A ação de espécies reativas de oxigênio e/ou de nitrogênio (ERO/ERN), produzidas por neutrófilos através do complexo NADPH oxidase (Nox2) e óxido nítrico sintase (iNOS), estão diretamente associadas à capacidade microbicida de fagócitos. Entretanto, se a produção destes oxidantes escapa ao controle da homeostase bioquímica, ocorrem danos oxidativos aos tecidos circunvizinhos do foco inflamatório, quando, então, há necessidade de intervenção farmacológica. Neste trabalho foi testado o efeito dos nitróxidos piperídinicos 2,2,6,6-tetrametil-1-piperidina-1-oxil (Tempo) e 4-((9 acridinecarbonil)amino)-2,2,6,6-tetrametil-1-piperidina-1-oxil (Ac-Tempo) sobre a atividade de Nox2 de neutrófilos inflamatórios e suas consequências sobre a capacidade fungicida dos fagócitos. Neutrófilos isolados de cavidade peritoneal de camundongos foram incubados (37°C, 10 min) com Tempo (50-400 μM) ou Ac-Tempo (25-200 μM) e estimulados com Candida albicans (ATCC 10231) opsonizadas (107 C. albicans/105 neutrófilos). A atividade de Nox2 foi determinada polarograficamente, pelo consumo de oxigênio mensurado através de eletrodo de Clark, ou espectrofotometricamente, medindo-se a redução de citocromo c (550nm). A atividade de Nox2 (119,311,6 nmol de O2-•.min-1) foi diminuída pelo tratamento com ambos os nitróxidos, de forma dose-dependente, embora Ac-Tempo tenha sido mais eficaz (ED50 31 M) que Tempo (ED50 160 M). Paralelamente, foi verificada a fluorescência (λexc361, λemi440) emitida pelo grupo acridina após reação de espécies radicalares com Ac-Tempo, corroborando a hipótese de que nitróxidos depletam radicais produzidos por neutrófilos estimulados com C. albicans. O nitróxido Tempo não mostrou nenhuma ação direta sobre culturas de C. albicans, conforme análises de antifugigramas padrões. Entretanto, testes de capacidade microbicida de neutrófilos mostraram que o tratamento prévio das células com Tempo (300 µM) causou decréscimo de 80% na efetividade fungicida sobre C. albicans. Testes in vivo mostraram que Tempo administrado em dose oral única (26,52 mg/kg) foi capaz de diminuir o edema de pata originado em resposta à inoculação do fungo (107 C. albicans) cujo ponto máximo de efeito foi 18 horas após a administração do nitróxido, bem como determinou significativa diminuição de nitração de proteínas em tecido muscular das patas dos animais, conforme resultados de ensaios com anticorpos anti-nitrotirosina. Em conjunto, os resultados corroboram a premissa de que a atividade de Nox2 é essencial para a resposta do hospedeiro a C. albicans e que a administração de nitróxidos para modular a produção de oxidantes nos focos infecciosos pode diminuir a resposta do sistema imune à infecção fúngica. / Production of oxidants species by neutrophils through NADPH oxidase complex (Nox2) is directly associated with the microbicidal activity of phagocytes. In this work we tested the effect of the nitroxides 2,2,6,6-tetramethylpiperidine-1-piperidine-1-oxil (Tempo) and 4-((9 acridinecarbonil)amino)-2,2,6,6-tetramethyl-1-piperidine-1-oxil (Ac-Tempo) on the activity of inflammatory neutrophil Nox2 complex and the consequences on the fungicidal phagocytes capability. Neutrophils were incubated with Tempo (50-400 mM) or Ac-Tempo (25-200 mM) and stimulated with Candida albicans. Nox2 activity was determined polarographically by oxygen consumption measured by Clark electrode. Oxidase activity decreased by treatment with both nitroxides in a dose-dependent manner. However, Ac-Tempo was more effective (ED5044M) that Tempo (ED50160M). In parallel, the fluorescence intensity elicited by acridine after reaction with free radicals was determined. Neutrophils stimulation elicited significant fluorescent response in direct correlation with Ac-Tempo doses (R2=0.997), supporting the hypothesis that nitroxide is consumed by free radicals neutrophils-released. Fungicidal neutrophils activity assay explicited that pretreatment of cells with Tempo caused 80% decrease in the effectiveness on C. albicans death. Oral administration of a Tempo single dose was able to reduce paw edema elicited in response to inoculation of the fungus. Also, a significant decrease in protein nitration in paw tissues was found in this condition. Together, the results support the premise that Nox2 activity is essential for the host response against C. albicans and that nitroxides cause a decrease on the oxidant immune response to fungal infection.

Durante algumas das reações que levam à formação de AGEs, espécies reativas de oxigênio são geradas e concorrem paralelamente com o estresse oxidativo e com os danos estruturais e funcionais às macromoléculas. A formação de AGEs in vivo pode envolver os neutrófilos, que após estímulo inflamatório, induzem importantes enzimas geradoras de ERO, como o sistema NADPH oxidase. Entretanto ainda permanecem questionamentos sobre a relação entre o Sistema NADPH oxidase de neutrófilos e os AGEs. O presente trabalho visa contribuir para o entendimento do papel do sistema NADPH oxidase e da geração de ERO na formação de AGEs e o impacto da formação destes produtos na função renal destes animais. Foi utilizado um modelo experimental de diabetes induzido pela injeção i.p. de aloxano em ratos Wistar que foram divididos em 5 grupos: controle, diabético, diabético tratado com aminoguanidina (AG), diabético tratado com apocinina (AP) e diabético tratado com apocinina e aminoguanidina (AP+AG). A AG é um conhecido inibidor da formação de AGEs, e a AP é inibidor do sistema NADPH oxidase. Os inibidores, ou água foram administrados aos animais por gavagem por 50 dias após a instalação do DM. A formação de ERO reflete a atividade de NADPH oxidase e foi mensurada em neutrófilos peritoneais por quimiluminescência e pelo ensaio de redução do citocromo C. A expressão das subunidades p47phox e a p67phox do sistema NADPH oxidase foi avaliada por imunofluorescência. Os AGEs circulantes foram avaliados por fluorescência e a função renal foi avaliada pela dosagem da uréia e creatinina séricas e análises histológica dos rins. Pelos parâmetros avaliados neste estudo, verificamos que o tratamento com AP, diminuiu a geração de ERO tanto no grupo controle, quanto diabético, mas não afetou a via de formação de AGEs. O que indica que, provavelmente, não há interrelação entre as vias do sistema da NADPH oxidase de neutrófilos e a formação de AGEs, no nosso modelo experimental. O tratamento com a AP atenuou a glomerulonefrite do grupo diabético. A AG, na dose e no tempo administrado, 100mg/Kg por 50 dias, melhorou o perfil glicêmico dos animais diabéticos, preveniu a formação de AGEs e o dano glomerular. Além disso, o tratamento com a AG aumentou a atividade do sistema NADPH oxidase e a expressão das subunidades citosólicas p47phox e a p67phox do sistema. Não podemos afirmar que o aumento da atividade e expressão do sistema NADPH oxidase seja devido à diminuição dos AGEs no nosso sistema ou um efeito isolado da aminoguanidina, ainda não descrito na literatura. A compreensão do papel desta enzima em modelo experimental de diabetes pode ajudar na compreensão do processo inflamatório persistente que ocorre devido ao acúmulo desses produtos nas complicações da doença, permitindo uma melhor adequação da abordagem clínica e tratamento desses pacientes. / Chronic hyperglycemia observed in diabetes disease leads to the advanced glycation end products (AGEs) formation, which are able to damage the patient organs, especially the kidney. Some AGEs formation reactions may also generate reactive oxygen species (ROS) and they are able to compete with oxidative stress and structural and functional macromolecules damages. Neutrophils are immune cells capable of expressing great amount of important ROS generator enzymes such as NADPH oxidase just after inflammatory stimuli. There are still questions about the relationship between the neutrophil NADPH oxidase system and AGEs despite the information that in vivo AGEs formation may involve neutrophils. The present study aims to contribute to the understanding of NADPH oxidase role and its ROS generation in AGEs formation and what is the impact of these products formation in renal function of rat model. Our experimental diabetes model was reached by alloxan i.p. injection in rats. These animals were divided in five groups: control, diabetic, diabetic treated with aminoguanidine (AG), diabetic treated with apocynin (AP) and diabetic treated with apocynin and aminoguanidine (AG + AP). AG is an AGEs formation inhibitor, and AP is a NADPH oxidase inhibitor. The inhibitors compounds or the vehicle (water) were administered by gavage for 50 days after DM confirmation. ROS formation reflects the NADPH oxidase activity and it was measured by chemiluminescence assay and by cytochrome C reduction in peritoneal neutrophils. The expression of NADPH oxidase p47phox and p67phox subunits was assessed by immunofluorescence. Circulating AGEs were analyzed by fluorescence and the renal function was evaluated by serum urea and creatinin concentration and by kidney histological sections. Our results showed that AP treatment decreased ROS generation in both control and diabetic groups, but did not affect the process of AGEs formation. This may indicate that in our experimental model there is no interrelation between the neutrophil NADPH oxidase system and the AGEs formation. It was also observed that the AP attenuated glomerulonephritis in diabetic mice. AG, at the administered dose and time, 100mg/Kg for 50 dias, improved glycemic control, prevented the AGEs formation and prevented glomerular damage. Furthermore, AG treatment increased the NADPH oxidase activity and its p47phox and p67phox cytosolic subunits expression. We are not able to confirm if the increased activity and expression of NADPH oxidase were caused by the AGEs decrease in our system or by an isolated effect of aminoguanidine not yet described. Understanding the NADPH oxidase role in experimental diabetes could improve the knowledge about the persistent inflammatory process that occurs due to these products accumulation during the disease complication, allowing a better clinical approach and treatment of these patients. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Já foi demonstrado que a aminoguanidina (AG), um conhecido inibidor de AGEs (Produtos Finais de Glicação Avançada), aumenta a atividade de NOX2 (Sistema NADPH oxidase fagocítico) em neutrófilos peritoneais de ratos diabéticos. Experimentos adicionais demonstraram que o mesmo efeito ocorre em neutrófilos de ratos não diabéticos. Visando elucidar o mecanismo e a importância deste aumento de NOX2 pela AG em neutrófilos, utilizou-se um modelo experimental de ratos diabéticos e não diabéticos e um modelo in vitro com neutrófilos humanos isolados de sangue periférico ou HUVEC, uma linhagem de células endoteliais. Para isso, ratos machos Wistar diabéticos e não diabéticos foram tratados ou não por gavagem com AG (100mg/kg/dia) por 50 dias. Os neutrófilos foram recrutados e isolados do peritônio após 4 horas da injeção de caseinato de sódio e foram usados para avaliar a produção de superóxido, ERO e as atividades fagocítica e candicida destas células. No modelo in vitro, neutrófilos isolados de sangue periférico ou HUVEC foram ou não incubados com AG ou Metformina (MET) por 18h em estufa de CO2. O tratamento in vivo com a AG, mostrou aumento na produção de superóxido em comparação com grupos tratados com água, em ambos os grupos, diabéticos e não diabéticos. O aumento de atividade e expressão de NOX2 (p47phox e p67 phox) pela AG aumentou as atividades fagocíticas e candicida de Candida albicans, entretanto este aumento foi menos pronunciado no grupo diabético. Adicionalmente, os resultados demonstram a eficácia do tratamento com a AG na prevenção de glicação de proteínas, reduzindo os níveis de HbA1C e AGEs, além de reduzir os níveis de ureia e manter os níveis de creatinina no soro dos animais. O tratamento com AG reduziu a pressão arterial e a produção de nitratos no soro dos animais, mas a atividade das enzimas antioxidantes foi mantida. Verificou-se que as HUVEC tratadas com AG ou MET no estado basal ou estimuladas com LPS, tiveram menor produção de ERO. Neutrófilos humanos incubados com AG aumentaram a produção de ERO e de superóxido, enquanto o tratamento com MET manteve a produção de ambos. Além disso, o tratamento com AG aumentou a fagocitose e a atividade candicida de neutrófilos humanos nos tempos avaliados. O tratamento de neutrófilos humanos com MET manteve a expressão das subunidades avaliadas e o tratamento com AG aumentou a expressão de gp91phox. A liberação de IL-8 foi diminuída nos neutrófilos humanos tratados com a MET, e a AG aumentou a liberação de IFN-y . Em HUVEC a liberação de IL-8 e INF-y foram mantidas com ambos os tratamentos. Pode-se sugerir que a AG aumentou a atividade e a expressão de NOX2 em neutrófilos humanos e de ratos e que isso contribuiu diretamente para o aumento da atividade microbicida dos neutrófilos. Além disso, nos neutrófilos humanos, o aumento da liberação de IFN-y pode ter contribuído para o aumento da expressão de gp91phox. Adicionalmente, a influência da AG em células em processo de diferenciação para um estado mais maduro pode ser diferente dos seus efeitos in vitro, em células já maduras. / It has been showed that aminoguanidine (AG), a known inhibitor of AGE (Advanced Glycation End Products) increases the activity of NOX2 (phagocyte system NADPH Oxidase) peritoneal neutrophils of diabetic rats. Additional experiments demonstrated that the same effect occurs in non-diabetic rat neutrophils. To elucidate the mechanism and significance of NOX2 increase in neutrophils by AG, it was used an experimental model of diabetic and non-diabetic rats and an in vitro model in which human neutrophils isolated from peripheral blood or HUVEC endothelial cell lineage. For this, Wistar rats diabetic and non-diabetic were treated or not with AG by gavage (100 mg / kg / day) for 50 days. Neutrophils were recruited and isolated from the peritoneum 4h after injection of sodium caseinate and were used to evaluate superoxide generation, ROS and phagocytic and candicida activity of these cells. In vitro model, neutrophils isolated from peripheral blood or HUVEC were incubated or not with AG or Metformin (MET) for 18h in CO2 incubator. In vivo treatment with AG showed an increase in superoxide production compared with groups treated with water, in both groups diabetics and non-diabetics. The increased activity and expression of NOX2 (p47phox and p67phox) by AG increased phagocytic and candicida activity of Candida albicans, however this increase was less pronounced in the diabetic group. Additionally, the results demonstrate the effectiveness of treatment with AG on preventing glycation of proteins, reducing HbA1c and AGEs, in addition to reducing levels of urea and maintain the creatinine levels in serum of animals. The treatment with AG reduced the blood pressure and the production of nitrates and antioxidant enzymes activity were maintained in animals. It was found that HUVEC treated with AG or MET at baseline, or stimulated with LPS had lower production of ROS. Human neutrophils incubated with AG increase ROS and superoxide while treatment with MET remained the production of both. In addition, treatment with AG increased phagocytosis and candicidal activity of human neutrophils at times evaluated. In addition, treatment with AG increased phagocytosis and candicida activity of human neutrophils at times evaluated. The MET treatment maintained the expression of subunits assessed, and treatment with AG caused increased expression of gp91. The release of IL-8 by human neutrophils was decreased by treatment with MET, and AG increased the release of IFN-y The release of IL-8 was reduced in human neutrophils treated with MET, and AG increased IFN-y . In HUVEC the release of IL-8 and IFN-y were maintained with both treatments. It can be suggested that the AG used to inhibit the formation of AGEs increased activity and NOX2 expression in human and rat neutrophils and that directly contributed to the increased microbicidal activity by neutrophils. In addition, the human neutrophils the increased of IFN-y may have contributed to the increased expression of gp91phox. In addition, the influence of AG in cells in differentiation process to more mature state may be different of in vitro effects on cells already mature.

Lors d’une infection, les polynucléaires neutrophiles phagocytent l’agent pathogène et le détruisent grâce à la production de formes réactives de l’oxygène (FRO) par la NADPH oxydase. Cette enzyme est constituée de sous-unités membranaires (Nox2, p22phox) et cytosoliques (p67phox, p47phox,p40phox, Rac) qui s’assemblent soit à la membrane plasmique, lors de l’activation des cellules par un stimulus soluble comme le fMLF, soit à la membrane du phagosome, lors de la phagocytose de particules. La régulation de la NADPH oxydase implique divers facteurs comme la signalisation calcique et les lipides, notamment les phospholipides anioniques. En effet, il a été montré que l’activation et la translocation de la petite protéine G Rac peuvent être dépendantes du calcium. D’autre part les sous unités Rac et p47phox peuvent interagir avec les phospholipides anioniques tels que la phosphatidylsérine,grâce à des interactions stéréosélectives et/ou électrostatiques.L’objectif de ce travail est donc d’évaluer le rôle du calcium et de la phosphatidylsérine dans le recrutement de p47phox et/ou Rac lors de l’assemblage de NADPH oxydase. Pour suivre la dynamique des deux protéines, nous avons exprimé ces sous-unités marquées avec des protéines fluorescentes dans des lignées phagocytaires mimant les neutrophiles (HL-60 et PLB-985). Nous avons ainsi pu suivre, par vidéomicroscopie, le déplacement des sous-unités marquées lors d’une stimulation par fMLF ou PMA etdurant la phagocytose de particules opsonisées. Après stimulation par fMLF, Rac1 transloque du cytosol à la membrane plasmique, alors que le mutant constitutivement actif de Rac1 est constamment localisé àla membrane plasmique, indépendamment de la stimulation par fMLF. De plus après stimulation parPMA, le mutant constitutivement actif de Rac2 transloque à la membrane plasmique, suggérant que sa translocation pourrait être possible en absence d’un influx de calcium extracellulaire. Lors de la phagocytose, en masquant la phosphatidylsérine avec le domaine C2 discoïdine de la lactadhérine qui lie spécifiquement ce phospholipide, nous avons pu montrer que la phosphatidylsérine régule la production initiale des FRO en favorisant le recrutement de p47phox et de Rac2 au phagosome. De plus, ces deux sous-unités se détachent du phagosome alors que la production intraphagosomale de FRO continue,suggérant que leur départ n’est pas un signal de terminaison pour l’activité oxydase. Plus précisément,p47phox et Rac2 sont recrutées de manière transitoire, pendant seulement 1 à 3 minutes après la fermeture du phagosome. Ceci est en accord avec le modèle qui propose que p47phox servirait principalement à transporter p67phox au phagosome, et que les deux sous-unités p47phox et Rac2 faciliteraient le positionnement de p67phox dans le complexe membranaire. / During phagocytosis, neutrophils internalize pathogens in a phagosome and kill them through the production of reactive oxygen species (ROS) by the NADPH oxidase enzyme. The cytosolic NADPHoxidase subunits (p67phox p47phox, p40phox, Rac2) and the membranous subunits (Nox2, p22phox) assemble either at the plasma membrane after stimulation with soluble agonist, or at the phagosomal membrane during particle phagocytosis. The regulation of this enzyme involves several actors like calciumsignalling and anionic phospholipids. Actually Rac activation and translocation was found to be calciumdependent and, on the other hand, p47phox and Rac can interact with anionic phospholipids such asphosphatidylserine through stereoselective and/or electrostatic interactions.Therefore we wanted to investigate the role of calcium and phosphatidylserine in p47phox and Rac membrane recruitment. To study this dynamic, we expressed the subunits tagged with a fluorescent protein in neutrophil-like cells (HL-60 and PLB-985), and followed them by videomicroscopy duringfMLF or PMA stimulation or during phagocytosis of opsonised particles. After fMLF stimulation, Rac1translocated from the cytosol to the plasma membrane whereas constitutively active form of Rac1 was permanently located at the plasma membrane, independently of fMLF stimulation. In addition, afterPMA treatment, constitutively active form of Rac2 translocated to the plasma membrane, suggesting that its translocation could occur without extracellular calcium entry. By using the specific phosphatidylserine binding discoïdine C2 domain of lactadherin, we could mask this phospholipid and found that phosphatidylserine is involved in NADPH oxidase activity by participating in the phagosomal recruitment of p47phox and Rac2. In addition we show that these two subunits detached from the phagosome while ROS production continued for a longer period, suggesting that their dissociation from the complex is not a termination signal for oxidase activity. More precisely, p47phox and Rac2 were briefly recruited to the phagosomal membrane, for 1 to 3 minutes after the phagosome closure. These results support the model in which p47phox serves as a carrier for p67phox and both p47phox and Rac2 are adapters that correctly position p67phox in the complex.

Le polynucléaire neutrophile (PN) humain est une cellule phagocytaire qui constitue une des premières barrières de défense de l’organisme contre les agents pathogènes. Sa stimulation par des facteurs chimioattractants, provoque sa migration de la circulation sanguine vers le foyer inflammatoire. Dans le site inflammatoire, les PN reconnaissent l’agent pathogène par l'intermédiaire d'opsonines, des fractions résultant de l'activation du complément et par l’intermédiaire de motifs de reconnaissance conservés au cours de l’évolution des agents pathogènes qui se lient à des récepteurs de la famille Toll (Toll-like receptors ; TLR). Le contact du pathogène avec le PN va provoquer sa phagocytose et sa destruction par la libération de molécules contenues dans les granules du PN et par la production de formes réactives de l’oxygène (FRO) par un complexe enzymatique la NADPH phagocytaire composée au repos de de protéines cytosoliques (p40phox, p47phox, p67phox et Rac 2) et membranaires (gp91phox et p22phox formant le cytochrome b558). Un des événements majeur de l’activation de la NADPH oxydase est la phosphorylation de certains composants cytosoliques comme la p47phox ou la p67phox ce qui conduit à la translocation de ces protéines vers le cytochrome b558 membranaire et permet d’activer l’enzyme pour la production de FRO. L’hyperactivation de cette enzyme ou son « priming » consiste en une pré-activation du PN par des agents dit « primants » tels que des cytokines (TNFα, GM-CSF, IL-1), des chimiokines comme l’IL-8, des molécules lipidiques (PAF et LTB4), ou encore des endotoxines bactériennes LPS, agoniste de TLR4. Les TLR sont des récepteurs exprimés à la surface de nombreuses cellules dont les cellules immunitaires ; ils détectent des motifs conservés au cours de l’évolution des agents pathogènes appelés PAMPs pour "pathogen-associated molecular patterns", des protéines modifiées reconnues comme étrangères, des lipides oxydés, des ligands endogènes. Quelques agonistes des TLR comme le LPS ont été décrits pour induire un priming de la production des FRO par les PN. D’autres ont été connus par leur pouvoir activateur de la NADPH oxydase des PN. Le CL097 (Imidazoquinoline : agoniste des TLR7/8) était l’agoniste des TLR induisant le plus fort effet de « priming » par les PN stimulés par le fMLP. Le CL097 induit la phosphorylation de la p47phox sur la sérine 345. Cette phosphorylation implique des MAPKinases ERK1/2 et de la p38MAPK. La phosphorylation de ce site induit le changement de conformation de la p47phox sous l’action d’une proline isomérase Pin1. Ce changement de conformation favorise la phosphorylation des autres sites (Ser-315, Ser-328) et par conséquent l’activation de la NADPH oxydase. La comparaison de l’effet du CL097 à deux agonistes reconnaissant l’un le TLR7, l’autre le TLR 8 a montré que l’action du CL097 dépendait du TLR8. Le zymosan non opsonisé (agoniste de TLR2) stimule l’activation de la NADPH oxydase des neutrophiles. IL induit la phosphorylation de la p47phox au niveau des Ser-345, -315 et -328. Ces phosphorylations font intervenir respectivement les MAPK ERK1/2 et p38, une protéine tyrosine kinase et les PKC. En plus cet agoniste active la petite protéine cytosolique Rac2, nécessaire à l’activation de la NADPH oxydase des PN. Ces données permettraient d’identifier de nouvelles cibles thérapeutiques de première importance afin de moduler les réponses inflammatoires pathologiques. / Superoxide anion production by the neutrophil NADPH oxidase plays a key role in host defense; however, excessive superoxide production is believed to participate to inflammatory reactions. Neutrophils express several TLR that recognize a variety of microbial motifs or agonists. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to eliminate the pathogen. However, the effects of some TLR agonists on the NADPH oxidase activation and the mechanisms controlling these effects have not been elucidated. In this study, we show that the TLR7/8 agonist CL097 by itself did not induce NADPH oxidase activation in human neutrophils, but induced a dramatic increase of fMLF-stimulated activation. Interestingly, CL097 induced cytochrome b558 translocation to the plasma membrane and the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315. Phosphorylations of Ser328 and Ser315 were significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser345, Ser328 and Ser315 was decreased by inhibitors of p38MAPK and the ERK1/2-pathway. Phosphorylation of Ser328 was decreased by a PKC inhibitor. Genistein, a braod range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that activation of TLR7/8 in human neutrophils induces hyper-activation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites, and suggest that p38MAPK, ERK1/2, PKC and Pin1 control this process.Zymosan a cell-wall preparation from saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan induced ROS production by human neutrophils. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315; and activation of the GTPase Rac2. Phosphorylation of p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor. Wortmannin a PI3Kinase inhibitor, inhibited phosphorylation of p47phox on Ser328 and Ser315 and Rac2 activation. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway respectively, inhibited phosphorylation of p47phox on Ser345. GF109203X a PKC inhibitor inhibited phosphorylation on Ser328 and Ser315. Zymosan-induced ROS production was inhibited by genistein, wortmannin, SB203580, UO126 and GF109203X. These results show that zymosan induced ROS production by NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation. Our results also suggest that a protein tyrosine kinase and PI3Kinase control p47phox phosphorylation and Rac2 activation while p38MAPK, ERK1/2 and PKC are involved in zymosan-induced p47phox phosphorylation.

La production d’anion superoxyde (O2-) par le complexe NADPH oxydase 2 (NOX2) du polynucléaire neutrophile (explosion oxydative, EO) contribue à l’élimination d’agents pathogènes. Cette fonction de défense est stimulée par divers agents pro-inflammatoires, notamment par des peptides bactériens (fMLP), qui déclenchent une cascade de signalisation impliquant différentes protéines kinases (PKC, AKT, MAP-Kinases) et aboutissant à l’activation de la NOX2 (également nommée gp91phox), le coeur catalytique du complexe. Dans cette thèse, j’ai identifié la protéine kinase mTOR comme un nouvel effecteur majeur de l’EO dans les neutrophiles sains et j’ai comparé son mode d’action transductionnel dans les neutrophiles de patients ayant une cirrhose alcoolique décompensée en vue de comprendre leur grande susceptibilité aux infections bactériennes.Une contribution majeure de mTOR dans la production d’O2- induite par le fMLP a pu être démontrée à l’aide de son antagoniste spécifique, le médicament Rapamycine et par une approche antisense. mTOR agit en amont de la p38-MAPK qui phosphoryle la p47phox, un composant majeur du complexe NADPH oxydase. Dans les neutrophiles de patients cirrhotiques, l’EO est fortement défaillante, associée à un défaut d’activation de la voie p38-MAPK/p47phox(S345). Ce déficit d’EO est aggravé par la Rapamycine. Les neutrophiles de patients cirrhotiques présentent également un déficit d’expression de la gp91phox (NOX2), p22phox, p47phox et mTOR. Un déficit d’expression de la NOX2 a pu être reproduit en traitant les neutrophiles sains par le fMLP ou du plasma des patients. De plus, ce phénomène met en jeu une dégradation protéolytique insoupçonnée de la gp91phox impliquant l’élastase. Enfin, la déficience fonctionnelle des neutrophiles de patients a pu être corrigée dans des neutrophiles isolés et dans du sang total des patients cirrhotiques à l’aide d’un agoniste de récepteurs « Toll-like receptor (TLR) » qui agit en favorisant la transcription du gène de la gp91phox et sa synthèse protéique.En conclusion, mTOR émerge comme un nouvel effecteur transductionnel majeur de l’EO des neutrophiles, favorisant l’activation de la voie p47phox/gp91phox via les MAPK. Cette nouvelle voie de signalisation est fortement impactée au cours de la cirrhose alcoolique, ce qui favorise la susceptibilité des patients aux infections bactériennes. Bien que notre étude soulève ainsi des inquiétudes quant à l’utilisation d’inhibiteurs de mTOR chez les patients immunodéprimés, elle suscite par ailleurs la perspective de pouvoir corriger les déficits fonctionnels des neutrophiles à l’aide d’agents capables de stimuler des TLR intracellulaires. / Superoxide anion (O2-) production by NADPH oxidase 2 (NOX2) complex of polymorphonuclear neutrophil (i.e respiratory burst, RB) contributes to efficient elimination of pathogens. This defense function is stimulated by various pro-inflammatory agents, especially by bacterial peptides (fMLP) which trigger a signaling cascade involving many protein kinases (PKC, AKT, MAP-Kinases) resulting in activation of NOX2, also called gp91phox, the catalytic core of the complex. In this thesis, I identified the protein kinase mTOR as a novel major RB effector of healthy neutrophils and I compared its transductional activity in neutrophils from patients suffering from alcoholic decompensated liver cirrhosis, aiming at understanding their high susceptibility to bacterial infections.A major contribution of mTOR to fMLP-induced neutrophil superoxide production was demonstrated using its specific drug antagonist Rapamycin, and by an antisens strategy. mTOR is activated upstream of p38-MAPK which phosphorylates p47phox, a major component of the NADPH oxidase complex. In neutrophils from cirrhotic patients, the RB is dramatically impaired and this was associated with a deficient activation of the p38-MAPK/p47phox(S345) signaling pathway. This RB deficiency was aggravated by Rapamycin. Neutrophils from cirrhotic patients also exhibited a deficient expression of gp91phox (NOX2), p22phox, p47phox and mTOR. A deficient NOX2 expression can be reproduced by treating healthy neutrophils with fMLP or plasma from cirrhotic patients. Furthermore, this phenomenon involved an unexpected proteolytic degradation of gp91phox mediated by elastase. Finally, this deficient superoxide production by neutrophil from cirrhotic patients can be corrected ex vivo in isolated neutrophils and in patients’ whole blood, using a Toll-like receptor agonist that acts by promoting the transcription and traduction of gp91phox .In conclusion, mTOR emerges as a novel and major signaling effector of neutrophil RB, promoting the activation of p47phox/gp91phox through MAPKs. This novel signaling pathway is strongly impaired during alcoholic liver cirrhosis, which increases patients’ susceptibility to bacterial infections. Although our study raises concerns about the use of mTOR inhibitors in immunocompromised patients, it also provides therapeutic propects for correcting neutrophil functional deficiencies using agents capable of stimulating intracellular TLR .

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Evidências têm mostrado que as espécies reativas de oxigênio (ROS) geradas pela NAD(P)H oxidase são importantes moduladores de diversas funções celulares como migração, crescimento, proliferação e sobrevivência. Estudos recentes demonstraram o envolvimento da atividade da NAD(P)H oxidase no crescimento e sobrevivência de células de melanoma. Neste trabalho, investigamos o efeito da inibição da NAD(P)H oxidase por difenileneiodônio (DPI) sobre o crescimento das células de melanoma humano MV3 e observamos que este composto reduziu o crescimento destas células em aproximadamente 50%. A inibição da NAD(P)H oxidase induziu mudanças no formato celular, com arredondamento, diminuição do espraiamento e descolamento celular. Esta redução foi acompanhada por um rearranjo do citoesqueleto de actina, diminuição da fosforilação no resíduo Tyr397 da quinase de adesão focal (FAK) e redução na associação de FAK com actina e com a tirosina quinase c-Src. Isto indica que a inibição da geração de ROS está modulando negativamente vias de sinalização ativadas por integrinas, o que freqüentemente conduz a um tipo particular de morte celular conhecida por anoikis. Comprovando a ocorrência deste fenômeno, observamos que a inibição da atividade da NAD(P)H oxidase aumentou a apoptose das células de melanoma e induziu a ativação da caspase-3. Nossos resultados mostram ainda que a inibição da viabilidade celular por DPI foi revertida com o pré-tratamento das células MV3 com um inibidor de tirosina fosfatases (ortovanadato de sódio). Em resumo, este estudo mostra que a geração de ROS por NAD(P)H oxidase está envolvida nos mecanismos de sobrevivência em células de melanoma, uma vez que afetam as vias de sinalização dependentes de FAK-Src, através da inibição da atividade de proteína tirosina fosfatases. / NAD(P)H oxidase-derived reactive oxygen species (ROS) have emerged as critical mediators of several cell functions as diverse as migration, growth, proliferation and survival. Recent evidence has show that NAD(P)H oxidase activity is essential to melanoma proliferation and survival. We reported that NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) inhibited melanoma growth. NAD(P)H oxidase inhibition induced changes in cell shape with cell spreading decrease, rounding up and detachment. These phenomena were accompanied by rearrangement of actin network and a decrease in both focal adhesion (FAK) phosphorylation in Tyr397 residue and in FAK association to actin and c-Src, indicating that inhibition of ROS generation would down- modulate integrin-mediated signaling, what often results in a particular type of apoptotic cell death, known as anoikis. We observed that NAD(P)H oxidase inhibitor induced apoptosis in melanoma cells with activation of caspase-3. We results show that the effects promoted by NAD(P)H oxidase inhibition on melanoma growth were completely abolished by the pre-treatment of MV3 cells with the protein tyrosine phosphatases inhibitor sodium orthovanadate. In conclusion, our results strongly suggest that ROS generated by NAD(P)H oxidase complex transmit cell survival signals in melanoma cells through the FAK-Src pathway, probably inhibiting protein tyrosine phosphatases.

A fotobiomodulação (PBM) utiliza a luz nas porções do espectro visível e infravermelho para estimular a produção de trifosfato de adenosina (ATP), síntese de ácidos nucleicos, geração de espécies reativas de oxigênio (ROS) e proliferação celular, promovendo resultados teraupêuticos benéficos, como a aceleração do reparo tecidual, analgesia e ativação de células do sistema imune. Deste modo, a presente pesquisa visou elucidar os efeitos da PBM sobre monócitos humanos in vitro, a fim de possivelmente estimular o burst oxidativo e, consequentemente, potencializar a defesa imune celular contra micro-organismos, além de analisar a expressão gênica em nível de RNA mensageiro (mRNA) de CD68, CD80, CD163, CD204, IL-6, TNF-α, IL-10 e SOD1 em macrófagos derivados destas células. Neste contexto, culturas primárias de monócitos humanos foram irradiadas com o laser de diodo InGaAlP (660nm)/ GaAlAs (780nm) - Twin Flex® (MMO, São Carlos, SP, Brasil), operando com potência de 40mW, área de feixe de 0,04cm2, densidade de potência de 1W/cm2 e doses independentes de 200J/cm2, 400J/cm2 e 600J/cm2. Após isto, as células foram submetidas ao ensaio de quimioluminescência para avaliação do burst oxidativo e quantificação da produção de ROS intracelulares e extracelulares. Ensaio da atividade microbicida foi realizado contra o fungo Candida albicans. Como controles positivo e negativo, utilizou-se o forbol 12-miristato 13-acetato (PMA) e o diphenyleneiodonium (DPI), respectivamente. A viabilidade celular foi verificada por meio do reagente azul de Trypan. Adicionalmente, os monócitos humanos irradiados foram cultivados por 72 horas, a fim de observar a diferenciação destas células em macrófagos mediante estímulos relacionados a macrófagos ativados pela via clássica (M1) (LPS e Candida albicans) e a macrófagos ativados pela via alternativa (M2) (M-CSF). O RNA total foi extraído de cada grupo experimental e submetido à transcrição reversa e PCR em tempo real. GAPDH foi utilizado como controle endógeno e a expressão relativa de cada gene foi calculada utilizando o método 2-ΔCt. A produção de nitrito (NO2) também foi mensurada através da reação de Griess. Os resultados obtidos foram analisados por ANOVA e teste de Tukey ao nível de significância de 5%. Para dados com variâncias desiguais, utilizou-se o Kruskal-Wallis e pós-teste de Newman-Keuls. Desta forma, os monócitos irradiados apresentaram um aumento significativo na produção de ROS intracelulares e extracelulares comparativamente ao grupo controle (P < 0,001), sendo o comprimento de onda de 660nm e a dose de 400J/cm2, os parâmetros que mais se destacaram (P < 0,001). Como consequência deste perfil funcional elevado dos monócitos irradiados, a capacidade fungicida dos mesmos contra Candida albicans mostrou-se bastante aumentada (P < 0,001). Além disso, observou-se que a PBM (660nm; 400J/cm2) não causou danos à viabilidade celular dos monócitos nos dias subsequentes à irradiação laser. A análise da expressão gênica revelou que a PBM (660nm; 400J/cm2) aumentou significativamente a expressão da citocina pró-inflamatória TNF-α pelos monócitos irradiados (P = 0,0302), aproximando-os de uma resposta imune Th1. Complementarmente, um aumento significativo na produção de NO2 pelos monócitos irradiados também foi observado (P < 0,05). Portanto, a PBM, como empregada neste estudo, foi capaz de aumentar a geração de ROS e NO2, intensificar a atividade microbicida contra Candida albicans e aumentar a expressão de TNF-α, sugerindo uma modulação da PBM na indução de agentes pró-inflamatórios relacionados ao perfil funcional de M1. Vislumbramos em um futuro próximo, a reprodução desses resultados em monócitos humanos in vivo, colaborando no tratamento de candidoses orais, por exemplo. / Photobiomodulation (PBM) comprises the use of light within the visible and infrared spectrum to stimulate the production of adenosine triphosphate (ATP), nucleic acid synthesis, generation of reactive oxygen species (ROS) and cell proliferation, thus promoting beneficial therapeutical results, such as the acceleration of tissue repair, analgesia and activation of cells of the immune system. In that way, the present research aimed to elucidate the effects of PBM on human monocytes in vitro by possibly stimulating the oxidative burst of these cells and, consequently, enhancing the cellular immune defense against microorganisms, in addition to analyzing the gene expression at the messenger RNA (mRNA) level of CD68, CD80, CD163, CD204, IL-6, TNF-α, IL-10 and SOD1 in macrophages derived from these cells. Thus, primary cultures of human monocytes were irradiated with an InGaAlP (660nm)/ GaAlAs (780nm) - Twin Flex® diode laser (MMO, São Carlos, SP, Brazil), operating with power of 40mW, beam area of 0.04cm2, power density of 1W/cm2 and independent doses of 200J/cm2, 400J/cm2 e 600J/cm2. Cells were then submitted to the chemiluminescence assay for oxidative burst evaluation and quantification of intracellular and extracellular ROS production. A microbicidal activity assay was performed against the fungus Candida albicans. As positive and negative controls, phorbol 12-myristate 13-acetate (PMA) and diphenyleneiodonium (DPI) were used, respectively. Cell viability was verified by Trypan blue reagent. Besides, irradiated human monocytes were cultured for 72 hours in order to observe the differentiation of these cells into macrophages by stimuli related to macrophages activated by the classical pathway (M1) (LPS and Candida albicans) and macrophages activated by the alternative pathway (M2) (M-CSF). Total RNA was extracted from each experimental group and submitted to reverse transcription and real-time PCR. GAPDH was used as endogenous control and the relative expression of each gene was calculated using the 2-ΔCt method. The production of nitrite (NO2) was also measured by the Griess reaction. The results obtained were analyzed by ANOVA and Tukey's test at a significance level of 5%. For data with unequal variances, Kruskal-Wallis and Newman-Keuls post-test were used. In that way, irradiated monocytes presented a significant increase in intracellular and extracellular ROS production compared to the control group (P < 0.001). The wavelength of 660nm and the dose of 400J/cm2 were the most relevant parameters (P < 0.001). As a consequence of this high functional profile of the irradiated monocytes, the fungicidal capacity of the monocytes against Candida albicans was shown to be greatly increased (P < 0.001). In addition, it was observed that PBM (660nm; 400J/cm2) did not cause damage to the cell viability of monocytes in the days following laser irradiation. Analysis of the gene expression revealed that PBM (660nm; 400J/cm2) significantly increased the expression of the proinflammatory cytokine TNF-α by the irradiated monocytes (P = 0.0302), bringing them closer to a Th1 immune response. Additionally, a significant increase in NO2 production by irradiated monocytes was also observed (P < 0.05). Therefore, PBM, as employed in this study, was able to increase ROS and NO2 generation, enhance microbicidal activity against Candida albicans and increase TNF-α expression, suggesting a modulation of PBM in the induction of related pro-inflammatory agents to the functional profile of M1. We envisage in a near future, the reproduction of these results in human monocytes in vivo, which would collaborate to the treatment of oral candidoses, for example. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments. Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2. Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME. Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells. Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.