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

Infection with Influenzavirus A in a murine model induces epithelial bronchial lesions and distinct waves of innate immune-cell recruitment

IntroductionInflammatory lesions after Influenza A viruses (IAV) are potential therapeutic target for which better understanding of post-infection immune mechanisms is required. Most studies to evaluate innate immune reactions induced by IAV are based on quantitative/functional methods and anatomical exploration is most often non-existent. We aimed to study pulmonary damage and macrophage recruitment using two-photon excitation microscopy (TPEM) after IAV infection.MethodsWe infected C57BL/6 CD11c+YFP mice with A/Puerto Ricco/8/34 H1N1. We performed immune cell analysis, including flow cytometry, cytokine concentration assays, and TPEM observations after staining with anti-F4/80 antibody coupled to BV421. We adapted live lung slice (LLS) method for ex-vivo intravital microscopy to analyze cell motility.ResultsTPEM provided complementary data to flow cytometry and cytokine assays by allowing observation of bronchial epithelium lesions and spreading of local infection. Addition of F4/80-BV421 staining allowed us to precisely determine timing of recruitment and pulmonary migration of macrophages. Ex-vivo LLS preserved cellular viability, allowing us to observe acceleration of macrophage motility.ConclusionAfter IAV infection, we were able to explore structural consequences and successive waves of innate immune cell recruitment. By combining microscopy, flow cytometry and chemokine measurements, we describe novel and precise scenario of innate immune response against IAV

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

Cytoskeleton as an Emerging Target of Anthrax Toxins

Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT) and edema toxin (ET). So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity

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

L’éradication des maladies infectieuses virales mise en danger par les avancées de la biologie synthétique

L’éradication des maladies infectieuses est un des vieux rêves de l’humanité, qui ne s’est pour l’instant matérialisé qu’une seule fois dans l’histoire avec la variole en 1980. Des efforts très importants sont consentis contre les virus de la poliomyélite depuis 1988, mais l’objectif final n’est pas encore atteint. Paradoxalement, alors que le but d’éradiquer ces deux maladies virales est presque atteint, la biologie de synthèse multiplie les prouesses permettant la néosynthèse de virus, mettant ainsi en péril, au moins virtuellement, la pérennité de ces avancées. Cet article met en regard les potentialités de cette nouvelle biologie et la difficile réalité de la lutte contre les infections

Influence du stress sur l'établissement de la réponse vaccinale (étude in vivo chez la souris dans un modèle de stress de contention et modélisation in vitro sur des cultures dentritiques)

Le stress chronique induit des perturbations immunologiques. L'objectif de ce travail a été de caractériser les modulations de la réponse vaccinale induites par le stress. Dans une première partie, nous avons étudié la réponse immunitaire au vaccin anti-tétanique sur des souris soumises à un stress chronique de contention. Nos résultats montrent que le stress induit une altération majeure de la réponse cellulaire T anti-tétanique, associée à une diminution et un retard de la réponse humorale spécifique. Dans un deuxième temps nous avons mis au point un modèle in vitro. Nous avons développé un modèle de culture de cellules dendritiques murines dérivées de la moelle osseuse, sur lequel nous avons appliqué un stress physique, l'hyperthermie, composante du stress de contention. Nous montrons que l'hyperthermie régule la sécrétion des cytokines et les fonctions des cellules dendritiques. A 39 ʿC,la production d'interleukine-12 est augmentée, alors que celle de tumor necrosis factor-a et d'interleukine-10 est diminuée, ce qui est corrélé avec une capacité plus importante des cellules dendritiques à activer des lymphocytes T allogéniques. A 40 ʿC, la sécrétion de l'ensemble des trois cytokines est diminuée ce qui est corrélé avec une diminution de leur capacité à activer des lymphocytes T allogéniques. Ces résultats permettent de proposer que l'hyperthermie puisse jouer un rôle dans la régulation de la réponse immunitaire adaptative.Chronic stress is known to induce immunological disorders. The aim of this work is to characterise the stress-induced modulation of the vaccine response.We first investigated the consequences of chronic restraint stress on the immune response to tetanus toxin in mice. Our results have shown that stress induces a severe disruption of T-cell specific response associated with decreased and delayed humoral specific response. Next, we investigated the effects of environmental stress or of mediators of stress on antigen-presenting cells in vitro. We developed a model of murine bone marrow-derived dendritic cells. Dendritic cells were exposed to hyperthermia which is a component of the contention stress.We have shown that hyperthermia regulate cytokines secretion and functions of dendritic cells. At 39ʿC interleukin-12 secretion increased, but tumour necrosis factor-a and interleukin-10 secretions decreased. Moreover, 39ʿC-exposed dendritic cells activated more efficiently allogeneic T-cells. Dendritic cells exposed at 40ʿC decreased their overall secretion, which is correlated with a decrease of their ability to activate T-cell reponses.GRENOBLE1-BU Médecine pharm. (385162101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Les adjuvants vaccinaux actuels et futurs (évaluation de l'efficacité de nanoparticules de poly (acide lactique) (PLA) comme adjuvants dans un vaccin contre Bacillus anthracis)

LYON1-BU Santé (693882101) / SudocSudocFranceF