A ocorrência de trombose está freqüentemente associada com a presença de um ou mais fatores de riscos, os quais podem ser genéticos e/ou adquiridos, tais como as mudanças hormonais que ocorrem durante a gravidez, a terapia de reposição hormonal e o uso de contraceptivos hormonais combinados (CHC). A inflamação, por sua vez, é uma importante resposta do organismo às agressões e envolve vários mecanismos biológicos relacionados entre si e altamente regulados, tais como: coagulação, fibrinólise, ativação do sistema complemento (SC), antioxidação e regulação hormonal. Fisiologicamente, os sistemas complemento e da coagulação compartilham componentes. A ativação do fator XII da coagulação é controlada pela mesma proteína reguladora da ativação do sistema complemento, o inibidor de C1. A deficiência do inibidor de C1 leva a uma patologia conhecida como angioedema hereditário. No entanto, uma manifestação clínica similar ao angioedema tem sido descrita em mulheres que usam CHC ou recebem terapia de reposição hormonal com estrogênio (E). Esta influência do estrogênio na coagulação e no SC também é evidenciada pela ação regulatória do E sobre a expressão do fator XII e dos seus níveis plasmáticos. Considerando o efeito pleiotrópico do E, e as interações do SC e da hemostasia, o objetivo desse estudo foi avaliar o efeito de diferentes CHC sobre: a) a atividade hemolítica (AH) do SC e ativação das vias clássica/lectina e alternativa; b) a atividade opsonizante do SC em mediar o burst oxidativo dos neutrófilos; e c) a função dos receptores para complemento (CR) em mediar o burst oxidativo dos neutrófilos. Nós estudamos 5 CHC diferentes e observamos que a) drospirenona + 30g E mostrou uma tendência a aumentar o burst oxidativo mediado por CR; b) gestodeno + 20g E mostrou redução da capacidade opsonizante do SC; c) levonorgestrel + 30g E e gestodeno + 20g E promoveram uma redução no número de neutrófilos positivos para a expressão de CR1; d) drospirenona + 30g E e drospirenona + 20g E promoveram um aumento da AH da via clássica (VC) do SC; e) levonorgestrel + 30g E promoveu uma redução da AH da VC do SC; f) drospirenona + 30g E, gestodeno + 20g E e levonorgestrel + 30g E promoveram uma diminuição do nível sérico de C4d, produto da ativação das vias clássica/lectina do SC; g) levonorgestrel + 30g E apresentou um aumento da concentração sérica de inibidor de C1; h) nenhum dos CHC mostrou diferenças na ativação da via alternativa do SC. Os resultados mostram a importância de considerar os diferentes grupos de CHC nas comparações com o Grupo Controle, uma vez que algumas diferenças foram significativas apenas para CHC em particular. Estas observações podem contribuir para o entendimento dos mecanismos envolvidos na fisiopatologia dos processos inflamatórios associados ao uso de estrogênios. / The occurrence of thrombosis is often associated with the presence of one or more risk factors, which may be genetic and/or acquired, such as hormonal changes that occur during pregnancy, hormone replacement therapy and the use of combined hormonal contraceptives (CHC). The inflammation in turn, is an important body\'s response to the aggression and involves several biological mechanisms related and highly regulated, such as coagulation, fibrinolysis, activation of the complement system (CS), oxidation and hormonal regulation. Physiologically, the complement and coagulation systems share components. Activation of coagulation factor XII is controlled by the same regulatory protein activation of the complement inhibitor C1. The deficiency of C1 inhibitor leads to a condition known as hereditary angioedema. However, a clinical manifestation similar to angioedema has been reported in women using CHC or receiving hormone replacement therapy with estrogen (E). The influence of E on coagulation and the CS is also evidenced by the regulatory action of E on the expression of factor XII and its plasma levels. Considering the pleiotropic effects of E, and the interactions of CS and hemostasis, the goal of this study was to evaluate the effect of different CHC on: a) hemolytic activity (HA) CS and activation of classical/lectin and alternative pathways, b) the opsonizing activity of the CS in mediating the oxidative burst of neutrophils, and c) the function of receptors for complement (CR) in mediating the oxidative burst (OB) of neutrophils. We studied 5 different CHC and data showed: a) drospirenone + 30g E increase of the OB neutrophils mediated by CR; b) gestodene + 20g E had a reduced opsonizing ability; c) levonorgestrel + 30g E and gestodene + 20g E promoted a reduction of neutrophils positive for the expression of CR1, d) drospirenone + 30g E and drospirenone + 20g E promoted an increase in HA for classical pathway (CP); e) levonorgestrel + 30g E reduced the HA for CP; f) drospirenone + 30g E and gestodene + 20g E and levonorgestrel + 30g E reduced the serum level of C4d; g) levonorgestrel + 30g E showed an increase of the serum level of C1 inhibitor; h) none of CHC showed differences in activation of the alternative pathway in CS. The results show the importance of considering the different groups of CHC in comparison with the control group, since some differences were significant only for CHC in particular. These observations may contribute to the understanding of the mechanisms involved in the pathophysiology of inflammatory processes associated with estrogen use.

A peritonite neutrofílica induzida por glicogênio acarreta antinocicepção em camundongos submetidos ao teste de contorção abdominal, a qual é mediada por uma proteína ligante de cálcio, com peso molecular de 14 kDa, denominada S100A9. O objetivo do presente trabalho foi aprofundar o estudo sobre o envolvimento dos neutrófilos na antinocicepção induzida pelo glicogênio em ratos submetidos ao teste de pressão de pata e avaliar a expressão da proteína S100A9 nos tempos onde foi detectado esse efeito. O glicogênio induz antinocicepção em ratos entre 2 e 12 horas após sua injeção intraplantar. O pré-tratamento dos animais com fucoidina, um inibidor de selectinas, não só reverte o efeito antinociceptivo observado como também induz hiperalgesia entre 2 e 6 horas após a injeção do glicogênio. Após 8 horas do tratamento com glicogênio, a fucoidina apenas inibiu a antinocicepção induzida pelo agente inflamatório. A análise histológica demonstrou um aumento na migração de células polimorfonucleares entre 2 e 8 horas após a administração de glicogênio, a qual foi inibida pelo pré-tratamento com fucoidina. Tanto a injeção subcutânea como intraplantar de naloxona, um inibidor inespecífico de receptores opioides, não interferiram no efeito antinociceptivo induzido pelo glicogênio, em nenhum dos tempos avaliados. Quanto à expressão da S100A9, analisada por "Western Blotting", foi observado que as amostras obtidas do coxim plantar dos animais injetados com o glicogênio, entre 2 e 12 horas, apresentaram uma banda com peso molecular aproximado de 14 kDa, o qual equivale ao peso da proteína S100A9. A quantificação das bandas marcadas com o anticorpo anti-S100A9, nos tempos entre 2 e 12 horas, demonstrou um aumento significativo da expressão dessa proteína nas amostras obtidas dos animais tratados com glicogênio, em comparação com os tratados com salina. A injeção intraperitoneal de glicogênio induziu um aumento significativo no número total de células presentes na cavidade abdominal dos animais entre a 2º e a 12º hora após o tratamento, representado pelo aumento do número de células polimorfonucleares migradas. Os sobrenadantes obtidos do exsudato peritoneal entre 2 e 12 horas após a injeção de glicogênio, administrados via intraplantar, não só reverteram a hiperalgesia induzida pela carragenina (Cg) como induziram efeito antinociceptivo. Já, o sobrenadante obtido após 24 horas da injeção de glicogênio reverteu apenas parcialmente o efeito hiperalgésico induzido pela Cg. O tratamento do sobrenadante obtido 4 horas após a injeção do glicogênio com o anticorpo anti-S100A9 aboliu totalmente o efeito antinociceptivo observado com esse sobrenadante sobre a hiperalgesia induzida pela Cg. Esses dados sugerem que a antinocicepção acarretada pelo glicogênio em ratos submetidos ao modelo de pressão de pata é dependente da migração neutrofílica, não está relacionado à liberação de peptídeos opioides, mas possivelmente à secreção da proteína S100A9 por essas células. Ainda, os resultados obtidos com os sobrenadantes do exsudato peritoneal após a injeção do glicogênio, demonstram que durante a peritonite neutrofílica é secretada uma molécula capaz tanto de inibir a hiperalgesia acarretada pela carragenina quanto induzir antinocicepção, a qual possivelmente é a proteína S100A9. / Neutrophilic peritonitis induced by glycogen causes antinociception in mice subjected to the writhing test, which is médiated by a calcium-binding protein with a molecular mass of 14 kDa, named S100A9. The purpose of this study was to deepen the study on the involvement of neutrophils in glycogen-induced antinociception in rats subjected to the paw pressure test and evaluate the expression of S100A9 protein in time periods when this effect was detected. Glycogen induces antinociception in rats between 2 and 12 hours after intraplantar injection. Pretreatment of animals with fucoidan, a selectin inhibitor, not only reversed the antinociceptive effect, but also induces hyperalgesia between 2 and 6 hours after glycogen injection. Eight hours after treatment with glycogen, fucoidan only inhibited the antinociception induced by the inflammatory agent. Histological analysis showed an increased migration of polymorphonuclear cells between 2 and 8 hours after glycogen administration, which was inhibited by pretreatment with fucoidan. Both intraplantar and subcutaneous injection of naloxone, a nonspecific inhibitor of opioid receptors, did not affect the antinociceptive effect induced by glycogen at all evaluated times. In relation to the expression of S100A9 analyzed by Western blotting, it was observed that the samples obtained from the footpad injected with glycogen, between 2 and 12 hours, had a band with a molecular weight of 14 kDa, which is similar to molecular weight of S100A9. Relative quantification of the bands marked with anti-S100A9 in the time periods between 2 and 12 hours showed a significant increase in protein expression in samples obtained from animals treated with glycogen, compared with those treated with saline. Intraperitoneal injection of glycogen induced a significant increase in the total number of cells in the abdominal cavity of animals between 2 and 12 hours after treatment, represented by increased numbers of migrated polymorphonuclear cells. The supernatants obtained from peritoneal exudate between 2 and 12 hours after injection of glycogen, administered intraplantarly, not only reversed the hyperalgesia induced by carrageenan (Cg) but also induced antinociceptive effect. Already, the supernatant obtained 24 hours after injection of glycogen only partially reversed the hyperalgesic effect induced by Cg. The treatment of the supernatant obtained 4 hours after injection of glycogen with anti-S100A9 abolished the antinociceptive effect observed with the supernatant on hyperalgesia induced by Cg. These data suggest that antinociception entailed by glycogen in rats submitted to the paw pressure is dependent on neutrophil migration. Moreover, this effect is not related to the release of opioid peptides but possibly to the S100A9 protein secretion by these cells. In addition, the results obtained with the supernatants of peritoneal exudate after glycogen injection show that during neutrophilic peritonitis a molecule able to inhibit carrageenan-induced hyperalgesia is secreted and induce antinociception entailed by glycogen, which is possibly the S100A9 protein.

Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas. / Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.

A peritonite neutrofílica induzida por glicogênio acarreta antinocicepção em camundongos submetidos ao teste de contorção abdominal, a qual é mediada por uma proteína ligante de cálcio, com peso molecular de 14 kDa, denominada S100A9. O objetivo do presente trabalho foi aprofundar o estudo sobre o envolvimento dos neutrófilos na antinocicepção induzida pelo glicogênio em ratos submetidos ao teste de pressão de pata e avaliar a expressão da proteína S100A9 nos tempos onde foi detectado esse efeito. O glicogênio induz antinocicepção em ratos entre 2 e 12 horas após sua injeção intraplantar. O pré-tratamento dos animais com fucoidina, um inibidor de selectinas, não só reverte o efeito antinociceptivo observado como também induz hiperalgesia entre 2 e 6 horas após a injeção do glicogênio. Após 8 horas do tratamento com glicogênio, a fucoidina apenas inibiu a antinocicepção induzida pelo agente inflamatório. A análise histológica demonstrou um aumento na migração de células polimorfonucleares entre 2 e 8 horas após a administração de glicogênio, a qual foi inibida pelo pré-tratamento com fucoidina. Tanto a injeção subcutânea como intraplantar de naloxona, um inibidor inespecífico de receptores opioides, não interferiram no efeito antinociceptivo induzido pelo glicogênio, em nenhum dos tempos avaliados. Quanto à expressão da S100A9, analisada por "Western Blotting", foi observado que as amostras obtidas do coxim plantar dos animais injetados com o glicogênio, entre 2 e 12 horas, apresentaram uma banda com peso molecular aproximado de 14 kDa, o qual equivale ao peso da proteína S100A9. A quantificação das bandas marcadas com o anticorpo anti-S100A9, nos tempos entre 2 e 12 horas, demonstrou um aumento significativo da expressão dessa proteína nas amostras obtidas dos animais tratados com glicogênio, em comparação com os tratados com salina. A injeção intraperitoneal de glicogênio induziu um aumento significativo no número total de células presentes na cavidade abdominal dos animais entre a 2º e a 12º hora após o tratamento, representado pelo aumento do número de células polimorfonucleares migradas. Os sobrenadantes obtidos do exsudato peritoneal entre 2 e 12 horas após a injeção de glicogênio, administrados via intraplantar, não só reverteram a hiperalgesia induzida pela carragenina (Cg) como induziram efeito antinociceptivo. Já, o sobrenadante obtido após 24 horas da injeção de glicogênio reverteu apenas parcialmente o efeito hiperalgésico induzido pela Cg. O tratamento do sobrenadante obtido 4 horas após a injeção do glicogênio com o anticorpo anti-S100A9 aboliu totalmente o efeito antinociceptivo observado com esse sobrenadante sobre a hiperalgesia induzida pela Cg. Esses dados sugerem que a antinocicepção acarretada pelo glicogênio em ratos submetidos ao modelo de pressão de pata é dependente da migração neutrofílica, não está relacionado à liberação de peptídeos opioides, mas possivelmente à secreção da proteína S100A9 por essas células. Ainda, os resultados obtidos com os sobrenadantes do exsudato peritoneal após a injeção do glicogênio, demonstram que durante a peritonite neutrofílica é secretada uma molécula capaz tanto de inibir a hiperalgesia acarretada pela carragenina quanto induzir antinocicepção, a qual possivelmente é a proteína S100A9. / Neutrophilic peritonitis induced by glycogen causes antinociception in mice subjected to the writhing test, which is médiated by a calcium-binding protein with a molecular mass of 14 kDa, named S100A9. The purpose of this study was to deepen the study on the involvement of neutrophils in glycogen-induced antinociception in rats subjected to the paw pressure test and evaluate the expression of S100A9 protein in time periods when this effect was detected. Glycogen induces antinociception in rats between 2 and 12 hours after intraplantar injection. Pretreatment of animals with fucoidan, a selectin inhibitor, not only reversed the antinociceptive effect, but also induces hyperalgesia between 2 and 6 hours after glycogen injection. Eight hours after treatment with glycogen, fucoidan only inhibited the antinociception induced by the inflammatory agent. Histological analysis showed an increased migration of polymorphonuclear cells between 2 and 8 hours after glycogen administration, which was inhibited by pretreatment with fucoidan. Both intraplantar and subcutaneous injection of naloxone, a nonspecific inhibitor of opioid receptors, did not affect the antinociceptive effect induced by glycogen at all evaluated times. In relation to the expression of S100A9 analyzed by Western blotting, it was observed that the samples obtained from the footpad injected with glycogen, between 2 and 12 hours, had a band with a molecular weight of 14 kDa, which is similar to molecular weight of S100A9. Relative quantification of the bands marked with anti-S100A9 in the time periods between 2 and 12 hours showed a significant increase in protein expression in samples obtained from animals treated with glycogen, compared with those treated with saline. Intraperitoneal injection of glycogen induced a significant increase in the total number of cells in the abdominal cavity of animals between 2 and 12 hours after treatment, represented by increased numbers of migrated polymorphonuclear cells. The supernatants obtained from peritoneal exudate between 2 and 12 hours after injection of glycogen, administered intraplantarly, not only reversed the hyperalgesia induced by carrageenan (Cg) but also induced antinociceptive effect. Already, the supernatant obtained 24 hours after injection of glycogen only partially reversed the hyperalgesic effect induced by Cg. The treatment of the supernatant obtained 4 hours after injection of glycogen with anti-S100A9 abolished the antinociceptive effect observed with the supernatant on hyperalgesia induced by Cg. These data suggest that antinociception entailed by glycogen in rats submitted to the paw pressure is dependent on neutrophil migration. Moreover, this effect is not related to the release of opioid peptides but possibly to the S100A9 protein secretion by these cells. In addition, the results obtained with the supernatants of peritoneal exudate after glycogen injection show that during neutrophilic peritonitis a molecule able to inhibit carrageenan-induced hyperalgesia is secreted and induce antinociception entailed by glycogen, which is possibly the S100A9 protein.

A sepse é uma resposta inflamatória sistêmica resultante da inabilidade do sistema imune em controlar uma infecção, onde a taxa de sobrevida está associada ao recrutamento de neutrófilos para o local da infecção. Tem sido demonstrado que a expressão de receptores quimiotáticos pode ser alterada durante a sepse. Neutrófilos de animais naives respondem às quimiocinas CXC, mas são irresponsivos às quimiocinas CC. Entretanto, dados do nosso laboratório mostram que a expressão de CXCR2 é reduzida na sepse, prejudicando a migração de neutrófilos para o foco da infecção. Além disso, ocorre o aparecimento do receptor CCR2 nos neutrófilos, levando à infiltração dessas células no pulmão e outros órgãos. Nesse contexto, o nosso objetivo foi investigar a possível expressão do receptor CCR5 em neutrófilos e seu papel na evolução da sepse. Demostramos que animais sham-operados expressam baixos níveis de CCR5 e altos níveis de CXCR2. Entretanto, sob a condição de sepse experimental induzida por ligação e perfuração do ceco (CLP), neutrófilos circulantes e que migraram para a cavidade peritoneal expressam altos níveis de CCR5 em paralelo com a internalização de CXCR2. Além disso, animais deficientes para CCR5 (CCR5-/-), submetidos à CLP, apresentam diminuição na taxa de sobrevida, redução na migração de neutrófilos para o foco da infecção, aumento da disseminação bacteriana, aumento no infiltrado de neutrófilos no pulmão e aumento nos níveis de marcadores de lesão do coração e rim, quando comparados com animais selvagens (WT). Adicionalmente, a incubação de neutrófilos isolados da medula óssea com LPS aumentou a expressão de CCR5 e os tornou responsivos à MIP-1? (ligante de CCR5), induzindo quimiotaxia. Também demonstramos que o receptor CCR5 possui importante papel durante a adesão de neutrófilos ao endotélio vascular para posterior migração. Em conjunto, esses resultados indicam que durante a CLP, o aumento da expressão de CCR5 em neutrófilos tem um papel protetor, visto que animais CCR5-/- sépticos apresentam reduzida migração de neutrófilos para o foco infeccioso, inflamação sistêmica acentuada e baixa taxa de sobrevivência. / Sepsis is a systemic inflammatory response resulted from the inability of the innate immune system to control infections, being the survival rate associated to the recruitment of neutrophils to the infection site. It has been demonstrated that chemokine receptors expression profile can be altered under sepsis conditions. Neutrophils from naïve mice respond to CXC chemokines, but are usually unresponsive to CC chemokines. However, data from our laboratory show that CXCR2 expression is down regulated, impairing the neutrophil migration to infection focus. In addition, CCR2 appears on the surface of neutrophils, mediating the accumulation of these cells in the lung and other organs. In this context, we aimed to investigate the possible expression of CCR5 receptor on neutrophils and its role on sepsis evolution. We showed that neutrophils from sham mice express high levels of CXCR2 and low levels of CCR5. However, during experimental sepsis, induced by cecal ligation and puncture (CLP), in parallel with CXCR2 internalization, neutrophils from the circulation or from the peritoneal cavity express higher levels of CCR5. Interestingly, deficient mice for the CCR5 receptor (CCR5-/-), undergone to CLP show decreased survival rate, reduction in the neutrophil migration to the site of infection, increase in the numbers of bacteria, increase in the neutrophil infiltration in lung and heart and increase in the levels of markers of injuries in heart and kidney, when compared to wild type mice (WT).In addition, the incubation of bone marrow derivedneutrophils with LPS enhances the expression of CCR5 and renders them responsive to CCL4 (a ligant of CCR5)-induced chemotaxis. Moreover, we demonstrated that CCR5 receptor has an important role during neutrophil adhesion to the vascular endothelium before transmigration. Together, these results indicate that during CLP-induced sepsis, the increase of the expression of CCR5 on neutrophils plays a host protective role, since CCR5-/- mice under sepsis present reduced neutrophil migration to infection focus, high systemic inflammation and low survival rate.

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

Identificamos em 12 pacientes deficientes de CD40L uma incidência de infecções fúngicas de 83%, incluindo os primeiros casos de paracoccidioidomicose (PCM) e aspergilose. As células dendríticas imaturas (DCs) dos pacientes apresentaram expressão reduzida das moléculas HLA-DR, CD80 e CD86 e as DCs maduras (mDCs) pulsadas com os fungos P. brasiliensis ou C albicans apresentaram baixa expressão das moléculas HLA-DR e CD80. As mDCs dos pacientes secretaram baixa concentração de IL-12 e alta de IL-10. Nas co-culturas das mDCs com os linfócitos autólogos, observamos que a produção de IFN-<font face=\"Symbol\">g foi reduzida e a produção de IL-4 e IL-5 foi aumentada. Após tratamento com CD40L solúvel os defeitos observados foram revertidos. Os neutrófilos dos pacientes falharam na atividade fungicida em resposta ao P. brasiliensis, reduzida produção de H2O2 e sinalização via TLR1/TLR2 e TLR2/TLR6 foram observadas. Falhas na resposta de DCs e neutrófilos sugerem que esses são mecanismos imunopatológicos subjacentes à suscetibilidade às infecções fúngicas em pacientes deficientes de CD40L. / We identified 12 CD40L-deficient patients with an incidence of fungal infections of 83%, including the first case of paracoccidioidomycosis and aspergillosis. The immature DCs (iDCs) from the patients present reduced protein expression of HLA-DR, CD80 and CD86 and mature DCs (mDCs) pulsed with P. brasiliensis (mDCs) presented low expression of HLA-DR and CD80 molecules The mDCs from the patients secreted low levels of IL-12 and high of IL-10. In co-cultures of mDCs and autologous T lymphocytes we found low production of IFN-<font face=\"Symbol\">g and high production of IL-4 and IL-5. After treatment with soluble CD40L, the defects were reversed. The neutrophils from these patients showed impaired fungicidal activity in response P. brasiliensis and low production of H2O2. Additionally defective TLR1/TLR2 and TLR2/TLR6 signaling were observed. Failure in DCs and neutrophils responses are imunophatological mechanisms underlying the suscetibility to fungal infections in CD40L-deficient patients.

La drépanocytose est une maladie génétique du globule rouge, due à une mutation ponctuelle du gène β de la chaine de l’hémoglobine. Néanmoins, la physiopathologie de la maladie va bien au-delà des anomalies érythrocytaires, avec notamment un dysfonctionnement vasculaire et leucocytaire qui font toute la complexité de la maladie. Au cours de ce travail, nous avons étudié le rôle de l’endothéline-1, peptide vaso-constricteur impliqué de nombreuses pathologies notamment vasculaires, sur l’activation et l’adhérence des polynucléaires neutrophiles dans la drépanocytose. Nous avons travaillé à la fois sur un modèle murin de drépanocytose (les souris SAD) et sur des échantillons sanguins de patients. Sur le modèle animal, nous avons réalisé des expériences de microscopie intravitale, permettant de tester in vivo l’effet des antagonistes des récepteurs ETA et ETB à l’endothéline sur le recrutement des polynucléaires neutrophiles. Sur les échantillons de sang de patients drépanocytaires, nous avons testé in vitro l’adhérence en flux des neutrophiles à l’endothélium vasculaire en réponse au blocage des récepteurs ETA et ETB. Nous avons enfin étudié l’expression des récepteurs ETA et ETB à la surface des neutrophiles et les voies de signalisations découlant de leur activation.Sur le modèle de souris drépanocytaire, l’inhibition des récepteurs ETA, mais surtout ETB permet de limiter le recrutement leucocytaire important provoqué par un stimulus inflammatoire. Ces résultats confirment le rôle d’ETA et celui, plus inattendu, d’ETB dans toutes les étapes d’adhérence des polynucléaires neutrophiles et dans leur transmigration tissulaire en contexte drépanocytaire.Sur des échantillons humains, nous avons confirmé le rôle crucial d’ETB dans l’adhérence des polynucléaires neutrophiles. Nous avons également confirmé la présence de récepteurs ETA et ETB à la surface des neutrophiles. Le récepteur ETB active une voie de signalisation responsable d’une mobilisation du calcium intra cytoplasmique, mais indépendante de l’activation de la phospho-inositide 3-kinase. Enfin, nous avons montré la capacité de ces cellules à synthétiser et excréter elles-mêmes de l’endothéline-1, pérennisant ainsi probablement la réponse inflammatoire et le recrutement leucocytaire. En conclusion, notre travail a permis de mettre en évidence le rôle important du récepteur ETB dans le recrutement des polynucléaires neutrophiles dans la drépanocytose. Ces données suggèrent que les antagonistes des récepteurs à l’endothéline pourraient être bénéfiques en prévention des phénomènes vaso-occlusifs chez les patients drépanocytaires. / Sickle cell disease is a genetic disorder affecting red blood cells, due to a point mutation in the β chain of the hemoglobin gene. However, the pathophysiology of the disease goes well beyond the erythrocyte abnormalities, including vascular and white blood cell dysfunctions that contribute to the complexity of the disease. In this project, we investigated the role of endothelin-1, a powerful vasoconstrictor peptide involved in many vascular diseases, on activation and adhesion of neutrophil in sickle cell disease.We worked on both a mouse model of sickle cell disease (SAD mice) and blood samples from patients. In mice, we performed intravital microscopy experiments, to test the in vivo effect of endothelin receptor antagonists ETA and ETB on neutrophils recruitment. On blood samples from patients with sickle cell disease, we tested in vitro adhesion of neutrophils to vascular endothelium in response to the blocking of ETA and ETB receptors. Finally, we studied the expression of ETA and ETB receptors on neutrophils and the signaling pathways resulting in their activation.In our mouse model of sickle cell disease, the inhibition of both endothelin receptors ETA and ETB limits the major leukocyte recruitment caused by an inflammatory stimulus. These results confirm the role of ETA and the more unexpected important role of ETB in all stages of neutrophil adhesion and transmigration in sickle cell context.On human samples, we demonstrated the crucial role of ETB in neutrophils adhesion. We also confirmed the expression of ETA and ETB receptors on neutrophils. ETB receptor activates a signaling pathway responsible for intracytoplasmic calcium mobilization, but not involving the activation of phosphoinositide 3-kinase. Finally, we have shown the ability of neutrophils to synthesize and secrete endothelin-1, which can contribute to sustain the inflammatory stimulation and increased leukocyte recruitment.In conclusion, our work has highlighted the important role of ETB receptor in the recruitment of neutrophils in sickle cell disease. These data suggest that the antagonists of endothelin could be beneficial in prevention of vaso-occlusive events in sickle cell patients.

Les neutrophiles jouent un rôle central dans la première ligne de défense mise en place contre les agents pathogènes. L’une de leur fonction essentielle est caractérisée par la capacité à rejoindre rapidement la circulation sanguine et de migrer vers les sites inflammatoires, où ils seront activés et exerceront des fonctions effectrices extrêmement puissantes. Ces processus doivent être finement contrôlés afin de moduler leur effet dévastateur dans l'organisme. L'hypothèse de travail de ce travail de thèse est que le CD31, un corécepteur ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif), exprimé de manière constitutive à la surface des neutrophiles, joue un rôle essentiel dans la régulation du recrutement et l'activation des neutrophiles au sein des sites inflammatoires. Nous avons pu mettre en évidence que le CD31 membranaire était clivé lors de l’activation neutrophilaire, et pouvait perdre une grande partie de son domaine extracellulaire, tout en conservant sa portion membranaire. Afin d’étudier le rôle du gain de fonction du CD31 neutrophilaire, nous avons utilisé un peptide synthétique homotypique ayant la capacité de se lier spécifiquement au domaine persistant du CD31 clivé, et permettant le maintien de sa fonction moléculaire. Parallèlement, des souris invalidées pour CD31 ont été utilisées dans divers modèles expérimentaux d'inflammation aiguë afin d’étudier le rôle de sa suppression. Dans leur ensemble, nos données suggèrent que le CD31 joue un rôle complexe et important dans la régulation du recrutement des neutrophiles au sein des sites d'inflammation aiguë. Nous avons pu montrer que des lymphocytes isolés à partir de souris invalidées pour CD31 adhèrent plus fortement aux cellules endothéliales activées, bien que leur progression hors du vaisseau est retardée. A l’inverse, l’ajout du peptide agoniste CD31 diminue leur adhérence, tout en favorisant leur détachement. La littérature rapporte que des neutrophiles isolés à partir de souris déficientes en CD31 sont incapables de migrer hors des vaisseaux, ce qui suggère un rôle important du CD31 dans les processus de transmigration. Cependant, les mécanismes sous-jacents restent inconnus. Nous avons pu montrer qu’au sein du front de migration neutrophilaire, le CD31 empêche l’ouverture des intégrines associées en clusters, par le biais du recrutement de phosphatase. Ce processus abouti à un diminution de l’adhérence neutrophilaires à l'endothélium. A l’inverse, la polarisation du CD31 à l'uropode semble être nécessaire à leur détachement. Cette dernière étape implique la fermeture des intégrines et semble être favorisée par la signalisation CD31. Dans la deuxième partie de ce travail de thèse, nos résultats suggèrent que le CD31 régule également l'activation des neutrophiles au sein des sites inflammatoires. Nous avons tenté de comprendre le rôle du CD31 dans le burst oxydatif et la dégranulation des neutrophiles. SHIP1, une phosphatase impliquée dans la signalisation de CD31, découple l'activation neutrophilaire induite par le récepteur Fc. En utilisant le modèle auto-immun de glomérulonéphrite, nous avons pu montrer que la signalisation du CD31 module les effets délétères causés par les protéases neutrophilaires. Dans ce modèle l’ajout du peptide agoniste CD31 prévient les lésions induites, alors que les souris déficientes en CD31 possèdent des neutrophiles plus activés et des lésions glomérulaires plus importantes. En conclusion, les résultats des expériences effectuées lors de ce travail de thèse suggèrent (1) que CD31 peut moduler le trafic neutrophilaire en contrôlant les intégrines et (2) que CD31 établit un seuil immunologique impliqué dans l'activation des neutrophiles, limitant leur activation excessive pendant la phase aiguë de l'inflammation / Neutrophils play a crucial role in the first line of defense against noxious agents. Central to their function, is their ability to rapidly exit the circulation and migrate to the site of inflammation where they get activated and exert extremely efficient effector functions. A tight regulation of these processes is mandatory to restrict their devastating effects in the organism. The working hypothesis of my thesis was that CD31, an ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) co-receptor constitutively expressed at the surface of neutrophils, plays a critical role in the regulation of the recruitment and activation of neutrophils at sites of inflammation. We found that, upon cell activation, the surface CD31 molecules underwent a cleavage and shedding of a large part of the extracellular portion, but leaving a portion that lingers at the cellular surface. A synthetic homotypic peptide that has the ability to specifically bind and maintain functional molecular clusters of CD31 was used to study the role of CD31 in neutrophils biology, by “gain-of-function” approach, whereas CD31 genetically invalidated mice were used as a “loss-of-function” approach both in vitro and in vivo, in experimental mouse models of acute inflammation. Altogether our data unveil a complex and important role for CD31 in the regulation of the recruitment of the neutrophils at the site of acute inflammation. We show that CD31-/- neutrophils established stronger adhesion on activated endothelial cells but their progression out of the vessel was delayed. At the opposite, the use of the CD31 agonist peptide prevented the attachment while it favored the detachment of the migrating neutrophils. It was previously observed that CD31-/- neutrophils are unable to migrate out of the vessels and it had been proposed that CD31 is necessary for the transmigration but the underlying mechanism had remained unknown. We show that, by co-clustering with the integrins at the migration front, the phosphatases recruited and activated by CD31 prevented their opening and hence inhibited the adhesion of neutrophils on the endothelium. At the opposite, CD31 polarization at the uropod appeared to be crucial for the detachment of the neutrophils. We found that this step, that relies upon the closure of the integrins, was favored by the CD31 signaling. Furthermore, our findings showed that CD31 not only regulates the recruitment but also the activation of neutrophils at sites of inflammation. Indeed, in the second part of my thesis, I extensively studied how the engagement of CD31 also regulates the oxidative burst and the degranulation of neutrophils. The phosphatase involved in the function of CD31 is reported to be the SHIP1 inositol phosphatase which uncouples the Fc-receptor dependent neutrophil activation. By using the autoimmune glomerulonephritis model that develops antibody-dependent acute inflammation, we showed that CD31 signaling is crucial for limiting the collateral damage caused by neutrophil-derived proteases: the CD31 agonist peptide prevented the damage whereas the induction of the disease in mice invalidated for the CD31 resulted in greater neutrophil activation and glomerular damage. In conclusion, the results of the experiments performed during my thesis work suggest that CD31 is an integrin regulator that controls neutrophil trafficking. In addition, CD31 sets an immunologic threshold for neutrophil activation that limits their excessive activation during the acute phase of inflammation

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.