A doença infecciosa que causa o maior número de mortes no mundo é a tuberculose, causada pelo bacilo Mycobacterium tuberculosis. Um dos fatores de risco que aumenta três vezes o desenvolvimento de tuberculose é a diabetes, sendo a obesidade associada com predisposição à diabetes. A obesidade gera inflamação de baixo grau que agrava a progressão de doenças crônicas. Estudos que avaliaram a associação da obesidade com tuberculose são controversos, e o tema merece maior investigação. No presente estudo, usamos um modelo experimental para determinar a interface da obesidade e da tuberculose. Camundongos C57BL/6 foram alimentados com dieta hiperlipídica (HFD - High Fat Diet) durante 60 dias, quando foram infectados com M. tuberculosis (HFD/Mtb) por via intra-traqueal. Como controles experimentais, animais foram alimentados com dieta padrão (LFD - Low Fat Diet) e infectados (LFD/Mtb). Paralelamente, um grupo recebeu HFD e outro LFD, e seguiram sem infecção. Após 30 dias de infecção, totalizando 90 dias de dieta, os diferentes grupos foram avaliados. Os animais obesos e infectados (HFD/Mtb) apresentaram aumento do peso corporal e do peso dos tecidos adiposos, aumento da expressão gênica de IL-1? no tecido adiposo, intolerância à glicose, deficiência na produção de insulina e aumento dos níveis séricos de IFN-? comparados aos animais LFD/Mtb. Além disso, o grupo HFD/Mtb foi mais suscetível e apresentou maior inflamação pulmonar comparado ao grupo LFD/Mtb. A inflamação foi caracterizada por aumento na expressão gênica para IL-17, IFN-?, TNF, IL-1?, IL-1?, NLRP3, caspase-1, IL-18, IL-6, aumento de células CD4+ produtoras de IFN-? e/ou IL-17 nos pulmões, e foi também acompanhada por aumento de células CD8+ e células CD4+Foxp3+ quando comparado ao grupo LFD/Mtb. Como NLRP3 é uma molécula chave na metainflamação induzida pela obesidade, mas seu papel ainda não está bem definido na tuberculose, animais deficientes de NLRP3 receberam HFD e foram infectados (NLRP3-/- HFD/Mtb). Esse grupo NLRP3-/- HFD/Mtb foi mais resistente e exibiu redução da inflamação pulmonar comparado ao grupo WT (Wild Type) HFD/Mtb. Sabendo que a obesidade está associada à disbiose e que produtos bacterianos derivados da dieta alimentar ou da microbiota podem estimular a liberação de IL-1? pela ativação de NLRP3, avaliamos o papel da microbiota na comorbidade obesidade e tuberculose. Encontramos disbiose intestinal, caracterizada por aumento do Filo Firmicutes e redução dos Filos Bacteroidetes e Proteobacteria, além do aumento de butirato e redução de acetato e propionato nos intestinos do grupo HFD/Mtb comparado ao grupo LFD/Mtb. O aumento na expressão de claudina-2 sugere alteração na permeabilidade intestinal e possível translocação bacteriana, caracterizada pela disbiose nos pulmões, nos quais foi detectado aumento de Firmicutes, Bacteroidetes e Actinobacteria, e redução de Proteobacteria no grupo HFD/Mtb. Em conclusão, a obesidade aumenta a magnitude da inflamação pulmonar e a suscetibilidade à infecção por M. tuberculosis por um mecanismo dependente de NLRP3. Ambos, aumento da suscetibilidade à infecção e da inflamação pulmonar estão associadas com disbiose intestinal e pulmonar, e aumento da permeabilidade intestinal. / The infectious disease that causes the largest number of deaths in the word is tuberculosis, caused by Mycobacterium tuberculosis bacilli. One of the risk factors that increases the devolpment of tuberculosis three times is diabetes. Obesity generates lowgrade inflammation that magnify the progression of chronic disease. Studies that have evaluated the association between obesity and tuberculosis are controversial, and the issue requires further investigation. In this study, we used an experimental model to determine the interface between obesity and tuberculosis. C57BL/6 mice were fed a highfat diet (HFD) for 60 days and infected by M. tuberculosis (HFD/Mtb) via intratracheal. As experimental control, animals were fed a light-fat diet (LFD) and were infected (LFD/Mtb). In parallel, one group was fed with HFD and another LFD, and they remained without infection. After 30 days of diet completing 90 days of feeding, the different groups were evaluated. Obese and infected animals (HFD/Mtb) showed increased body mass and adipose tissue weight, increased of IL-1? gene expression in adipose tissue, glucose intolerant, impaired insulin production and increased of serum levels of IFN-? compared to LFD/Mtb animals. In addition to, HFD/Mtb animals were more susceptible and exhibited higher lung inflammation compared to LFD/Mtb animals. The inflammation was characterized by increased of IL-17, IFN-?, TNF, IL-1?, IL-1?, NLRP3, caspase-1, IL-18, IL-6 gene expression and increase of IFN-? and/ or IL-17- producing CD4+ cells in the lungs, and was also accompanied by increased CD8+ and CD4+Foxp3+ cells compared to the LFD/Mtb group. As NLRP3 is a key molecule in obesity-induced meta-inflammation, but its role is still not well defined in tuberculosis, NLRP3 deficient animals fed with HFD and were infected (NLRP3-/- HFD/Mtb). This NLRP3-/- HFD/Mtb group was more resistant and exhibited reduction of lung inflammation compared to the WT (Wild Type) HFD/ Mtb group. Considerate that obesity-associated dysbiosis and that bacterial products derived from diet or microbiota can stimulate the release of IL-1? by the activation of NLRP3, we evaluated the microbiota role in obesity and tuberculosis comorbidity. We found intestinal dysbiosis characterized by increased Firmicutes phylum and reduction of Bacteroidetes and Proteobacteria phylum, as well as increased butyrate and diminished acetate and propionate in the intestine of the HFD/Mtb group compared to the LFD/Mtb group. An increase of claudin-2 expression suggests an alteration in intestinal permeability and a possible bacterial translocation characterized by dysbiosis in the lungs, with increased of Firmicutes, Bacteroidetes and Actinobacteria and diminished of Proteobacteria in the HFD/Mtb group. In conclusion, obesity increases the magnitude of pulmonary inflammation and susceptibility to M. tuberculosis infection by an NLRP3-depedent mechanism. Both increased susceptibility to infection and pulmonary inflammation are associated with intestinal and pulmonary dysbiosis, and increased intestinal permeability.

Embora já tenha sido comprovada a ação adjuvante da SBA-15, pouco se sabe sobre o seu mecanismo molecular que leva a modulação positiva da resposta imunológica. Foi avaliada a ativação do inflamassoma NLRP3, sobre estímulos de SBA-15, em macrófagos de camundongos C57BL/6. Como parâmetro dessa ativação, foi analisada a produção de IL-1β por ELISA. A SBA-15 foi capaz de induzir a produção de IL-1β a níveis semelhantes quando comparado com um agonista de NLRP3 (Nano-SiO2), sugerindo a ativação do inflamassoma. Para avaliar o envolvimento da caspase-1, nos resultados obtidos com a SBA-15, os macrófagos foram estimulados com sílica na presença do inibidor de caspase-1, e como esperado, a produção de IL-1β foi restaurada para o seu nível basal. A ativação do inflamassoma, por estímulos da SBA-15, parece ser parcialmente dependente da fagocitose e da produção das espécies reativas do oxigênio. Além disso, foi visto que a SBA-15 não induz a produção de IL-6, confirmando que essa sílica está envolvida na via do inflamassoma e não em outras vias, como por exemplo, NF-κB. / Although it has already been proven adjuvant action of SBA-15, little is known about its molecular mechanism leading to positive modulation of the immune response. NLRP3 inflammasome activation was evaluated on SBA-15 stimulation in C57BL/6 mice macrophages. As this parameter activation, it analyzed the production of IL-1β by ELISA. The SBA-15 was able to induce the production of IL-1β at levels similar when compared to an agonist of NLRP3 (Nano-SiO2), suggesting the activation of the inflammasome. To assess the involvement of caspase-1, the results obtained with SBA-15, the macrophages were stimulated with silica in the presence of caspase-1 inhibitor, and as expected, IL-1β production was restored to its baseline level. Activation of the inflammasome, by stimuli of SBA-15, appears to be partly dependent on phagocytosis and production of reactive oxygen species. In addition, it was seen that the SBA-15 does not induce IL-6 production, confirming that silica is involved inflammasome the path of and not in other ways, eg, NF-κB.

Dermatofitoses são infecções fúngicas de natureza crônica cujo principal agente etiológico é Trichophyton rubrum. Apesar de sua alta ocorrência mundial, pouco se sabe sobre os mecanismos imunológicos envolvidos nestas infecções. Neste trabalho investigamos a participação de duas classes de receptores de imunidade inata (NLRs e CLRs) na resposta a T.rubrum e avaliamos o perfil proteômico de macrófagos quando estimulados com o fungo. Observamos que T.rubrum foi capaz de induzir a produção de IL-1β dependente do inflamassomo NLRP3 e destacamos o papel da sinalização de IL-1 na modulação da resposta de IL-17. Determinamos os CLRs dectina-1 e dectina-2 como receptores essenciais na produção de citocinas inflamatórias e para o controle da infecção experimental. Curiosamente, a IL-17 e os linfócitos T e B foram dispensáveis para a eliminação do fungo. Também identificamos a proteína CLEC1A como uma novo receptor para fungos, envolvido no reconhecimento de glicolipídeos de T.rubrum. Por fim, a análise proteômica de macrofagos revelou a vimentina e a plastina-2 como duas proteínas potencialmente envolvidas na relação patógeno-hospedeiro. / Dermatophytosis are chronic fungal infections whose main causative agent is Trichophyton rubrum. Despite its high incidence worldwide, the immunological mechanisms underlying these infections remain largely unknown. Here we investigated the involvement of two classes of innate immune receptors (NLRs and CLRs) in the reponse to T.rubrum and performed a proteomic profiling of macrophages upon T.rubrum stimulation. We observed that T.rubrum was able to drive NLRP3 inflammasome-derived IL-1β production and highlighted IL-1 signaling as an important component in the shaping of the IL-17 response. We defined the CLRs dectin-1 and dectin-2 as key receptors for the induction of inflammatory cytokines and for the infection control in the in vivo settings. Curiously, IL-17 cytokines and T and B lymphocytes were dispensable for fungal clearance. In addition, we uncovered CLEC1A as a new receptor in fungal sensing, involved in the recognition of T.rubrum glycolipids. Finally, the proteomic analysis revealed Vimentin and Plastin-2 as two proteins potentially involved in the host-pathogen interaction.

A inibição crônica do óxido nítrico com Nw-nitroargininemethylester (L-NAME), associado à sobrecarga de sal, leva a hipertensão grave, albuminúria, glomeruloesclerose, isquemia glomerular e fibrose intersticial, caracterizando um modelo de doença renal crônica (DRC). Achados anteriores deste laboratório e de outros sugerem que a ativação de pelo menos duas vias da imunidade inata, TLR4/NF-kB e NLRP3/IL-1beta, ocorre em vários modelos experimentais de DRC e que a progressão da lesão renal pode ser atenuada com a inibição destas vias. No presente estudo, investigamos se a ativação da imunidade inata, através da via TLR4/NF-kB ou NLRP3/IL-1beta, está envolvida na patogênese da lesão renal em outro modelo de DRC, o de inibição crônica do NO com sobrecarga de sal. Ratos Munich-Wistar machos adultos receberam sobrecarga de sal (2% Na+ na dieta e 0,5% Na+ na água do bebedouro) e L-NAME (32 mg/Kg/dia) dissolvido na salina do bebedouro (Grupo HS+N) ou tratados com alopurinol (Alo, 36 mg/Kg/dia, v.o), usado como inibidor de NLRP3 (grupo HS+N+Alo) ou tratados com ditiocarbamato de pirrolidina (PDTC, 60 mg/Kg/dia, v.o), um inibidor de NF-kB (Grupo HS+N+PDTC). Após 4 semanas, os ratos HS+N desenvolveram hipertensão arterial, albuminúria e lesão renal, juntamente com inflamação renal, estresse oxidativo e ativação de ambas as vias NLRP3/IL1-beta e TLR4/NF-kB. Alo reduziu o ácido úrico renal e inibiu a via NLRP3/IL-1beta. Esses efeitos foram associados à atenuação da hipertensão arterial, albuminúria e inflamação/fibrose intersticial, mas não à lesão glomerular. O PDTC diminuiu o ácido úrico renal e inibiu as vias NLRP3 e NF-kB, promovendo um efeito antiinflamatório e nefroprotetor mais eficiente que o Alo. As vias NLRP3/IL-1beta e TLR4/NF-kB atuam paralelamente para promover lesão/inflamação renal e devem ser simultaneamente inibidas para obter nefroproteção maior nesse modelo de DRC / Nitric oxide inhibition with Nk-nitroargininemethylester (L-NAME) along with salt overload leads to severe hypertension, albuminuria, glomerulosclerosis, glomerular ischemia and collapse, together with interstitial fibrosis, characterizing a model of chronic kidney disease (CKD). Previous findings of this laboratory and elsewhere suggest that activation of at least two pathways of innate immunity, TLR4/NF-kB and NLRP3 inflammasome/IL-1beta, occurs in several experimental models of CKD, and that progression of renal injury can be slowed with inhibition of these pathways. In the present study, we investigated whether activation of innate immunity, through either the TLR4/NFkB or NLRP3/IL-1beta pathway, is involved in the pathogenesis of renal injury in yet another CKD model, chronic NO inhibition with salt overload. Adult male Munich-Wistar rats receiving L-NAME in drinking water and salt overload (Group HS+N) were treated with Allopurinol (ALLO), used as an NLRP3 inhibitor (Group HS+N+ALLO), or PyrrolidineDithiocarbamate (PDTC) a NF-kB inhibitor (Group HS+N+PDTC). After 4 wks, HS+N rats developed hypertension, albuminuria and renal injury, along with renal inflammation, oxidative stress and activation of both the NLRP3/IL1-beta and TLR4/NF-kB pathways. ALLO lowered renal uric acid and inhibited the NLRP3 pathway. These effects were associated with amelioration of hypertension, albuminuria and interstitial inflammation/fibrosis, but not glomerular injury. PDTC lowered renal uric acid and inhibited both the NLRP3 and NF-kB pathways, promoting a more efficient anti-inflammatory and nephroprotective effect than ALLO. NLRP3/IL-1beta and TLR4/NF-kB act in parallel to promote renal injury/inflammation and must be simultaneously inhibited for best nephroprotection

L'inflammasome est une plateforme moléculaire composée d'un récepteur de l'immunité innée tels que NLRP3 ou NLRP1b, de la protéine adaptatrice ASC et de la caspase-1. Il joue un rôle essentiel dans le déclenchement de la réponse inflammatoire via l'activation de la caspase-1 qui mène à la sécrétion de cytokines pro-inflammatoires telle que l'IL-1ß. Dans un premier axe de recherche, nous avons mis en évidence que les macrophages de souris déficientes pour ASC traités avec l'activateur de l'inflammasome NLRP1b, la toxine létale de bacillus anthracis, étaient capables de sécréter la forme mature de l'IL-1ß en absence de clivage de la caspase-1 pourtant décrit comme un événement indispensable à son activité. En reconstituant des macrophages caspase-1 KO avec une forme mutante non-clivable de la caspase-1, nous avons démontré que la forme entière de la caspase-1 est capable d'induire la sécrétion d'IL-1ß en réponse à la toxine létale alors qu'elle n'est pas fonctionnelle au sein de l'inflammasome NLRP3.Dans un second axe de recherche, mon travail de thèse s'est intéressé à comprendre le rôle de l'inflammasome au cours de la progression tumorale. En effet, l'IL-1ß est une cytokine fréquemment retrouvée dans le microenvironnement tumorale mammaire suggérant donc l'activation de l'inflammasome au sein des tumeurs. Au moyen d'un modèle de tumeurs in vivo, nous avons montré que l'absence de la caspase-1 et de ASC dans les cellules immunitaires chez la souris conduit à une réduction de la croissance tumorale. De plus, l'absence de ces deux protéines provoque également un plus fort recrutement et une meilleure activité des cellules NK au sein des tumeurs.En plus d'identifier un nouveau mécanisme d'activation de la caspase-1, mon travail de thèse a permis de mettre en évidence le rôle de l'inflammasome dans l'altération des cellules NK au cours de la progression tumorale mammaire. Ces données permettent d'envisager l'inflammasome comme une cible thérapeutique dans le cancer / The inflammasome is a molecular platform composed of an innate immune receptor such as NLRP3 or NLRP1b, of the adaptor protein ASC and of the caspase-1. It plays an essential role in triggering inflammatory response through the activation of caspase-1 that leads to the secretion of pro-inflammatory cytokines such as IL-1ß.In a first research axis, we showed that ASC deficient mice macrophages treated with an NLRP1b inflammasome activator, the lethal toxin of Bacillus anthracis, were able to secrete the mature form of IL-1ß in absence of any cleavage of caspase-1 previously described as an essential event for its function. By reconstituting caspase-1 KO macrophages with an uncleavable mutant form of caspase-1, we showed that the entire form of the protein is able to induce IL-1ß secretion upon lethal toxin treatment but is nonfunctional upon NLRP3 inflammasome activation.In a second research axis, my PhD work focused on underlying the inflammasome role during tumor progression. Indeed, IL-1ß is frequently found within breast tumor microenvironment suggesting that inflammasome is activated in tumors. Using in vivo tumor model, we showed that the absence of caspase-1 of ASC in immune cells lead to a delay tumor growth. In addition, the absence of these two proteins also causes a stronger recruitment and an enchanced activity of NK cells within mammary tumors

Le développement de la réponse immunitaire innée de l’hôte au cours des infections respiratoires nécessite la mise en place rapide d'un réseau moléculaire et cellulaire relativement complexe ayant pour but de contrôler la croissance microbienne ainsi que permettre son éradication. Dans certaines circonstances, et malgré l’existence de vaccins et d'antibiotiques efficaces, l’infection par Streptococcus pneumoniae peut aboutir à des pathologies graves telles qu'une pneumonie, une méningite et/ou une septicémie. Ainsi, à l'heure actuelle, les maladies associées au pneumocoque sont encore loin d'être sous contrôle. Dans ce contexte, une meilleure compréhension de la réponse immunitaire innée de l’hôte contre ce pathogène est nécessaire.Mes travaux de thèse ont permis pour la première fois de mettre en évidence la fonctionnalité et la relevance biologique de l’inflammasome NLRP3 au sein des neutrophiles pulmonaires in vivo dans un modèle d’infection respiratoire par S. pneumoniae.Ainsi, de façon inattendue, les neutrophiles jouent un rôle accessoire original à des temps précoces de l’infection via leur capacité à produire de l’IL-1β. Cette synthèse protéique est possible grâce à la combinaison de 2 signaux à la fois dérivés de l’hôte (TNF-α des macrophages alvéolaires) et de la bactérie (toxine). Ces deux signaux permettent l’assemblage et l’activation de l’inflammasome NLRP3 neutrophilique. D’un point de vue translationnel, nous avons été capables de démontrer un mécanisme similaire avec des neutrophiles humains.Cette production d’IL-1β par les neutrophiles participe à l’activation des lymphocytes T γδ producteurs d’IL-17; une cytokine essentielle dans le contrôle de l’infection bactérienne via sa capacité à induire rapidement le recrutement d’une deuxième vague de neutrophiles participant directement à l’élimination et la clairance bactérienne.Sur la base de ces travaux fondamentaux, nous avons émis l’hypothèse qu’une augmentation du pool de cellules innées sécrétrices d’IL-17A pourrait avoir un effet bénéfique sur le contrôle d’une infection respiratoire à pneumocoque. Ainsi via l’administration prophylactique et locale d’IL-7, nous avons été capables d’augmenter la fréquence et le nombre de lymphocytes innés producteurs d’IL-17A résultant en un meilleur contrôle de la charge bactérienne pathogène associée à une augmentation du recrutement neutrophilique. A ce stade, ces résultats encourageants, nous pousse à mieux comprendre les mécanismes moléculaires et cellulaires associés à cet effet dans l’éventualité de proposer à terme une nouvelle approche thérapeutique dans le contrôle des infections respiratoires pulmonaires basée sur la manipulation de la biologie de l’IL-7. / The mounting of an appropriate host innate immune response in the lungs requires the rapid establishment of a complex cellular and molecular networking that allows the containment and clearance of pathogens during respiratory infections. Both neutrophils and γδT cells are central players in the host response during mucosal infections. Using a model of invasive pneumococcal disease, we illustrated a role for Interleukin-17A in controlling neutrophil recruitment, bacterial loads and survival. Following Streptococcus pneumoniae infection, we defined pulmonary γδT cells, especially the lung resident Vγ6Vδ1+ subset, as the primary source of IL-17A in an IL-23/IL- 1β-dependent manner. Using gene-targeted mice, we demonstrated that γδT cells largely contributed to neutrophilia and to the control of the pathology. Furthermore, we now defined a second and unexpected early role for neutrophils as accessory cells in γδT17 cell activation through IL-1β secretion. Neutrophil-derived IL-1β was dependent on NLRP3 inflammasome activity and required alveolar macrophage-secreted TNF-α for priming and bacterial pneumolysin for NLRP3- dependent caspase-1 activation. This report thus brings to light the sequential molecular/cellular events leading to γδT17 cell activation and highlights the existence of a biologically relevant and fully functional NLRP3 inflammasome in pulmonary neutrophils that regulates a key immune axis in the development of protective innate response to respiratory bacterial infection.Based on these observations, we hypothesized that an increase in the pool of IL-17A-producing innate-like T lymphocytes might play a protective role during pneumococcal infection. As recently suggested, we demonstrated that intranasal IL-7/M25 complex administration into naïve mice allowed the expansion of the cellular pool of innate immune cells presenting a Th17-like phenotype in the lungs especially T cells. Moreover, we showed during S. pneumoniae infection that prophylactic IL-7/M25 treatment increased the capacity of Vγ6Vδ1+ T cells to produce IL-17A. Interestingly, this phenotype led to higher neutrophil recruitment and a better control of bacterial burden in the lungs as well as systemic dissemination. Thus, we report a critical role of IL-7 in creating an IL-17-enriched microenvironment which improves the early development of host innate immune response to respiratory bacterial infection. This observation might pave the way to the development of future innovative cytokine/cell-based strategies against Streptococcus pneumoniae.

Des études menées au laboratoire avaient démontré un rôle critique de l’inflammasome NLRP3 dans l’asthme allergique en réponse à l’ovalbumine en absence d’adjuvant. Mes travaux de thèse ont porté sur le rôle de NLRP3 et de la caspase-1 dans un modèle murin d’inflammation pulmonaire induite par l’allergène d’acarien HDM. Nous avons montré un rôle régulateur de la caspase-1 dépendant de l’inflammasome NLRP3 et la molécule adaptatrice ASC mais pas de l’inflammasome NLRC4. Cette régulation de la réponse allergique se caractérise par une augmentation de l’infiltration des éosinophiles, de l’hyperréactivité bronchique et de la production des cytokines de type Th2 telles que l’IL-4, l’IL-5, l’IL-13 et l’IL-33 dans les poumons. Nous avons montré que les mécanismes responsables de cette régulation sont associés à l’IL-33 produite par les macrophages et que la neutralisation de l’IL-33 par administration locale de la protéine de fusion au récepteur ST2 (muST2-Fc) atténue les caractéristiques de l’asthme allergique. Ces résultats suggèrent que l’activation de la caspase-1 réduit la production d’IL-33 in vivo et régule ainsi la réponse l’inflammation pulmonaire induite par HDM et la réponse Th2. D’autre part, nous nous sommes intéressés au rôle de la Protéine Kinase C thêta (PKC-θ) dans ce même modèle d’inflammation pulmonaire. Nous avons démontré que PKC-θ joue non seulement un rôle protecteur dans l’asthme allergique mais également un rôle critique pour la prolifération et l’activation des cellules lymphoïdes innées (ILC2). D’autre part, l’inhibition de PKC-θ in vivo par administration orale de son inhibiteur spécifique C20 (BIX02656) atténue l’inflammation pulmonaire et la production d’IL-5 et d’IL-13. Nous suggérons que PKC-θ est impliquée dans la différenciation des Th2 et des ILC2 via un mécanisme dépendant des facteurs de transcription IRF4 et NFAT-1. Au total, mes travaux de thèse mettent en exergue deux molécules IL-33 et PKC-θ qui pourraient constituer des cibles thérapeutiques potentielles. / Studies from our laboratory have shown a critical role of NLRP3 inflammasome in response to ovalbumin allergen. In the present study we investigate the role of NLRP3 and caspase-1 in a mouse model of pulmonary inflammation induced by HDM. We have shown a regulatory role of caspase-1 dependant of the NLRP3 inflammasome and the adaptator molecule ASC but not NLRC4. The regulation of the allergic response is characterized by an increase of eosinophilia, bronchial hyperreactivity and Th2 cytokines production (IL-4, IL-5, IL-13 and IL-33) in lungs. We have shown that mechanisms responsible of this regulation are associated with IL-33 production by macrophages and that neutralization of IL-33 by local administration of a fusion protein of the ST2 receptor (muST2-Fc) reduce characteristics of asthma. These results suggest that caspase-1 activation reduce IL-33 production in vivo regulating lung inflammation and Th2 response induced by HDM. Moreover, we investigate the role of the Protein Kinase C theta (PKC-θ) in allergic airway inflammation. We have demonstrated that PKC-θ plays a protective role in allergic asthma but is critical for the activation and proliferation of innate lymphoid cells (ILC2). In addition, in vivo inhibition by oral administration of PKC-θ specific inhibitor C20 (BIX02656) reduces pulmonary inflammation with IL-5 and IL-13 production. We suggest that PKC-θ is implicated in Th2 and ILC2 differenciation by a mechanism dependant on transcription factors IRF4 and NFAT-1. Finally, my thesis projects describe IL-33 and PKC-θ as potential therapeutic targets for allergic lung inflammation.

La pandémie de l'obésité entraine une augmentation de la prévalence des maladies chroniques du foie ou stéatopathies métaboliques (NAFLD). Le spectre des NAFLD va de la stéatose caractérisée par une accumulation de lipides dans le foie à la stéatohépatite (NASH) associant une inflammation, de la mort hépatocytaire et de la fibrose. Lors de l'obésité, l'élévation de signaux de dangers métaboliques perturbe les fonctions du réticulum endoplasmique (RE) essentielles pour l’homéostasie cellulaire. Les perturbations sont transmises par 3 senseurs : IRE1α, ATF6 et PERK pour activer une réponse adaptative. Si ce stress est sévère ou devient chronique, la cellule enclenchera une réponse terminale apoptotique. La protéine Bax Inhibitor-1 (BI-1) pourrait jouer un rôle hépatoprotecteur en inhibant l’hyperactivation de la voie de signalisation IRE1α.En combinant des études chez l’homme et dans des modèles animaux, l’objectif de cette étude était de mieux caractériser l'activation chronique du stress du RE dans les NAFLD. Ce travail a émis l’hypothèse qu’une déficience en BI-1 entrainerait l’activation soutenue de la voie IRE1α qui serait responsable de la transition de la stéatose à la NASH. Cette étude s'intéresse au dialogue potentiel entre le stress du RE et l’activation de l'inflammasome NLRP3, qui induit la sécrétion des cytokines pro-inflammatoires (IL-1β, IL-18) grâce aux caspases pro-inflammatoires (caspase-1, caspase-4/11). L’utilisation d’un inhibiteur global du stress du RE ou des inhibiteurs pharmacologiques spécifiques à la voie IRE1α améliorerait les caractéristiques pathophysiologiques de la NASH et pourrait ouvrir de nouvelles perspectives thérapeutiques. / Due to the obesity pandemic, the last decades have been marked by a constantly increasing prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD covers a spectrum of hepatic disorders ranging from steatosis, characterized by the ectopic accumulation of lipids in the liver, to steatohepatitis (NASH), featuring inflammation, hepatocellular death and fibrosis. During obesity, an increase in metabolic danger signals leads to disrupted endoplasmic reticulum (ER) function, essential for cellular homeostasis. The resulting ER stress activates a signaling network involving three sensors: IRE1α, ATF6 and PERK to enforce adaptive programs. If this stress is severe or becomes chronic, the cell will trigger a terminal apoptotic response. The protein Bax Inhibitor-1 (BI-1), as a negative endogenous regulator of the IRE1α signaling pathway in the liver, may play a hepatoprotective role.By combining data from obese patients with liver complications and experimental approaches in mice, this thesis aimed to better characterize the chronic activation of ER stress in NAFLD pathogenesis. This work also emitted the hypothesis that a deficiency in BI-1 leads to unrestrained IRE1α signaling that may be responsible for the steatosis to NASH transition. This study further investigated the potential dialogue between ER stress and the activation the NLRP3 inflammasome, which induces the secretion of pro-inflammatory cytokines (IL-1β, IL-18) by activating pro-inflammatory caspases (caspase-1, caspase-4/11). The administration of a broad spectrum ER stress inhibitor or specific inhibitors of IRE1α improved the pathophysiological features of NASH and may open novel therapeutic perspectives.

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS / Als Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS

Atherosclerosis is complex inflammatory disease of the arterial wall with progressive accumulation of lipids and narrowing of the vessel. Increasing evidence suggest that inflammation plays an important role in plaque stability and often accelerate cardiovascular events such as myocardial infarction (MI). Among the vast number of inflammatory cytokines, IL-1β is known to be a key modulator in vessel wall inflammation and acceleration of the atherosclerotic process. The biologically active IL-1β is regulated by a multiprotein complex known as the NLRP3 inflammasome complex. In this thesis, we have focused on polymorphisms in the NLRP3 and CARD8 genes and their possible association to atherosclerosis and/or MI. We have also investigated the expression of inflammasome components NLRP3 and CARD8 in atherosclerosis and the role of genetic variants for the expression of these genes. The expression of NLRP3, CARD8, ASC, caspase-1, IL-1β, and IL-18 were found significantly upregulated in atherosclerotic lesions compared to normal arteries. Human carotid plaques not only express the NLRP3 inflammasome, but also release IL-1β upon exposure to lipopolysaccharide (LPS), adenosine triphosphate (ATP) and cholesterol crystals, which suggest NLRP3 inflammasome activation in human atherosclerotic lesions. Also, CARD8 was found to be important in the regulation of several inflammatory markers in endothelial cells, like RANTES, IP10 and ICAM-1. We further assessed the potential association of a CARD8 polymorphism and polymorphisms located downstream of the NLRP3 gene to the risk of MI in two independent Swedish cohorts. The CARD8 variant exhibited no association to risk of MI in either of the two cohorts. Some of the minor alleles of NLRP3 variants were associated with increased IL-1β levels and to NLRP3 mRNA levels in peripheral blood monocytic cells (PBMC). Taken together, the present thesis shows that NLRP3 inflammasome activation and increased expression of CARD8 in the atherosclerotic plaque might be possible contributors to the enhanced inflammatory response and leukocyte infiltration in the pathophysiology of atherosclerosis.