont les principaux responsables. Nous montrons que le recrutement et l'activation des éosinophiles, et l'effet anti-tumoral de l'IL-33 sont dépendants de l'IL-5, une cytokine jouant un rôle clé dans les réponses immunitaires de type 2. Nous proposons que dans ce modèle de cancer du côlon, l'IL-33 libérée par les cellules endothéliales, ou l'épithélium transformé est capable d'activer les ILC2, qui vont libérer de grandes quantités d'IL-5, ce qui conduit au recrutement et à l'activation des éosinophiles qui vont à leur tour pouvoir exercer une réponse anti-tumorale. Ainsi, nos résultats suggèrent un rôle clé de la voie IL-33/ILC2 dans l'immunité anti-tumorale et le maintien de l'homéostasie tissulaire
Interleukin-33 or IL-33 is a recently discovered nuclear cytokine of the Interleukin-1 family. IL-33 is expressed by several epithelia in contact with the environment and its full length form can be released during necrosis. This active form can interact with the ST2 receptor, a member of the IL-1 receptor family (IL-1R4). ST2 is expressed by several immune cell types involved in innate immunity (Type 2 Innate lymphoid cells or ILC2, mast cells, Natural Killer cells. . . ) and adaptive immunity (Th2 lymphocytes, CD8+T cells,. . . ). All together, these properties suggest that IL-33 is an alarm signal (or alarmin) able to alert the immune system after tissue or cell damage. The IL-33/ST2 pathway plays an important role in chronic inflammatory diseases, infectious and allergic diseases. Since other alarmins and IL-1 family members have been implicated in tumorigenesis, IL-33 could also be involved in tumor progression. However, the potential role of endogenous IL-33 during tumorigenesis has not yet been studied. For this work, we developed an Il-33 knock-out (Il-33 KO) mice model using a "Gene Trap" strategy. In the first part of our study, we characterized the expression profile of endogenous IL-33 in mice. We found that, similar to its human counterpart, murine IL-33 is constitutively expressed in high levels by epithelia from barrier tissues. Surprisingly, IL-33 was not found in the endothelium in mice, but its expression in endothelial cells could be induced during inflammation, for instance during colitis. Then, we used the "Il-33 Gene Trap" model to describe and study the potential role of IL-33 during tumorigenesis. We observed that in a tumor context associated with chronic inflammation, the expression of IL-33 is strongly induced in the nuclei of endothelial cells and mesenchymal cells of the tumor microenvironment as well as tumor cells. In the second part of our studies, we used a chemically induced colon carcinogenesis model in our Il-33 KO mice to show that a deficiency in Il-33 increased the development of colon cancer. Rescue of IL-33 levels in "Il-33 Gene Trap" mice inhibited the development of colonic tumors. We sought to identify which cell types are involved in this anti-tumor response initiated by IL-33 and discovered that eosinophils are the major players. We showed that the recruitment and activation of eosinophils and the anti-tumor effects of IL-33 are dependent on IL-5, a cytokine with a key role in type 2 immune responses. We suggest that in this model of colon cancer, IL-33 is released by endothelial cells and/or transformed epithelial cells, activates the ILC2 population that secretes high levels of IL-5, which in turn, induces the recruitment and activation of eosinophils that contribute to the anti-tumor response. Together, our data suggest a key role of the IL-33/ILC2 pathway in the anti-tumor response and the maintenance of tissue homeostasis

Les traitements actuels de la leucémie myéloïde chronique (LMC) ne permettent pas d’éliminer la totalité des cellules leucémiques. Dans le but de développer un traitement curatif, il est donc nécessaire de parvenir à une meilleure compréhension des mécanismes sous-jacents des réponses partielles aux traitements, Dans ce travail, nous avons postulé qu’il existe des mécanismes de contrôle extrinsèques de la LMC pouvant influencer l’efficacité des différents traitements. Nous avons choisi d’étudier le rôle potentiel dans la LMC des lymphocytes iNKT, cellules T de type « inné » auxquelles la littérature attribue de nombreuses fonctions antitumorales et des facteurs moléculaires, la cytokine/alarmine IL-33, produite dans la niche hématopoïétique, et son récepteur ST2 à la surface des cellules hématopoïétiques comme cibles de l’IL-33.La première partie de notre travail a ainsi permis de mettre en évidence de profondes altérations fonctionnelles des lymphocytes iNKT chez les patients atteints de LMC ainsi qu’une correction partielle de ces défauts après traitement par l’Imatinib (IM) ou l’IFN-. L’ensemble de ces résultats permet de proposer que l’altération des fonctions des cellules iNKT au cours du développement de la LMC pourrait participer aux mécanismes d’échappement de la tumeur au contrôle par le système immunitaire. La deuxième partie de notre travail a permis de mettre en évidence une expression de la molécule ST2, chaîne spécifique du récepteur à l’IL-33, à la surface des cellules CD34+ de patients atteints de LMC, expression non décelée chez les sujets sains et les patients en rémission après traitement par l’IM. De plus, contrairement aux cellules CD34+ de sujets sains, les cellules progénititrices de patients en phase chronique prolifèrent en réponse à l’IL-33. Enfin, nous avons montré que l’IL-33 est capable de contrecarrer in vitro les effets antiprolifératifs de l’IM. Ainsi nous pouvons émettre l’hypothèse selon laquelle l’IL-33, une cytokine/alarmine, puisse participer aux phénomènes conduisant à la persistance de progéniteurs hématopoïétiques leucémiques chez les patients sous traitement par IM
To date, treatment of Chronic Myeloid Leukaemia (CML) is not sufficient to completely eradicate leukaemia cells. Hence, in order to develop a curative treatment, it is necessary to have a better understanding of the underlying mechanisms explaining why response to treatment is only partial..We therefore addressed the question whether the extrinsic mechanisms of CML control can affect the effectiveness of different treatments. We first provided evidence of profound functional impairments of iNKT cells in patients with CML. Interestingly these impairments were partially corrected after treatment with Imatinib (IM) or IFN-. Consequently our results suggest that altered functions of iNKT cells during the development of CML could facilitate tumour escape from immune destruction. . The second part of our work revealed that CD34+ progenitors from CML patients upregulate their cell surface expression of the IL-33-specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34+ cells from healthy individuals. Moreover, ST2 overexpression is normalized following IM therapy, while IL-33 counteracts in vitro IM-induced growth arrest in CML CD34+ progenitors. From these findings, it can be surmised that IL-33, a cytokine/alarmin likely expressed in the hematopoietic niche, facilitates the development of CML and IM resistance

La transplantation rénale (TR) est la stratégie thérapeutique de choix pour les insuffisances rénales terminales. L'utilisation de greffons de donneurs à critères étendus, tels les sujets diabétiques, est devenue une nécessité face à la pénurie de greffons, malgré le risque accru de dysfonction du greffon. L'ischémie/reperfusion (I/R) est une étape inhérente à la TR. Le diabète est un facteur de risque pour le développement de lésions rénales aiguës mais les mécanismes d'action mis en jeu restent à élucider. Il existe peu de données expérimentales sur les conséquences délétères du diabète sur les lésions d'I/R rénales. Nous avons fait l'hypothèse que l'IL-33, en tant qu'alarmine, joue un rôle clé au carrefour qui lie l'hyperglycémie à l'I/R.L'objectif de ce travail a été d'étudier l'effet de la délétion du récepteur ST2 de l'IL-33 sur les lésions d'I/R rénales dans un contexte hyperglycémique. Nous avons utilisé un modèle d'I/R rénale chez des souris transgéniques dépourvues du récepteur ST2. Le diabète type 1 a été induit par la streptozotocine. Nous avons démontré que la délétion du récepteur ST2 a un effet néphroprotecteur en situation euglycémique, suggérant un effet exacerbateur de l'IL-33 sur les lésions d'I/R rénales. Cette protection rénale a été perdue en condition hyperglycémique. L'ensemble de ces résultats suggère que l'IL-33, en tant qu'alarmine, a des effets différentiels en fonction de l'environnement hyperglycémique. Sur le plan clinique, compte tenu de l'impact néfaste du diabète sur le rein, nos résultats conduisent à proposer d'étudier la place d'IL-33 dans l’inflammation chronique associée au diabète et de ses conséquences sur la fonction rénale
Renal transplantation (RT) is the therapeutic strategy of choice for end-stage renal failure. The use of graft from extended criteria donors, including patients with diabetes, has become mandatory to cope with the shortage of grafts, despite an increased risk of graft dysfunction. Ischemia reperfusion (I/R) is a key step in RT. Diabetes is a risk-factor for the development of acute kidney injury (AKI), which mecanisms are not fully elucidated. Moreover, there are few experimental data on the deleterious effects of diabetes on ischemic AKI. We hypothesized that IL-33, as an alarmin, plays a key role at the crossroads linking hyperglycemia to I/R injury.The objective of this work was to study the effect of the deletion of the ST2 receptor of IL-33 on I/R injury in the context of hyperglycemic environment. We thus used a renal I/R model in transgenic mice lacking the IL-33 receptor (ST2). Type 1 diabetes was induced by streptozotocin. We demonstrated that the deletion of ST2 has a renal protective effect in euglycemic conditions, suggesting an exacerbating effect of IL-33 in renal I/R injury. The protective effect induced by the absence of ST2 was lost in hyperglycemic conditions. Taken together, these findings indicate that IL-33 exerts differential effects depending on hyperglycemia. Clinically, given the deleterious impact of diabetes on the kidney, our results lead to propose to further study the place of IL-33 in chronic inflammation associated with diabetes and its effects on renal function

Regulatory T cells (Treg) play a crucial role in controlling immune homeostasis. Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Treg. While several mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain unknown. The Treg pool consists of highly diverse subpopulations, indicating that there is a potential to optimise Treg-targeted therapies if disease-relevant mechanisms can be established. Microarray data from our lab suggests a marked upregulation of the integrin αv as well as the IL-33 receptor ST2 in Treg retrieved from the inflamed central nervous system (CNS) during experimental autoimmune encephalomyelitis compared to peripheral lymphoid organs. These two molecules were further investigated within this PhD project with the aim to understand their role in Treg function during chronic inflammatory disease. αvβ integrins have been reported to be needed for effector T cell migration to inflamed sites through binding of extracellular matrix components and are involved in TGF-β activation by a variety of cell types. Conditional knockout mice lacking the integrin αv specifically in Foxpγ+ Treg were generated to address the role of αv integrins on regulatory T cells in inflammatory disease. αv-/- Treg showed a deficiency in activating latent TGF-β, but were able to suppress responder T cell proliferation in vitro as well as in vivo. αv-/- Treg were also able to migrate to the inflamed CNS during EAE and resolve disease. However, αv-/- Treg were detected at significantly lower numbers and proportions in the inflamed gut during a curative T cell transfer model of colitis; this led to a quantitative impairment in the ability of αv-/- Treg to cure colitis when compared to wild-type (WT) Treg. Whether this is a deficit in migration, survival, proliferation, or Foxp3 stability, remains to be investigated. IL-33 acts as an alarmin and is best studied as a cytokine released upon tissue damage that induces a potent type 2 immune response by acting on a multitude of immune cells. Expression of the IL-33 receptor ST2 on Treg has recently been associated with positive metabolic parameters in visceral adipose tissue, protection from gut inflammation, and tissue-restorative function in other inflamed tissues such as injured muscle or lung. This project showed that in steady state, ST2+ Treg expressed high levels of several markers which have been associated with potent regulatory function. When stimulated in vitro, ST2+ Treg showed a better survival and expansion rate compared to their ST2- counterparts, even more so in the presence of IL-33. T-bet deficiency in Treg resulted in an increased ST2+ Treg pool, and T-bet-associated cytokine IFN-γ was found to antagonise IL-33-induced expansion of the ST2+ Treg pool in a T-bet-independent manner. When ST2+ and ST2- Treg were tested for their respective suppressive capacity in vivo, ST2+ Treg were able to suppress responder T cell expansion despite being found only at low numbers in secondary lymphoid organs compared to ST2- Treg. However, in a curative model of T cell transfer colitis, ST2+ Treg were less capable of controlling the ongoing immune response than ST2- Treg. A possible explanation for the superiority of ST2- Treg in this setting can be found in the fact that injected ST2- Treg acquired a distribution of ST2 expression reminiscent of WT Treg over the course of disease. On the other hand, an increased starting pool of ST2+ Treg as occurs in T-bet-/- Treg significantly enhanced the capacity of Treg to control colitis compared to WT Treg. In conclusion, both ST2- and ST2+ Treg are likely to have a distinct, non-redundant role in suppressing T cell activation in secondary lymphoid organs and controlling ongoing inflammation in peripheral tissue, respectively.

Die Pathophysiologie der systemischen Sklerose (Sklerodermie, SSc) ist bisher unvollständig erforscht. Die Sklerodermie ist eine immun-mediierte Systemerkrankung, gekennzeichnet durch eine Fibrose des Bindegewebes. Die unklare Pathophysiologie erschwert eine frühzeitige Diagnose und therapeutische Intervention bei den Patienten. Es wird angenommen, dass die vaskulären Endothelzellen, insbesondere die der mikrovaskulären Gefäße, eine wichtige Rolle zu Beginn der Erkrankung einnehmen. Erste sichtbare Anzeichen einer gestörten Mikrovaskulatur sind in über 90 % der SSc-Patienten bereits vor einer gesicherten Diagnose mittels Kapillarmikroskopie nachweisbar (1, 2). Inwiefern die Moleküle des Sphingolipid-Signalwegs und der IL-33/ST2-Achse sich für die Sklerodermie als serologische Biomarker eignen, wurde in der vorliegenden Arbeit untersucht. Dabei lag das Augenmerk auf der Evaluation eines Biomarkers(-Panels) zur Diagnose der Sklerodermie oder der Verlaufskontrolle bei Medikation der Sklerodermie-Patienten. Zum einen wurden Serumproben von 69 Patienten mit limitierter systemischer Sklerose (lcSSc), zwanzig Patienten mit diffuser SSc sowie zehn Patienten im frühen Stadium der SSc (LeRoy) aus Schweden und Deutschland untersucht. Als Kontrollgruppen fungierten gesunde, dem Alter und Geschlecht der SSc-Patienten angepasste, Individuen (n = 38) sowie elf Patienten mit dem idiopathischen, primärem Raynaud-Phänomen. Bei den deutschen Individuen wurde eine Kapillar-mikroskopie durchgeführt. Es wurden Plasmaproben von 175 schwedischen lcSSc-Patienten und entsprechenden 112 gesunden Probanden analysiert. Die Sphingolipid- und Glucosyl-Ceramide-Profile wurden mittels LC-MS/MS erhoben, während die Konzentrationen weiterer Proteinparameter, IL-33, sST2, DKK-1 und den Dermatofibrosemarkern COMP sowie Lysyl-Oxidase, mit spezifischen ELISA untersucht wurden. Ebenso wurde der modifizierte Rodnan-Skin-Score (mRSS) erhoben. Die statistische Analyse erfolgte mit dem Mann-Whitney-U-Test, Kruskal-Wallis-Test oder dem Spearmans Korrelationskoeffizient. Die deutschen und schwedischen lcSSc-Patienten (n = 77) zeigten einen lcSSc-typischen Auto-Antikörpertiter. Bei den deutschen lcSSc-Patienten konnten keine Auffälligkeiten in der klinischen Chemie und Hämatologie gefunden werden. Bei diesen 26 Patienten konnte bei der kapillarmikroskopischen Untersuchung die, für die SSc-charakteristische Verringerung der Kapillardichte und die Megakapillaren im Vergleich zu elf 1.RP-Patienten und drei gesunde Probanden nachgewiesen werden. Die schwedischen lcSSc-Patienten (n = 44) wiesen im frühen Stadium der lcSSc vermehrt einen frühen oder aktiven und bei fortgeschrittener Krankheit den späteren Kapillarstatus auf. Im Serum der lcSSc-Patienten war IL-33 nicht erhöht. Der sST2-Serumspiegel war bei lcSSc-Patienten mit einer Krankheitsdauer von mehr als neun Jahren (n = 29) im Vergleich zu dcSSc-Patienten (n = 20) oder früheren Stadien der lcSSc (Krankheitsdauer ein bis drei Jahren, n = 15) deutlich höher. Der sST2-Spiegel korrelierte in SSc-Patienten (n = 95) positiv mit der Krankheitsdauer. In einem auf Zytokine spezialisiertem Assay konnte gezeigt werden, dass auch PDGF-AB/BB, IL-4, EMMPRIN, IL-1ra und ENA-78 in vier lcSSc-Patienten verglichen mit vier alters- und geschlechtsübereinstimmenden Kontrollindividuen erhöht waren. Im Serum zeigte sich ein länderspezifischer Unterschied zwischen deutschen und schwedischen Individuen. In der deutschen Kontrollgruppe (n = 10) waren die S1P-Spiegel signifikant niedriger als in der schwedischen (n = 10). Die gemessenen Ceramid-Konzentrationen unterschieden sich hingegen nicht. Die Sphingosin-Serumkonzentrationen waren, normiert auf die Mittelwerte der jeweiligen gesunden Probanden, bei lcSSc-Patienten (Krankheitsdauer ein bis drei Jahren, n = 15; Krankheitsdauer vier bis neun Jahren, n = 14) im späteren Krankheitsstadium im Vergleich zu den 1.RP-Patienten erhöht. Der S1P-Spiegel der SSc-Patienten (n = 95) korrelierte mit der Erkrankungsdauer, dem COMP-, dem DKK-1- (n jeweils 95) und dem Lysyl-Oxidase-Serumspiegel (n = 33). Ein Zusammenhang zwischen der Erkrankungsdauer dem S1P-, COMP-, DKK-1- (n jeweils 95) und dem Lysyl-Oxidase-Serumkonzentrationen (n = 33) wurde nachgewiesen. Untersuchungen der Plasmaproben von schwedischen 175 lcSSc-Patienten und 112 gesunden Probanden zeigten, dass C18:0- sowie C24:1-Ceramid und das C16:0-Dihydroceramid bei den Patienten erhöht war. Ebenso war S1P und das Glucosyl-Ceramid C24:1 lcSSc-Patienten im Vergleich zur Kontrollgruppe höher. Die Sphingosin-Serumspiegel waren bei den lcSSc-Patienten leicht erhöht. Erste Ergebnisse einer klinische Studie mit sieben lcSSc-Patienten, die eine vasodilatorische Therapie (Iloprost) erhielten, zeigten eine deutliche Zunahme der S1P- und dhS1P-Spiegel am Ende der Behandlung. Damit könnte sich S1P als serologischer Marker für den Behandlungserfolg nach einer Iloprost-Therapie eignen. Die hier untersuchten Sphingolipide, u. a. S1P, und sST2 unterstreichen, insbesondere unter Einbezug ihrer Serumspiegelveränderungen nach einer Iloprost-Therapie, die pathophysiologische Bedeutung der Endothelzellen bei der Sklerodermie. S1P, welches sowohl pro- als auch anti-fibrotisch agiert und zu großen Teilen in Endothelzellen produziert wird (3) und sST2, welches bereits als Marker für fibrotische Erkrankungen beschrieben wurde (4), könnten über das direkt auf Endothelzellen wirkende Iloprost modifiziert werden. Zusammenfassend lässt sich darstellen, dass sST2 und spezifische Sphingolipide neue Zielmoleküle für ein Biomarkerpanel zur exakteren und möglicherweise frühzeitigeren Diagnose der lcSSc sind und die hier dargestellten Ergebnisse deutlich auf eine Beteiligung der Sphingolipide und der IL-33/ST2-Achse in der Pathophysiologie der Sklerodermie hinweisen.