As células dendríticas (DCs) são as principais células apresentadoras de antígeno do sistema imune, e há evidências da participação na tolerância imunológica. Neste estudo avaliamos as alterações ocorridas na população de células CD4+CD25+Foxp3+ após co-cultura de células de linfonodo com BMDCs, na presença de timócitos alogênicos ou singênicos em apoptose. Após cultura na presença de células alogênicas a população de Tregs mostra-se aumentada, e essa expansão é dependente de contato entre a DC e o linfócito T, já que o isolamento das culturas diminuiu a expressão destes marcadores. A internalização de células em apoptose foi induziu caráter tolerogênico nas DCs com baixa expressão de moléculas co-estimuladoras e resistência à maturação por LPS. As células CD4+CD25+ geradas in vitro foram capazes de conter a proliferação de esplenócitos de camundongos BALB/c estimulados por esplenócitos de C57BL/6 irradiados, anti-CD3 e OVA. No ensaio in vivo, as Tregs geradas in vitro também foram capazes de suprimir a proliferação de células CD25- quando transferidas para animais Nude, impedindo também o infiltrado inflamatório no estômago, cólon, fígado e rins, sendo assim capazes de suprimir a resposta imune in vitro e in vivo. / The dendritic cell (DC) plays a very important role in antigen presentation in the immune system and recent articles have shown the involvement in maintaining peripheral tolerance. Here we evaluated the changes in the CD4+CD25+Foxp3+ after co culture of DCs with lymph node cells. BMDCs were pulsed with apoptotic cells and co-cultured with lymph-node cells. Our results show an increase of CD4+CD25+Foxp3+ T cells after co-culture with DCs pulsed with apoptotic cells. Furthermore, the DCs did not change the pattern of co-stimulatory molecules expression due to phagocytosis of syngeneic or allogeneic apoptotic cells and further stimulation with LPS. The CD4+CD25+ cells sorted from the in vitro culture were able to suppress the proliferation of splenocytes in vitro in a specific and non-specific manner. As expected, the co-transfer of CD4+CD25- and CD4+CD25+, both sorted from the in vitro culture, was able to control the cell infiltrates in the target organs and the total cell count in the lymph-nodes. Thus, the Tregs expanded in vitro are able to suppress the immune response in vitro and in vivo assays.

O veneno da subespécie de cascavel Crotalus durissus terrificus (C.d.terrificus) altera a coagulação, tem ação neurotóxica, miotóxica e efeito imunossupressivo. O veneno de C.d.terrificus e a sua fração majoritária, crotoxina (CTX), inibem a resposta celular e humoral, sendo esse efeito independente da indução de morte celular. O objetivo foi investigar os mecanismos envolvidos na imunossupressão exercida pela CTX. A CTX induz a produção de IL-10, TGF-<font face=\"Symbol\">&#946 e prostaglandina E2 (PGE2) pelas células dos camundongos que a receberam. Menor secreção de IFN-<font face=\"Symbol\">&#947 e IL-12 foi observada nas culturas de células de camundongos imunizados e que receberam CTX. A CTX aumenta a expressão da enzima indoleamina 2,3 dioxigenase e a geração de células T reguladoras em camundongos imunizados com OVA e que receberam CTX. A CTX foi capaz de inibir a expressão de CD40, CD80, CD86 e de MHC de classe II em células de camundongos imunizados e nas células dendríticas (DCs) purificadas destes animais. Em culturas de DCs, a CTX induz alta secreção de IL-10, TGF-<font face=\"Symbol\">&#946 e PGE2 e menor de IL-12. / The venom of the rattlesnake Crotalus durissus terrificus (C.d.terrificus) changes the coagulation system and presents neurotoxic, myotoxic and immunosuppressive effect. The venom and its main fraction, crotoxin (CTX) inhibited both the cellular and humoral response. This effect is not due to induction of cell death. The objective was to investigate the immunosuppressive mechanisms of CTX. CTX induces production of IL-10, TGF-<font face=\"Symbol\">&#946 and prostaglandin E2 (PGE2) by cells of mice that received it. Lower secretion of IFN-<font face=\"Symbol\">&#947 and IL-12 was observed in the cultured cells from mice immunized that received CTX. CTX promotes increased expression of the enzyme indoleamine 2,3 dioxygenase and generation of regulatory T cells in mice immunized with OVA and receiving the toxin. CTX was able to inhibit the expression of CD40, CD80, CD86 and MHC II in mice immunized and in dendritic cells (DCs) purified from these animals. In cultures of DCs, CTX increased secretion of IL-10, TGF-<font face=\"Symbol\">&#946 and PGE 2 and lower IL-12.

Estima-se que a malária seja responsável por um milhão de mortes anuais, atingindo principalmente crianças. O sistema imune participa tanto na proteção contra a infecção pelo plasmódio, como no desenvolvimento das síndromes associadas à malária (anemia, malária cerebral, acidose metabólica e choque sistêmico). Recentemente, tem sido mostrado que a imunidade inata é capaz de detectar sinais liberados por células ou tecidos danificados como o ATP e o ácido úrico. Esses sinais de perigo parecem ser importantes para promover a regulação da inflamação após o trauma ou injúrias ocasionadas pelos patógenos. Porém, a relevância fisiológica desses sinais na resposta imune e seu mecanismo de ação ainda não estão claros. Na malária, é provável que o ATP seja liberado no momento da ruptura dos eritrócitos, a partir de células danificadas do endotélio vascular ou de células do sistema imune mortas por ativação. Assim, neste estudo nós avaliamos o papel do ATP na ativação do sistema imune e no desenvolvimento da doença durante a infecção com P. chabaudi. Nossos resultados sugerem que, a concentração de ATP no soro e permeabilização de linfócitos do sangue é maior após a ruptura dos eritrócitos. Durante a infecção, as células T e as células dendríticas possuem uma capacidade exacerbada de resposta ao ATP, que pelo menos em parte depende do P2X7R. Além disso, a presença funcional de receptores purinérgicos parece ser importante para a fase inicial da resposta imune ao P. chabaudi. A inibição farmacológica do receptor reduziu o número de células, produção de IFN-<font face=\"Symbol\">g e injúria hepática. Desta forma a utilização do antagonistas do P2X7R poderia ser benéfico na resolução de problemas ocasionados pelo aumento acentuado do sistema imune. / It is estimated that malaria accounts for one million deaths annually, affecting mainly children. The immune system participates in both the protection against infection by the plasmodium, as in the development of the syndromes associated with malaria (anemia, cerebral malaria, metabolic acidosis and systemic shock). Recently, it has been shown that innate immune is able to detect signals released by damaged cells or tissues as ATP and uric acid. These danger signals appear to be important to promote the regulation of inflammation after trauma or injuries caused by pathogens. However, the physiological relevance of these signals in the immune response and its mechanism of action remain unclear. In malaria, it is likely that ATP is released upon rupture of erythrocytes, vascular endothelial cells damaged or dead cells of the immune system activation. In this study we evaluated the role of ATP in the activation of the immune system and the development of disease during infection with P. chabaudi. Our results suggest that ATP concentration serum and permeabilization of blood lymphocytes is higher after the rupture of erythrocytes. During infection T cells and dendritic cells have a heightened ability to respond to ATP which is partly dependent on P2X7R. Moreover, the presence of functional purinergic receptors appears to be important for the initial phase of the immune response to P. chabaudi. The pharmacological inhibition of the receptor reduced the number of cells, liver damage and IFN-<font face=\"Symbol\">g, thus the use of the P2X7 receptor antagonists could be beneficial in solving problems caused by the sharp increase in the immune system.

Este estudo consistiu em avaliar comparativamente se camundongos neonatos e adultos, quando expostos ao poluente 1,2-NQ exibiriam susceptibilidade aumentada à inflamação alérgica (ovalbumina; OVA). Camundongos adultos ou neonatos foram expostos à 1,2-NQ ou veículo (15 min por três dias). Os camundongos adultos, após 24 h, ou neonatos, após 59 dias, foram sensibilizados/desafiados com OVA e os parâmetros funcionais e inflamatórios foram avaliados 24 h após. Em animais expostos a 1,2-NQ na fase neonatal, o desafio alérgico na idade adulta causou potente influxo de leucócitos no pulmão, sangue periférico e maturação eosinofílica na medula óssea, mas não afetou a hiperresponsividade brônquica. Isto foi consistente com maior biossíntese de citocinas Th2 e apresentação de CD11c esplênica. Em animais adultos, a 1,2-NQ não amplificou a inflamação alérgica. Conclui-se que, a exposição de camundongos neonatos à 1,2-NQ aumentou a susceptibilidade destes à inflamação alérgica na idade adulta via maior apresentação de CD11c esplênica e da biossíntese de citocinas Th2. / We have comparatively investigated whether exposure of neonates and adults mice to 1,2-NQ increases their susceptibility to allergic inflammation evoked by ovalbumin (OVA). Adults or neonate mice were nebulized with 1,2-NQ or corresponding vehicle. Following 24 h or eight weeks, adult or neonate mice were sensitized and challenged with OVA and the functional and inflammatory parameters were evaluated 24 h later. The allergic challenge in mice exposed to 1,2-NQ as neonates caused a potent influx of leukocytes in bronchoalveolar lavage, peripheral blood and increased eosinophil maturation in the bone marrow, without affecting Penh response. This was correlated with increased presentation of splenic CD11c and biosynthesis of Th2 cytokines in the lung. Adult mice exposure to 1,2-NQ failed to significantly increase OVA-induced allergic responses. Exposure to 1,2-NQ during neonatal period is critical to enhance susceptibility of asthma at a later stage of life, and that increased expression of splenic CD11c and inflammatory mediators contribute to this effect.

A transmissão vertical do HIV-1 da mãe para criança foi significativamente reduzida com o uso de terapias antirretrovirais, resultando um aumento do número de crianças que foram expostas ao vírus. Estudos evidenciam a expressão de receptores Toll-like (TLR) em células trofoblásticas que pode ser uma ferramenta importante para regular a diferenciação e ativação de células do sistema imunológico, orquestrando um ambiente imunorregulador, contribuindo para o sucesso gravidez e proteção do feto. Considerando que as consequências da infecção por HIV-1 no microambiente placentário, na mãe e no recém-nascido (RN) não são bem entendidos, salienta-se a necessidade de investigar estratégias que possam potencializar a resposta imune inata, neste contexto. Com isso, no presente trabalho, foram avaliados as células mononucleares (CMN) do sangue periférico e cordão umbilical (RN) de parturientes infectadas por HIV-1 e parturientes-RN não infectados, a secreção de TNF-a, IL-10 e IFN-a induzida por agonistas de TLRs extracelular (TLR2, TLR4 e TLR5) e agonistas de TLRs intracelulares (TLR3, TLR7, TLR7/8, TLR9). Além disso, como sensor da resposta imune inata foi avaliado o perfil de resposta de células dendríticas mielóides (mDC) e plasmocitóides (pDC), após ativação com ligantes relacionados com resposta antiviral (TLR7, TLR7/8 e TLR9). No microambiente placentário foi verificada a expressão de CD123, TLR8, CD14/CD16 e HLA-G de amostras da decídua e vilo de mães infectadas por HIV-1 e grupo controle. Os resultados mostraram que as CMNs de mães infetadas por HIV-1 e de seus RNs têm um déficit na secreção TNF-a após ativação pelas vias de TLR2, TLR5, TLR3, TLR7, contudo, preservada para estimulação TLR7/TLR8 (composto CL097), principalmente pelas células de recém-nascidos. Além disso, apenas a ativação com CL097 foi eficaz para induzir a secreção IL-10 e IFN-a pelo grupo infectado, tanto na mãe quanto no RN. O CL097 também foi capaz de induzir a expressão de RNAm para o fator regulador de interferon-7 (IRF-7), IFN-a e TNF-a em níveis similares entre os grupos, confirmando o potencial de ativação TLR7/8. A ativação via TLR7/TLR8 foi capaz de controlar o déficit na da produção de TNF-a pelas mDC, mas não reverteu a resposta funcional de IFN-a por pDCs nas mães e RN infectados por HIV-1. No tecido placentário infectado por HIV-1, houve aumento da expressão de TLR8, CD123, e HLA-G. Os achados mostraram a importância da via TLR8 tanto na resposta sistêmica como no microambiente placentário, ressaltando a importância dos ligantes naturais e/ou sintético no papel adjuvante para melhorar a resposta antiviral na interação materno-fetal. / Mother-to-child transmission of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting an increased number of HIV1-exposed uninfected infants. Studies have been evidenced that expression of Toll-like receptors (TLR) in trophoblast cells may be a relevant tool to regulate the differentiation and activation of immune cells, orchestrating an immunoregulatory environment, contributing to successful pregnancy and fetal protection. Since the consequences of HIV-infection in the immune innate system from placental microenvironment, mother, and newborn are not well understood, emphasize to investigate the strategies to potentiate the innate immune immune response. We evaluated in mononuclear cells (CMN) of peripheral blood and cord blood from HIV-1-infected pregnant and uninfected mother-cord blood the TNF-a, IL-10 and IFN-a secretion induced by agonists of extracellular Toll-like receptor (TLRs) (TLR2, TLR4 and TLR5) and agonists for intracellular TLRs (TLR7, TLR7/8, TLR9). Moreover, as checkpoint of innate immune response we evaluate the myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) responsinevess to TLRs related to antiviral response (TLR7, TLR7/8 e TLR9). Were also evaluated expression of CD123, TLR8, CD14/CD16 and HLA-G of maternal samples of decidua and villi both infected by HIV-1.The results showed that HIV-1 infected mother-cord blood have a deficit in the TNF-a response induced by TLR2, TLR5, TLR3, TLR7 PBMC activation, but preserved for TLR7/8 (CL097) stimulation, mainly by the newborn cells. Moreover, only CL097 activation was efficacious to induce IL-10 and IFN-a secretion by the infected group, in both mother and cord blood cells. Up-regulation of IFN-a secretion level was achieved with CL097 by cord blood from HIV-infected mother compared to control mothers. CL097 stimulation was also able to induce Interferon-regulator factor-7, IFN-a and TNF-a mRNA expression in PBMC at similar levels between groups, confirming the potential of TLR7/8 activation. TLR7/TLR8 activation overcomes the impairment of TNF-a production by mDC, but maintained the dysfunctional type I IFN response by pDCs in HIV-infected pairs. Expression of TLR8, CD123 and HLA-G was increased in placental tissue from infected-mother compare to uninfected control. Our findings highlight the dysfunction of innate immune response in HIV-treated mother-newborn. Adjuvant potential of TLR8, in the systemic response as well as in the placental microenvironment, emphasizes the use of natural and /or synthetic TLR agonists to improve antiviral response at the maternal-fetal interface.

Le Virus de l’Immunodéficience Humaine de type I (VIH-I) est majoritairement dégradé dans les cellules dendritiques dès son entrée. Des travaux précédemment réalisés dans l’équipe montrent que le mécanisme de l’autophagie contribue à la dégradation virale (virophagie) et promeut les réponses immunitaires innées et adaptatives. Etant donné que cette virophagie est par la suite inhibée par le virus dans les cellules dendritiques, mieux comprendre la mise en place de cette autophagie antivirale est primordial afin de pouvoir favoriser et surtout stabiliser les défenses cellulaires. Nos résultats montrent que DC-SIGN, un récepteur lectine de type C (CLR) qui reconnait des carbohydrates mannosylés ou fucosylés, pourrait être impliqué dans la mise en place de cette autophagie antivirale. Ainsi, nous montrons qu’après reconnaissance du VIH, ce récepteur induit l’autophagie et interagit rapidement avec plusieurs protéines autophagiques dont certaines impliquées dans les voies endosomales. En effet, nous montrons pour la première fois l’association de la protéine Atg9 avec le récepteur DC-SIGN internalisé. La protéine Atg9 est un facteur essentiel de l’initiation de la voie autophagique régulant notamment l’apport de membranes issues de la membrane plasmique pour la nucléation de vésicules liées à la voie autophagique. D’autre part, après une étude par spectrométrie de masse de l’intéractome du récepteur DC-SIGN internalisé, nous montrons qu’une E3 ligase faisant partie de la famille des TRIM, TRIM25, est recrutée lors de l’endocytose du récepteur. Cette protéine a été rapportée comme étant impliquée dans la régulation des réponses innées antivirales issues du récepteur de reconnaissance de pathogène (PRR) RIG-I suggérant une fonction essentielle de TRIM25 dans les réponses immunes innées. De manière intéressante, certains membres de la famille TRIM ont été montrés récemment comme étant essentiels pour l’induction d’une forme d’autophagie sélective parfois antivirale, l’autophagie de précision. Dans ce sens, nous montrons qu’un complexe est formé entre le récepteur DC-SIGN, Atg9 et TRIM25, suggérant que l’autophagie mise en place très tôt lors de l’engagement du récepteur DC-SIGN pourrait s’avérer être sélective. L’ensemble de ces éléments constitue une première étape pour une meilleure compréhension des étapes précoces de l’entrée du VIH dans les cellules dendritiques avec la caractérisation d’une virophagie sélective induite lors de l’engagement d’un récepteur de l’immunité innée et qui représente une cible particulièrement intéressante afin d’améliorer certaines stratégies thérapeutiques développées actuellement. / The Human Immunodeficiency Virus type I (HIV-I) is mostly degraded in dendritic cells as soon as it enters. Previous work in the team shows that the mechanism of autophagy contributes to viral degradation (virophagy) and promotes innate and adaptive immune responses. Since this virophagy is subsequently inhibited by the virus in dendritic cells, a better understanding of the implementation of this antiviral autophagy is essential in order to promote and above all stabilize cellular defenses. Our results show that DC-SIGN, a C-type lectin receptor (CLR) that recognizes mannosylated or fucosylated carbohydrates, may be involved in the development of this antiviral autophagy. Thus, we show that after recognition of HIV, this receptor induces autophagy and interacts rapidly with several autophagic proteins, some of which are involved in the endosomal pathways. Indeed, we show for the first time the association of the protein Atg9 with the internalized DC-SIGN receptor. The Atg9 protein is an essential factor in the initiation of the autophagic pathway regulating in particular the supply of membranes originating from the plasma membrane for the nucleation of vesicles linked to the autophagic pathway. On the other hand, after a mass spectrometric study of the internalized DC-SIGN receptor interbody, we show that an E3 ligase belonging to the TRIM family, TRIM25, is recruited during endocytosis of the receptor. This protein has been reported to be involved in the regulation of antiviral innate responses from the RIG-I pathogen recognition receptor suggesting an essential function of TRIM25 in innate immune responses. Interestingly, some members of the TRIM family have recently been shown to be essential for the induction of a form of selective autophagy, sometimes antiviral, precision autophagy. In this sense, we show that a complex is formed between the DC-SIGN, Atg9 and TRIM25 receptors, suggesting that autophagy early on engagement of the DC-SIGN receptor could be selective. All these elements constitute a first step for a better understanding of the early stages of the entry of HIV into dendritic cells with the characterization of a selective virophagy induced when a receptor of the innate immunity And which represents a particularly interesting target in order to improve certain therapeutic strategies currently being developed.

Les cellules dendritiques (DC) sont une charnière entre immunité innée et adaptative. Les DC du sous-type CD8a+ excellent à la présentation croisée d’antigènes exogènes pour activer les lymphocytes T (LT) CD8+. Mon projet de thèse consistait à analyser la contribution de gènes sélectivement exprimés par les DC CD8a+ à leur spécialisation fonctionnelle. Nous avons d’abord analysé le rôle du récepteur de chimiokine XCR1 que les DC CD8a;+ expriment spécifiquement. XCR1 reconnait XCL1, une chimiokine qui a un pouvoir attractant sur les DC CD8a+ in vitro, et qui est exprimée par les cellules NK, les lymphocytes innés de type 1 et les LT CD8+ activés. Nous avons montré que XCR1 était indispensable à l’hôte pour résister à une infection par le cytomégalovirus murin, en permettant l’activation optimale des cellules NK. L’axe XCL1-XCR1 est donc un moyen privilégié de communication entre les cellules NK et les DC CD8a+. Ensuite, nous avons utilisé un modèle murin ciblant spécifiquement les DC CD8a+ pour montrer leur rôle majeur dans la réactivation des LT CD8+ mémoires au cours d’infections secondaires. Enfin, nous avons tenté d’analyser si une autre molécule préférentiellement exprimée par les DC CD8a+, la GTPase Rab7b, était impliquée dans la présentation croisée des antigènes. Dans d’autres cellules, Rab7b interviendrait dans l’échange de matériel entre le réseau trans-Golgien et les endosomes tardifs. Dans les DC CD8a+, cette activité pourrait favoriser le trafic intracellulaire des antigènes vers les endosomes où s’effectue la présentation croisée. Mon travail de thèse a permis de mieux comprendre comment les DC CD8a+ activent les lymphocytes effecteurs innés et adaptatifs. / Dendritic cells (DC) link innate and adaptive immunity. The CD8α+ DC subtype is particularly efficient in activating CD8+ T cells through cross-presentation of exogenous antigens. The aim of my PhD project was to analyze the role of genes specifically expressed by CD8a+ DC in the functional specialization of these cells. First we investigated the role of the chemokine receptor XCR1 in the biology of CD8a+ DC. In mice, XCR1 is a powerful chemo-attractive molecule for CD8a+ DC in vitro. It recognizes XCL1, a chemokine expressed by NK cells, type 1 innate lymphoid cells, and activated CD8+ T cells. We have shown that XCR1 is crucial for the resistance to murine cytomegalovirus (MCMV) infection, allowing optimal activation of NK cells. Thus, the XCL1-XCR1 axis is a privileged way of communication between NK cells and CD8a+ DC. We then used a mouse model which specifically targets CD8a+ DC, to show their major role in the reactivation of memory CD8+ T cells upon diverse secondary infections. Finally, we tried to investigate the potential role in antigen cross-presentation of another molecule selectively expressed by CD8a+ DC, the Rab7b GTPase. ,. Indeed, in another cell type, Rab7b could be involve in material exchange between the trans-golgi network and late endosomes. In CD8a+ DC, this process could favor the intracellular trafficking of endocytosed antigens towards the endosomes where cross-presentation occurs. In summary, my PhD work advanced our understanding of how CD8a+ DC activate innate and adaptive effector lymphocytes

Light entering the eye is transformed by the retina into electrical signals. Extensive processing takes place in the retina before these signals are transmitted to the brain. Beginning in the outer retina, light-evoked electrical signals are distributed into parallel pathways specialized for different visual tasks, such as the detection of dark vs. bright ambient light, the onset or offset of light, and the direction of stimulus motion. Pathway diversity is a consequence of cell type diversity, differential cell connectivity, synapse organization, receptor expression, or any combination thereof. Cell connectivity itself can be accomplished through excitatory or inhibitory chemical synapses, or electrical coupling via gap junctions. Gap junctions are further specialized based on the expression of different connexin subunit isoforms. In aggregate, this diversity gives rise to ganglion cells with highly specialized functions, including ON and/or OFF responses, contrast-tuning and direction-selectivity (DS). The directionally-selective circuit, a circuit specialized for the encoding of stimulus motion, makes use of many of these circuit specializations. Bipolar cells, in response to glutamate release from cone photoreceptors, provide highly-sensitive glutamatergic input to amacrine cells and DS ganglion cells (DSGCs) in this circuit, while amacrine cells provide cholinergic and directionally-tuned GABAergic input to DSGCs. One population of DSGCs also transmit signals laterally to one another via gap junctions. Thus numerous specializations in bipolar cells, amacrine cells and ganglion cells endow DSGCs with their unique encoding abilities. In Chapters 2 and 3 of this dissertation I focus on synchronized firing between gap junction-coupled DSGCs. sDSGCs exhibit fine-scale correlations, with action potentials in an sDSGC more likely within ~2ms of action potential firing in a coupled neighbour. I first characterize electrical coupling of DSGCs through the identification of the molecular composition of DSGC gap junctions (Chapter 2). Physiological and immunohistochemical methods allowed me to demonstrate an important role for connexin 36 subunits in DSGC electrical coupling. Next (Chapter 3) I investigate the sub-cellular mechanisms underlying neuronal correlations between electrically coupled DSGCs. Using paired recordings, I show that chemical input (from bipolar cells and amacrine cells), electrical input (from gap junctions), and Na+ channel activity in DSGC dendrites underlie the generation of correlated spiking activity. While a common feature of electrically coupled networks, the mechanisms underlying correlations were previously unclear. In Chapter 4 I focus on the mechanisms within the DS circuit that endow these neurons with impressive sensitivity to stimulus contrast. Using physiological and pharmacological methods I first assess the relative contrast sensitivity of ganglion cells and starburst amacrine cells (SACs) in the DS circuit. The sensitivity of DSGC and SAC excitatory currents to antagonists of Na+ channels suggests an important role for these channels in amplifying low contrast responses and other weak inputs to the circuit. This role is later attributed to the differential expression of voltage-gated Na+ channels in specific bipolar cell populations. In aggregate, this dissertation describes several novel circuit mechanisms within the well-studied DS circuit. I also provide specific roles for such specializations in visual coding. / Graduate

Le porc est un modèle d’étude présentant à la fois une grande importance agronomique et un fort potentiel comme modèle biomédical pour différentes pathologies respiratoires. Pourtant, son système immunitaire pulmonaire est peu connu, limitant l’approfondissement de ces études. Notamment, les cellules dendritiques (DCs) et macrophages (Mθs) tissulaires, qui sont à l’interface entre immunités innée et acquise, n’étaient pas caractérisés. L’objectif de cette thèse était de décrire ces cellules dans le poumon porcin à l’état basal et durant des infections par différentes souches de virus Influenza A (VIA), un pathogène capable d’infecter le porc et l’Homme. Nous avons caractérisé pour la première fois les DCs conventionnelles (cDCs) et dérivées de monocytes (moDCs), ainsi que des macrophages dérivés de monocytes (moMθs) et les macrophages alvéolaires (AMs) porcins. Les cDC1 et cDC2 se sont révélées proches de leurs équivalentes murines et humaines de par leur phénotype et leurs capacités de migration et de présentation de l’antigène in vitro. Les moDCs sont, comme chez la souris, pro-inflammatoires et recrutées lors d’une infection par le VIA in vivo. Nous avons également étudié des cellules tissulaires fortement représentées dans le poumon porcin et dont le phénotype est très similaire à celui des AMs : les cellules AM-like. Ces dernières sont pro-inflammatoires lors d’infections in vitro par un virus H3N2 porcin ou une souche Lena du PRRSV. Nous avons enfin étudié ces différents types cellulaires dans le cadre d’infections par deux souches porcines du VIA, l’une du sous-type H3N2 et l’autre du sous-type H1N2, la première étant plus pathologique en élevage et induisant plus d’inflammation. Il se pourrait que les cellules AM-like aient un rôle important dans cette différence de pathogénicité. En conclusion, nous avons mis en place des moyens pour étudier plus précisément le rôle des DCs et Mθs dans le poumon porcin, et ainsi, de mieux comprendre l’inflammation et la réponse immunitaire pulmonaire. Ce travail ouvre des voies, en santés vétérinaire et biomédicale, qui étaient jusqu’à présent réservées au modèle murin. / Pig is a research model with a major agronomic importance and a great potential as a biomedical model for different respiratory pathologies. Yet, its pulmonary immune system is little known, limiting the deepening of these studies. In particular, tissular Dendritic Cells (DCs) and Macrophages (Mθs), which are at the interface between innate and adaptive immune systems, were not characterized. The aim of this thesis was to describe DCs and Mθs in the porcine lung at steady-state and during infections with different strains of influenza virus A (IAV), a pathogen capable of infecting pigs and humans. We characterized for the first time conventional DCs (cDCs) and monocyte derived (moDCs), as well as monocyte-derived macrophages (moMθs) and alveolar macrophages (AMs) in pig. The cDC1 and cDC2 were phenotypically close to their murine and human equivalents. They also had the same in vitro capabilities of migration and antigen presentation. The moDCs are, like murine ones, pro-inflammatory and are recruited during an IAV infection in vivo. We also found a highly represented interstitial population whose phenotype is very similar to that of AMs: AM-like cells. They are pro-inflammatory upon in vitro infection by a swine H3N2 strain or a Lena strain of PRRSV. Finally, we studied these different cell types through infection by two strains of porcine IAV, an H3N2 and an H1N2 strains, the first one being more pathological and inducing more inflammation. The AM-like cells might play an important role in this pathogenicity difference. In conclusion, we found a strategy to study more precisely the roles of DCs and Mθs in the porcine lung, and thus to better understand lung inflammation and immune response. This work opens pathways, in veterinary and biomedical health, which were previously reserved for mouse model.

Au sein des synapses comme dans les régions extra synaptiques, la diffusion latérale joue un rôle critique dans la densité membranaire des récepteurs. En face des zones actives, l’accumulation de récepteurs détermine en particulier l’efficacité de la transmission synaptique. Il est important de comprendre les paramètres cellulaires qui jouent sur l’accès au compartiment synaptique, qu’ils soient d’origine moléculaires ou morphologiques. Dans les synapses excitatrices, la tige de l’épine dendritique se comporte comme une barrière à la diffusion. Cette barrière pourrait être fonction de la longueur et du diamètre de la tige (paramètre géométrique), ou résider dans la présence d’éléments spécifiques constituant des obstacles à la diffusion. Une sous-population d’épines contient dans sa tige une forme spécialisée de réticulum endoplasmique, appelé appareil épineux et constituée d’un empilement des accules de réticulum. Une protéine liant l’actine, nommée synaptopodine, est associée de façon étroite à l’appareil épineux et participe aux mécanismes de plasticité synaptique. La question centrale de ce travail de thèse était de définir si la présence de synaptopodine influait sur les caractéristiques de la diffusion dans la tige de l’épine, et d’identifier les mécanismes sous-jacents. Afin d’étudier la diffusion membranaire, j’ai utilisé trois protéines recombinantes différentes: une protéine associée au feuillet extérieur de la membrane plasmique (GFP-GPI), une protéine avec un domaine transmembranaire et une courte séquence intracellulaire (TMD-pHluorin), et la sous-unitéGluR5 du récepteur métabotropique (mGluR5) contenant 7 domaines transmembranaires et une séquence intracellulaire volumineuse. Les trois constructions portent une étiquette (GFP ou pHluorin) du côté extracellulaire. Les propriétés diffusives de ces molécules ont été mesurées par un suivi de particules uniques, à base de quantum dots. Ces expériences ont révélé que la diffusion des protéines membranaires est fonction du diamètre de la structure cylindrique considérée, et par conséquent moins rapide dans la tige de l’épine que dans le tronc du dendrite. Mais les propritétés diffusives dépendent aussi de la taille et delà complexité des molécules membranaires considérées. En effet, la diffusion de molécules comportant des domaines transmembranaires est particulièrement faible dans les tiges contenant de la synaptopodine. Cet aspect a été approfondi par l’utilisation de traitements pharmacologiques, qui ont permis de modifier la structure interne de la tige dendritique. Les variations des tailles des domainesoccupés par l’actine-F, et par lesaggrégats de synaptopodine, ont été observées à l’échelle nanoscopique en utilisant l’imagerie PALM/STORM. En conditions contrôle, la synaptopodine occupe la partie centrale de la tige. La dépolymérisation indirecte de l’actine-F par le 4-Aminopyridineentraîne une diminution des zones occupées par ces deux composants, corrélée à une augmentation de la vitesse de diffusion de mGluR5. En revanche, la dépolymérisation par la latrunculin-A (effet direct sur l’actine) induit une augmentation de la taille des clusters de synaptopodine et donc de la surface occupée par ceux-ci dans la tige. Les mesures de la diffusion de la sous-unité mGluR5 réalisées dans ces conditions montrent une accélération de la vitesse de diffusion, indiquant que la mobilité de mGluR5 n’est pas régulée par une interaction directe avec la synaptopodine. En conclusion, je propose un rôle de stabilisation mutuel pourl’actine-F et la synaptopodinedans la tige des épines dendritiques de neurones d’hippocampe en culture. Les épines contenant de la synaptopodine dans leur tige auraient une organisation unique du cytosquelette qui agirait comme une barrière additionnelle pour la diffusion de récepteurs aux neurotransmetteurs. / Lateral diffusion in and outside synapses plays a key role in the accumulation of receptors at synapses, which critically determines the efficacy of synaptic neurotransmission. Therefore, to better understand the trapping of neurotransmitter receptors in synapses, it is important to investigate the mechanisms that may affect receptors diffusion and their capacity to reach synapses. The neck of dendritic spine imposes a diffusional barrier that is considered to depend on the length and diameter of the spine neck. The origin of this barrier could be purely geometrical or could be induced by the presence of specific barriers/obstacles for diffusion. A subpopulation of spines contains a specialized form of endoplasmic reticulum in the spine neck called spine apparatus. The actin-binding protein synaptopodin (SP) is tightly associated with the spine apparatus and participates in synaptic plasticity mechanisms. The central question of my research was to assess whether the presence of the SP affects the diffusion of receptors in the spine neck and to characterize the underlying molecular mechanisms. To study membrane diffusion, I have developed three different probes: a construct associated with the outer leaflet of the plasma membrane (GFP-GPI), a construct with one transmembrane domain and a short intracellular sequence (TMD-pHluorin), and a recombinant metabotropic mGluR5 receptor construct containing an extracellular domain tagged with pHluorin, seven transmembrane domains, as well as a large intracellular region. The diffusion properties of these molecules were measured by single particle tracking using quantum dots. My experiments revealed that the diffusion of membrane proteins was slower in the spine neck than in the dendrite as a result of the different diameter of the two compartments. Furthermore, the diffusion properties depended on the molecular size and complexity of the membrane proteins. Interestingly, the diffusion of membrane proteins with transmembrane domains was particular slow in spine necks containing SP. This could be the result of direct molecular interactions between the membrane proteins and SP or due to spatial constraints that are related to the structural organization of spine necks expressing SP. To address these questions further I used pharmacological treatments to change the internal organization of the spine neck, and measured their effect on the diffusion properties of mGluR5. The distribution of SP and F-actin in the spine neck was determined on the nanoscopic scale using PALM/STORM imaging. This showed that under control condition SP occupies only the central region of the spine neck. Activity-dependent depolymerization of F-actin by 4-Aminopyridine led to a simultaneous decrease of the amount of F-actin and SP and enhanced the diffusion of mGluR5 in all analyzed neck regions. Disruption of F-actin by latrunculin A induced the re-distribution of SP and the formation of larger SP clusters, occupying an increased region within the spine neck. The recruitment of SP was accompanied by an acceleration of mGluR5 diffusion in SP-positive spines, demonstrating that the mobility of mGluR5 is not controlled by direct interactions with SP. Instead, the diffusion of mGluR5 is dependent on the organization of the spine cytoskeleton. In conclusion, I propose that SP and the polymerization of actin filaments have a reciprocal effect on the stability of each other in the spine neck of cultured hippocampal neurons. Spine necks bearing SP have a unique F-actin cytoskeletal organization that acts as an additional diffusion barrier for neurotransmitter receptors such as mGluR5.