Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischemic tissues become contaminated with C. perfringens. C. perfringens quickly multiplies in ischemic tissues and spreads to healthy areas, leading to high levels of morbidity and mortality. As a species, the bacterium can synthesize thirteen different toxins. The alpha toxin (PLC) and perfringolysin O (PFO) are thought to be important virulence factors in gangrene. We wished to understand how C. perfringens is capable of avoiding killing by the host immune system, and determine if PLC and PFO play a role in this avoidance. We found C. perfringens was not killed by J774-33 cells or mouse peritoneal macrophages under aerobic or anaerobic conditions. Using electron microscopy, we showed that C. perfringens could escape the phagosome of J774-33 and mouse peritoneal macrophages. We believe the ability of C. perfringens to survive in the presence of macrophages is due to its ability to escape the phagosome. Using a variety of inhibitors of specific receptors, we identified those used by J774-33 cells to phagocytose C. perfringens. The scavenger receptor, mannose receptor(s), and complement receptor (CR3) were involved in the phagocytosis of C. perfringens. To determine if PFO or PLC were involved in the ability of C. perfringens to survive in the presence of macrophages, we constructed C. perfringens strains lacking these toxins. The ability of C. perfringens to survive in the presence of J774-33 cells is dependent on PFO, while survival in mouse peritoneal macrophages is dependent on PFO and PLC. The ability of C. perfringens to escape the phagosome of J774-33 cells and mouse peritoneal macrophages is mediated by either PFO or PLC. Using a mouse model, we found that PFO and PLC were necessary for C. perfringens to survive in vivo using infectious doses 1000 times lower than those required to initiate a gangrene infection. We propose that PFO and PLC play a critical role in the survival of C. perfringens during the early stages of gangrene infections, when phagocytic cells are present and bacterial numbers are low. / Ph. D.

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. / Polymorphonuclear neutrophils (PMN) are innate immune cells rapidly recruited to the inflammatory sites where they play a major role during the acute phase response but also contribute to resolution and repair. They can also modulate the adaptive response by interacting with lymphocytes (Ly) or dendritic cells (DC) via soluble mediators or cell-cell contacts. Activated PMN release Neutrophil Extracellular Traps (NETs) that could play a role in this context. NETs are decondensed chromatin fibers associated with granule and cytoplasmic proteins. As they are mainly involved in host defense against infection, they contribute to some autoimmune and inflammatory diseases. The aim of this work was to evaluate NET effects on DC maturation in an inflammatory context where PMN and DC co-exist, thus bridging innate and adaptive immunity. We first developed a model to induce, isolate and characterize NETs from human PMN. Calcium ionophore A23187 was chosen to induce NETs and the restriction enzyme AluI allowed the recovery of heterogeneous-sized fragments of NETs. Some of their components were quantified (DNA, elastase, histone 3, in particular) and we found that they retained their bactericidal activity. These NETs samples thus constitute a new and important biological tool to study their effects on immune cells or tissues. In the second part of this work, we found that isolated NETs were able to down-regulate LPS-induced moDC maturation as evidenced by the expressions of HLA-DR, CD80, CD83, CD86 and cytokine release (TNFα, Il-6, IL-12, Il-23). Moreover, the presence of NETs during moDC maturation lead to a decrease capacity of these moDC to induce T lymphocyte proliferation and modulated polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and decreasing Th1 cytokines (INFγ) and Th17 (IL- 17). Interestingly, moDC migration capacity was not modified when moDC maturation was done in the presence of NETs. In summary, these data suggest that NETs could downregulate DC activation. NETs produced by activated PMN could thus participate to the regulation of inflammation.

La Polyarthrite Rhumatoïde est caractérisée par une synovite à l’origine de lésions progressives ostéo-articulaires induites par les formes réactives de l’oxygène (ROS) produites par la NADPH oxydase des polynucléaires neutrophiles (PMN). La NADPH oxydase des phagocytes, est formée d’un centre catalytique membranaire, le cytochrome b558, sur lequel vient s’associer des protéines cytosoliques régulatrices (p67phox, p47phox, p40phox et Rac1/2). Nous avons étudié la spécificité de l’interaction entre la (6-phosphofructokinase 2) et de la 6PGDH (6-phosphogluconate déshydrogénase) et la NADPH oxydase des PMN. D’autre part, nous avons caractérisé les domaines de l’hétérocomplexe S100A8/A9 impliqués dans l’activation de la NADPH oxydase phagocytaire. Par ailleurs, une étude de la signature protéique dans le liquide synovial a été menée afin de rechercher l’empreinte de l’activation du PMN dans la PR.Après stimulation par le PMA, la 6PGDH et la PFK2 co-imunoprécipitent avec les facteurs cytosoliques p67phox, p47phox and p40phox. Les expériences de microscopie confocale suggèrent une co-localisation de ces deux enzymes du métabolisme du glucose avec la NADPH oxydase, dans des micro-domaines membranaires : les radeaux lipidiques. La 6PGDH est impliquée dans l’activation de la NADPH oxydase phagocytaire en élevant la concentration du NADPH cytosolique mais également en augmentant l’affinité de cette enzyme pour son substrat, le NADPH. PFK2 est l’enzyme majeure de la régulation de la glycolyse, voie est essentielle pour la production d’ATP du PMN. L’utilisation du complexe S100A8/A9 et de protéines chimères de fusion nous a permis de révéler que la partie C-terminale de S100A8 est impliquée dans la liaison avec le cytochrome b558 et l’activation de la NADPH oxydase phagocytaire. In vivo, le profil protéique du liquide articulaire de PR a révélé l’empreinte de l’activation du PMN dans cette pathologie avec une surexpression des protéines S100A8 et S100A9. Une production ectopique de S100A8/A9 par les synoviocytes de type fibroblastique a été mise en évidence.En conclusion, la 6PGDH, la PFK2 et l’hétérodimère S100A8/A9 sont de nouveaux partenaires d’activation de la NADPH oxydase des phagocytes. Dans la PR, l’activation des PMNs conduit à la sécrétion de S100A8/A9 qui semblent constituer à la fois des biomarqueurs pertinents, mais également des cibles thérapeutiques potentielles. / Rheumatoid Arthritis (RA) is caused by an inflammation of the synovial membrane leading to progressive joint destruction and deformation, related to the production NADPH oxidase related-reactive oxygen species (ROS) production. The phagocyte NADPH oxidase is a multi-protein complex formed by a catalytic core, i.e. the transmembrane cytochrome b558 and cytosolic regulators (p67phox, p47phox, p40phox and Rac1/2). We aimed at better analyzing the NADPH oxidase activation through the evaluation of the specificity of the interaction with 6PGDH or PFK2 and through the further analysis of the association with the S100A8/A9 heterocomplex. The RA-specific protein profiling was conducted in order to determine whether a PMN activation fingerprint could be revealed among RA specific proteins.Upon PMA stimulation, both 6PGDH and PFK2 co-imunoprecipitated with cytosolic factors p67phox, p47phox and p40phox. At the plasma membrane level, confocal microscopy experiments suggested a co-localization of either 6PGDH or PFK2 with the phagocyte NADPH oxidase in lipid rafts. 6PGDH enhanced the phagocyte NADPH oxidase activity by both improving the availability of cytosolic NADPH content and by increasing the affinity of the NADPH oxidase for its substrate. PFK2 also augmented the NADPH oxidase activity. PFK2 modulated the ATP concentration available for the phosphorylation of the phagocyte NADPH oxidase components and for the NDP Kinase related-Rac activation. The generation of truncated S100A8/S100A9 heterodimer chimera could reveal that the C-terminal region of S100A8 is involved in both the interaction and the activation of the phagocyte NADPH oxidase.In vivo, synovial fluid of RA patients was remarkably labelled with the PMN activation fingerprint. S100A8 and S100A9 proteins clearly distinguished RA synovial fluid from osteoarthritis and non RA-synovial fluids. An ectopic production of S100A8/S100A9 was shown in RA fibroblast like synoviocyte.In conclusion, 6PGDH, PFK2 and S100A8/A9 proteins are surrogate activating partners of the phagocyte NADPH oxidase. In RA, the activation of PMNs leads to the release of S100A8/A9 proteins which may constitute interesting biomarkers and promising therapeutic targets.

Les médiateurs lipidiques de l’inflammation dont le leucotriène B4 (LTB4) et le facteur d’activation plaquettaire (PAF) permettent la régulation de la migration des neutrophiles polymorphonucléaires (PMNs) et l’extravasation plasmatique au site inflammatoire. Afin de déterminer leurs rôles dans la régulation de la migration des PMNs au site inflammatoire, nous avons étudié leur effet potentiellement coopératif en utilisant une approche pharmacologique à l’aide d’antagonistes sélectifs des récepteurs du LTB4 et du PAF dans un modèle d’inflammation dermique chez le lapin. Les résultats montrent un effet inhibiteur additif des antagonistes des deux médiateurs lipidiques, lorsque utilisés de façon concomitante, sur la migration des neutrophiles induite par le LTB4, le PAF et aussi sur des médiateurs non-chimiquement apparentés comme le facteur nécrosant des tumeurs (TNFα), ainsi que sur l'inhibition de l’extravasation plasmatique induite par le leucotriène D4, suggérant un rôle régulateur des récepteurs du LTB4 et du PAF dans la migration des PMNs au site inflammatoire. Nous avons déterminé le rôle de ces médiateurs dans la régulation de la migration des PMNs en réponse à une ischémie-reperfusion des membres inferieurs chez le lapin. Les résultats appuient l’hypothèse selon laquelle le LTB4 et le PAF exercent un rôle important dans l’accumulation des PMNs au site inflammatoire. En effet l’administration concomitante des antagonistes des récepteurs de ces deux médiateurs lipidiques a réduit de façon significative la migration des PMNs aux poumons, intestins et foie. Nos résultats contribuent à élucider le rôle du LTB4 et du PAF dans la régulation de l’extravasation des PMNs et du plasma au site inflammatoire. / Inflammatory lipid mediators including leucotriene B4 (LTB4) and platelet activating factor (PAF) regulate the trafficking of polymorphonuclear neutrophils (PMNs) and plasma extravasation at inflammatory sites. To delineate their role in regulating PMNs extravasation, we studied the effect of PAF and/or LTB4 selective receptor antagonists in dermal inflammation induced by a variety of agonists in a rabbit bioassay model. The results show that there is an additive inhibitory effect when the two antagonists are used concomitantly on PMNs dermal accumulation induced by LTB4 and PAF, as well by chemically unrelated agonists including TNFα, in addition to inhibiting plasma extravasation induced by LTD4. These results support a regulatory role of LTB4 and PAF in regulating PMNs trafficking and plasma extravasation at inflammatory sites. Next, we studied the regulatory role of lipid mediators in regulating PMNs trafficking in response to hind limb ischemia-reperfusion. The results show that the administration of both PAF and LTB4 receptor antagonists reduced significantly PMNs migration to the lung, the liver and the intestine. Our results contributed to elucidate the role of LTB4 and PAF in the regulation of PMNs migration and oedema formation at inflammatory sites.

La leishmaniose viscérale est une maladie systémique mortelle en l’absence de traitement. Elle est due aux protozoaires Leishmania donovani et L. infantum, parasites des phagocytes mononucléés, capables d’envahir les organes lymphoïdes et le foie. Le contrôle de l’infection hépatique repose sur la mise en place d’une réponse granulomateuse efficace, promue par une réponse immunitaire Th1, dans un environnement tissulaire Th2. L’objectif de ce travail était l'étude du rôle d’une cytokine Th2 récemment décrite, l’IL-33, dans cette réponse hépatique complexe encore partiellement incomprise. Des dosages d’IL-33 sur des sérums de patients et la détection de cellules IL-33+ dans le foie d’un patient rennais ont placé l’IL-33 comme un biomarqueur possible de la maladie active. L’IL-33 étant exprimée dans les cellules étoilées du foie au cours d’hépatites chroniques, ces cellules ont été exposées à L. donovani. Leur permissivité aux leishmanies sans toxicité apparente ni perturbation de leurs propriétés fonctionnelles, ainsi que la persistance des leishmanies sur une culture de plusieurs semaines, nous ont conduit à proposer les cellules étoilées comme cellules sanctuaires possibles pour les leishmanies viscérotropes, contribuant donc potentiellement au portage asymptomatique. En revanche, elles ne sont pas apparues comme une source majeure d'IL-33 au cours de la leishmaniose viscérale. Chez des souris C57BL/6 et BALB/c infectées par L. donovani, l'IL-33 a été observée dans des cellules ne s'apparentant pas à des cellules étoilées, et principalement localisées dans les granulomes. Des cellules exprimant son récepteur ST2 ayant été également observées dans le foie, un rôle de l’axe IL-33/ST2 a été recherché. Les résultats obtenus chez des souris BALB/c déficientes en ST2 ou traitées par de l'IL-33 recombinante suggèrent que l'IL-33 régule négativement l'expression de cytokines Th1 (IL-12, IFN-γ) et l'infiltrat de neutrophiles et monocytes dans le foie, limitant ainsi le contrôle de la charge parasitaire. Ainsi, l'IL-33 semble être un facteur de susceptibilité pour la leishmaniose viscérale. En parallèle, des travaux entrepris sur des souris C57BL/6 infectées par L. donovani suggèrent de possibles rôles différentiels de l'IL-33 en fonction de l'environnement immunitaire inhérent au fond génétique de l'hôte. / Visceral leishmaniasis is a life-threatening systemic disease caused by Leishmania protozoans, L. donovani and L. infantum, which invade mononuclear phagocytes in the lymphoid organs and the liver. The control of the hepatic parasite burden depends on the granuloma formation, which is favored by a Th1 immune response in a Th2 tissue microenvironment. The aim of this work was to study the role of the recently described Th2 cytokine IL-33 in this complex immune response, which remains partially misunderstood. IL-33 dosages in different patient sera and IL-33+ cells detected in the liver of a patient from Rennes suggested that IL-33 could be a biomarker for active visceral leishmaniasis. As IL-33 was described in hepatic stellate cells during chronic hepatitis, these cells were exposed to L. donovani in primary culture. The cell permissivity to L. donovani and the parasite persistence during a long term culture led us to propose hepatic stellate cells as a new type of sanctuary cells, which could partially explain asymptomatic carriage. However, these cells were apparently not the main source of IL-33 during visceral leishmaniasis. In infected BALB/c and C57BL/6 mice, IL-33 was detected in the liver in non stellate cells preferentially localized in granulomas. The presence of cells expressing its specific receptor ST2 in the liver led us to explore the role of the IL-33/ST2 axis. BALB/c mice deficient in ST2 or treated with recombinant IL-33 and infected with L. donovani revealed that IL-33 downregulates the expression of Th1 key cytokines (IL-12, IFN-γ) and the recruitment of neutrophils and monocytes. Finally, IL-33 acts as a susceptibility factor during visceral leishmaniasis. Besides, the model of L. donovani infected C57BL/6 mice deficient in IL-33 or treated with recombinant IL-33 suggests possible differential roles of IL-33 depending on the immune environment related to the host genetic background.

Estudos em nosso laboratório caracterizaram camundongos B10.A como susceptíveis e camundongos A/J como resistentes à infecção pulmonar pelo P. brasiliensis. Para investigar o papel das células PMN na paracoccidioidomicose (PCM) pulmonar, camundongos B10.A e A/J foram depletados destas células através da inoculação in vivo por via intraperitoneal (i.p.) do anticorpo monoclonal anti-células PMN e infectados pela via intratraqueal (i.t.) com um milhão de leveduras viáveis. Camundongos-controle receberam doses equivalentes de IgG normal de rato. A depleção de granulócitos diminuiu o tempo de sobrevida dos animais B10.A, mas não dos animais A/J. Quando comparados com os animais não depletados, camundongos resistentes apresentaram aumento da carga fúngica no pulmão somente no dia 7 pós-infecção. Ao contrário, camundongos susceptíveis depletados de PMN apresentaram números mais elevados de células leveduriformes no pulmão, fígado e baço nos dias 7, 15, 30 e 120 pós-infecção, com relação aos seus grupos-controle tratados com IgG. A depleção dos granulócitos, entretanto, não alterou as reações de hipersensibilidade do tipo tardio (HTT) desenvolvidas por ambas as linhagens de animais. Considerando a resposta imune humoral, a depleção de células PMN levou à maior produção de anticorpos específicos em animais B10.A (Ig Total, IgG1, IgA e IgG3) e em animais A/J (Ig Total, IgG2a, IgG2b e IgG3). A depleção também alterou o padrão de citocinas pulmonares. Nos animais B10.A-tratados foram encontradas concentrações mais elevadas de IL-12 aos 15 dias e de IL-4 aos 120 dias pós-infecção, em comparação aos animais controle. Níveis de IL-12 significativamente mais altos foram detectados no grupo de animais A/J-depletados aos 7 e 120 dias e o IFN-γ foi detectado em níveis mais elevados em todo o curso da doença. Então, a depleção de PMN induz níveis mais altos de anticorpos e um ambiente mais pró-inflamatório no local da infecção. De acordo com esses dados, pudemos verificar que os neutrófilos são células importantes na defesa do hospedeiro à infecção pelo P.brasiliensis. Entretanto, o efeito protetor desta população celular depende do patrimônio genético do hospedeiro e é mais marcante na linhagem susceptível de camundongos. Neste trabalho também investigamos o efeito da depleção in vivo de leucócitos PMN na imunidade adquirida e protetora desenvolvida pela pré-imunização de animais B10.A. Assim, os camundongos foram previamente imunizados pela via s.c., depletados ou não de células PMN e desafiados i.t. com 1 milhão de células leveduriformes. Não foram detectadas diferenças significativas na contagem de fungos viáveis do pulmão, fígado e baço, entre os grupos imunizados tratados ou não com o AcM anti-PMN. A depleção não alterou a produção de anticorpos específicos, porém aumentou significativamente a síntese de IL-3, bem como a reatividade de HTT. Portanto, nossos resultados mostraram que, diferentemente da imunidade natural, os leucócitos PMN não exercem um papel protetor na fase adquirida da resposta imune à infecção com o P.brasiliensis. / Previous studies in our laboratory defined susceptible (B10.A) and resistant (A/J) mice to pulmonary P.brasiliensis infection. To investigate the role of PMN cells in pulmonary PCM, resistant and susceptible mice were depleted in vivo of these cells by intraperitoneal (i.p.) injection of a granulocyte-depleting monoclonal antibody and infected intratracheally (i.t) with one million yeast cells. Control mice received equivalent doses of normal rat IgG. PMN depletion decreased survival times of B10.A, but not of A/J infected mice. When compared with the non-depleted counterparts, resistant mice presented increased fungal loads in the lung only at day 7 after infection. On the contrary, PMN-depleted susceptible mice presented higher number of yeast cells in the lung, liver and spleen at days 7, 15, 30 and 120 after infection than their IgG-treated controls. PMN cells depletion, however, did not alter the DTH reaction developed by both mouse strains. Regarding humoral immune response, PMN cells depletion caused increased production of specific antibodies in B10.A (Total Ig, IgG1, IgA and IgG3) and A/J (Total Ig, IgG2a, IgG2b and IgG3) mice. Levels of pulmonary cytokines were also altered after PMN depletion. B10.A-treated mice presented increased levels of IL-12 and IL-4 at days 15 and 120 post-infection, respective/y. In A/J-depleted mice, augmented levels of IL-12 were detected at days 7 and 120 after infection; IFN-γ, however, was produced in higher levels during whole course of infection. Thus, PMN depletion induces higher levels of specific antibodies and enhanced pro-inflammatory milieu at the site of infection. As a whole, our data on PMN depletion at the onset of infection showed that neutrophils are important cells in host defense to P.brasiliensis infection. However, the effect of PMN depletion depends on the genetic background of the host and has a more pronounced effect in the susceptible strain of mice. We have also assessed the effect of in vivo depletion of the leukocytes on the acquired phase of immunity developed by B10.A mice previously immunized by the subcutaneous (s.c.) route were depleted or not of PMN cells and challenged i.t. with one million yeast cells. No differences were detected in the CFU counts in the lung, liver and spleen between untreated and PMN depleted vaccinated mice. PMN depletion also did not alter the production of specific antibodies but enhanced IL-3 synthesis as well as DTH reactivity. In conclusion, our results showed that, differently from natural immunity, PMN cells do not play a protective role in the acquired phase of immune response to P.brasiliensis infection.

O recrutamento de neutrófilos para o sítio da infecção é um evento crucial para o combate aos microrganismos e sobrevivência na sepse. A migração destes polimorfonucleares é dirigida através de um gradiente quimiotático por meio do reconhecimento de quimiocinas por receptores acoplados a proteína G (GPCRs), os quais são regulados por quinases específicas (GRKs). Estudos prévios demonstraram que na sepse ocorre uma falência na migração de neutrófilos para o foco infeccioso em função da dessensibilização de receptores quimiotáticos via GRKs induzida pela ativação de receptores toll-like (TLRs), TLR2 e TLR4. Apesar de a ausência de TLR9 em células dendriticas ter sido relacionada a maior sobrevivência de camundongos sépticos, o papel do TLR9 atuando diretamente em neutrófilos não foi avaliado. Objetivando preencher esta lacuna, propôs-se avaliar o papel direto de TLR9 na falência de migração de neutrófilos na sepse. Os camundongos TLR9-/- apresentaram maior sobrevivência a sepse polimicrobiana avaliada por meio do modelo de ligadura e perfuração do ceco (CLP). A deficiência de TLR9 também acarretou em aumento na migração de neutrófilos para o foco da infecção, menor seqüestro de neutrófilos no pulmão, bem como, menor número de bactérias no lavado peritoneal e sangue. A ativação de TLR9 por oligodeoxinucleotídeo contendo o dinucleotídeo CpG não metilado (ODN CpG) nos neutrófilos reduziu a quimiotaxia destes em direção a quimiocina CXCL2 e expressão do receptor quimiotático CXCR2. Além disso, neutrófilos estimulados com ODN CpG apresentaram aumento na expressão da quinase tipo 2 relacionada a receptores acoplados a proteína G (GRK2). Dessa forma, a ativação de TLR9 em neutrófilos circulantes no sangue é prejudicial na sepse por reduzir a quimiotaxia destes para o foco da infecção ao induzir a dessensibilização de CXCR2 via GRK2. / The recruitment of neutrophils to the site of infection is a crucial event for combating the microorganisms and survival on sepsis. The neutrophil migration is directed by a chemotactic gradient through the recognition of chemokines by G protein-coupled receptors (GPCRs), which are regulated by specific kinases (GRKs). Previous studies have shown a failure of neutrophil migration into infectious focus on sepsis due to chemotactic receptor desensitization via GRKs induced by activation of toll- like receptors (TLRs), TLR2 and TLR4. Despite the absence of activation of TLR9 in dendritic cells have been related to increase survival of septic mice, the role of TLR9 acting directly on neutrophils was not evaluated. We proposed to verify the direct role of TLR9 in the failure of neutrophil migration on sepsis. The TLR9 knockout mice (TLR9-/-) showed high survival to polymicrobial sepsis using cecal ligation and puncture model (CLP). TLR9-/- mice had high neutrophil migration to the focus of infection, low neutrophil sequestration in the lung, as well as, few bacteria in the peritoneal exudates and blood. The activation of TLR9 by oligodeoxinucleotide containing unmethylated dinucleotide CpG (CpG ODN) in neutrophils also reduced chemotaxis toward CXCL2 and the expression of chemokine receptor CXCR2. In addition, neutrophils stimulated with CpG ODN showed increased expression of kinase-related G protein-coupled receptor type 2 (GRK2). Thus, the activation of TLR9 in blood circulating neutrophils is harmful on sepsis by reducing their chemotaxis into the site of the infection by inducing CXCR2 desensitization via GRK2.

One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.

Estudos em nosso laboratório caracterizaram camundongos B10.A como susceptíveis e camundongos A/J como resistentes à infecção pulmonar pelo P. brasiliensis. Para investigar o papel das células PMN na paracoccidioidomicose (PCM) pulmonar, camundongos B10.A e A/J foram depletados destas células através da inoculação in vivo por via intraperitoneal (i.p.) do anticorpo monoclonal anti-células PMN e infectados pela via intratraqueal (i.t.) com um milhão de leveduras viáveis. Camundongos-controle receberam doses equivalentes de IgG normal de rato. A depleção de granulócitos diminuiu o tempo de sobrevida dos animais B10.A, mas não dos animais A/J. Quando comparados com os animais não depletados, camundongos resistentes apresentaram aumento da carga fúngica no pulmão somente no dia 7 pós-infecção. Ao contrário, camundongos susceptíveis depletados de PMN apresentaram números mais elevados de células leveduriformes no pulmão, fígado e baço nos dias 7, 15, 30 e 120 pós-infecção, com relação aos seus grupos-controle tratados com IgG. A depleção dos granulócitos, entretanto, não alterou as reações de hipersensibilidade do tipo tardio (HTT) desenvolvidas por ambas as linhagens de animais. Considerando a resposta imune humoral, a depleção de células PMN levou à maior produção de anticorpos específicos em animais B10.A (Ig Total, IgG1, IgA e IgG3) e em animais A/J (Ig Total, IgG2a, IgG2b e IgG3). A depleção também alterou o padrão de citocinas pulmonares. Nos animais B10.A-tratados foram encontradas concentrações mais elevadas de IL-12 aos 15 dias e de IL-4 aos 120 dias pós-infecção, em comparação aos animais controle. Níveis de IL-12 significativamente mais altos foram detectados no grupo de animais A/J-depletados aos 7 e 120 dias e o IFN-γ foi detectado em níveis mais elevados em todo o curso da doença. Então, a depleção de PMN induz níveis mais altos de anticorpos e um ambiente mais pró-inflamatório no local da infecção. De acordo com esses dados, pudemos verificar que os neutrófilos são células importantes na defesa do hospedeiro à infecção pelo P.brasiliensis. Entretanto, o efeito protetor desta população celular depende do patrimônio genético do hospedeiro e é mais marcante na linhagem susceptível de camundongos. Neste trabalho também investigamos o efeito da depleção in vivo de leucócitos PMN na imunidade adquirida e protetora desenvolvida pela pré-imunização de animais B10.A. Assim, os camundongos foram previamente imunizados pela via s.c., depletados ou não de células PMN e desafiados i.t. com 1 milhão de células leveduriformes. Não foram detectadas diferenças significativas na contagem de fungos viáveis do pulmão, fígado e baço, entre os grupos imunizados tratados ou não com o AcM anti-PMN. A depleção não alterou a produção de anticorpos específicos, porém aumentou significativamente a síntese de IL-3, bem como a reatividade de HTT. Portanto, nossos resultados mostraram que, diferentemente da imunidade natural, os leucócitos PMN não exercem um papel protetor na fase adquirida da resposta imune à infecção com o P.brasiliensis. / Previous studies in our laboratory defined susceptible (B10.A) and resistant (A/J) mice to pulmonary P.brasiliensis infection. To investigate the role of PMN cells in pulmonary PCM, resistant and susceptible mice were depleted in vivo of these cells by intraperitoneal (i.p.) injection of a granulocyte-depleting monoclonal antibody and infected intratracheally (i.t) with one million yeast cells. Control mice received equivalent doses of normal rat IgG. PMN depletion decreased survival times of B10.A, but not of A/J infected mice. When compared with the non-depleted counterparts, resistant mice presented increased fungal loads in the lung only at day 7 after infection. On the contrary, PMN-depleted susceptible mice presented higher number of yeast cells in the lung, liver and spleen at days 7, 15, 30 and 120 after infection than their IgG-treated controls. PMN cells depletion, however, did not alter the DTH reaction developed by both mouse strains. Regarding humoral immune response, PMN cells depletion caused increased production of specific antibodies in B10.A (Total Ig, IgG1, IgA and IgG3) and A/J (Total Ig, IgG2a, IgG2b and IgG3) mice. Levels of pulmonary cytokines were also altered after PMN depletion. B10.A-treated mice presented increased levels of IL-12 and IL-4 at days 15 and 120 post-infection, respective/y. In A/J-depleted mice, augmented levels of IL-12 were detected at days 7 and 120 after infection; IFN-γ, however, was produced in higher levels during whole course of infection. Thus, PMN depletion induces higher levels of specific antibodies and enhanced pro-inflammatory milieu at the site of infection. As a whole, our data on PMN depletion at the onset of infection showed that neutrophils are important cells in host defense to P.brasiliensis infection. However, the effect of PMN depletion depends on the genetic background of the host and has a more pronounced effect in the susceptible strain of mice. We have also assessed the effect of in vivo depletion of the leukocytes on the acquired phase of immunity developed by B10.A mice previously immunized by the subcutaneous (s.c.) route were depleted or not of PMN cells and challenged i.t. with one million yeast cells. No differences were detected in the CFU counts in the lung, liver and spleen between untreated and PMN depleted vaccinated mice. PMN depletion also did not alter the production of specific antibodies but enhanced IL-3 synthesis as well as DTH reactivity. In conclusion, our results showed that, differently from natural immunity, PMN cells do not play a protective role in the acquired phase of immune response to P.brasiliensis infection.

O recrutamento de neutrófilos para o sítio da infecção é um evento crucial para o combate aos microrganismos e sobrevivência na sepse. A migração destes polimorfonucleares é dirigida através de um gradiente quimiotático por meio do reconhecimento de quimiocinas por receptores acoplados a proteína G (GPCRs), os quais são regulados por quinases específicas (GRKs). Estudos prévios demonstraram que na sepse ocorre uma falência na migração de neutrófilos para o foco infeccioso em função da dessensibilização de receptores quimiotáticos via GRKs induzida pela ativação de receptores toll-like (TLRs), TLR2 e TLR4. Apesar de a ausência de TLR9 em células dendriticas ter sido relacionada a maior sobrevivência de camundongos sépticos, o papel do TLR9 atuando diretamente em neutrófilos não foi avaliado. Objetivando preencher esta lacuna, propôs-se avaliar o papel direto de TLR9 na falência de migração de neutrófilos na sepse. Os camundongos TLR9-/- apresentaram maior sobrevivência a sepse polimicrobiana avaliada por meio do modelo de ligadura e perfuração do ceco (CLP). A deficiência de TLR9 também acarretou em aumento na migração de neutrófilos para o foco da infecção, menor seqüestro de neutrófilos no pulmão, bem como, menor número de bactérias no lavado peritoneal e sangue. A ativação de TLR9 por oligodeoxinucleotídeo contendo o dinucleotídeo CpG não metilado (ODN CpG) nos neutrófilos reduziu a quimiotaxia destes em direção a quimiocina CXCL2 e expressão do receptor quimiotático CXCR2. Além disso, neutrófilos estimulados com ODN CpG apresentaram aumento na expressão da quinase tipo 2 relacionada a receptores acoplados a proteína G (GRK2). Dessa forma, a ativação de TLR9 em neutrófilos circulantes no sangue é prejudicial na sepse por reduzir a quimiotaxia destes para o foco da infecção ao induzir a dessensibilização de CXCR2 via GRK2. / The recruitment of neutrophils to the site of infection is a crucial event for combating the microorganisms and survival on sepsis. The neutrophil migration is directed by a chemotactic gradient through the recognition of chemokines by G protein-coupled receptors (GPCRs), which are regulated by specific kinases (GRKs). Previous studies have shown a failure of neutrophil migration into infectious focus on sepsis due to chemotactic receptor desensitization via GRKs induced by activation of toll- like receptors (TLRs), TLR2 and TLR4. Despite the absence of activation of TLR9 in dendritic cells have been related to increase survival of septic mice, the role of TLR9 acting directly on neutrophils was not evaluated. We proposed to verify the direct role of TLR9 in the failure of neutrophil migration on sepsis. The TLR9 knockout mice (TLR9-/-) showed high survival to polymicrobial sepsis using cecal ligation and puncture model (CLP). TLR9-/- mice had high neutrophil migration to the focus of infection, low neutrophil sequestration in the lung, as well as, few bacteria in the peritoneal exudates and blood. The activation of TLR9 by oligodeoxinucleotide containing unmethylated dinucleotide CpG (CpG ODN) in neutrophils also reduced chemotaxis toward CXCL2 and the expression of chemokine receptor CXCR2. In addition, neutrophils stimulated with CpG ODN showed increased expression of kinase-related G protein-coupled receptor type 2 (GRK2). Thus, the activation of TLR9 in blood circulating neutrophils is harmful on sepsis by reducing their chemotaxis into the site of the infection by inducing CXCR2 desensitization via GRK2.