La dégénérescence maculaire liée à l’âge (DMLA), une maladie neurodégénérative de la rétine, est considérée comme la principale cause de perte de vision chez les personnes âgées. Le CD36 est un récepteur éboueur (scavenger) exprimé à la surface des phagocytes mononucléés et de l’épithélium pigmentaire rétinien (EPR), qui agit également comme corécepteur de l’hétérodimère des récepteurs de type Toll-like (TLR) 2/6 dans l’induction de l’inflammation. Dans la DMLA, l’activation du CD36 favorise l’internalisation des lipides oxydés par les cellules de l’EPR, le recrutement et l’accumulation des phagocytes mononucléés (microglie, monocytes/macrophages), ainsi que l’induction de la réponse inflammatoire au niveau sous-rétinien. L’identification du CD36 comme récepteur de peptides synthétiques de la famille des sécrétines de l’hormone de croissance (GHRPs) par notre laboratoire, nous a conduit au développement de ligands plus sélectifs pour ce récepteur, qui ont été étudiés dans le contexte des maladies inflammatoires chroniques liées aux macrophages telle que l’athérosclérose. Étant donné les similitudes observées entre la pathologie de l’athérosclérose et de la DMLA, nous avons émis l’hypothèse selon laquelle les azapeptides, comme ligands du CD36, auraient des effets protecteurs contre la DMLA en modulant la réponse inflammatoire. Ainsi, l’objectif général de ce projet de doctorat était d’évaluer les effets anti-inflammatoires et immunomodulateurs des azapeptides, comme ligands du CD36, et d’élucider leurs mécanismes d’action au niveau des macrophages ainsi que dans des modèles murins de DMLA. Le premier objectif spécifique de cette thèse était de déterminer l’effet de l’azapeptide MPE-001 dans la modulation de la réponse inflammatoire cellulaire médiée par le TLR2 et les voies de signalisation associées. Pour ce faire, des macrophages péritonéaux de souris C57BL/6 ou déficientes en Cd36 (Cd36-/-) ont été stimulés avec des agonistes du TLR2. Le traitement avec le MPE-001 a induit une diminution significative de la production de cytokines pro-inflammatoires, tels que le TNFa, l’IL-6, l’IL-12 et l’IL-1b et la chimiokine CCL2, de même qu’une inhibition des voies de signalisation nuclear factor kB (NFkB), c-Jun N-terminal kinase (JNK) et P38. Ce premier objectif nous a permis de mettre en évidence les effets anti-inflammatoires du MPE-001 et son mécanisme d’action, en modulant la réponse inflammatoire médiée par l’hétérodimère TLR2/6. Afin de déterminer les effets du MPE-001 sur la modulation de la réponse inflammatoire chez un modèle murin de DMLA, les souris C57BL/6, Cd36-/-, Cx3Cr1-/- et Cx3Cr1-/-/Cd36-/- ont été soumises à un stress photo-oxydatif induit par la lumière bleue pendant 5 jours. Une journée après le début de l’illumination, les souris ont reçu une injection s.c. de MPE-001 répétée pendant 7 jours. Le modèle murin de Cx3Cr1-/- est connue pour exacerber la réponse inflammatoire et induire l’accumulation de phagocytes mononucléés, plus particulièrement les microglies dans la rétine. Les montages à plat des coupes des yeux prélevés ont montré que le traitement au MPE-001 a engendré une diminution importante de 60% de l’accumulation de phagocytes mononucléés dans l’espace sous-rétinien, de même qu’une diminution des marqueurs inflammatoires (iNOS, IL-12), ainsi que la préservation de l’intégrité et de la fonction des photorécepteurs. Ce deuxième objectif nous a permis de démontrer les effets in vivo du MPE-001, en entraînant une diminution de l’accumulation de phagocytes mononucléés sous-rétiniens et la préservation des photorécepteurs. Étant donné les effets inhibiteurs du MPE-001 sur la sécrétion d’IL-1b, notre troisième objectif était d’investiguer l’effet du MPE-001 sur la régulation de l’inflammasome au niveau des macrophages. Nous avons montré que le MPE-001 entraînait une diminution de la réponse inflammatoire en inhibant la sécrétion d’IL-1b au niveau des macrophages, tout en diminuant l’expression de nucleotide-binding domain leucin-rich repeat and pyrin-containing receptor 3 (NLRP3) et de la caspase-1. Ce troisième objectif nous a permis de montrer que le MPE-001 a modulé le profil inflammatoire des macrophages en atténuant l’inflammasome NLRP3. Étant donné que le phénotype des macrophages est régulé par leur métabolisme, notre 4e et dernier objectif était de déterminer si les effets anti-inflammatoires du MPE-001 pouvaient influencer la polarisation des macrophages. Pour ce faire, nous avons utilisé un modèle de bone marrow-derived macrophages (BMDM) induits en phénotype pro-inflammatoire M1 ou anti-inflammatoire M2. Bien que le MPE-001 n’ait eu aucune influence sur les marqueurs de surfaces phénotypiques, nous avons montré que ce dernier induisait un changement métabolique des macrophages pro-inflammatoires M1, en inhibant la glycolyse et en favorisant la phosphorylation oxydative, de façon dépendante du récepteur nucléaire peroxisome proliferator-activated receptor-γ (PPAR-γ). En conclusion, les travaux de cette thèse ont montré que l’azapeptide MPE-001 possède de puissants effets anti-inflammatoires et immunomodulateurs au niveau des macrophages dans un contexte d’inflammation rétinienne, pouvant constituer une nouvelle approche thérapeutique prometteuse pour le traitement de la DMLA. / Age-related macular degeneration (AMD), a neurodegenerative disease of the retina, is considered to be the main cause of vision loss in the elderly. CD36 is a scavenger receptor expressed on the surface of mononuclear phagocytes and retinal pigment epithelium (RPE), which also acts as a co-receptor for the Toll-like receptor (TLR)-2/6 heterodimer in the induction of inflammation. In AMD, CD36 activation promotes the internalization of oxidized lipids by RPE cells, the recruitment and accumulation of mononuclear phagocytes (microglia, monocytes / macrophages), as well as the induction of the inflammatory response at the subretinal level. The identification of CD36 as a receptor for synthetic peptides of the growth hormone releasing-peptides (GHRP) by our laboratory, led us to the development of more selective ligands for this receptor, which have been studied in the context of chronic inflammatory diseases related to macrophages such as atherosclerosis. Given the similarities observed between the pathology of atherosclerosis and AMD, we hypothesized that azapeptides, as CD36 ligands, protect against AMD by modulating the inflammatory response. Thus, the general objective of this doctoral project was to characterize the anti-inflammatory and immunomodulatory effects of azapeptides, as CD36 ligands, and to elucidate their mechanisms of action on macrophages as well as in murine models of AMD. The first specific objective of this thesis was to determine the effect of the azapeptide MPE-001 in the modulation of the cellular TLR2-mediated inflammatory response and the associated signaling pathways. To do so, peritoneal macrophages from C57BL/6 or Cd36-deficient (Cd36-/-) mice were stimulated with TLR2 agonists. Treatment with MPE-001 induced a significant decrease in the production of pro-inflammatory cytokines, such as TNFa, IL-6, IL-12 and IL-1b and the chemokine CCL2, as well as inhibition of the nuclear factor k B (NF kB), N-terminal c-Jun kinase (JNK) and P38 signaling pathways. This first objective allows us to highlight the anti-inflammatory effects of MPE-001 and its mechanism of action, by modulating the inflammatory response mediated by the TLR2/6 heterodimer. In order to determine the effects of MPE-001 on the modulation of the inflammatory response in a mouse model of AMD, the murine models C57BL/6, Cd36-/-, Cx3Cr1-/- and Cx3xr1-/-/Cd36-/- were subjected to photo-oxidative stress induced by blue light for 5 days. One day after the start of illumination, the mice received a repeated s.c. injection of MPE-001 for 7 days. The mouse model of Cx3Cr1-/- is known to exacerbate the inflammatory response and induce the accumulation of mononuclear phagocytes, more specifically microglia, in the retina. Flat mounts from collected eyes showed that MPE-001 treatment caused a significant 60% decrease in the accumulation of mononuclear phagocytes in the subretinal space, as well as a reduction in inflammatory markers (iNOS, IL-12), with the preservation of photoreceptor integrity and function. This second objective allows us to demonstrate the in vivo effects of MPE-001, leading to a decrease in the accumulation of sub-retinal mononuclear phagocytes and the preservation of photoreceptors. Given the inhibitory effects of MPE-001 on IL-1b secretion, our third objective was to investigate the effect of MPE-001 on inflammasome regulation in macrophages. We have shown that MPE-001 decreases the inflammatory response by inhibiting the secretion of IL-1b in macrophages, while decreasing the expression of nucleotide-binding domain leucin-rich repeat and pyrin-containing receptor 3 (NLRP3) and caspase-1. This third objective allows us to show that MPE-001 modulates the inflammatory profile of macrophages by attenuating the NLRP3 inflammasome. Since macrophage phenotypes are regulated by their metabolism, our fourth and final objective was to determine whether the anti-inflammatory effects of MPE-001 could influence the polarization of macrophages. To do so, we used a bone marrow-derived macrophages (BMDM) model induced to pro-inflammatory M1 or anti-inflammatory M2 phenotype. Although MPE-001 had no influence on phenotypic markers, we have shown that the latter induces a metabolic shift of the pro-inflammatory M1 macrophages, by inhibiting glycolysis and promoting oxidative phosphorylation of macrophages, in a peroxisome proliferator-activated receptor-γ (PPAR-g)-dependent manner. In conclusion, the work conducted in this thesis showed that the azapeptide MPE-001 has powerful anti-inflammatory and immunomodulatory effects on macrophages in a context of retinal inflammation, which could constitute a promising new therapeutic approach for the treatment of AMD.

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

Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.

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[ES] Esta tesis doctoral titulada "El microambiente tumoral inflamatorio como objetivo en el diseño de nanoconjugados para el tratamiento del cáncer de mama avanzado" se centra en la evaluación de un nuevo inhibidor del inflamasoma (MM01) como herramienta química para estudiar el papel del inflamasoma en modelos de inflamación y cáncer. El capítulo I incluye una descripción general del sistema inmune, los inflamasomas dependientes de ASC y el papel que juegan en el desarrollo de enfermedades. También se profundiza en el papel de los inflamasomas y de la proteína ASC en la progresión del cáncer de mama. Además, se incluyen conceptos básicos de nanotecnología, nanomedicina y polímeros terapéuticos. Finalmente, se abordan las ventajas de utilizar nanomedicinas como terapia, las interacciones de las nanomedicinas con los sistemas biológicos, los nanofármacos descritos en la literatura, así como sus posibilidades de traslación a la práctica clínica. En los capítulos de resultados, delineamos un novedoso mecanismo de acción para MM01, un modulador de la actividad del inflamasoma recientemente identificado: la inhibición de la oligomerización del ASC y el subsiguiente procesamiento reducido de la pro-caspasa-1 y la inhibición de la actividad de la caspasa-1. Demostramos que MM01 interrumpe el proceso de oligomerización de ASC asociado a la actividad de varios inflamasomas e inhibe la liberación de IL-1ß y la piroptosis en varios modelos celulares de inflamación. MM01 también reduce la infiltración de neutrófilos y la acumulación de citoquinas pro-inflamatorias en un modelo in vivo de peritonitis. Dada la implicación de la función de ASC en múltiples complejos del inflamasoma, el tratamiento con MM01 puede representar un enfoque terapéutico eficaz para tratar aquellas enfermedades en las que está implicada la activación de múltiples inflamasomas. Teniendo en cuenta los resultados obtenidos, empleamos nuestro inhibidor del inflamasoma, MM01, para estudiar el papel del inflamasoma en la progresión tumoral en diferentes modelos de cáncer de mama tanto in vitro como in vivo. Demostramos que diferentes líneas celulares de cáncer de mama responden de forma diferente al tratamiento con MM01. Desarrollamos un ensayo funcional que comprende la evaluación de la migración de las células de cáncer de mama en respuesta al secretoma pro-inflamatorio de los macrófagos M1 (estímulo inflamatorio) en presencia de MM01. Ciertas líneas celulares (como la línea celular EO771) mostraron un aumento de la migración en respuesta al estímulo inflamatorio y una disminución de la migración en respuesta al tratamiento con MM01; sin embargo, también identificamos líneas celulares que responden negativamente al tratamiento con MM01 (como la línea celular 4T1). Por último, demostramos la eficacia de este experimento funcional in vivo demostrando que el tratamiento con MM01 redujo el tamaño del tumor en el modelo ortotópico EO771 pero aumentó el tamaño del tumor y la metástasis pulmonar en el modelo ortotópico 4T1. Estos dos modelos, que recapitulan respuestas contradictorias al tratamiento con nuestro inhibidor del inflamasoma, podrán utilizarse en el futuro para determinar biomarcadores que predigan la respuesta. Por último, desarrollamos una estrategia sintética para obtener un nuevo nanomedicamento que mejora la solubilidad y la orientación tumoral del MM01 en un modelo de cáncer de mama. Implementamos un enfoque híbrido de conjugación-complejación que comprende la conjugación de ß-ciclodextrina con un ácido lineal poli-L-glutámico (PGA) (L-PGA-ßCD) para proporcionar la capacidad de atrapar MM01 dentro de los anillos de ciclodextrina. El nanosistema obtenido mostró una mejor solubilidad en soluciones acuosas en comparación con la forma libre de MM01. Nuestro nanosistema demostró una mejor eficacia en un modelo ortotópico de cáncer de mama al producir una mayor reducción del tamaño del tumor en aquellos ratones tratados con la nanomedicina L-PGA-CD-MM01. / [CA] Aquesta tesi doctoral titulada "El microambient tumoral inflamatori com a objectiu en el disseny de nanoconjugats per al tractament del càncer de mama avançat" se centra en l'avaluació d'un nou inhibidor del inflamasoma (MM01) com a eina química per a estudiar el paper del inflamasoma en models d'inflamació i càncer. El capítol I inclou una descripció general del sistema immune, els inflamasomas dependents de ASC i el paper que juguen en el desenvolupament de malalties. També s'aprofundeix en el paper dels inflamasomas i de la proteïna ASC en la progressió del càncer de mama. A més, s'inclouen conceptes bàsics de nanotecnologia, nanomedicina i polímers terapèutics. Finalment, s'aborden els avantatges d'utilitzar *nanomedicinas com a teràpia, les interaccions de les nanomedicines amb els sistemes biològics, els nanofármacs descrits en la literatura, així com les seues possibilitats de translació a la pràctica clínica. En els capítols de resultats, delineem un nou mecanisme d'acció per a MM01, un modulador de l'activitat del s*inflamasoma recentment identificat: la inhibició de la oligomerización del ASC i el subsegüent processament reduït de la pro-caspasa-1 i la inhibició de l'activitat de la caspasa-1. Vam demostrar que MM01 interromp el procés de oligomerización de ASC associat a l'activitat de diversos inflamasomas i inhibeix l'alliberament de IL-*1ß i la piroptosis en diversos models cel·lulars d'inflamació. MM01 també redueix la infiltració de neutròfils i l'acumulació de citocines pro-inflamatòries en un model in vivo de peritonitis. Donada la implicació de la funció de ASC en múltiples complexos del *inflamasoma, el tractament amb MM01 pot representar un enfocament terapèutic eficaç per a tractar aquelles malalties en les quals està implicada l'activació de múltiples *inflamasomas. Tenint en compte els resultats obtinguts, emprem el nostre inhibidor del *inflamasoma, MM01, per a estudiar el paper del *inflamasoma en la progressió tumoral en diferents models de càncer de mama tant in vitro com in vivo. Vam demostrar que diferents línies cel·lulars de càncer de mama responen de forma diferent del tractament amb MM01. Desenvolupem un assaig funcional que comprén l'avaluació de la migració de les cèl·lules de càncer de mama en resposta al secretoma pro-inflamatori dels macròfags M1 (estímul inflamatori) en presència de MM01. Unes certes línies cel·lulars (com la línia cel·lular EO771) van mostrar un augment de la migració en resposta a l'estímul inflamatori i una disminució de la migració en resposta al tractament amb MM01; no obstant això, també identifiquem línies cel·lulars que responen negativament al tractament amb MM01 (com la línia cel·lular 4T1). Finalment, vam demostrar l'eficàcia d'aquest experiment funcional in vivo demostrant que el tractament amb MM01 va reduir la grandària del tumor en el model ortotòpic EO771 però va augmentar la grandària del tumor i la metàstasi pulmonar en el model ortotòpic 4T1. Aquests dos models, que recapitulen respostes contradictòries al tractament amb el nostre inhibidor del *inflamasoma, podran utilitzar-se en el futur per a determinar biomarcadors que prediguen la resposta. Finalment, desenvolupem una estratègia sintètica per a obtindre un nou nanomedicament que millora la solubilitat i l'orientació tumoral del MM01 en un model de càncer de mama. Implementem un enfocament híbrid de conjugació-complexació que comprén la conjugació de ß-ciclodextrina amb un àcid lineal *poli-L-glutàmic (PGA) (L-PGA-ßCD) per a proporcionar la capacitat d'atrapar MM01 dins dels anells de ciclodextrina. El nanosistema obtingut va mostrar una millor solubilitat en solucions aquoses en comparació amb la forma lliure de MM01. El nostre nanosistema va demostrar una millor eficàcia en un model ortotòpic de càncer de mama en produir una major reducció de la grandària del tumor en aquells ratolins tractats amb la nanomedicina L-PGA-CD-MM01. / [EN] This PhD thesis entitled "The inflammatory tumor microenvironment as a target in the design of nanoconjugates for the treatment of advanced breast cancer" focuses on the evaluation of a novel inflammasome inhibitor (MM01) as a chemical tool to study the role of the inflammasome in models of inflammation and cancer. Chapter I includes an overview of the immune system, ASC-dependent inflammasomes and the role they play in disease development. It also delves into the role of inflammasomes and ASC protein in breast cancer progression. In addition, basic concepts of nanotechnology, nanomedicine and therapeutic polymers are included. Finally, the advantages of using nanomedicines as therapeutics, the interactions of nanomedicines with biological systems, the nanodrugs described in the literature, as well as their translation possibilities to clinical practice are discussed. In the results chapters, we delineate a novel mechanism of action for MM01, a recently identified modulator of inflammasome activity: inhibition of ASC oligomerization and subsequent reduced pro-caspase-1 processing and inhibition of caspase-1 activity. We demonstrate that MM01 disrupts the ASC oligomerization process associated with the activity of several inflammasomes and inhibits IL-1ß release and pyroptosis in several cellular models of inflammation. MM01 also reduces neutrophil infiltration and pro-inflammatory cytokine accumulation in an in vivo model of peritonitis. Given the involvement of ASC function in multiple inflammasome complexes, treatment with MM01 may represent an effective therapeutic approach to treat those diseases in which multiple inflammasome activation is involved. Considering the results obtained, we employed our inflammasome inhibitor, MM01, to study the role of the inflammasome in tumor progression in different breast cancer models both in vitro and in vivo. We demonstrate that different breast cancer cell lines respond differently to MM01 treatment. We developed a functional assay involving the assessment of breast cancer cell migration in response to the pro-inflammatory M1 macrophage secretome (inflammasome stimulus) in the presence of MM01. Certain cell lines (such as the EO771 cell line) showed increased migration in response to the inflammatory stimulus and decreased migration in response to MM01 treatment; however, we also identified cell lines that respond negatively to MM01 treatment (such as the 4T1 cell line). Finally, we demonstrated the efficacy of this functional experiment in vivo by showing that MM01 treatment reduced tumor size in the orthotopic EO771 model but increased tumor size and lung metastasis in the orthotopic 4T1 model. These two models, which recapitulate conflicting responses to treatment with our inflammasome inhibitor, may be used in the future to determine biomarkers predictive of response. Finally, we developed a synthetic strategy to obtain a novel nanomedicine that enhances the solubility and tumor targeting of MM01 in a breast cancer model. We implemented a hybrid conjugation-complexation approach comprising the conjugation of ß-cyclodextrin to a linear poly-L-glutamic acid (PGA) (L-PGA-ßCD) to provide the ability to trap MM01 within the cyclodextrin rings. The obtained nanosystem showed improved solubility in aqueous solutions compared to the free form of MM01. Our nanosystem demonstrated improved efficacy in an orthotopic breast cancer model by producing a greater reduction in tumor size in those mice treated with the L-PGA-CD-MM01 nanomedicine. / Soriano Teruel, P. (2023). Inflammatory Tumor Microenvironment as target in the design of nanoconjugates for the treatment of advanced breast cancer [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192498

Le prédiabète et le diabète de type 2 (DT2) affectent approximativement 9 millions de canadiens, soit près de 30% de la population. Le DT2 est caractérisé par une résistance à l’insuline (RI) qui ne peut être compensée par une sécrétion d’insuline augmentée. La dysfonction du tissu adipeux blanc (TAB) est centrale à ce phénomène et est caractérisée par de l’inflammation et un flux augmenté de lipides vers les tissus périphériques, dont on sait qu’ils favorisent le développement de RI et une hypersécrétion d’apoB-lipoprotéines (apoB) par le foie menant à une élévation de l’apoB plasmatique. En parallèle, les études épidémiologiques montrent que l’apoB plasmatique prédit le développement du DT2 de 3 à 10 ans avant l’apparition de la maladie, indépendamment de facteurs de risque classiques. Dans cette thèse, nous avons formulé l’hypothèse que l’augmentation de la sécrétion d’apoB-lipoprotéines secondaire à un TAB dysfonctionnel contribue à aggraver cette même dysfonction. En ce sens, les apoB-lipoprotéines et le TAB seraient le centre d’un cercle vicieux menant au développement de facteurs de risque cardiométaboliques. Au courant de cette investigation, nous avons combiné des expériences in vitro, ainsi que des analyses post-hoc sur des données in vivo et ex vivo au sein d’une population d’hommes et de femmes post-ménopausées recrutés à l’Institut de recherches cliniques de Montréal pour deux études métaboliques entre 2006 et 2019. En circulation, 90% de apoB-lipoprotéines sont des LDL. Le nombre d’apoB-lipoprotéines en circulation se mesure par l’apoB plasmatique, qui est associé au développement du TAB dysfonctionnel chez l’humain via divers mécanismes. Parmi ceux-ci, l’enrichissement postprandial des lipoprotéines riches en triglycérides (TG) par l’apolipoprotéine C-I (apoC-I) sécrétées par le TAB a été suggéré comme un facteur contributoire. Dans un premier manuscrit, nous montrons que les sujets (N=39) avec un TAB dysfonctionnel sécrètent de plus grandes quantités d’apoC-I, ce qui est associé spécifiquement à une clairance postprandiale réduite des chylomicrons. Cette dysfonction semble due à une diminution de l’hydrolyse des TG secondaire à une inhibition de la lipase lipoprotéique (LPL) des adipocytes, ce qui constitue un nouveau mécanisme par lequel le TAB contribue à l’augmentation de l’apoB plasmatique. La proprotéine convertase subtilisin-kexin type 9 (PCSK9) est une enzyme circulante qui cible les récepteurs aux apoB-lipoprotéines comme le récepteur aux LDL (LDLR) et le CD36. Une PCSK9 circulante faible combinée à un haut apoB plasmatique, donc un ratio apoB-sur-PCSK9 élevé, est fortement associée au TAB dysfonctionnel et à la RI, suggérant qu’une augmentation de l’internalisation des apoB-lipoprotéines par voie de récepteurs joue un rôle dans ces pathologies. En parallèle, les apoB-lipoprotéines, principalement les LDL natifs et oxydés, activent l’inflammasome NLRP3 (Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3), le récepteur intracellulaire responsable de la sécrétion d’interleukine 1 bêta (IL-1), dont on sait qu’elle joue un rôle majeur dans le développement de l’inflammation liée au DT2. Dans un deuxième manuscrit, nous montrons que le ratio apoB-sur-PCSK9 plasmatique est un index, dans les états à jeun et postprandial, de l’expression des récepteurs aux apoB-lipoprotéines LDLR et CD36 en surface du TAB chez une population en surpoids ou obèse (N=31). Ce même ratio est aussi indicateur de la régulation à jeun et postprandial de l’expression de l’inflammasome NLRP3 et de l’infiltration de macrophages au sein du TAB. Finalement, les études épidémiologiques récentes suggèrent que le risque de DT2 est aussi augmenté chez les sujets avec un LDL cholestérol (LDL-C) faible causé par des variants génétiques perte-de-fonction dans le gène de la PCSK9 ou suite à une thérapie hypocholestérolémiante. Dans un troisième manuscrit, nous montrons que, chez les sujets avec LDL-C faible (<3.5mM, N=28), une PCSK9 plasmatique plus basse identifie les sujets avec une expression augmentée de LDLR et CD36 en surface de leur TAB. Malgré le LDL-C faible, les sujets avec une PCSK9 faible montraient une dysfonction du TAB et à un indice de disposition diminué et une sécrétion augmentée d’IL-1, suggérant une progression vers le DT2. Dans une exploration mécanistique, une exposition chronique des adipocytes humains SGBS aux LDL natifs induit une différenciation anormale, marquée par une diminution de leur fonction. Bien que ce phénomène soit indépendant de l’inflammasome NLRP3, qui n’est pas exprimé chez ces adipocytes, les LDL natifs induisent une augmentation du ratio de sécrétion d’IL-1 actif relativement à la pro-IL-1 inactive qui suggère une activation du système NLRP3 chez les macrophages humains THP-1. En conclusion, ces données suggèrent que le TAB dysfonctionnel contribue en partie via une sécrétion d’apoC-I à la clairance réduite des lipoprotéines et, donc, à l’hyperapoB et au risque cardiométabolique. En retour, une internalisation plus grande d’apoB-lipoprotéines par voie des récepteurs semble associée au développement d’un TAB dysfonctionnel et aux facteurs de risque cardiométaboliques associés. Au sein du TAB, au niveau cellulaire, ceci pourrait être dû à un effet concomitant des LDL natifs, qui induiraient une baisse de différenciation des préadipocytes menant à leur dysfonction ainsi qu’une activation de l’inflammasome NLRP3 chez les macrophages. / Prediabetes and type 2 diabetes (T2D) affect approximately 9 million Canadians, which represents close to 30% of the population. T2D is characterized by insulin resistance (IR) that cannot be compensated by increased insulin secretion. White adipose tissue (WAT) dysfunction is at the root of this pathology and is characterized by increased lipid flux to peripheral tissues causing IR and hypersecretion of apoB-lipoproteins (apoB) by the liver, contributing to increased plasma apoB. In line, epidemiological studies show that plasma apoB is an independent predictor of T2D development 3 to 10 years before onset. In this thesis, we formulated the hypothesis that increased secretion of apoB-lipoprotein secondary to WAT dysfunction promotes further development of this dysfunction in a feed-forward cycle that contributes to increased metabolic risk. To investigate this, we have combined in vitro experiments as well as post hoc analyses of in vivo and ex vivo data from a cohort of men and postmenopausal women recruited from two metabolic studies conducted at Institut de recherches cliniques de Montréal between 2006 and 2019. In circulation, more than 90% of apoB-lipoproteins are in the form of LDL. The number of apoB-lipoproteins (measured by plasma apoB), is associated to the development of WAT dysfunction in humans via different mechanisms. Postprandial enrichment of triglyceride-rich lipoproteins (TRL) by WAT-secreted apoC-I has been proposed as one of them. In a first manuscript, we show that subjects (N=39) with dysfunctional WAT secrete greater amount of apoC-I, which is associated specifically to delayed postprandial chylomicrons clearance in a mechanism that appears to be dependent on apoC-I-mediated inhibition of adipocyte lipoprotein lipase. This constitutes a new mechanism linking adipose tissue dysfunction to increased plasma apoB. Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a circulatory enzyme that targets apoB-lipoprotein receptors, such as the LDLR and CD36, for degradation. Low circulating PCSK9 relative to high plasma apoB, expressed as a higher apoB-to-PCSK9 ratio, is strongly associated to WAT dysfunction and IR, suggesting that increased receptor-mediated uptake of apoB-lipoproteins plays an important role in these pathologies. In parallel, apoB-lipoproteins, mostly native and oxydized LDL, activate the NLRP3 inflammasome (Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3). The NLRP3 inflammasome is an intracellular receptor responsible for interleukin-1 beta (IL-1) secretion, which is known to be implicated in the pathogenesis of T2D. In a second manuscript, we demonstrate in overweight and obese subjects (N=31) that the apoB-to-PCSK9 is indeed an index of WAT surface-expression of LDLR and CD36 both at fasting and in the postprandial state. Similarly, the apoB-to-PCSK9 ratio is associated with chronic NLRP3 inflammasome priming at fasting and with postprandial macrophage infiltration and concomitant NLRP3 upregulation within WAT. Finally, recent epidemiological studies suggest an increased risk for T2D in subjects with low plasma LDL cholesterol (LDL-C) secondary to loss-of-function genetic variants in PCSK9, or secondary to cholesterol-lowering therapies. In a third manuscript, we show that in subjects with low LDL-C (<3.5mM, N=28), lower plasma PCSK9 identifies subjects with higher WAT LDLR and CD36 surface-expression. Despite having lower LDL-C, subjects with lower plasma PCSK9 show dysfunction WAT and decreased disposition index. Mechanistically, human SGBS adipocytes chronically exposed to native LDL show impaired differentiation and concomitant dysfunction. While this phenomenon cannot be described by NLRP3 inflammasome activation, since it is not expressed in these adipocytes, native human LDL increase the ratio of secreted active Il-1 relative to inactive pro-IL-1 suggesting activation of the NLRP3 inflammasome in human THP-1 macrophages. In conclusion, these observations suggest that dysfunctional WAT promotes delayed postprandial lipoprotein clearance via increased apoC-I secretion, thus promoting hyperapoB and increased cardiometabolic risk. In turn, upregulated receptor-mediated uptake of apoB-lipoproteins appears to be connected to the development of WAT dysfunction and associated cardiometabolic risk factors. At the cellular level within WAT, this could be secondary to a concomitant effect of LDL on preadipocytes inducing their reduced differentiation and function and on macrophage inducing activation of the NLRP3 inflammasome.

Le diabète de type 2 (DT2) est une maladie chronique affectant 3 millions de canadiens. Une augmentation progressive de la résistance à l'insuline (RI) et de la sécrétion d'insuline est observée chez des sujets normoglycémiques bien avant la survenue du DT2. La moindre fonction du tissu adipeux blanc (TAB) est centrale dans le développement du DT2 car elle accroît le flux d'acides gras vers les tissus périphériques, y induisant la RI, l'hyperinsulinémie et l’inflammation chronique. Durant ma maîtrise, nous avions démontré que les lipoprotéines de basses densité (LDL) natifs réduisent la différentiation et la fonction des adipocytes et induisent la dysfonction du TAB humain. De plus, nous avions montré qu'un taux plasmatique élevé d'apolipoprotéine B (apoB), indiquant un nombre élevé d'apoB-lipoprotéines dont principalement les LDL, est associé à la RI, la sécrétion d'insuline gluco-stimulée (SIGS) élevée, la clairance plasmatique retardée des gras alimentaires et la moindre fonction du TAB chez 81 sujets obèses non diabétiques. Afin de déterminer si l'apoB plasmatique permet aussi d'identifier les sujets obèses répondant le mieux à une diète hypocalorique en termes de réduction des facteurs de risque du DT2, nous avons testé l'effet d'une intervention hypocalorique de six mois. Parmis 59 sujets qui ont terminés l’intervention, nous avons mesuré une diminution de la SIGS et une amélioration de la fonction du TAB seulement chez les sujets avec apoB plasmatique élevée. Toutefois, les mécanismes de ces effets délétères possibles des apoB-lipoprotéines n'avaient pas été explorés. Des évidences suggèrent que l'activation chronique de l'inflammasome Nucleotide- binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) et la sécrétion d'interleukine-1b (IL-1b) promeuvent la dysfonction du TAB et la RI systémique. Cependant, les signaux métaboliques induisant l'inflammasome NLRP3 dans le TAB humain sont inconnus. Afin de tester si l'activation de l'inflammasome NLRP3/système IL-1b participe au mécanisme précédemment identifié liant les apoB-lipoprotéines et les facteurs de risque du DT2, nous avons investigué l'association et l'effet direct des apoB-lipoprotéines sur le système IL-1b. Nous avons démontré chez 81 sujets obèses non-diabétiques que les individus avec apoB plasmatique élevée montrent un taux élevé d'antagoniste du récepteur à l'IL-1 (IL-1Ra) plasmatique, un marqueur de l'activation systémique de la voie IL-1b. Aussi, les associations entre l'apoB plasmatique élevée et la RI et SIGS étaient statistiquement dépendantes des niveaux d'IL-1Ra plasmatique. Dans une autre population de 32 sujets, nous avons démontrés que ceux avec apoB plasmatique élevée ont une sécrétion augmentée d'IL-1b par le TAB ex vivo. Les relations entre l'apoB plasmatique, la clairance plasmatique retardée des gras alimentaires et la sécrétion de C-peptide glucostimulée étaient statistiquement dépendantes de la sécrétion d'IL- 1b du TAB. Puis, les LDL natifs ajoutés au TAB ex vivo induisaient la sécrétion d'IL-1b, y agissant en tant que signaux d'amorçage (1er signal de l'inflammasome NLRP3/système IL-1b). En conclusion, ces résultats suggèrent que les LDL natifs, forme principale d'apoB- lipoprotéines, régulent positivement l'inflammasome NLRP3 du TAB humain. Ceci pourrait expliquer la dysfonction du TAB, l'hyperinsulinémie et l'incidence élevée du DT2 présents chez les sujets avec apoB plasmatique élevée. En outre, ils suggèrent que l'apoB plasmatique élevée pourrait être un biomarqueur permettant d'identifier les sujets obèses qui répondraient le mieux à la diète hypocalorique afin de réduire le risque de DT2. / Type 2 diabetes (T2D) is chronic disease affecting 3 million Canadians and a new case is diagnosed every 3 minutes in Canada. Long before the onset of T2D, a progressive increase in insulin resistance (IR) and insulin secretion is observed in normoglycemic subjects. A decreased white adipose tissue (WAT) function is central to the development of T2D as it promotes an increased fatty acid flux to peripheral tissues, inducing IR, hyperinsulinemia and chronic inflammation. During my MSc, we reported that low density lipoproteins (LDL) reduce the differentiation and function of adipocytes and induce the dysfunction of human WAT. Moreover, we showed that elevated plasma apolipoprotein B (apoB), indicating high numbers of circulating apoB-lipoproteins mainly in the form of LDL, is associated to IR, elevated glucose-induced insulin secretion (GIIS), delayed postprandial plasma clarance of fat and reduced WAT function in 81 non-diabetic obese subjects. To explore whether apoB also identifies obese subjects who best respond to weight loss to reduce risk factors for T2D, we tested the effect of a 6 months hypocaloric diet. We showed in the 59 completers of the hypocaloric intervention that the decrease in GIIS and increase in WAT function were significant in subjects with high plasma apoB but not in subjects with low plasma apoB. However, the mechanism underlying the negative effects apoB-lipoproteins was yet unexplored. Chronic activation of the Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasome and secretion of interleukin-1b (IL-1b) promote WAT dysfunction and systemic IR. However, endogenous metabolic signals that induce the activation of WAT NLRP3 inflammasome are unknown. To test if the activation of the NLRP3 inflammasome/ IL-1b system is an underlying mechanism linking apoB- lipoproteins to risk factors for T2D, we examined the association and direct effect of apoB- lipoproteins on the IL-1b system. We observed in our cohort of 81 non-diabetic obese subects that subjects with high plasma apoB have higher plasma IL-1 receptor antagonist (IL-1Ra), which is an marker of systemic activation of the Il-1b pathway. Furthermore, the associations between high plasma apoB and IR and GIIS were statistically dependent on plasma IL-1Ra. Additionnaly, in a separate population of 32 subjects, we demonstrated that subjects with high plasma apoB have higher ex vivo WAT IL-1b secretion. The relation between plasma apoB and delayed postprandial plasma fat clearance and elevated glucose-induced C-peptide secretion were statistically dependent on WAT IL-1b secretion. Finally, native LDLs directly induce IL- 1b secretion from ex vivo WAT, acting primarily as priming signals (i.e. the first signal leading to activation of the NLRP3 inflammasome/ IL-1b system). In conclusion, the findings from this thesis suggest that native LDL, the main form of apoB-lipoproteins, upregulate human WAT NLRP3 inflammasome. This may explain WAT dysfunction, hyperinsulinemia and higher incidence of T2D in subjects with high plasma apoB. Moreover, they suggest that high apoB may serve as biomarker to identify obese subjects who best respond to a hypocaloric-intervention to reduce the risk of T2D.

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

A lesão tecidual é um dos efeitos locais descritos nos envenenamentos botrópicos. Toxinas isoladas, como: a jararagina (JAR) e bothropstoxina-I (BthTX-I), obtidas dos venenos de B. jararaca e B. jararacussu, respectivamente, induzem intensa resposta inflamatória e lesão tecidual, porém apresentam diferentes mecanismos de ação. Como descrito, a resolução da lesão tecidual envolve a interação entre mecanismos de reparo do tecido e o sistema imune. Neste contexto, a resposta inflamatória é iniciada por meio da detecção de sinais de dano tecidual agudo devido a distúrbios da homeostasia resultantes ou não de agentes microbianos (DAMPs) e/ou por reconhecimento de padrões moleculares associados a patógenos (PAMPs). Uma vez induzida, a resposta inflamatória está envolvida tanto no processo de lesão visando a eliminação do agente indutor, assim como no reparo tecidual. Diversos receptores estão envolvidos no reconhecimento de PAMPs e DAMPs como os transmembrânicos representados pelos do tipo Toll, e os citosólicos que compreendem complexos proteicos que formam os inflamassomas. Estes complexos multiproteicos citosólicos podem participar da indução da resposta imune inata por ativação de caspase-1 com consequente liberação de IL-1&#946, que pode resultar em morte celular. Considerando o exposto, o objetivo deste trabalho foi estudar a participação de inflamassoma na resposta inflamatória no tecido muscular de injeção das toxinas, a capacidade da BthTX-I e JAR de induzir a ativação de inflamassoma em macrófagos e os mecanismos moleculares envolvidos nesse processo. O estudo da migração de neutrófilos e macrófagos para o músculo gastrocnémio de animais C57BL/6 ou deficientes em caspase 1/11 (Caspase 1/11-/-) ou NLRP3 (NLRP3-/-) injetados com JAR e BthTX-I permitiu verificar que o inflamassoma NLRP3 participa da migração destas células inflamatórias para local de injeção das toxinas. A análise da produção de IL-1&#946 nas culturas de macrófagos peritoneais incubados com JAR e BthTX-I (6 e 24h) mostrou que somente a BthTX-I foi capaz de induzir a secreção desta citocina por um mecanismo dependente de caspase 1/11, ASC e NLRP3 e independente de IPAF. A incubação de macrófagos humanos com as toxinas permitiu verificar que ambas as toxinas induziram a secreção de IL-1&#946 dependente de caspase 1, porém esta produção foi significativamente maior em resposta à BthTX-I. Nos macrófagos peritoneais de camundongos observamos a relação entre a secreção de IL-1&#946 e morte celular em ensaio de incorporação do brometo de etídio nas culturas incubadas com BthTX-I por 24h e não com a JAR. Visto que ambas as toxinas injetadas via intramuscular induzem resposta inflamatória intensa, foi analisado o efeito delas sobre a viabilidade e secreção de IL-6 e MCP-1 em miotubos C2C12. Os resultados mostraram que somente a BthTX-I induz efeito miotóxico sobre esta linhagem celular. Além disso, pudemos verificar que BthTX-I induz altos níveis de IL-6 e MCP-1 quando comparados aos obtidos com a JAR. Visto que a BthTX-I induziu alta secreção de MCP-1 pelos miotubos C2C12, em experimento in vivo realizado em camundongos C57BL/6 ou deficientes em CCR2 (CCR2-/-) pode ser observada a dependência da interação entre MCP-1 e o CCR2 para a migração dos macrófagos em resposta a injeção de BthTX-I. Em culturas de miotubos incubados com as toxinas pôde ser observada alta liberação de ATP induzida pela BthTX-I in vitro. Em outros experimentos foi estudada a capacidade do sobrenadante de miotubos incubados com BthTX-I de induzir a secreção de IL-1&#946 pelos macrófagos in vitro. Os resultados mostraram a produção de IL-1&#946 nessas culturas de macrófagos, assim como a produção desta citocina em macrófagos incubados com BthTX-I juntamente com ATP. A hidrólise do ATP no sobrenadante da cultura de C2C12 estimulada com BthTX-I aboliu a secreção de IL-1 pelos macrófagos in vitro. Além disso, foi observada a inibição da produção de IL-1&#946 nas culturas de macrófagos primados com LPS e incubados com a BthTX-I em condições de altas concentrações de KCl sugerindo papel relevante do efluxo de K+ na produção de IL-1&#946 induzida pela BthTX-I. Em conjunto, os resultados acrescentam novas informações sobre o potencial inflamatório da JAR e BthTX-I, quanto à ação destas toxinas em macrófagos, ativação de inflamassoma e células musculares. / Tissue damage is one of the local effects described in bothropic envenomations. Isolated toxins, such as jararhagin (JAR) and bothropstoxin-I (BthTX-I), obtained from B. jararaca and B. jararacussu venoms, respectively, induce intense inflammatory response and tissue injury, however mediated by distinct mechanisms. As described, resolution of tissue injury involves the interaction between mechanisms of tissue repair and the immune system. In this context, the inflammatory response is initiated by detecting of signs of acute tissue damage due to disorders of homeostasis resulting from distinct agents (DAMPs) and / or recognition of pathogens associated molecular patterns (PAMPs). Once induced, the inflammatory response is involved both in the injury process for the elimination of the pathogenic agent, as well as in the tissue repair. Several receptors are involved in the recognition of PAMPs and DAMPs as the transmembrane receptors such as the Toll-like, and the cytosolic ones that comprise protein complexes - inflammasomes. These cytosolic multiprotein complexes may participate in the induction of the innate immune response by activation of caspase-1 with consequent release of IL-1&#946, which may result in cell death. Thus, we aimed to study the role of inflammasome on the inflammatory response in muscular tissue of JAR and BthTX-I injection, the ability of BthTX-I and JAR to induce the activation of inflammasome in macrophages and the molecular mechanisms involved in this process. The analyses of the neutrophils and macrophages migration in gastrocnemius muscle of C57BL/6 or Caspase 1/11 (Caspase 1/11-/-) or NLRP3 (NLRP3-/-) deficient mice injected with JAR e BthTX-I allow us to verify that NLRP3 inflammasome participates in these cells migration for the local of the toxins injection. The detection of IL-1&#946 on supernatants from macrophage cultures incubated with JAR or BthTX-I (6 e 24h) showed that only BthTX-I was able to induce this cytokine secretion by a mechanism dependent of caspase 1/11, ASC and NLRP3 and independent of IPAF. The incubation of human macrophages with the toxins demonstrated that both toxins induced IL-1&#946 secretion, however this production was significantly higher in response to BthTX-I. On murine macrophage cultures it was verified the correlation between the IL-1&#946 secretion and the cell death in the incorporation of ethidium bromide assay of cultures incubated with BthTX-I during 24h and not with JAR. Since that both toxins injected in the muscle induced intense inflammation, it was analyzed the effect of both toxins on the viability and the secretions of IL-6 and MCP-1 by C2C12 myotubes. The results showed that only BthTX-I induces a myotoxic effect on this cell line. Furthermore, it was verified that BthTX-I induces high secretion of IL-6 and MCP-1 when compared with those induced by JAR. Considering that BthTX-I induces high levels of MCP-1, in in vivo experiment using C57BL/6 and CCR2 deficient (CCR2-/-) mice it was observed that the interaction of MCP-1 and CCR2 is essential for the macrophage recruitment for the toxin injection. High release of ATP was detected in C2C12 myotube cultures incubated with BthTX-I but not with JAR. In another experiments it was studied the ability of the supernatants of C2C12 myotubes incubated with BthTX-I to induce the IL-1&#946 secretion by peritoneal macrophages in vitro. The results showed the IL-1&#946 secretion in the macrophage cultures as well as the cytokne secretion in macrophages incubated with BthTX-I and ATP independent of the priming with LPS. The hydrolysis of the ATP on the supernatants of C2C12 incubated with the BthTX-I abolished the IL-1&#946 production by macrophages in vitro. In addition, it was not observed IL-1&#946 production by macrophages primed with LPS and incubated with BthTX-I in the presence of high concentration of KCl suggesting a relevant role of K&#43 efflux for this cytokine secretion in response to BthTX-I. Taken together, the results show new findings about the inflammatory effect of JAR and BthTX-I, concerning about the action of these toxins on macrophages, inflammasome activation and muscle cell.

Toll-like Rezeptoren (TLR) und Nod-like Rezeptoren (NLR) sind Muster-erkennende Rezeptoren des angeborenen Immunsystems, die bakterielle Zellwandbestandteile erkennen können. Interleukin (IL)-1beta ist ein streng reguliertes Zytokin. Durch eine erste Stimulation wird der TLR-Rezeptor ausgelöst und führt zur Expression des Vorläuferproteins proIL-1beta. Durch einen zweiten Stimulus wird ein zytoplasmatischer NLR-Rezeptor zur Caspase1-Aktivierung angeregt. Dies führt zur post-translationalen Reifung von proIL-1beta zu reifem IL-1beta und zur Aktivierung weiterer Mechanismen der Pathogen-Eliminierung während einer bakteriellen Meningitis. Im ersten Teil dieser Arbeit wurde die synergistische Beziehung zwischen TLRs und NOD2 in Bezug auf die IL-1beta-Sekretion in Astrozyten und Mikroglia untersucht. Primäre murine WT-Astrozyten und eine humane Zelllinie, die mit Lipopolysaccharid (LPS) oder Lipopeptid sowie Muramyldipeptid (MDP) stimuliert wurden, zeigten signfikant erhöhte IL-1beta-Werte. IL-1beta war in NOD2-/- Astrozyten nicht erhöht. NOD2 trägt demnach als MDP-ausgelöster Rezeptor in Astrozyten, vermutlich zusammen mit dem Inflammasom-Komplex, zur Caspase-1-Aktivierung bei. In Mikrogliazellen lässt sich der bei Astrozyten gezeigte Effekt nicht reproduzieren. Zum ersten Mal wurde gezeigt, dass die TLR-abhängige IL-1beta-Antwort durch NOD2-Beteiligung in murinen und humanen Astrozyten synergistisch erhöht wird. In einem weiteren Versuchsteil wurde in primären murinen Peritonealmakrophagen von adulten Mäusen der TLR/NLR-Synergismus untersucht. Es stellte sich überraschenderweise heraus, dass weibliche NOD2-/- Mäuse zu einer synergistisch erhöhten IL-1beta-Sekretion fähig waren. SiRNA-Versuche mit in Östrogen vorinkubierten RAW264.7-NOD2-/- Zellen zeigten eine eindeutige Synergie der TLR4- und NOD2-Rezeptoren in der IL-1beta-Ausschüttung. Östrogen scheint weiblichen Individuen einen protektiven Vorteil vor Infektionen bei NOD2-Defizienz zu verschaffen. / Toll-like receptors (TLR) and nod-like receptors (NLR) are pattern-recognition receptors that recognize lipopolysaccharide (LPS), lipopeptides and myramyldipeptide (MDP) derived from bacterial cell wall. We focus our question on the regulation of the pro-inflammatory cytokine interleukin (IL)-1beta during bacterial meningitis in primary murine astrocytes and microglia as well as cell lines and the synergism of TLR4 or TLR2 and NOD2 to amplify IL-1beta-expression. ProIL-1beta is expressed by TLR-stimulation and activation of NF-kB signal transduction. Through the activation of Caspase-1, possibly through NOD2 and the inflammasome, proIL-1beta is cleaved on post-translational level and obtains its activated status, leading to pathogen elimination during bacterial meningitis. Primary murine WT-astrocytes and a human cell line primed with LPS or lipopeptide and stimulated with MDP show significantly increased IL-1beta levels in the supernatant. NOD2-/- astrocytes do not show elevated IL-1beta levels. After screening of cytoplasmic proCaspase-1 and activated Caspase-1 by Western blot it became clear, that stimulation of NOD2 with MDP led to Caspase-1 activation and thus to IL-1beta maturation in primary murine WT-astrocytes. We demonstrate for the first time that the synergism between TLR4 and NOD2 leads to significantly elevated IL-1beta levels and that NOD2 is capable of activating caspase-1 in primary murine astrocytes. Another part of the work was to test the TLR/NLR-synergism on primary peritoneal macrophages from adult mice. Surprisingly, female NOD2-/- mice showed significantly elevated IL-1beta levels. SiRNA- and stimulation-experiments with RAW264.7-NOD2-/- cells pre-incubated in estrogen show a clear synergy in IL-1beta secretion through TLR4 and NOD2 receptors. Estrogen seems to protect females from infection when having a NOD2 deficiency.