Apports de la microscopie biphotonique intravitale pulmonaire à l'étude de la physiopathologie de la maladie du charbon

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires.Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

International audience: Dynamics of the lung immune system at a microscopic level are largely unknown because of inefficient methods to rid chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process

Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms

Contribution of in vivo two-photon lung microscopy to the study of anthrax pathophysiology.

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires.Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections

Apports de la microscopie biphotonique intravitale pulmonaire à l'étude de la physiopathologie de la maladie du charbon

Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires

Dendritic Spines in Early Postnatal Fragile X Mice Are Insensitive to Novel Sensory Experience

Autism spectrum disorders are often associated with atypical sensory processing and sensory hypersensitivity, which can lead to maladaptive behaviors, such as tactile defensiveness. Such altered sensory perception in autism spectrum disorders could arise from disruptions in experience-dependent maturation of circuits during early brain development. Here, we tested the hypothesis that synaptic structures of primary somatosensory cortex (S1) neurons in Fragile X syndrome (FXS), which is a common inherited cause of autism, are not modulated by novel sensory information during development. We used chronic in vivo two-photon microscopy to image dendritic spines and axon "en passant" boutons of layer 2/3 pyramidal neurons in S1 of male and female WT and Fmr1 KO mice, a model of FXS. We found that a brief (overnight) exposure to dramatically enhance sensory inputs in the second postnatal week led to a significant increase in spine density in WT mice, but not in Fmr1 KO mice. In contrast, axon "en passant" boutons dynamics were impervious to this novel sensory experience in mice of both genotypes. We surmise that the inability of Fmr1 KO mice to modulate postsynaptic dynamics in response to increased sensory input, at a time when sensory information processing first comes online in S1 cortex, could play a role in altered sensory processing in FXS.SIGNIFICANCE STATEMENT Very few longitudinal in vivo imaging studies have investigated synaptic structure and dynamics in early postnatal mice. Moreover, those studies tend to focus on the effects of sensory input deprivation, a process that rarely occurs during normal brain development. Early postnatal imaging experiments are critical because a variety of neurodevelopmental disorders, including those characterized by autism, could result from alterations in how circuits are shaped by incoming sensory inputs during critical periods of development. In this study, we focused on a mouse model of Fragile X syndrome and demonstrate how dendritic spines are insensitive to a brief period of novel sensory experience

Dynamics of rapid spore capture by dendritic cells in the lung alveolus.

International audienc

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

International audienceImaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals

Maximal Distance of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. C, D: Overlay of Round-shaped cell tracks after late PBS (C) and LPS injection (D), after aligning their first coordinates. One color by track. Values of black circle radii in µm, equal to average cell Maximal Distance, are indicated ± standard deviation. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p