Le choc anaphylactique est une réaction allergique systémique qui survient en quelques minutes et pouvant être fatale. Mon travail de thèse s’articule autour de deux projets dont la finalité est de mieux comprendre le mécanisme physiopathologique. La première partie de ce travail consiste à étudier in vivo chez la souris les contributions des récepteurs Fc aux IgG (FcγRs), des cellules effectrices et des médiateurs contribuant dans un modèle d’anaphylaxie systémique passive induit par une sous-classe particulière d’IgG : des IgG1, des IgG2a ou des IgG2b monoclonales dirigées contre un même antigène. Cette étude a permis de démontrer que le FcγRIII, les neutrophiles et les monocytes/macrophages sont les acteurs majoritaires quelque soit la sous-classe d’IgG de souris ; en revanche, la participation des basophiles ainsi que la contribution relative des médiateurs histamine et PAF sont dépendantes de la sous-classe d’IgG utilisée. La deuxième partie de ce travail consiste à étudier plus particulièrement la population plaquettaire dans un modèle de souris humanisées. Contrairement à la souris, les plaquettes humaines expriment un FcγR, le FcγRIIA déjà identifié comme acteur clé de l’anaphylaxie. Un modèle de choc allergique induit par des IgG humaines dans des souris transgéniques pour le FcγRIIA m’a permis de tester l’hypothèse suivant laquelle les plaquettes participent à l’initiation et/ou à la propagation de la réaction. Ce modèle a permis de mettre en évidence une thrombocytopénie sévère, des complexes plaquettes-leucocytes circulants et de montrer que le transfert de plaquettes ou de leur surnageant restaure les signes cliniques du choc allergique. / Anaphylaxis is a systemic hyperacute allergic reaction that occurs within minutes and can be fatal. The aim of my PhD project is to investigate the physiopathological mechanisms underlying anaphylaxis induction. The first part of my work focused on the contribution of FcγRs, effector cells and mediators in passive murine models of systemic anaphylaxis induced by the different subclasses of mouse specific IgG ; directed against the same antigen: IgG1, IgG2a or IgG2b. This study demonstrated that FcγRIII, neutrophils and monocytes/macrophages are the key players of anaphylaxis induction whatever the mouse IgG subclasses used. On the contrary, basophil participation and the relative contribution of histamine and PAF are IgG subclass dependent. The second part of this work examined the role of platelets in anaphylaxis using a humanized mouse model. Opposing the murine situation, human platelets express an IgG receptor, FcγRIIA. This receptor has already been identified as a key player in anaphylaxis. Using aggregated human IgG to induce anaphylaxis in mice transgenic for FcγRIIA, we tested our hypothesis that platelets contribute to the initiation and/or the propagation of this reaction. Anaphylaxis in this model was accompanied by a severe thrombocytopenia, the presence of circulating platelet-leukocyte complexes and activated platelets. I further demonstrated that the transfer of platelets or their activated supernatent into resistant mice restored features of anaphylactic shock.

No description available.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes. / Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103ΔexoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous injectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103-infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-α in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli and interalveolar septa. The tissue factor-dependent procoagulant activity detected in BALF from PA103-infected mice did not depend on decreased production of tissue factor pathway inhibitor (TFPI) production, but was associated with increased concentration of plasminogen activator inhibitor-1 (PAI-1). To investigate the role of platelet activating factor (PAF) in PAI-1 release, we compared the activity of the enzyme PAF-acetylhydrolase (PAF-AH) in BALF from control and infected mice, and we observed that PAF-AH activity was significantly elevated in BALF from PA103-infected animals. Mice treatment with a PAF inhibitor prior to infection resulted in a significant reduction of PAI-1 concentration, as well as of BALF total leukocytes and procoagulant activity. In vitro, ExoU induced higher expression of PAI-1 m RNA and increased release of PAI-1 protein in supernatants from A549 epithelial respiratory cell cultures. A549 cell treatment with an anti-PAF receptor prior to infection significantly reduced PAI-1 concentration in supernatants from cells infected with the wild type strain. These results show a novel mechanism by which a baterial product can favor a prothrombotic activity and ExoU production by infecting P. aeruginosa isolates may have prognostic implications for patients.

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-selectina em sua superfície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-α nos LBAs. A análise imunohistoquímica mostrou intensa deposição de fibrina nos alvéolos e septos interalveolares. A atividade pró-coagulante dependente do fator tissular detectada nos LBAs dos camundongos infectados com PA103 foi independente da produção do inibidor da via de ativação do fator tissular (TFPI), mas associada ao aumento da produção do inibidor do ativador do plasminogênio-1 (PAI-1). Para investigar a participação do fator de ativação plaquetária (PAF) na liberação de PAI-1, foi pesquisada a atividade da enzima PAF-acetil-hidrolase (PAF-AH) nos LBAs dos camundongos. A atividade de PAF-AH apresentou-se significativamente elevada nos LBA dos camundongos infectados com PA103. O tratamento dos animais com um inibidor do PAF, antes da infecção, resultou na diminuição significativa das concentrações de PAI-1 e de leucócitos totais, bem como da atividade pró-coagulante dos LBAs. In vitro, ExoU induziu maior expressão do RNA mensageiro de PAI-1 e maior liberação da proteína PAI-1 nos sobrenadantes de células epiteliais respiratórias da linhagem A549. O tratamento das células A549 com um anticorpo anti-receptor de PAF, antes da infecção, reduziu significativamente a concentração de PAI-1 nos sobrenadantes de células infectadas com a cepa selvagem. Estes resultados demonstraram um novo mecanismo de virulência de P. aeruginosa através da atividade pró-trombótica de ExoU e a possibilidade de utilização da identificação de ExoU em isolados clínicos de pacientes graves como um marcador prognóstico para estes pacientes. / Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103ΔexoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous injectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103-infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-α in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli and interalveolar septa. The tissue factor-dependent procoagulant activity detected in BALF from PA103-infected mice did not depend on decreased production of tissue factor pathway inhibitor (TFPI) production, but was associated with increased concentration of plasminogen activator inhibitor-1 (PAI-1). To investigate the role of platelet activating factor (PAF) in PAI-1 release, we compared the activity of the enzyme PAF-acetylhydrolase (PAF-AH) in BALF from control and infected mice, and we observed that PAF-AH activity was significantly elevated in BALF from PA103-infected animals. Mice treatment with a PAF inhibitor prior to infection resulted in a significant reduction of PAI-1 concentration, as well as of BALF total leukocytes and procoagulant activity. In vitro, ExoU induced higher expression of PAI-1 m RNA and increased release of PAI-1 protein in supernatants from A549 epithelial respiratory cell cultures. A549 cell treatment with an anti-PAF receptor prior to infection significantly reduced PAI-1 concentration in supernatants from cells infected with the wild type strain. These results show a novel mechanism by which a baterial product can favor a prothrombotic activity and ExoU production by infecting P. aeruginosa isolates may have prognostic implications for patients.