Sepse é uma síndrome, na qual, o paciente apresenta lesões de órgãos com risco a vida, em decorrência de uma inflamação exagerada desencadeada por uma infecção. Estima-se uma ocorrência anual de 31,5 milhões de casos de sepse e 19,4 milhões de casos de choque séptico no mundo, causando potencialmente 5,3 milhões de mortes. Esses índices alarmantes fizeram com que em 2017, a Organização Mundial da Saúde (OMS) adotasse uma resolução com o objetivo de aperfeiçoar a prevenção, diagnóstico e tratamento dessa condição clínica que vem sendo negligenciada. A iniciação da sepse, ocorre quando há um descontrole da infecção, acarretando excessiva ativação de células do sistema imune inato. Isso resulta em uma inflamação sistêmica danosa que é responsável pela maioria das alterações fisiopatológicas da sepse. Nesse contexto do sistema imune inato, o papel de neutrófilos já é bem compreendido da patogênese da sepse. Contudo, a função dos monócitos inflamatórios ainda não é bem estabelecida. Ao mesmo tempo que essas células podem participar do controle de infecções, elas também podem contribuir com a inflamação sistêmica e a lesão de órgãos. Deste modo, a compreensão do papel dessas células se faz importante para determinação de novos alvos terapêuticos para essa condição clínica. Nossos resultados demonstraram, em modelo experimental de sepse, que o aumento da emigração de monócitos inflamatórios da medula óssea está relacionado com maior taxa de mortalidade dos animais e exacerbação da inflamação sistêmica. A migração dessas células para órgãos, como rim e pulmão, está relacionado com inflamação e aumento de lesões, nesses locais. Deste modo, conclui-se que monócitos inflamatórios possuem um papel deletério na patogênese da sepse / Sepsis is a syndrome in which the patient has life-threatening organ damage due to an exaggerated inflammation triggered by an infection. The annual occurrence is 31.5 million cases of sepsis and 19.4 million cases of septic shock in the world, which potentially cause 5.3 million deaths. In concern of these alarming reports in 2017, the World Health Organization (WHO) adopted a resolution aimed at improving the prevention, diagnosis and treatment of this neglected clinical condition. The initiation of sepsis occurs when the infection was not controlled, causing excessive activation of the innate immune cells. This excessive activation causes a systemic inflammation that is responsible for most pathophysiological phenomena in sepsis. In this context of the innate immune system, the role of neutrophils is already well understood in the pathogenesis of sepsis. However, the role of inflammatory monocytes is not yet well established. These cells can participate in the control of infections, or can also contribute to systemic inflammation and organs damage. Thus, the understanding of the roles of these cells become important for the development of new therapeutic targets for this clinical condition. Our results demonstrated that the systemic increase of the inflammatory monocytes frequency is related to higher mortality rate, exacerbation of systemic inflammation, increased migration to organs (lung and kidney), and in these sites, are related to inflammation and lesions. Thus, we concluded that these cells have a deleterious role in the pathogenesis of sepsis

O vírus ZIKA pertence ao gênero Flavivírus e diversos trabalhos têm demonstrado que a infecção por esse vírus está associada com o desenvolvimento de anomalias congênitas fetais e doenças neurológicas como casos de microcefalia e de síndrome de Guillan-Barré. Com intuito de entender os mecanismos imunológicos de controle da replicação viral e na patogênese da doença, o objetivo do trabalho foi estabelecer um modelo experimental de infecção por vírus ZIKV em animais imunocompetentes e investigar a migração de leucócitos para o sistema nervoso central durante a infecção por esse vírus. Os animais C57BL/6 infectados por ZIKV apresentaram um aumento da carga viral no sistema nervoso central (SNC) após 24 horas de infecção e uma redução significativa após 48 e 72 horas de infecção. No entanto, os animais deficientes do receptor de IFN tipo I (IFNAR-/-) apresentaram uma alta carga viral após 48 e 72 horas de infecção. Nossos dados mostram que os animais IFNAR-/- são altamente suscetíveis a infecção com 100% de mortalidade na fase aguda da doença, enquanto os animais C57BL/6 são resistentes a infecção com 100% sobrevida por mais de 30 dias de infecção. A infecção com ZIKA induziu o recrutamento de duas subpopulações de monócitos CD11b+Ly6Chi e CD11b+Ly6Clo para o SNC de maneira dependente da sinalização de IFN tipo I. Uma vez que, os animais C57BL/6 apresentaram um aumento de monócitos no SNC após 48 horas de infecção, enquanto que os animais IFNAR-/- apresentaram o recrutamento dessas células somente após 72 horas de infecção. De maneira interessante, o recrutamento dos monócitos culminou com um aumento da expressão das citocinas IL-6 e IL-18 no SNC de animais C57BL/6, enquanto que a expressão dessas citocinas não foi encontrada no SNC de animais IFNAR-/-, sugerindo a importância da via de sinalização de IFN tipo I para a produção dessas citocinas. E por fim, demostramos que a migração dos monócitos para SNC de animais infectados com vírus ZIKA é dependente da produção de CCL2 produzida no cérebro de animais infectados e da expressão dos receptores de quimiocinas CCR2 e CCR5. Uma vez que, animais deficientes para CCR2 (CCR2-/-) e CCR5 (CCR5-/-) apresentaram uma redução da frequência dos monócitos CD11b+Ly6Chi para o SNC em comparação aos animais C57BL/6 após a infecção. De maneira geral, nossos dados mostram que os animais C57BL/6 são resistentes à infecção por ZIKV, uma vez que a via de sinalização de IFN do tipo I está envolvida no recrutamento de monócitos inflamatórios para SNC e consequente controle da replicação viral. O desenvolvimento de um modelo experimental de infecção por vírus ZIKV abre perspectivas para o entendimento dos mecanismos de controle da replicação viral e sobre a patogênese da doença. / The ZIKA virus belongs to the genus Flavivirus, several studies have demonstrated that the infection by this virus is associated with the development of fetal congenital anomalies and neurological diseases, such as microcephaly and Guillan-Barré syndrome. In order to elucidate the immunological mechanisms of the viral replication control and the disease pathogenesis, the study aim was to establish an experimental model of ZIKV infection in immunocompetent animals and investigate the leukocytes migration to the central nervous system during this virus infection. ZIKV infected C57BL/6 animals demonstrated an increased viral load in the central nervous system (CNS) after 24 hours of infection and a significant reduction after 48 and 72 hours of infection. However, type I INF receptor deficient mice (IFNAR-/-) had a high viral load after 48 and 72 hours of infection. Our data demonstrated that IFNAR-/- animals are highly susceptible to infection with 100% mortality during the acute phase of the disease, while C57BL/6 mice are resistant with 100% survival for more than 30 days of infection. ZIKA infection induced the recruitment of two monocytes subpopulation CD11b+Ly6Chi and CD11b+Ly6Clo to the CNS in a dependent manner of type I INF signaling. C57BL/6 mice showed an increase of monocytes in the CNS after 48 hours of infection, whereas IFNAR-/- mice recruited these cells only after 72 hours of infection. Interestingly, the monocytes recruitment culminated in an increased expression of IL-6 and IL-18 in C57BL/6 mice CNS, while these cytokines expression was not observed in the CNS of IFNAR-/- animals, thus suggesting the importance of type I INF signaling pathway for these cytokines production. Finally, we have demonstrated that the monocytes migration to the CNS from animals infected with ZIKA virus is dependent of CCL2 production in the brain of infected animals, as well as the expression of the chemokine receptors CCR2 and CCR5. Given that CCR2 (CCR2-/-) and CCR5 (CCR5-/-) deficient animals demonstrated a frequency reduction of CD11b+Ly6Chi monocytes to the CNS compared to C57BL/6 mice after infection. Overall, our data demonstrate that C57BL/6 animals are resistant to ZIKV infection, since the type I INF signaling pathway is involved in the inflammatory monocytes recruitment to the CNS and subsequent viral replication control. The development of an experimental model of ZIKV infection open perspectives for the understanding of viral replication control mechanisms and disease pathogenesis.

O vírus ZIKA pertence ao gênero Flavivírus e diversos trabalhos têm demonstrado que a infecção por esse vírus está associada com o desenvolvimento de anomalias congênitas fetais e doenças neurológicas como casos de microcefalia e de síndrome de Guillan-Barré. Com intuito de entender os mecanismos imunológicos de controle da replicação viral e na patogênese da doença, o objetivo do trabalho foi estabelecer um modelo experimental de infecção por vírus ZIKV em animais imunocompetentes e investigar a migração de leucócitos para o sistema nervoso central durante a infecção por esse vírus. Os animais C57BL/6 infectados por ZIKV apresentaram um aumento da carga viral no sistema nervoso central (SNC) após 24 horas de infecção e uma redução significativa após 48 e 72 horas de infecção. No entanto, os animais deficientes do receptor de IFN tipo I (IFNAR-/-) apresentaram uma alta carga viral após 48 e 72 horas de infecção. Nossos dados mostram que os animais IFNAR-/- são altamente suscetíveis a infecção com 100% de mortalidade na fase aguda da doença, enquanto os animais C57BL/6 são resistentes a infecção com 100% sobrevida por mais de 30 dias de infecção. A infecção com ZIKA induziu o recrutamento de duas subpopulações de monócitos CD11b+Ly6Chi e CD11b+Ly6Clo para o SNC de maneira dependente da sinalização de IFN tipo I. Uma vez que, os animais C57BL/6 apresentaram um aumento de monócitos no SNC após 48 horas de infecção, enquanto que os animais IFNAR-/- apresentaram o recrutamento dessas células somente após 72 horas de infecção. De maneira interessante, o recrutamento dos monócitos culminou com um aumento da expressão das citocinas IL-6 e IL-18 no SNC de animais C57BL/6, enquanto que a expressão dessas citocinas não foi encontrada no SNC de animais IFNAR-/-, sugerindo a importância da via de sinalização de IFN tipo I para a produção dessas citocinas. E por fim, demostramos que a migração dos monócitos para SNC de animais infectados com vírus ZIKA é dependente da produção de CCL2 produzida no cérebro de animais infectados e da expressão dos receptores de quimiocinas CCR2 e CCR5. Uma vez que, animais deficientes para CCR2 (CCR2-/-) e CCR5 (CCR5-/-) apresentaram uma redução da frequência dos monócitos CD11b+Ly6Chi para o SNC em comparação aos animais C57BL/6 após a infecção. De maneira geral, nossos dados mostram que os animais C57BL/6 são resistentes à infecção por ZIKV, uma vez que a via de sinalização de IFN do tipo I está envolvida no recrutamento de monócitos inflamatórios para SNC e consequente controle da replicação viral. O desenvolvimento de um modelo experimental de infecção por vírus ZIKV abre perspectivas para o entendimento dos mecanismos de controle da replicação viral e sobre a patogênese da doença. / The ZIKA virus belongs to the genus Flavivirus, several studies have demonstrated that the infection by this virus is associated with the development of fetal congenital anomalies and neurological diseases, such as microcephaly and Guillan-Barré syndrome. In order to elucidate the immunological mechanisms of the viral replication control and the disease pathogenesis, the study aim was to establish an experimental model of ZIKV infection in immunocompetent animals and investigate the leukocytes migration to the central nervous system during this virus infection. ZIKV infected C57BL/6 animals demonstrated an increased viral load in the central nervous system (CNS) after 24 hours of infection and a significant reduction after 48 and 72 hours of infection. However, type I INF receptor deficient mice (IFNAR-/-) had a high viral load after 48 and 72 hours of infection. Our data demonstrated that IFNAR-/- animals are highly susceptible to infection with 100% mortality during the acute phase of the disease, while C57BL/6 mice are resistant with 100% survival for more than 30 days of infection. ZIKA infection induced the recruitment of two monocytes subpopulation CD11b+Ly6Chi and CD11b+Ly6Clo to the CNS in a dependent manner of type I INF signaling. C57BL/6 mice showed an increase of monocytes in the CNS after 48 hours of infection, whereas IFNAR-/- mice recruited these cells only after 72 hours of infection. Interestingly, the monocytes recruitment culminated in an increased expression of IL-6 and IL-18 in C57BL/6 mice CNS, while these cytokines expression was not observed in the CNS of IFNAR-/- animals, thus suggesting the importance of type I INF signaling pathway for these cytokines production. Finally, we have demonstrated that the monocytes migration to the CNS from animals infected with ZIKA virus is dependent of CCL2 production in the brain of infected animals, as well as the expression of the chemokine receptors CCR2 and CCR5. Given that CCR2 (CCR2-/-) and CCR5 (CCR5-/-) deficient animals demonstrated a frequency reduction of CD11b+Ly6Chi monocytes to the CNS compared to C57BL/6 mice after infection. Overall, our data demonstrate that C57BL/6 animals are resistant to ZIKV infection, since the type I INF signaling pathway is involved in the inflammatory monocytes recruitment to the CNS and subsequent viral replication control. The development of an experimental model of ZIKV infection open perspectives for the understanding of viral replication control mechanisms and disease pathogenesis.

<p>Cancer metastasis is the cause of about 90% of cancer patients' deaths. Despite significant improvements in the past three decades in understanding the molecular bases of oncogenic transformation of cancer cells, little is known about the molecular mechanisms underlying tumour cells' alteration of their microenvironment, entrance into the circulation, and colonization of distant organs. In recent years, accumulating evidence has indicated that tumour microenvironment, which consists of a variety of cell types and extracellular matrix components&#65292;plays an important role in regulating the metastatic abilities of carcinoma cells. Co-opted by cancer cells, those stromal cells promote tumour progression via multiple mechanisms, including enhancement of tumour invasiveness, elevation of angiogenesis, and suppression of immune surveillance activity. </p><p>Using a series of human breast cancer cell lines with different metastatic potentials <italic>in vivo</italic>, we performed an unbiased screen examining expression of miRNAs, and found that miR-126 and miR-126*, whose expression are regulated by methylation of the promoter of their host gene Egfl7 inside tumour cells, were significantly negatively correlated with metastatic potential. Using both mouse xenograft models and <italic>in vitro</italic> assays, we showed that this pair of miRNAs suppressed breast cancer metastasis through shaping the tumour microenvironment without changing tumour cell autonomous properties. Specifically, miR-126 and miR-126* act independently to suppress the sequential recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes into the primary tumour stroma, consequently inhibiting lung metastasis by breast tumour cells. Mechanistically, these miRNAs directly inhibit the production of stromal cell-derived factor-1 alpha (Sdf-1&alpha;, also known as Cxcl12), and indirectly suppress the expression of chemokine (C-C motif) ligand 2 (Ccl2) by the cancer cells within the tumour mass in an Sdf-1&alpha;-dependent manner. In addition, in contrast with the majority of reports which have shown incorporation of only the guiding strand of the miRNA duplex into the mRNA-targeting RNA induced silencing complex (RISC), both strands of the miR-126 RNA duplex are maintained at a similar level and suppress Sdf-1&alpha; expression independently. </p><p>Collectively, we have determined a dynamic process by which the composition of the primary tumour microenvironment could be altered via a change in the expression of two tumour-suppressive miRNAs derived from a single miRNA precursor to favor metastasis by breast cancer cells. Importantly, this work provides a prominent mechanism to explain the clinical correlation between reduced expression of miR-126/126* and poor metastasis-free survival of breast cancer patients.</p> / Dissertation

Les monocytes inflammatoires Ly-6Chigh murins, et leurs homologues humains CD14+CD16-, jouent un rôle important dans l'initiation et la persistance des maladies inflammatoires chroniques. Leur action délétère dans ces pathologies a mené au développement de stratégies thérapeutiques visant à les éliminer ou empêcher leur recrutement aux sites inflammatoires. Toutefois, ces méthodes se sont avérées peu spécifiques des monocytes et surtout d'une faible efficacité compte tenu de l'aspect hautement inflammatoire des monocytes. Le besoin de développer de nouvelles stratégies est donc nécessaire. Les objectifs de ma thèse ont donc été dans un premier temps de caractériser in vivo le ciblage spécifique des monocytes inflammatoires par la formulation liposomale DMAPAP. Dans un second temps, l'utilisation de DMAPAP pour formuler des siRNA a permis d'évaluer l'efficacité thérapeutique d'une stratégie basée sur l'inhibition spécifique de gènes jouant un rôle clef dans l'inflammation dans les monocytes inflammatoires. Mon travail a permis de montrer dans un modèle préclinique d'arthrite que l'inhibition de gènes régulateurs de l'inflammation dans les monocytes Ly-6Chigh est une approche thérapeutique efficace permettant d'induire une immunomodulation des réponses pathogéniques des lymphocytes T effecteurs, aboutissant au défaut de recrutement des cellules immunitaires dans les articulations et à une amélioration des signes cliniques. J'ai également validé le transfert de cette technologie ex vivo sur cellules humaines primaires. L'inhibition de gènes clefs dans les monocytes inflammatoires représente donc une stratégie prometteuse pour le développement de futures thérapies dans la polyarthrite rhumatoïde. Par ailleurs, mes résultats confirment le rôle central des monocytes inflammatoires dans les pathologies inflammatoires chroniques. / Inflammatory mouse Ly6Chigh monocyte subset and its human counterpart, defined as CD14+ CD16-, play key roles in the initiation and chronicization of immune-mediated inflammatory disorders (IMID). Deleterious effects of monocytes led to the development of therapeutic strategies aiming at depleting them or preventing their recruitment to inflamed tissues. However, these methods are poorly specific with weak efficacy considering the high number of inflammatory monocytes and their marked level of activation. The need for developing new therapeutic approaches is obvious. The aim of my thesis was to characterize selective delivery of a siRNA-containing lipid formulation to the Ly-6Chigh monocyte population and at evaluating the therapeutic potential of this targeted strategy. Using the cationic lipid-based DMAPAP vehicle for in vivo RNAi-mediated gene silencing, my work allowed demonstrating, in a preclinical mouse model of arthritis, the efficacy to inhibit master genes of inflammation specifically within Ly-6Chigh monocytes upon systemic injection. Reduced disease severity in mice was associated with an overall systemic immunomodulation of the pathogenic T cell populations and led to defective mobilization of immune cells to arthritic joints. Importantly, the formulation was successfully optimized in a perspective of clinical application and the targeting of human CD14+CD16- inflammatory monocytes was validated ex vivo. Overall, my findings demonstrate that the silencing of a key gene within Ly-6Chigh monocytes is a promising strategy for future therapeutic intervention in the context of IMID and reinforces the pivotal role of Ly-6Chigh monocytes in inflammatory processes.

No description available.