O objetivo geral deste trabalho foi avaliar a influência dos leucócitos do colostro bovino na imunidade inata, desenvolvimento da microbiota intestinal e ocorrência de diarreias em bezerras Holandesas recém-nascidas. Para isso, 20 bezerras Holandesas foram acompanhadas nos seguintes momentos: antes da mamada do colostro (D0); 1-2 (D2); 7 (D7); 14 (D14); 21 (D21) e 28 (D28) dias após nascimento e foram distribuídas em dois grupos experimentais: grupo COL+ recebeu colostro fresco (4L) proveniente de suas respectivas mães; e grupo COL- recebeu colostro congelado e acelular (4L) de vacas doadoras. Capítulo 1 O objetivo deste capítulo foi avaliar a presença e influência dos leucócitos do colostro na colonização do trato gastrintestinal. Para isso, amostras de colostro foram semeadas em TSA e MacConkey para CBT e CCT; e amostras de fezes em agar sangue, MacConkey e Salmonella-Shiguella. Bezerras apresentaram maior frequência de diarreia no D14 (COL+=78%; COL-=60%). O grupo COL- apresentou maior CBT e CCT/mL (3,93x106 ufc/mL e 3,01x105 ufc/mL) em relação ao COL+ (0,94x106 ufc/mL e 0,78x105ufc/mL). Sobre as espécies bacterianas isoladas, as que foram mais frequentes no COL+ foram Proteus mirabilis (29,91%), Escherichia coli (28,04%), Citrobacter freundii (5,61%) e Staphylococcus spp (5,61%), no COL- foram Escherichia coli (28,70%), Proteus mirabilis (27,78%), Klebsiella pneumoniae (11,32%) e Morganella morganii (5,66%). O momento com maior isolamento em ambos os grupos foi o D2 (COL+ = 26 cepas; COL- = 27 cepas), período em que iniciou a diarreia nas bezerras. Não foi possível detectar diferenças entre as frequências de microrganismos entre os grupos COL+ e COL-. A administração de colostro COL+ e COL- não influenciou na proporção de bactérias aeróbias presentes nas fezes e na ocorrência de diarreia das bezerras durante o período neonatal. Capítulo 2 O objetivo deste capítulo foi avaliar a influência dos leucócitos do colostro na resposta imune inata em bezerras Holandesas. Os animais foram submetidos ao exame clínico, seguido da colheita das amostras sanguíneas para realização das provas laboratoriais. As concentrações de cortisol sérico e haptoglobina não apresentaram diferenças estatísticas, enquanto a concentração de ferro foi diferente entre os grupos no D7. No leucograma não foi detectada diferença para os leucócitos totais e neutrófilos absolutos, mas houve tendência entre os grupos no D7 para os valores de neutrófilos relativos. O marcador CH138+CD62L+ não apresentou diferença entre os grupos, apesar da maior expressão ter sido detectada no COL-. A expressão de CH138+CD62L- foi maior no COL+ no D14. A fagocitose e produção de H2O2 pelos neutrófilos sanguíneos apresentou perfil semelhante em relação ao S. aureus e E. coli entre os grupos, apesar do COL- apresentar maior intensidade de fagocitose do D7 ao D14. Houve tendência para o aumento da proporção de granulócitos liberando H2O2 basal no D14 para o COL-, entretanto o COL + apresentou tendência para maior intensidade da fagocitose no D21. A proporção de granulócitos (%), estimulados com S. aureus, liberando H2O2 foi maior no COL+ em D7, enquanto o estímulo com a E. coli não resultou em diferenças entre os grupos. A intensidade de fluorescência foi maior e gradual no COL+ quando as células foram estimuladas, porém não foram encontradas diferenças estatísticas. As células do colostro influenciaram no perfil sanitário apresentado pelas bezerras, observando-se maior intensidade de diarreias, menores teores de ferro sérico e anemias no grupo que recebeu colostro congelado. A migração dos granulócitos sanguíneos foi mais rápida e intensa no COL+ em relação ao COL-, após exposição natural aos patógenos causadores de diarreia. O índice de fagocitose (%) dos granulócitos apresentou semelhança entre os grupos, entretanto a intensidade da fluorescência foi mais intensa no COL-. Em contrapartida, o COL+ demonstrou maior habilidade em produzir H2O2 / The aim of this study was to evaluate the influence of bovine colostrum leukocytes in innate immunity, in the development of the intestinal microbiota and the occurrence of diarrhea in newborn Holstein calves. For this, 20 Holstein calves were followed in the following moments: before colostrum intake (D0); 1-2 (D2); 7 (D7); 14 (D14); 21 (D21) and 28 (D28) days after birth. They were divided into two experimental groups: COL+ group, which received fresh colostrum (4L) from their mothers; and COL-group which received frozen and acellular colostrum (4L) of donor cows. Chapter 1 -The aim of this chapter was to evaluate the presence and influence of colostrum leukocytes in the colonization of the gastrointestinal tract. For this, colostrum samples were seeded in TSA and MacConkey for TPC and TCC; and stool samples on blood agar, MacConkey and Salmonella-Shiguella. Calves presented higher frequency of diarrhea in D14 (COL+ = 78%; COL- = 60%). The COL- group showed higher TPC and TCC/mL (3,93x106 cfu/mL and 3,01x105 cfu/mL) compared to COL+ (0,94x106 cfu/mL and 0,78x105 cfu/mL). Regarding the isolated bacterial species, those that were more frequent in the COL+ were Proteus mirabilis (29.91%), Escherichia coli (28.04%), Citrobacter freundii (5.61%) and Staphylococcus spp (5.61%); in the COL-were Escherichia coli (28.70%), Proteus mirabilis (27.78%), Klebsiella pneumoniae (11.32%) and Morganella morganii (5.66%). The moment with more insolation in both groups was the D2 (COL+ = 26 strains; COL- = 27 strains), period in which started the diarrhea in calves. It was not possible to detect differences between the frequencies of microorganisms between the COL+ and COL- groups. The COL+ and COL- colostrum management did not influence the proportion of aerobic bacteria present in feces and the occurrence of diarrhea in calves during the neonatal period. Chapter 2 The aim of this chapter was to evaluate the influence of colostrum leukocytes in the innate immune response in Holstein calves. The animals were submitted to clinical examination, followed by blood samples collection to perform laboratory tests. The serum cortisol and haptoglobin concentrations did not show statistical differences while the iron concentration was different between groups at D7. The leukogram did not show difference to total leukocytes and absolute neutrophils, but there was a trend between groups at D7 for relative values of neutrophils. The CH138+CD62L+ marker showed no difference between groups, despite an increased expression was detected in COL-. The expression of CH138+CD62L- was greater in COL+ at D14. The phagocytosis and production of H2O2 by blood neutrophils showed similar profile in relation to S. aureus and E. coli between groups, although COL- presented greater phagocytosis intensity from D7 to D14. There was a trend for the increase of granulocyte proportion, releasing basal H2O2 at D14 for COL-, however COL+ presented trend to a higher intensity of phagocytosis at D21. The proportion of granulocytes (%) stimulated with S. aureus releasing H2O2 was greater in COL+ at D7, while stimulation with E. coli resulted in no differences between the groups. The fluorescence intensity was higher and gradual in COL+ when cells were stimulated; however, it was not detected statistical differences. The colostrum cells influenced in the health profile presented by the calves, with a higher intensity of diarrhea, lower levels of serum iron and anemia in the group that received frozen colostrum. The migration of blood granulocytes was faster and more intense in COL+ in relation to COL- after natural exposure to pathogens that cause diarrhea. The phagocytosis rate (%) of granulocytes showed similarity between groups, although the fluorescence intensity of COL- was more intense. In contrast, the COL+ demonstrated greater ability in producing H2O2

O sistema imune inato é organizado por processos complexos que envolvem os diversos tipos celulares que culminam na eliminação de uma variedade de microorganismos a fim de proteger o organismo de infecções. Os neutrófilos são células importantes neste sistema, sendo capazes de migrar rapidamente ao foco da infecção. Estas células reconhecem e fagocitam partículas estranhas e atuam no sentido de elimina-los através da produção de espécies reativas de oxigênio (EROs), proteínas antimicrobianas e enzimas proteolíticas dos grânulos, que são liberadas no fagossomo e no espaço extracelular. Entretanto, em algumas doenças inflamatórias e autoimunes têm sido encontradas lesões nos tecidos envolvendo a produção excessiva de EROs pelos neutrófilos, desencadeada por imunocomplexos (ICs) via receptores Fc gama (Fc) e de complemento (CR). Assim, a modulação da ativação destes receptores e da produção de EROs é importante na manutenção da homeostasia. Nas últimas décadas, flavonóides são considerados como metabólitos secundários promissores mostrando atividade antioxidante e imunomodulatória. Neste trabalho foi avaliado o efeito modulatório dos flavonóides galangina, kaempferol, quercetina e miricetina em algumas funções efetoras de neutrófilos humanos, como o metabolismo oxidativo estimulado via receptores de membrana Fc e/ou CR, fagocitose e atividade microbicida, além do efeito citotóxico, antioxidante e inibitório dessas substâncias sobre a atividade das enzimas mieloperoxidase (MPO) e NADPH oxidase. Os flavonóides analisados mostraram um efeito modulatório negativo no metabolismo oxidativo dos neutrófilos humanos na seguinte ordem: galangina> kaempferol> quercetina > miricetina. Embora a galangina tenha exercido maior atividade, sua ação antioxidante e inibitória na atividade das enzimas MPO e NADPH oxidase foi a menos efetiva dentre os flavonóides avaliados. Além disso, esta substância inibiu de maneira mais acentuada a resposta celular mediada por ambos os receptores Fc e CR quando comparado a estimulação destes receptores individualmente. As substâncias contendo o grupo catecol, quercetina e miricetina mostraram elevada eficiência na inibição da atividade das enzimas NADPH oxidase e MPO, e na redução do radical livre 2,2-difenil-1-picrilhidrazil (DPPH). De maneira geral, os flavonóides não interferiram no processo fagocítico e no mecanismo microbicida, e não induziram toxicidade e morte celular por apoptose ou necrose. Os resultados apresentados neste trabalho mostram uma possível aplicação destes flavonóides como fármacos de origem natural em doenças inflamatórias que apresentem acúmulo de neutrófilos, sendo uma ferramenta promissora para o tratamento efetivo destas doenças. / The innate immune system is composed of complex biological processes involving a variety of cell types that eliminate invading microorganisms to protect the host against infection. Neutrophils are key effector cells of this system, being able to migrate rapidly to infection sites. These cells recognize and engulf foreign particles by phagocytosis, and act to destroy them through production and release of reactive oxygen species (ROS), antimicrobial proteins and proteolytic granule enzymes, which are delivered to the phagossomes and to the extracellular environment. However, tissue damage related to excessive neutrophil ROS production triggered by immune complexes (ICs) via Fc-gamma (FcR) and complement receptors (CR) has been found in some inflammatory and autoimmune diseases. So, modulation of both the activation of these receptors and ROS generation are important to the maintenance of body homeostasis. In the last decades, flavonoids have been considered promising plant secondary metabolites with antioxidant and immunomodulatory activity. This study evaluated the modulator effect of four flavonoids (galangin, kaempferol, quercetin and myricetin) in some human neutrophil effector functions, such as the oxidative metabolism stimulated via FcR and CR classes of membrane receptors, phagocytosis and microbicidal activity, as well as the cytotoxic, antioxidant and inhibitory effect of these compounds in the MPO and NADPH oxidase activity. The flavonoids studied showed a negative modulator effect in the human neutrophils oxidative metabolism in the following ranking order: galangin > kaempferol > quercetin > myricetin. Although galangin had shown the highest activity, it was the least effective antioxidant and inhibitor of MPO and NADPH oxidase activity among the flavonoids tested herein. Furthermore, this compound inhibited more the cellular response mediated by both Fc and CR receptors than by each one of them individually. The flavonoids bearing the catechol group, quercetin and myricetin, had a high efficiency to inhibit NADPH oxidase and MPO activity, as well as to reduce the 2,2-diphenyl-1-picrylhidrazil (DPPH) free radical. In general, the flavonoids did not interfere in the phagocytic process and microbicidal mechanism, and also did not induce toxicity and cell death by apoptosis or necrosis. The group of results presented here provides a possible application of these flavonoids as drugs of natural source in inflammatory diseases with excessive neutrophil infiltration, being a promising tool for an effective treatment of these illnesses.

Avaliamos o papel dos leucotrienos (LTs), as vias de sinalização e o efeito da insulina na fagocitose via FcgR por macrófagos alveolares (MAs) de ratos sadios (RS) e diabéticos (RD). Vimos que a) MAs de RD fagocitam menos que os de RS; b) a fagocitose é dependente de LTs endógenos em RS mas não em RD; c) a adição de LTB4 ou LTD4 aos MAs em cultura aumenta a fagocitose em RS e RD; d) MAs de RS e RD produzem quantidades equivalentes de LTB4 e LTC4; e) a adição de insulina aos MAs aumenta a capacidade fagocitica em ambos os grupos; f) em RS, a fagocitose via FcgR induz fosforilação de Akt e PKC-d, que é amplificada por LTs endógenos, enquanto que em RD ocorreu fosforilação somente da PKC-d. A foforilação de Akt e PKC-d amplificada por LTs produzidos sob estímulo do FcgR em MAs de RS parece ser, de alguma forma, dependente da ação da insulina, pois MAs provenientes de RD fagocitam menos, a fagocitose não é dependente de LTs endógenos e o estímulo via FcgR não é capaz de ativar a Akt. / We evaluated the role of leukotrienes (LTs), the signaling pathways and the effect of insulin in phagocytosis via FcgR by alveolar macrophages (AMs) from healthy (HR) and diabetic (DR) rats. The results showed that: a) AMs from DR showed lower phagocytic capacity than AMs from healthy rats; b) the phagocytosis was dependent of endogenous LTs in AMs from HR but not DR; c) addition of LTB4 and LTD4 to cultures enhanced the phagocytosis by AMs from HR and DR; d) AMs from HR and DR rats produced similar levels of LTB4 and LTC4; e) addition of insulin to AMs enhanced the phagocytic capacity in HR and DR; f) in HR, the phagocytosis via FcgR induced Akt and PKC-d phosphorylation, which is amplified by endogenous LTs, whereas in DR, only PKC-d was phosphorylated. The phosphorylation of Akt and PKC-d, amplified by LTs produced under FcgR engagement in AMs from HR, seems to be, in a way, dependent of insulin action, because AMs from DR have lower phagocytic capacity, the phagocytosis is not dependent of endogenous LTs and the FcgR engagement is not capable to activate Akt.

As linhagens de camundongos selecionadas para a máxima (AIRmax) ou mínima (AIRmin) reatividade inflamatória aguda, demonstram diferenças quanto a capacidade de infiltrar neutrófilos. O projeto tem como objetivo caracterizar a atividade de macrófagos residentes ou induzidos com tioglicolato no exsudato peritoneal nestas linhagens. Nas 6h do estímulo, o tioglicolato induz migração de neutrófilos sendo o máximo de migração de macrófagos após 96h. Em ambas as linhagens, macrófagos induzidos por tioglicolato fagocitavam mais partículas de zimosan em relação a macrófagos residentes. Nos resultados pudemos observar que macrófagos da linhagem AIRmax respondem mais ao LPS em relação à expressão de TNF-a, IL-6, IL-12, IL-1b, TREM1, DAP12, e síntese de H2O2 e NO, que condiz com a alta inflamação dos AIRmax. Observamos que células residentes da linhagem AIRmin sintetizavam maiores quantidades de IL-10 e TGF-b em relação à linhagem AIRmax. Após o estímulo de tioglicolato, macrófagos da linhagem AIRmax produziam maiores quantidades de citocinas anti-inflamatórias. / Lines of mice genetically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction (AIR) demonstrated differences in the capacity to infiltrate neutrophil cells. The aim of this work is the characterization of the activity of resident or thioglicollate-induced macrophages in the peritoneal exudates in these mice. After 6h, thioglicollate induces the migration of neutrophils being the maximal macrophage migration achieved at 96h. On both lines, thioglicollate-induced macrophages showed higher phagocytic activity of zymosan particles than resident macrophages. Macrophages from AIRmax mice produced higher response with LPS, than AIRmin cells, regarding the expression of TNF-a, IL-6, IL-12, IL-1b, TREM1, DAP12, and synthesis of H2O2 and NO, which is consistent with the high inflammation selected phenotype of AIRmax mice. We observed that resident cells from AIRmin mice secret higher amounts of IL-10 and TGF-b than AIRmax cells. After thioglicollate stimulus, macrophages from AIRmax mice produced higher levels of the anti-inflammatory cytokines.

Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB4 e LTD4 aumentam a fagocitose via FcγR de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da PI3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via FcγR e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB4 envolveu predominantemente a via ERK1/2 e PKCα. E com menor intensidade da PKCδ. A ação do LTD4 envolveu exclusivamente a via p38 e PKCδ. Ambos leucotrienos utilizaram a PI3K para aumentar a fagocitose via FcγR. Na fagocitose de C.albicans, os macrófagos alveolares produziram LTB4. Tanto o LTB4 quanto o LTD4 endógenos contribuíram para a fagocitose da C.albicans que ocorre predominantemente via receptor manose. A fagocitose via receptor manose foi potencializada pela adição tahto do LTB4 quanto do LTD4 cγ de modo dependente da ativação da PKCδ e da PI3K. As vias da PKCα., ERK1/2 e p38 não estão envolvidas na potencialização da fagocitose de C.albicans por leucotrienos. O esclarecimento das moléculas sinalizadoras envolvidas na interação de macrófagos alveolares com patógenos poderá contribuir para o desenvolvimento de alvos específicos e refinados no controle e tratamento de infecções pulmonares. Analisados em conjunto, os resultados mostram que os leucotrienos são importantes moduladores das funções dos macrófagos alveolares, por aumentar a fagocitose tanto através dos receptores para Fcγ como do receptor manose. Além disso, os LTB4 e LTD4 ativam programas de sinalização distintos para potencializar a fagocitose via receptor Fcγ, enquanto na fagocitose via receptor manose os LTB4 e LTD4 usam as mesmas vias. / Leukotrienes enhance phagocytosis and microbicidal activity against several pathogens. In alveolar macrophages, LTB4 and cysteinyl LT both enhance Fcγ receptor - mediated phagocytosis. While both LTB4 and LTD4 enhances phagocytosis dependent on PKC activity, only LTB4 requires the protein tyrosine kinase syk to do so. However, the role of specific PKC isoforms, MAPKs (p38 and ERK1/2), and PI3K in mediating LT-enhanced FcγR-mediated phagocytosis is unknown. In addition, the importance of LT in the phagocytosis via other receptors is also understudied. In the present work we sought to determine the importance of the above kinases during IgG-opsonized phagocytosis and if both classes of LT enhance the ingestion of C. albicans; the receptors and the molecular mechanisms involved. Studies with isoform-selective inhibitors indicated that LTB4 effects were dependent on both PKC&#945. And PKCδ, while LTD4 effects were exclusively due to PKCδ activation. Although both exogenous LTB4 and LTD4 enhanced p38 and ERK1/2 activation, LTB4 required only ERK1/2, while LTD4 required only p38 activation. Activation by both LT was dependent on PI3K activation. It was found that exogenously added LTB4 and LTD4 both enhanced PKC& and PKCα. Phosphorylation during FcγR engagement. Regarding the studies concerning the importance of LT in the ingestion of C.albicans, both endogenous and exogenous LTB4 and LTD4 enhanced C.albicans phagocytosis. LT effects were dependent exclusively on PKCδ and P13K. We also demonstrated that the LT effect on C.albicans was due to the mannose receptor activation. Taken together, leukotrienes are potente immunomodulators of the phagocyte function, by enhancing phagocytosis both via FcγR and mannose receptor. Moreover, LT receptors activate different signaling programs to enhance FcγR-mediated phagocytosis, while the signaling elicited to enhance C.albicans ingestion is similar for both classes of LTs. Thus the nature of the signaling elicited by the phagocytic receptors dictates the signaling induced by LT.

Life events and one's ability to adapt to these events has significant effects on immune functioning. Immunosuppression has been related to a high magnitude of life stress and low adaptive ability. While studies have explored immune response in stressed individuals, no study has approached the area of prevention with low-immunity individuals. The purpose of this study was to investigate whether subjects who self-report stressful lives have lower immunity, and whether "low" immunity subjects under "high" stress could enhance phagocytic activity through biofeedback-assisted relaxation.

L’extension des activités humaines est responsable du rejet de molécules et de perturbations climatiques pouvant affecter la physiologie des organismes aquatiques. La moule zébrée possède des caractéristiques biologiques faisant d’elle une espèce intéressante en surveillance environnementale. Chez cet organisme, les hémocytes constituent une cible privilégiée pour la mise en place d’une approche multi-biomarqueurs. En effet, ces cellules à fonctionnalités multiples sont impliquées dans les grandes fonctions physiologiques de l’espèce et la régulation de l’homéostasie des individus. L’objectif de ce travail est de développer les outils analytiques permettant d’étudier les réponses hémocytaires de la moule zébrée. Les expérimentations menées ont permis de caractériser la structure des populations hémocytaires ainsi que leurs fonctionnalités propres en lien avec le processus de phagocytose. L’utilisation de ces biomarqueurs dans divers contextes indique une forte adaptabilité de l’espèce aux conditions environnementales. Les résultats montrent l’intérêt d’analyser les activités hémocytaires à l’échelle des sous populations comparativement à l’approche globale ne tenant pas compte de la diversité cellulaire. Il a été observé que certains facteurs comme le statut reproducteur ou l’espèce échantillonnée (D. polymorpha vs D. bugensis) constituent des facteurs de confusion importants. Il ressort également un positionnement fort du test de phagocytose en tant que marqueur de sensibilité aux contaminants. Ce travail constitue un ensemble de données voué à être utilisé dans des contextes multiples aussi bien en écotoxicologie qu’en écophysiologie. / Extension of human activities is responsible of molecule releases and climate changes that may affect physiology of aquatic organisms. The zebra mussel has biological traits making it an interesting species for environmental monitoring. In this organism, hemocyte cells constitute an interesting target to develop a multi-biomarker approach. These cells possess multiple functionalities and are involved in all major physiological functions of the species and in homeostasis regulation. The objective of this work was to develop analytical tools to study hemocyte responses of zebra mussels. Experiments allowed characterizing structure of hemocyte populations and their functionalities linked with phagocytosis process. Use of these biomarkers in various contexts indicated an important adaptation capacity of the species to environmental conditions. Results highlighted interest to analyze hemocyte activities at sub-population scale comparatively to global approach that does not consider hemocyte diversity. It was demonstrated that factor such as reproductive status or sampled species (D. polymorpha vs D. bugensis) constitute important confounding factors. Studies also demonstrated a strong positioning of phagocytosis assay as a sensitive marker to contaminants. This work constitutes a data set destined to be used in multiple contexts such as ecotoxicology or ecophysiology.

La résistance aux antibiotiques est un problème majeur en santé publique qui mène à développer de nouvelles stratégies thérapeutiques, notamment en ciblant des facteurs de virulence bactériens. MgtC est un facteur de virulence impliqué dans la survie intra-macrophagique chez plusieurs pathogènes intracellulaires. La protéine MgtC est également présente chez le pathogène extracellulaire Pseudomonas aeruginosa. De plus, un peptide MgtR a été identifié comme étant un antagoniste naturel potentiel de MgtC. Pour étudier le rôle de MgtC dans la virulence des mycobactéries, j'ai analysé un mutant mgtC de Mycobacterium marinum dans les modèles d'infection d'embryons de danio et de macrophages en culture. Cela a permis de mettre en évidence un nouveau rôle de MgtC au niveau de l'étape de phagocytose qui est augmentée avec le mutant mgtC. Chez P. aeruginosa, nous avons montré qu'un mutant mgtC est atténué dans l'embryon de danio et présente une sensibilité accrue à l'action bactéricide du macrophage. Par ailleurs, j'ai montré que les gènes mgtC de M. marinum et de P. aeruginosa sont régulés par le magnésium. De plus, l'expression de mgtC de P. aeruginosa est fortement induite dans les macrophages. Enfin, concernant les propriétés antagonistes de MgtR, des souches de Mycobacterium bovis BCG ou de P. aeruginosa exprimants mgtR semblent se comporter de la même manière que les mutants mgtC, suggérant des propriétés anti-virulence prometteuses pour MgtR et confirmant le choix de MgtC en tant que cible dans le cadre d'une stratégie anti-virulence. / Antibiotic resistance is a major problem in public health, which leads to develop new therapeutics strategies, including strategies targeting bacterial virulence factors. MgtC is a virulence factor involved in intramacrophage survival in several intracellular pathogens. MgtC protein is also present in extracellular pathogen Pseudomonas aeruginosa. Moreover, a peptide MgtR has been identified as a potential and natural antagonist of MgtC.To study the role of MgtC in mycobacterial virulence, I have analysed a Mycobacterium marinum mgtC mutant in zebrafish embryo and macrophage infection models. This approach allowed us to uncover a new role of MgtC in phagocytosis, which is increased with mgtC mutant. In P. aeruginosa, we have shown that a mgtC mutant is attenuated in zebrafish embryos. In ex vivo experiments, mgtC mutant is more sensitive to macrophage killing. In parallel, I have shown that M. marinum and P. aeruginosa mgtC genes are regulated by magnesium. In addition, expression of P. aeruginosa mgtC is highly induced in macrophages.Finally, regarding MgtR antagonistic properties, Mycobacterium bovis BCG or P. aeruginosa strains expressing mgtR appear to mimic the behaviour of mgtC mutants, suggesting promising anti-virulence properties for MgtR and supporting the choice of MgtC as a suitable target of an anti-virulence strategy.

Abstract Innate immunity provides the first line of defense against invading, pathogenic microorganisms in all multicellular organisms. The fruit fly Drosophila melanogaster has turned out to be an excellent model organism to elucidate mechanisms of innate immune responses because of the highly conserved intracellular signaling cascades mediating these ancient immune functions in flies and mammals. In the present study, RNA interference (RNAi) -based functional genomics were utilized to identify novel components of Drosophila’s immune reactions. Mediators of bacterial phagocytosis, nuclear factor kappa B (NF-κB) signaling and the antiviral RNAi pathway were screened in hemocyte-like S2 cells. Follow-up studies were executed in mammalian cells as well as in Drosophila larvae and adult flies to gain broader significance for the results. Seven novel components essential for efficient phagocytosis of bacteria were identified. Eater was defined as Drosophila’s most important phagocytic receptor showing novel epidermal growth factor (EGF)-repeat -based microbial recognition properties. Additionally, Abelson interacting protein (Abi), capping protein alpha (cpa), 14-3-3ζ, tousled-like kinase (tlk), CG2765 and CG15609 were determined as intracellular effectors of phagocytosis, the three former ones executing their evolutionarily conserved functions through remodeling of the actin cytoskeleton. Eater, together with Scavenger receptor class C, type I (Sr-CI), was demonstrated to be responsible for double-stranded RNA (dsRNA) uptake into S2 cells and, when ectopically expressed, into mammalian cells via clathrin-mediated endocytosis. Proteasome component Pros45 and RNA helicase Belle were established as mediators of the intracellular RNAi pathway, whereas essential roles in antimicrobial signaling via the immune deficiency (Imd) pathway were addressed for Inhibitor of apoptosis 2 (Iap2) and Tak1-associated binding protein (TAB). Iap2 and TAB were shown to affect nuclear translocation of NF-κB -like transcription factor Relish. The present study identifies several novel mediators of the Drosophila immune response and provides insight into mechanisms of fly host defense. As insects serve as vectors of human diseases (e.g. malaria), knowledge about Drosophila immune mechanisms may help to better understand the transmission and pathogenesis of these diseases and develop treatments to fight these infections. Additionally, knowledge gained from model organisms serves as valuable background information, often conducting human research into new tracks. / Tiivistelmä Synnynnäinen immuniteetti on elintärkeä puolustusjärjestelmä taudinaiheuttajia vastaan. Kodeissakin yleinen banaanikärpänen, Drosophila melanogaster, on osoittautunut erinomaiseksi synnynnäisen immuniteetin tutkimusmalliksi, erityisesti teknisesti yksinkertaisen ja eettisesti ongelmattoman geneettisen muunneltavuutensa ansiosta. On myös havaittu, että solunsisäiset, immunologisia signaaleja välittävät mekanismit ovat evoluutiossa hyvin säilyneitä. Hyvin usein samankaltaiset geenituotteet toimivat signaalinsiirtäjinä sekä kärpäsen että ihmisen soluissa. Tämän työn tarkoituksena oli RNA-häirintää (RNAi) sekä muita nykyaikaisia solu- ja molekyylibiologisia tutkimusmenetelmiä hyödyntäen tunnistaa uusia kärpäsen synnynnäiselle immuunipuolustukselle välttämättömiä geenituotteita. Bakteerien fagosytoosille, viruspuolustukselle ja tumatekijä nuclear factor kappa B:n (NF-κB) välittämälle signaloinnille välttämättömiä signalointimolekyylejä pyrittiin identifioimaan laajan mittakaavan RNA-häirintään perustuvilla seuloilla kärpäsen soluissa. Saatujen tulosten merkitystä nisäkkäiden immuunipuolustukselle tutkittiin myös hiiren soluissa. Seitsemän geenituotteen osoitettiin olevan bakteerien fagosytoosille tärkeitä kärpäsen soluissa. Aiemmin tuntematon geenituote, joka nimettiin Eateriksi, osoitettiin kärpäsen tärkeimmäksi bakteereja fagosytoivaksi reseptoriksi. Eaterin solun ulkoisen osan osoitettiin tunnistavan taudinaiheuttajia uudella epidermaalisen kasvutekijän (epidermal growth factor, EGF) kaltaisella toistosekvenssillä. Myös useiden solun tukirankaan, sytoskeletoniin, liittyvien proteiinien (Abi, cpa, 14-3-3ζ) sekä aiemmin vähemmän tunnettujen geenituotteiden (CG2765, CG15609, tlk) osoitettiin osallistuvan bakteerien fagosytoosiin. Näistä kolmen ensinmainitun immunologinen tehtävä havaittiin evoluutiossa säilyneeksi, kärpäsestä hiireen. Eaterin, yhdessä kärpäsen toisen scavenger reseptorin (Sr-CI) kanssa, havaittiin myös toimivan kaksijuosteisen RNA:n (dsRNA) reseptoreina kärpäsen soluissa, mahdollistaen helpon ja tehokkaan RNA-häirinnän. RNA-häirinnän, ja siten mahdollisesti myös viruspuolustuksen, välittäjiksi identifioitiin proteasomin alayksikkö Pros45 ja RNA-helikaasi Belle. Lisäksi Inhibitor of apoptosis 2 (Iap2) ja Tak1-associated binding protein (TAB) todettiin kärpäsen immune deficiency (Imd) signalointireitin komponenteiksi, jotka osallistuvat antimikrobisten peptidien tuotantoon välittämällä NF-κB:n kaltaisen kärpäsen transkriptiotekijän (Relish) siirtymisen tumaan aktivoimaan immuunipuolustusta välittävien geenien ilmentymistä. Tämän tutkimuksen tulokset valottavat banaanikärpäsen immuunipuolustuksen mekanismeja. Koska hyönteiset toimivat monien ihmisten infektiotautien välittäjinä, kärpäsen immuniteetin tuntemus luo mahdollisuuksia kehittää hoitoja näitä tauteja vastaan. Lisäksi malliorganismeista saatu tieto luo uusia teorioita ja näkökulmia, johtaen usein myös lääketieteellistä tutkimusta uusille raiteille.

La formation des circuits cérébraux, dont l’altération est associée à divers troubles neurologiques et psychiatriques, commence pendant l’embryogenèse. Les microglies sont les macrophages du cerveau et répondent à une inflammation ou une infection par une production de facteurs et une phagocytose active. En plus de leurs fonctions immunitaires, ces cellules ont été récemment impliquées dans l’émergence de troubles associés à des maladies neurodéveloppementales. Elles agissent également sur le remodelage post-natal des circuits neuronaux en phagocytant des synapses et des neurones immatures. Étant donné que les microglies colonisent précocement le cerveau embryonnaire, elles pourraient également participer au développement cérébral précoce. Mes travaux de thèse ont montré que les microglies embryonnaires, par le biais d’une localisation hétérogène, modulent la formation du cerveau antérieur. En effet, l’étude phénotypique comparative in vivo de modèles murins dans lesquels les microglies sont absentes ou leurs fonctions perturbées montrent des défauts de positionnement d’interneurones corticaux et de progression des axones dopaminergiques. De plus, les microglies sont impliquées dans la progression d’un autre faisceau axonal, la capsule externe. Après avoir observé une association très forte entre les microglies et des axones spécifiques, j’ai utilisé deux approches ex vivo et in vivo afin de déterminer si les microglies pourraient agir par phagocytose. Mes résultats montrent que malgré la présence de caractéristiques morphologiques phagocytaires, les microglies ne phagocytent pas les axones de manière intense. Ces travaux suggèrent ainsi que la phagocytose des axones à elle seule ne peut pas expliquer les rôles des microglies sur la croissance axonale. Ce travail, qui montre un rôle anténatal des microglies, révèle une interaction nouvelle entre le développement des systèmes nerveux et immunitaires, et ouvre des perspectives nouvelles pour l’étude sur les mécanismes contrôlant la formation du cerveau en condition physiologique et pathologique. / Brain functioning relies on complex neural circuits that are built during embryogenesis and defects in this process can lead to neurologic or psychiatric disorders. Microglia are the brain resident macrophages, which respond to inflammation and infection by active phagocytosis and secretion of various molecules. In addition to these immune-related functions, microglia were recently involved in the onset of several neuropsychiatric disorders, as well as in postnatal neuronal plasticity, notably through the phagocytosis of developing neurons and synapses. Since microglia colonize the brain during early embryogenesis, they might also participate to the elaboration of neural networks. Here, we reveal that embryonic microglia, via a heterogeneous localization, are modulators of forebrain wiring. Using a cross-comparative analysis of phenotypes induced by either an absence or a perturbation of microglia activity, we showed that microglia directly regulate interneuron positioning as well as dopaminergic axons outgrowth. Moreover, microglia are involved in the formation of another tract, the external capsule. Having observed a strong association between microglia and axons, we combined ex vivo and in vivo approaches in order to decipher whether microglia could act by means of phagocytosis. Despite showing some clear morphological features of intense phagocytosis, our results suggest that microglia do not perform active phagocytosis of axons in vivo. Thus, the phagocytic activity of embryonic microglia by itself is unlikely to explain their role on axonal progression. This study shows for the first time an early prenatal role for microglia and reveal a novel interplay between the central and nervous systems during embryonic development.