Les polynucléaires neutrophiles (PN) sont des cellules essentielles au cours de la réponse immunitaire innée ; recrutés rapidement au site inflammatoire où ils participent à la phase aigüe, ils vont aussi contribuer à la résolution de l'inflammation. Ils peuvent en effet moduler la réponse adaptative par interaction avec les lymphocytes (Ly) ou les cellules dendritiques (DC) via des médiateurs solubles ou des interactions cellulaires directes. Les Neutrophil Extracellular Traps (NETs) libérés par les PN activés pourraient jouer un rôle important dans ce contexte. Les NETs sont des filaments de chromatine décondensée associés à des protéines issues principalement des granulations et du cytoplasme. Ils sont essentiels dans la réponse anti-infectieuse mais semblent également impliqués dans la physiopathologie de certaines maladies auto-immunes et inflammatoires. L'objectif de ce travail a été d'évaluer les effets des NETs sur la maturation des DC dans un contexte inflammatoire au cours duquel les PN et les DC peuvent co-exister, assurant ainsi un pont entre immunité innée et immunité adaptative. La première partie de ce travail a consisté à développer un modèle de production, isolement et caractérisation des NETs issus de PN sanguins humains. L'ionophore de calcium A23187 a été choisi pour induire les NETs et l'enzyme de restriction AluI a permis la récupération de fragments de NETs de taille hétérogène. Certains des composants de ces NETs sont quantifiables (ADN, élastase, histone 3 en particulier), et nous avons montré qu'ils conservaient leurs capacités bactéricides in vitro. Ces échantillons de NETs constituent donc un outil biologique standardisé, permettant d'évaluer leurs effets sur des cellules ou des tissus. Dans la deuxième partie de ce travail, nous avons mis en évidence que ces NETs purifiés régulaient négativement la maturation de moDC induites par le LPS (expression de HLA-DR, CD80, CD83, CD86 et production de TNFα, IL-12, IL-6, IL-23). De plus, les NETs diminuent la capacité de ces moDC à induire la prolifération des LyT, et leur polarisation est modulée en favorisant la production de cytokines de type Th2 (IL-5 et IL-13) aux dépens de cytokines de types Th1 (INFγ) et Th17 (IL-17). De manière intéressante, la capacité de migration des moDC activées par le LPS n'est pas modifiée en présence de NETs. En résumé, ces résultats suggèrent que les NETs pourraient jouer un rôle immunorégulateur sur la maturation des moDC dans des conditions inflammatoires. Les NETs produits par les PN activés pourraient ainsi participer à la régulation indispensable de la réponse inflammatoire.

Hemostasis, thrombosis, and inflammation are tightly interconnected processes which may give rise to thrombo-inflammation, involved in infectious and non-infectious acute and chronic diseases, including cardiovascular diseases (CVD). Traditionally, due to its hemostatic role, blood coagulation is isolated from the inflammation, and its critical contribution in the progressing CVD is underrated, until the full occlusion of a critical vessel occurs. Underlying vascular injury exposes extracellular matrix to deposit platelets and inflammatory cells. Platelets being key effector cells, bridge all the three key processes (hemostasis, thrombosis, and inflammation) associated with thrombo-inflammation. Under physiological conditions, platelets remain in an inert state despite the proximity to the endothelium and other cells which are decorated with glycosaminoglycan (GAG)-rich glycocalyx (GAGs). A pathological insult to the endothelium results in an imbalanced blood coagulation system hallmarked by increased thrombin generation due to losses of anticoagulant and cytoprotective mechanisms, i.e., the endothelial GAGs enhancing antithrombin, tissue factor pathwayinhibitor (TFPI) and thrombomodulin-protein C system. Moreover, the loss of GAGs promotes the release of mediators, such as von Willebrand factor (VWF), platelet factor 4 (PF4), and P-selectin, both locally on vascular surfaces and to circulation, further enhancing the adhesion of platelets to the affected sites. Platelet-neutrophil interaction and formation of neutrophil extracellular traps foster thrombo-inflammatory mechanisms exacerbating the cardiovascular disease course. Therefore, therapies which not only target the clotting mechanisms but simultaneously or independently convey potent cytoprotective effects hemming the inflammatory mechanisms are expected to provide clinical benefits. In this regard, we review the cytoprotective protease activated protein C (aPC) and its strong anti-inflammatory effects thereby preventing the ensuing thrombotic complications in CVD. Furthermore, restoring GAGlike vasculo-protection, such as providing heparin-proteoglycan mimetics to improve regulation of platelet and coagulation activity and to suppress of endothelial perturbance and leukocyte-derived pro-inflammatory cytokines, may provide a path to alleviate thrombo-inflammatory disorders in the future. The vascular tissue-modeled heparin proteoglycan mimic, antiplatelet and anticoagulant compound (APAC), dual antiplatelet and anticoagulant, is an injury-targeting and locally acting arterial antithrombotic which downplays collagen- and thrombin-induced and complement-induced activation and protects from organ injury.

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.

Sepsis is a clinical syndrome characterized by the systemic activation of inflammatory and coagulation pathways in response to microbial infection of normally sterile parts of the body. Despite considerable advances in our understanding of sepsis pathophysiology, sepsis remains the leading cause of death in non-coronary intensive care units (ICU) with a global disease burden between 15 and 19 million cases per year (Dellinger et al., 2008). Severe sepsis, defined as sepsis associated with organ dysfunction is associated with mortality rates of 33% to 45%. The incidence of severe sepsis continues to increase by 1.5% per annum due to the aging population, a rise in the prevalence of comorbidities, and the wider use of immunosuppressive agents and invasive procedures (Angus et al., 2001). Over the past several decades, many potential treatments for sepsis have shown early promise, yet have failed to improve survival in over 100 Phase II and Phase III clinical trials (Marshall, 2014) suggesting that some fundamental knowledge is lacking in our understanding of sepsis pathophysiology. Emerging studies on cell-free DNA (cfDNA), DNA released extracellularly into the circulation, demonstrate that cfDNA is a crucial link between inflammation and coagulation . In various conditions characterized by excessive inflammatory responses or aberrant prothrombotic responses, cfDNA has been implicated in exacerbating disease pathology (Atamaniuk, Kopecky, Skoupy, Säemann, & Weichhart, 2012; Fuchs, Brill, & Wagner, 2012; Swystun, Mukherjee, & Liaw, 2011). In clinical sepsis, levels of cfDNA upon admission into the ICU have strong prognostic value in predicting mortality (Dwivedi et al., 2012; Saukkonen et al., 2008). However, it is unclear whether these increases in cfDNA are an epiphenomenon during sepsis progression, or whether cfDNA actively plays a role in sepsis pathophysiology. In this work, in vivo studies were conducted to characterize the role of cfDNA in sepsis, the effects of DNase administration, and the potential mechanism by which cfDNA is released during experimental sepsis. In addition, mortality studies were conducted to identify surrogate markers of death to promote the design of humane and ethical animal studies in conducting sepsis research. Polymicrobial sepsis was induced via a surgical procedure whereby the cecum is exteriorized, ligated and punctured twice to introduce a continuous source of microorganisms, a model termed cecal ligation and puncture (CLP). In our CLP sepsis model, levels of cfDNA increased in a time-dependent manner. These increases accompanied an early pro-inflammatory response marked by increased pro-inflammatory IL-6, a transient increase in anti-inflammatory IL-10, and elevated lung myeloperoxidase (MPO) activity. Septic mice with elevated cfDNA levels also had high bacterial loads in the lungs, blood, and peritoneal cavity fluid. Organ damage was also observed in mice following CLP surgery versus mice subjected to the non-septic sham control surgery marked by increased levels of creatinine and alanine aminotransferase (ALT) indicative of kidney and liver injury, respectively. Histological analyses further confirmed lung and kidney damage following CLP surgery. Changes in coagulation were also observed in septic mice as mice subjected to CLP had sustained increases in thrombin-antithrombin (TAT) complexes. In addition, plasma from CLP-operated mice had increased thrombin generation (i.e. increased endogenous thromin potential, increased peak thrombin, decreased time to peak, and decreased lag time) mediated by FXIIa and enhanced by platelets. Following CLP-induced sepsis, elevations in cfDNA levels accompanied pro-inflammatory and pro-coagulant responses. The effects of in vivo DNase treatment in septic mice were time-dependent. Early DNase treatment when cfDNA levels were low resulted in an exaggerated pro-inflammatory response marked by increased plasma IL-6 levels and increased lung damage. In contrast, delayed DNase treatment at time-points when cfDNA levels were elevated suppressed inflammation characterized by an increase in anti-inflammatory IL-10 and reductions in cfDNA, IL-6, lung MPO, and ALT activity. Furthermore, delayed DNase administration resulted in decreased bacterial load in the lungs, blood, and peritoneal cavity fluid. Delayed DNase treatment also resulted in blunted pro-coagulant responses as levels of TAT complexes were suppressed and thrombin generation from septic mouse plasma was normalized. Moreover, DNase treatment when cfDNA levels were elevated increased survival in CLP-operated mice by 80% and reduced lung and liver damage. These findings suggest that administration of DNase when cfDNA levels are elevated may reduce pro-inflammatory and pro-coagulant responses and that delayed DNase treatment may infer protection in the CLP model of sepsis. One mechanism by which cfDNA is released is via the formation of neutrophil extracellular traps (NETs). Upon inflammatory stimulation, some neutrophils release chromatin material and antimicrobial proteins (i.e. neutrophil elastase, MPO, and histones) in an active process termed NETosis. Although NETs ensnare bacteria and exert antimicrobial properties, NETs may also exert harmful effects on the host by activating inflammation and coagulation. While some in vitro evidence suggest that neutrophils are the main source of cfDNA released following inflammatory stimulation, others have reported that neutrophils are not the main source of circulating cfDNA following septic challenge. To determine whether NETs contribute to cfDNA released during CLP sepsis, genetically modified mice that are incapable of forming NETs, PAD4-/- mice, were used. Levels of cfDNA in PAD-/- mice were significantly lower than cfDNA levels in C57Bl/6 mice following CLP surgery, suggesting that NETs were a source of cfDNA in our model. Levels of IL-6, MPO, and bacterial load in the lungs, blood, and peritoneal cavity were significantly reduced, indicating that NETs exert pro-inflammatory effects in CLP sepsis. Thrombin generation was also suppressed in PAD4-/- mice which suggests that NETs contribute to thrombin generation following CLP sepsis. NETs contribute to increases in circulating cfDNA and may exacerbate pathology by driving pro-inflammatory and pro-coagulant responses in CLP-induced sepsis. Appreciating the implications of conducting research using animals, it is pertinent that researchers ensure the highest ethical standards and design animal studies in the most humane, yet scientifically rigorous manner. Using mortality studies, we validated the utility of physiological and phenotypic markers to assess disease severity and predict death in murine sepsis. Temperature via a rectal probe monitor and sepsis scoring systems which assess components such as orbital tightening, level of consciousness, and activity were effective surrogate markers of death. These tools offer a non-invasive assessment of disease progression which do not artificially exacerbate sepsis pathology and immediate information regarding any changes in the health status. Surrogate markers of death also provide reliable monitoring to meet increasing standards of ethical, humane animal research and a feasible and cost-efficient means to obtain vital signs in small rodents. We have proposed a scoring system which can be used for assessing disease severity, endpoint monitoring, and predicting death to obviate inhumane methods of using death as an endpoint in sepsis studies. In summary, cfDNA levels are elevated in CLP-induced sepsis and these elevations accompany pro-inflammatory and pro-coagulant responses. NETosis may be a mechanism by which cfDNA is released and NETs may drive inflammation and coagulation in CLP sepsis. Delayed DNase administration may suppress inflammation and coagulation and may be protective in polymicrobial sepsis. In future animal sepsis studies, surrogate markers of death and a sepsis scoring system can be used in place of death as an endpoint to raise the standards in conducting ethical, humane sepsis research. / Thesis / Doctor of Philosophy (PhD)

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat. / Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.

Les granulocytes de faible densité (LDGs) sont un sous-type de neutrophiles mis en évidence initialement dans le sang de patients atteints de différentes maladies telles que le lupus érythémateux systémique ou le psoriasis. Cependant, des études rapportent également leur présence chez des individus sains. On connait mal à ce jour les caractéristiques des LDGs, notamment en ce qui a trait à leur profil inflammatoire. De plus, leur biogenèse demeure toujours mal connue. Mes travaux de maîtrise visaient à comparer les propriétés des LDGs à celles des neutrophiles de densité normale (NDNs). Pour ce faire, 8 chevaux atteints d’asthme équin sévère et 11 chevaux sains bien caractérisés ont été sélectionnés pour l’étude et sur lesquels des isolations de NDNs ainsi que des LDGs ont été réalisées. La morphologie des neutrophiles a ensuite été évaluée par microscopie optique. Le contenu en myéloperoxidase, un composant des granules primaires azurophiles des neutrophiles, et la présence de récepteurs du N-formylméthionine-leucyl-phénylalanine (fMLP-R) ont été évalués par cytométrie de flux et immunofluorescence, respectivement. Enfin, la capacité fonctionnelle de ces cellules à produire spontanément des pièges extracellulaires des neutrophiles (NETs) a été étudiée in vitro par microscopie confocale. Les résultats démontrent que le nombre de LDGs est augmenté dans le sang des chevaux asthmatiques lors d'exacerbation de la maladie. De plus, ces cellules présentent une morphologie différente puisqu’elles sont de taille plus petite et contiennent plus de fMLP-R que les NDNs. Le contenu en myéloperoxidase est cependant similaire dans les deux populations de neutrophiles. Enfin, les LDGs produisent plus de NETs, et sont plus sensibles aux stimuli activateurs que les NDNs. Ces caractéristiques sont similaires dans les 2 groupes de chevaux suggérant ainsi que ce sont des propriétés intrinsèques des LDGs et qu’ils représentent une population cellulaire préactivée et qui de plus, est majoritairement mature. Cette étude caractérise et compare pour la première fois les LDGs chez des animaux sains et ceux retrouvés chez des animaux atteints d’une maladie inflammatoire chronique. / Low-density granulocytes (LDGs) are a subset of neutrophils first described in the bloodstream upon pathological conditions. However, several studies also reported the presence of these cells in the blood of healthy patients. Whether LDGs characteristics, especially their enhanced pro-inflammatory profile, are specific to this subset of neutrophils and not related to disease states is unknown. Thus, we sought to compare the properties of LDGs to those of autologous normal-density neutrophils (NDNs), in both health and disease. We studied 8 horses with severe equine asthma and 11 healthy animals. Neutrophil morphology was studied using optical microscopy, and content in myeloperoxidase and N-formylmethionine-leucyl-phenylalanine receptors (fMLP-R) evaluated using flow cytometry and immunofluorescence, respectively. Confocal microscopy was used to determine their functional capacity to spontaneously release neutrophil extracellular traps (NETs) stimulating with phorbol-12-myristate-13-acetate (PMA). LDGs were smaller and contained more fMLP-R than NDNs, but myeloperoxidase content was similar in both populations of neutrophils. They also had an increased capacity to produce NETs, and were more sensitive to activation stimuli. These characteristics were similar in both healthy and diseased horses, suggesting that these are intrinsic properties of LDGs. Furthermore, these results suggest that LDGs represent a population of primed and predominantly mature cells. Our study is the first to characterize LDGs in health, and to compare their characteristics with those of animals with a naturally occurring disease.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-11-01T18:12:18Z No. of bitstreams: 1 disserta??o Josiane Silva Silveira vers?o final corrigida.pdf: 4036302 bytes, checksum: cdea806bf90b79da9a4047282152d203 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-11-05T12:55:20Z (GMT) No. of bitstreams: 1 disserta??o Josiane Silva Silveira vers?o final corrigida.pdf: 4036302 bytes, checksum: cdea806bf90b79da9a4047282152d203 (MD5) / Made available in DSpace on 2018-11-05T13:36:59Z (GMT). No. of bitstreams: 1 disserta??o Josiane Silva Silveira vers?o final corrigida.pdf: 4036302 bytes, checksum: cdea806bf90b79da9a4047282152d203 (MD5) Previous issue date: 2018-10-26 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / INTRODUCTION: asthma is a chronic inflammatory disease characterized by secretion of elevated levels of cytokines (interleukin (IL)-4, IL-5 and IL-13), reactive oxygen species (ROS), autophagy and eosinophil extracellular traps (EETs) release in airway. Moreover, respiratory syncytial virus (RSV) infection may facilitate allergic sensitization development as well as exacerbate asthma symptoms. Recently, studies have demonstrated an increase of autophagy in eosinophils of asthmatic patients, contributing to an increase in inflammatory response. In asthma, an increase in EETs release may cause tissue damage and an increase in mucus viscosity, which contribute to airway obstruction and reduction of lung function. However, the mechanism of EETs formation and its pathophysiologic role in asthma are poorly understood. OBJECTIVE: the aim of this dissertation was to elucidate some mechanisms involved in EETs release in asthma. We investigated whether the respiratory syncytial virus (RSV) could induce EETs in vitro in bronchoalveolar lavage fluid (BALF) eosinophils of an experimental asthma model. Moreover, we evaluated ROS and autophagy participation in mechanisms involved in EETs formation. METHODS: in order to perform the experimental model of asthma, BALB/cJ mice were sensitized with two subcutaneous injections of ovalbumin (OVA) on days 0 and 7, followed by three intranasal challenges with OVA on days 14, 15 and 16 of the protocol. In paper 1, BALF eosinophils of OVA group and control group were stimulated with RSV (103 PFU/mL) in vitro for 3 hours. After that, culture supernatant was collected in order to perform the analyses proposed in this study which were evaluated according to the specific objectives of this paper. In paper 2, during the experimental asthma protocol, mice were treated intranasally with a nicotinamide adenine dinucleotide phosphate oxidase (NDPH oxidase) inhibitor, diphenyleneiodonium (DPI), or a glutathione precursor, N-acetylcysteine (NAC). In paper 3, mice were treated intranasally with an autophagy inhibitor, 3-Methyladenine (3-MA). Treatments were performed 45 minutes before of the three intranasal administrations with OVA. At the end of the protocol, BALF and lung tissue were collected to perform the techniques discribed in each of the papers, according to their specific objectives. RESULTS: in paper 1, we verified an increase in EETs release in BALF eosinophils from OVA group stimulated with RSV in vitro. RSV in vitro decreased IFN-? in BALF cells when compared to the OVA group. In paper 2, we showed that in NAC-treated OVA group there was a decrease in the inflammatory cells in BALF and lung tissue. DPI or NAC treatments reduced EPO activity, goblet cells hyperplasia, inflammatory cytokines and NF?B p65 immunocontent in lung, and they helped in decreasing ROS production in lung. Furthermore, NAC increased catalase (CAT) activity in lung. However, only NAC treatment improved mitochondrial energy metabolism in lung. We showed that DPI or NAC reduced EETs formation in BALF from the OVA group. In paper 3, we showed that in 3-MA-treated OVA group there was a decrease in the inflammatory cells, EPO activity, goblet cells hyperplasia, inflammatory cytokines, NF?B p65 immunocontent, and oxidative stress in airway. Moreover, 3-MA was able to improve mitochondrial energy metabolism and increase Na+,K+-ATPase activity. We also demonstrated that 3-MA decreased light chain 3B (LC3B) in BALF cells and lung tissue as well as reduced EETs formation in BALF. CONCLUSION: our results verified an important role for RSV in the induction of EETs release. Moreover, DPI, NAC and 3-MA treatments decreased airway inflammation, oxidative stress and EETs release in asthma. Our data suggested that RSV, ROS and autophagy participate in the mechanisms for EETs release in asthma. Thus, identification of mechanisms that regulate EETs formation in asthma may contribute to a better understanding of the pathogenesis of this chronic inflammatory disease which damages patients? quality of life and is responsible for a high economic cost for the Brazilian Single Health System (SUS). / INTRODU??O: a asma ? uma doen?a inflamat?ria cr?nica caracterizada pela secre??o de elevados n?veis de citocinas do perfil T helper 2 (Th2) como interleucina (IL)-4, IL-5 e IL-13, esp?cies reativas de oxig?nio (EROs), aumento da autofagia e forma??o redes extracelulares de eosin?filos (EETs). A infec??o pelo v?rus sincicial respirat?rio (VSR) pode facilitar o desenvolvimento da sensibiliza??o al?rgica bem como exacerbar os sintomas da doen?a. Recentemente, estudos t?m demonstrado o aumento da autofagia em eosin?filos das vias de pacientes asm?ticos, contribuindo para o aumento da resposta inflamat?ria nas vias a?reas. Na asma, a produ??o excessiva de EETs pode causar dano tecidual e aumento da viscosidade do muco, podendo contribuir para o aumento da obstru??o da via a?rea e redu??o da fun??o pulmonar. Entretanto, os mecanismos de forma??o das EETs e seu papel fisiopatol?gico na asma s?o pouco compreendidos. OBJETIVO: esta disserta??o teve como objetivo elucidar alguns mecanismos envolvidos na libera??o de EETs na asma. Avaliamos a participa??o do VSR in vitro, das EROs e da autofagia nos mecanismos envolvidos na libera??o das EETs em eosin?filos do lavado broncoalveolar (LBA) em um modelo experimental de asma. METODOLOGIA: para o desenvolvimento do modelo experimental de asma, camundongos BALB/cJ foram sensibilizados com duas inje??es subcut?neas de ovalbumina (OVA) nos dias 0 e 7 seguidos por tr?s desafios intranasais com OVA nos dias 14, 15 e 16 do protocolo. No artigo cient?fico 1, eosin?filos do LBA de animais do grupo OVA e do grupo controle foram estimulados com VSR (103 PFU/mL) in vitro por 3 horas. Ap?s este per?odo, o sobrenadante da cultura foi coletado para a realiza??o das t?cnicas avaliadas conforme os objetivos espec?ficos deste artigo cient?fico. No artigo cientifico 2, durante o protocolo experimental de asma, os animais foram tratados via intranasal com um inibidor da nicotinamida adenina dinucleot?deo fosfato oxidase (NADPH oxidase), difenileno-iod?nio (DPI), ou com um precursor da glutationa, N-acetilciste?na (NAC), 45 minutos antes dos tr?s desafios intranasais com OVA. J? no artigo cientifico 3, os animais foram tratados via intranasal com um inibidor de autofagia, 3-metiladenina (3-MA), 45 minutos antes dos tr?s desafios intranasais com OVA. Ao final do protocolo o LBA e o tecido pulmonar foram coletados para a realiza??o das t?cnicas avaliadas em cada um dos artigos cient?ficos, conforme seus objetivos espec?ficos. RESULTADOS: no artigo cientifico 1, observamos um aumento na libera??o de EETs em eosin?filos do LBA de animais submetidos ao modelo experimental de asma e estimulados com VSR in vitro. Por outro lado, o VSR in vitro foi capaz de diminuir os n?veis de IFN-? no sobrenadante da cultura de eosin?filos do LBA. Em rela??o aos resultados do artigo cient?fico 2, verificamos que no grupo OVA tratado com NAC ocorreu uma diminui??o no n?mero de c?lulas inflamat?rias no LBA bem como uma redu??o no infiltrado inflamat?rio pulmonar. Al?m disso, os animais do grupo OVAM tratados com DPI ou NAC apresentaram uma redu??o da enzima EPO, hiperplasia de c?lulas caliciformes, citocinas inflamat?rias e da prote?na fator nuclear kappa B (NF?B p65). Os tratamentos com DPI ou NAC foram capazes de reduzir a forma??o de EROs, aumentar a atividade da enzima catalase antixidante (CAT). Por outro lado, par?metros do metabolismo energ?tico mitocondrial aumentaram somente com o tratamento com NAC. Por fim, demonstramos que os tratamentos com DPI ou NAC foram capazes de reduzir a forma??o de EETs do LBA. No artigo cient?fico 3, observamos que no grupo OVA tratado com o inibidor de autofagia, 3-MA, ocorreu uma diminui??o no n?mero de c?lulas inflamat?rias no LBA bem como uma redu??o do infiltrado inflamat?rio pulmonar. Al?m disso, os animais tratados com 3-MA apresentaram uma redu??o nos n?veis da enzima EPO, hiperplasia de c?lulas caliciformes, citocinas inflamat?rias e da prote?na NF?B p65. O tratamento com 3-MA foi capaz de reduzir a forma??o de EROs bem como aumentar os n?veis da enzima antioxidante CAT. O tratamento com 3-MA tamb?m melhorou par?metros do metabolismo energ?tico mitocondrial e a atividade da enzima Na+,K+ATPase. Demonstramos tamb?m que o tratamento com 3-MA diminuiu o imunoconte?do da prote?na light chain 3B (LC3B) em eosin?filos do LBA e no tecido pulmonar e reduziu a forma??o de EETs no LBA. CONCLUS?O: Nossos resultados demonstram um importante papel do VSR na indu??o da libera??o de EETs. Al?m disso, verificamos que os tratamentos com DPI, NAC e 3-MA foram capazes de reduzir a inflama??o das vias a?reas, o estresse oxidativo e a libera??o de EETs no LBA. Demonstramos que o VSR, as EROs e a autofagia participam dos mecanismos que regulam o processo de libera??o das EETs na asma. Assim, a identifica??o de alguns desses mecanismos envolvidos na libera??o de EETs na asma pode contribuir para uma melhor compreens?o da patog?nese desta doen?a inflamat?ria cr?nica que prejudica a qualidade de vida dos pacientes e ? respons?vel por um alto custo econ?mico para o Sistema ?nico de Sa?de (SUS).

No description available.

Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...

Les neutrophiles peuvent synthétiser et relâcher l’angiopoïétine 1 (Ang1), un facteur de croissance cytosolique impliqué dans l’angiogenèse, capable d’induire plusieurs activités pro-inflammatoires chez les neutrophiles. Ces derniers induisent la synthèse et la relâche de neutrophil extracellular traps (NETs), des filaments d’ADN nucléaires décondensés transportant des protéines telles que l’élastase, la myélopéroxydase (MPO), la protéinase (PR3) et la calprotectine (S100A8/S100A9) qui contribuent à la réponse immunitaire innée afin de combattre les pathogènes (i.e. bactéries). Les NETs ont des effets pro-inflammatoires, pro-thrombotiques, jouant un rôle dans la dysfonction endothéliale, et ont récemment été retrouvés dans l’insuffisance cardiaque (IC) et le diabète de type 2 (T2DM). Le but de cette étude est de déterminer la synthèse et la relâche d’Ang1 chez des patients T2DM et insuffisants cardiaques avec fraction d’éjection préservée (HFpEF) (stables ou en décompensation aigue (ADHFpEF)) avec ou sans T2DM en comparant à des volontaires sains (VS) comme groupe témoin. Nos résultats démontrent qu’en absence de traitement (PBS) et avec un traitement avec LPS, les niveaux de NETs augmentent chez les patients ADHFpEF + T2DM comparé aux VS. Nous observons également que le LPS, le PMA ou A23187 augmentent la synthèse de l’Ang1 (de 150 à 250%) chez des VS et cet effet est amplifié chez les patients T2DM et IC. L’Ang1 est relâchée à 100% par les neutrophiles de tous les groupes à l’étude et ne se lient pas sur les NETs, comparé à la calprotectine. Notre étude suggère que les patients en insuffisance cardiaque aigue (ADHFpEF + T2DM) synthétisent et relâchent plus de NETs et que l’exocytose de l’Ang1 est indépendante de la synthèse et la relâche de NETs. / Neutrophils can induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis capable of inducing several neutrophil-driven pro-inflammatory activities. Neutrophils also synthesize and release neutrophil extracellular traps (NETs) composed of decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9) which contribute to the innate immune response to combat pathogens (e.g., bacteria). NETs are also implicated in various pathological conditions through pro-inflammatory, pro-thrombotic properties, leading to endothelial dysfunction, and have recently been found in heart failure (HF) and type 2 diabetes (T2DM). The purpose of this study is to determine the synthesis and release of Ang1 in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated (ADHFpEF)) with or without T2DM, compared to healthy volunteers (HV) as control group. Our results show that in absence of agonist (PBS) and after LPS treatment, NETs levels are increased in ADHFpEF + T2DM patients compared to HV. We also observed that LPS, PMA or A23187 treatments increase the synthesis of Ang1 (from 150 to 250%) in HV and this effect is amplified in T2DM and HF patients. Ang1 is completely released (100%) by neutrophils isolated from all cohorts and does not bind to NETs compared to calprotectin. Our study suggests that severely ill patients with decompensated heart failure (ADHFpEF + T2DM) synthesize and release more NETs and that Ang1 exocytosis is independent from NETs synthesis.