Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.

Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.

No description available.

Le cerveau est un organe complexe composé de neurones et des cellules non-neuronales. La communication entre les neurones a lieu via les synapses, dont le remodelage morphologique est considéré essentiel pour le traitement et le stockage des informations dans le cerveau des mammifères. Récemment, ce point de vue neuro-centré de la fonction synaptique a évolué, en prenant également en compte les processus gliaux à proximité immédiate de la synapse. Cependant, comme leur structure est bien en deçà de la résolution spatiale de la microscopie optique conventionnelle, les progrès dans les enquêtes dans leur environnement physiologique, le cerveau intact, ont été entravés. En effet, on sait peu sur les variations nanométriques de la morphologie des épines dendritiques et l'interaction avec les processus gliaux, et, finalement, comment elles affectent la transmission synaptique in vivo. Dans cette thèse, nous cherchons à visualiser la dynamique de la nano-morphologie des épines dendritiques et les processus gliaux dans le cortex à tonneaux de souris in vivo. Nous avons donc mis en place l’imagerie super-résolution 2P-STED en temps réel, ce qui permet une haute résolution spatiale et la pénétration profonde des tissus, chez la souris anesthésiée in vivo. Nous montrons que la nano-morphologie des épines est diversifiée, variable, mais globalement stable, et que les différences dans la morphologie des épines peut avoir un effet sur leur compartimentation in vivo. En outre, la mise en œuvre de l’imagerie super-résolution en double couleur in vivo et le développement d'une approche de marquage astrocytaire, nous ont permis de fournir la caractérisation à l'échelle nanométrique des interactions neurone-glie. Ces résultats apportent un aperçu sans précédent dans la dynamique de la synapse à l'échelle nanométrique in vivo, et ouvrent la voie à une meilleure compréhension de la façon dont les réarrangements morphologiques des synapses contribuent à la physiologie du cerveau. / The brain is a complex organ consisting of neurons and non-neuronal cells. Communication between neurons takes place via synapses, whose morphological remodeling is thought to be crucial for information processing and storage in the mammalian brain. Recently, this neuro-centric view of synaptic function has evolved, also taking into account the glial processes in close vicinity of the synapse. However, as their structure is well below the spatial resolution of conventional light microscopy, progress in investigating them in a physiological environment, the intact brain, has been impeded. Indeed, little is known on the nanoscale morphological variations of dendritic spines, the interaction with glial processes, and how these affect synaptic transmission in vivo. Here, we aim to visualize the dynamic nano-morphology of dendritic spines in mouse somatosensory cortex in vivo. We implemented super-resolution 2P-STED time-lapse imaging, which allows for high spatial resolution and deep tissue penetration, in anesthetized mice, and show that the nano-morphology of spines is diverse, variable, but on average stable, and that differences in spine morphology can have an effect on spine biochemical compartmentalization in vivo. Moreover, implementation of dual color in vivo super-resolution imaging and a novel astrocytic labeling approach provided the first steps towards nanoscale characterization of neuron-glia interactions in vivo. These findings bring new insights in synapse dynamics at the nanoscale in vivo, and our methodological endeavors help pave the way for a better understanding of how nanoscale aspects of spine morphology and their dynamics might contribute to brain physiology and animal behavior.

Les cellules iNKT et T CDS innées sont présumées contribuer à l'irnmunosurveillance (IS) des cancers et sont fonctionnellement déficientes dans la leucémie myéloïde chronique (LMC). Notre hypothèse était que ces défauts résultent de l'incapacité des cellules dendritiques myéloïdes (mDC) à les activer. Des analyses par cytométrie en flux et microscopie confocale ont révélé une baisse de l'expression membranaire de CD 1 d, qui présente les antigènes aux cellules iNKT, à la surface des mDC des patients LMC, par comparaison aux sujets sains. Ce défaut n'est associé ni à un défaut de maturation des mDC, comme le montre l'expression normale de HLA-DR et de CDS6, ni à une baisse d'expression intracellulaire de CDld ou de son transcrit. Ces résultats sont conciliables avec une rétention intracellulaire. Le traitement in vitro des mDC des patients LMC avec un inhibiteur de la protéine ROCK restaure partiellement l'expression de surface de CD 1 d et la présentation antigénique par CD Id, alors qu'il n'a eu aucun effet sur les mDC des sujets sains. Nous proposons que la protéine ROCK, qui est activée par le domaine DH-PH de BCR-ABL, interfere avec la réponse immunitaire dépendant des lymphocytes iNKT au cours de la LMC par régulation négative de l'expression membranaire de CDld des mDC. Le fait que les cellules iNKT et T CDS innées retrouvent des fonctions normales après rémission complète de la LMC est en faveur d'une génération de cellules T CD8 innées dépendante des cellules iNKT, comme décrit chez la souris. Notre travail suggère une implication des cellules iNKT et T CD8 innées dans l'IS de la LMC et révèle l'axe ROCK/mDC comme une nouvelle cible thérapeutique dans la maladie. / CDld-restricted iNKT cells and innate CD8 T cells are believed to play a key role in cancer immune surveillance and are functionally deficient in chronic myeloid leukemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDC). Indeed, flow cytometry and confocal microscopy revealed that cell-surface expression of CDld was downregulated in CML mDC, relative to healthy donor (HD) controls. The decreased cell-surface display of CDld could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CDld or its mRNA transcripts, consistent with intracellular retention. ln vitro treatrnent of CML mDC with the Rho-associated protein Kinase (ROCK) inhibitor Y-27632 partially restored both cell-surface CDld expression and CDld-mediated antigen presentation, while it had no effect on HD mDC. We propose that ROCK, which is most likely activated by the DH-PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CDld expression on CML mDC. Remarkably, both iNKT cells and innate CD8 T cells retumed to nonnal after complete CML remission, a finding consistent with a iN KT cell-dependent generation of innate CD8 T cells, similarly to the observations in mice. Ali in ali, our study supports the possible contribution of iNKT/innate CD8 T cells to tumor surveillance in CML, and reveals the ROCK/mDC axis as a new potential target to restore immune surveillance in CML.

Dans les conditions physiologiques des robustes mécanismes immunologiques empêchent le développement des réponses exagérées aux antigènes alimentaires. En revanche, dans le cas de maladie céliaque, qui affecte environ 1% de la population occidentale, l’exposition au gluten alimentaire d’individus génétiquement prédisposés HLA-DQ2.5/DQ8 provoque l’entéropathie chronique de l’intestin grêle. Les études précédentes chez l’homme ont établi le rôle crucial de la réponse cellulaire T CD4+ restreinte par HLA-DQ2.5/DQ8 et spécifique du gluten. La réponse T CD4+ est nécessaire mais cependant insuffisante pour induire des lésions tissulaires. D’autres études ont suggéré le rôle de l’interleukine 15 (IL-15). Ainsi, l’IL-15 surexprimée dans la muqueuse des patients céliaques peut interférer avec les mécanismes d’immunorégulation et stimuler l’activation des lymphocytes intraépithéliaux T CD8+ cytotoxiques probablement induisant des lésions épithéliales. Comment les cellules T CD4+ spécifiques du gluten et l’IL-15 interagissent pour activer les lymphocytes intraépithéliaux T CD8+ et induisent des lésions n’a pas été toutefois établi. Pour répondre à cette question, nous avons créé un modèle murin basé en croisant des souris OTII possédant des cellules T CD4+ spécifiques de l’antigène modèle, ovalbumine, avec les souris transgéniques hétérozygotes surexprimant une forme secrétée de l’IL-15 humaine dans l’épithélium intestinale (souris hIL-15Tge). Les souris obtenues OTII+/- B6 and OTII+/- hIL-15Tge+/- ont été mises au régime riche en ovalbumine depuis la période prénatale jusqu’à l’âge de 3 mois. Les souris OTII+/- hIL-15Tge+/-, contrairement aux souris OTII+/- B6, exposées de façon chronique à l’ovalbumine ont développé un retard de croissance et une atrophie villositaire associée à l’expansion des cellules intestinales T CD8+ cytotoxiques, comme dans la maladie céliaque. En outre, nous avons démontré que l’IL-15 altérait l’immunorégulation par les cellules T FoxpP3+ et coopérait avec l’IL-2, produite par les cellules T CD4+ activées par l’OVA, pour l’expansion des cellules T CD8+ non-spécifiques de l’OVA. Nous suggérons que le scénario similaire pourrait opérer dans la maladie céliaque. Au cours de cette étude, j’ai observé que la surexpression chronique de l’IL-15 était associée avec l’expansion de cellules dendritiques CD103+CD11c+CD11b-. Dans la partie de résultats supplémentaires, j’ai démontré que cet effet dépend de la production de la cytokine GM-CSF secrétée par les cellules Natural Killer (NK) activées par l’IL-15 et que ces cellules dendritiques étaient enrichies en cellules CD103+ ayant une capacité accrue de cross-présentation in vitro. Ces derniers résultats illustrent comment l’IL-15 peut moduler les réponses immunes adaptatives en orchestrant la coopération entre les cellules NK et les phagocytes mononucléaires. / In physiological conditions, robust immunological mechanisms avoid adverse responses to food antigens. In contrast, in celiac disease that affects about 1% of Western populations, exposure to dietary gluten of genetically predisposed HLA-DQ2.5/ DQ8 individuals triggers a chronic small intestinal enteropathy. Previous studies in humans have established the crucial role of HLA-DQ2/DQ8 restricted gluten-specific intestinal CD4 T cell response. This CD4 T cell response is necessary but is however not sufficient to induce tissue damage. Other studies have pointed to the role of interleukin 15 (IL-15). Thus, IL-15 over-expressed in the mucosa of celiac patients can interfere with immunoregulatory mechanisms and stimulate the activation of cytotoxic CD8 T intraepithelial lymphocytes, thought to induce epithelial lesions. Whether and how gluten-specific CD4 T cells and IL-15 interact to activate CD8 T intraepithelial lymphocytes and to drive intestinal tissue damage has not been however established. To address this question, we have set up a mouse model based on the breeding of OTII mice possessing CD4 T cells specific of a model antigen, ovalbumin, with heterozygous transgenic mice overexpressing a secreted form of human IL-15 in intestinal epithelium (hIL-15Tge mice). Resulting OTII+/- B6 and OTII+/- hIL-15Tge+/- mice were exposed to dietary ovalbumin from the prenatal period until 3 months of age. Upon chronic exposure to ovalbumin, OTII+/- hIL-15Tge+ mice, contrary to their OTII+/- B6 littermates, developed growth retardation, and villous atrophy associated with expansion of intestinal cytotoxic CD8 T cells, as in celiac disease. Moreover, we showed that IL-15 impaired immunoregulation by FoxP3+ T cells and cooperated with IL-2 produced by OVA-activated CD4 T cells to stimulate the expansion of non-cognate cytotoxic CD8 T cells. We suggest that a comparable scenario can operate in celiac disease. During this study, I observed that chronic overexpression of IL-15 was associated with an expansion of CD103+CD11c+CD11b- mononuclear cells. In the Supplementary results, I have shown that this effect depends on the production of GM-CSF secreted by IL-15-activated NK cells and that CD11c+ DCs differentiated in mice overexpressing IL-15 were enriched in CD103+ cells and displayed enhanced cross-presentation abilities in vitro. The latter results illustrate how IL-15, by orchestrating a crosstalk between NK cells and mononuclear phagocytes, can modulate adaptive immune responses.

Il est maintenant bien établi que le système immunitaire joue un rôle majeur dans le contrôle des tumeurs, y compris dans les carcinomes pulmonaires. Cependant, les interactions entre les cellules tumorales et les cellules immunitaires du microenvironnement tumoral sont mal connues. Dans ce travail, nous avons étudié les caractéristiques morphologiques et moléculaires des cellules tumorales provenant d’adénocarcinomes pulmonaires et leur association avec la composition du microenvironnement immunitaire. Nous avons rapporté la valeur pronostique des paramètres morphologiques de ces tumeurs, comme le grade histologique des adénocarcinomes, et leur association avec le statut moléculaire EGFR et KRAS. Nous avons émis l’hypothèse que la diversité morphologique et moléculaire de ces tumeurs pouvait être associée à une signature immunitaire intra-tumorale spécifique et que cela pourrait avoir un impact pronostique. Nous avons mis en évidence que la densité des cellules dendritiques matures, situées au sein de structures lymphoïdes tertiaires, variait en fonction du statut moléculaire EGFR et KRAS des tumeurs. De même, l’impact pronostique des cellules dendritiques matures et des lymphocytes CD8+ variait en fonction du statut moléculaire des tumeurs. Nous avons également retrouvé la valeur pronostique de l’environnement immunitaire, représenté par la densité en cellules dendritiques matures et en lymphocytes CD8+, sur la survie à long terme des carcinomes pulmonaires de stade III-N2 opérés après chimiothérapie néoadjuvante. Enfin, nous avons démontré que la chimiothérapie n'est pas associée à de profondes modifications de l’infiltrat immunitaire, alors qu’elle entraîne des modifications des cellules tumorales. L’ensemble de ces résultats suggère que l’infiltrat immunitaire est intimement lié à la cellule tumorale et que la composition du microenvironnement immunitaire varie avec les caractéristiques de la tumeur. Cette interaction entre les cellules tumorales et les cellules immunitaires contribue au pronostic de ces tumeurs. Ces données démontrent l’intérêt d’utiliser des traitements combinant des drogues cytotoxiques, telle la chimiothérapie conventionnelle, à des traitements immunomodulateurs permettant de favoriser une réponse immunitaire anti-tumorale efficace. / The major role of the immune system against tumor development is now clearly established, including lung carcinoma. Nevertheless, interaction between tumoral cells and immune environment is less well-defined. In that study, we have studied morphological and molecular tumoral cells characteristics from lung adenocarcinoma and their role in the composition of immune environment. We reported the prognostic value of morphological parameters, as histological grade of adenocarcinoma, and their association with molecular EGFR and KRAS status. We hypothesized that morphological and molecular diversity of these tumors could be associated with a specific intratumoral immune signature, and could have an impact in prognosis. We showed that mature dendritic cells density, located in tertiary lymphoid structures, differed according to EGFR and KRAS status. Morever, molecular status of tumors modified the pronostic value of mature dendritics cells and CD8+ T cells. We found a prognostic value of immune environment, represented by dendritic cells and T CD8+ cells, in operated stage III-N2 lung carcinomas treated by neoadjuvant chemotherapy. At last, we demonstrated that chemotherapy is not associated with wide modifications in immune infiltrate, whereas it induced modifications in tumoral cells. All together, these data strongly argue for a close link between tumoral cells and immune environment, which seems to depend on tumoral cell characteristics. This interaction between tumoral cells and immune cells contribute to the prognosis of these tumors. These results show the evidence that combine cytotoxic treatment, like conventional chemotherapy, with immunomodulators, favour a protective anti-tumor immune response.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.

Background Growing evidence emphasizes the relevance of sphingolipids for metabolism and immunity of antigen-presenting cells (APC). APCs are key players in balancing tolerogenic and encephalitogenic responses in immunology. In contrast to the well-known prominent effects of sphingosine-1-phosphate (S1P) on lymphocyte trafficking, modulatory effects on APCs have not been fully characterized. Methods Frequencies and activation profiles of dendritic cell (DC) subtypes, monocytes, and T cell subsets in 35 multiple sclerosis (MS) patients were evaluated prior and after undergoing fingolimod treatment for up to 24 months. Impact of fingolimod and S1P on maturation and activation profile, pro-inflammatory cytokine release, and phagocytotic capacity was assessed in vitro and ex vivo. Modulation of DC-dependent programming of naïve CD4+ T cells, as well as CD4+ and CD8+ T cell proliferation, was also investigated in vitro and ex vivo. Results Fingolimod increased peripheral slanDC count—CD1+ DC, and monocyte frequencies remained stable. While CD4+ T cell count decreased, ratio of Treg/Th17 significantly increased in fingolimod-treated patients over time. CD83, CD150, and HLADR were all inhibited, but CD86 was upregulated in DCs after incubation in the presence of fingolimod. Fingolimod but not S1P was associated with reduced release of pro-inflammatory cytokines from DCs and monocytes in vitro and ex vivo. Fingolimod also inhibited phagocytic capacity of slanDCs and monocytes. After fingolimod, slanDCs demonstrated reduced potential to induce interferon–gamma-expressing Th1 or IL-17-expressing Th17 cells and DC-dependent T cell proliferation in vitro and in fingolimod-treated patients. Conclusions We present the first evidence that S1P-directed therapies can act additionally as immunomodulators that decrease the pro-inflammatory capabilities of APCs, which is a crucial element in DC-dependent T cell activation and programming.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.