Thèse Exploration d'Un Nouveau Réservoir Pulmonaire Humain de Réplication du Virus de la Grippe H/F - Doctorat.Gouv.Fr

Établissement : Université de Tours École doctorale : Santé, Sciences Biologiques et Chimie du Vivant - SSBCV Laboratoire de recherche : Centre d'Etudes des Pathologies Respiratoires Direction de la thèse : Antoine GUILLON ORCID 0000000248848620 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-04-27T23:59:59 Le virus Influenza A (IAV) constitue un enjeu majeur de santé publique mondiale. Les macrophages alvéolaires (AM) sont essentiels à l'immunité pulmonaire, mais leur interaction avec IAV reste peu explorée, les études portant principalement sur des modèles murins ou dérivés de monocytes. Il est admis que le cycle viral est abortif dans les cellules myéloides. Nous démontrons que les AM humains (hAM) infectés par IAV produisent des virions, révélant une seconde niche inattendue de réplication virale en plus des cellules épithéliales des voies respiratoires (AEC). Nous avons émis l'hypothèse que la structure des virions peut varier en fonction de leur cellule d'origine. En effet, nos résultats préliminaires montrent que les hAMs et les hAECs génèrent des virions de morphologie distinctes. L'objectif de cette thèse est de décrire les différences structurelles entre les virions dérivés des hAEC et ceux des hAM, et d'explorer si la source de production virale modifie les interactions hôte-pathogène. Influenza continues to be a global health concern, with annual epidemic outbreaks causing significant morbidity and mortality. Influenza A virus (IAV) is the leading viral strain. Alveolar macrophages (AM) are critical players in innate immunity involved in the maintenance of lung homeostasis, or, at the contrary, in inducing excessive inflammation (i.e. hypercytokinemia) in pathologic conditions. 1,2
The interaction between influenza and macrophages remains an ongoing area of research that is mostly explored in murine macrophage models or monocytes.3,4 To date, the main conclusive information are: (i) viral cycle is abortive in macrophage meaning that the virus may begin genome replication and protein synthesis but new virions cannot be formed, (ii) IAV induces early apoptosis of monocytes, thereby avoiding their antiviral activity, (iii) an important reduction in the AM population is seen in IAV-infected mice models and referred to as the macrophage disappearance reaction'' which is suspected to be responsive for an increased lung vulnerability.1,2,4,5 Overall, the balance between potential benefits and drawbacks of these phenomena are unknown for the host.
We recently analyzed bronchoalveolar lavages (BAL) from patients with influenza pneumonia and found that the macrophage disappearance reaction does not occur in IAV-infected patients. Next, we examined immune-trained mouse models with AM pathophysiology more similar to humans. We observed that the macrophage disappearance reaction was present in infected mice as reported by others but did not occur in these human-like mice. This significant discrepancy between mice and humans in AM pathophysiology challenges the prevailing consensus and underscores the urgent need to reconsider the AM-influenza interaction using more appropriate experimental models.
To investigate this interspecies difference, we examined whether IAV may differ in its ability to infect murine or human AM (called mAM and hAM, respectively). We performed ex vivo IAV infection of AM, collected from human or naïve mice. We did not observe difference regarding the ability of IAV to infect AM and to produce viral protein in both mAM and hAM. We assessed virus release and spread with neuraminidase activity and the count of infectious viral particles. We found that, in contrast to what is observed in mice, the viral cycle in human macrophages was not abortive: IAV-infected hAM produced virions. These cutting-edge findings question our understanding on influenza pathophysiology. So far, airway epithelial cells (AEC) were considered as the only significant replication niche for influenza viruses. We demonstrated that AM are a significant secund source for virions production in the human lungs.
We next wondered whether virions budding from cells with distinct membrane compositions, such as AEC or AM, had diverse morphologies. We analysed serial sections of IAV-infected hAEC (Beas2B cells) and hAM by TEM and observed that hAEC produced mostly filamentous particles whereas hAM produced mostly spherical particles (observed with two different IAV strains - H3N2 Scotland, H1N1 PR8). These results suggest that infected hAM and hAEC produced virions with distinct morphologies.Objective:
The dynamics of host-pathogen interactions during influenza infection necessitate reevaluation, given that human lungs contain two distinct reservoirs of influenza viruses, AEC and AM, each generating virions with divergent morphological characteristics. We aim: (i) to describe the structural differences between hAEC-derived and hAM-derived influenza virions; (ii) to explore whether the source of virus production alter the pathophysiology of the infection; (iii) to reveal if these distinct IAV sources are observed in influenza-infected human. Work package 1. To describe cell host-specific features of influenza virion architecture in human
Task 1.1. To compare the shape of the virions according to cell-origin. Particle size will be measured using a Malvern instrument based on light scattering in collaboration with Team 3 from Inserm U1100, as previously performed.6 Size distribution and key parameters such as mean diameter and polydispersity will be compared from particles produced by IAV-infected human epithelial cell models and hAM.
Task 1.2. To compare the protein and lipid composition of hAM-derived or hAEC-derived influenza virions. We will reveal the protein and lipid composition protein of hAM- or hAEC-derived influenza virions using spectral index normalization quantification from LC-MS/MS analysis and UHPLC-HRMS. A focus on the glycosylation profiles will be performed using lectin array.
Work package 2. To examine the pathophysiology of virion according to cell source
Task 2.1 Analysis of viral spread. Supernatant of IAV-infected hAMs and hAECs will be collected and calibrated to provide the same concentration of virions. Viral spread of hAM-derived or hAEC-derived influenza virions will then be compared on host cells (viral production, inflammatory response, cell viability).
Task 2.2 Microenvironment of IAV-infected hAM and hAEC. We hypothesized that the resulting microenvironment of IAV-infected hAMs and hAECs could drive different infection dynamics in neighboring cells. To recapitulate in vivo cell behaviors, we will used co-culture methods. hAM or hAEC will be seeded on cell culture inserts and infected with IAV for 12h. Secondly, inserts containing infected cells will be positioned above a layer of host epithelial cells (Beas-2B cells). Infection dynamics will be characterized through multiple assays. Subsequently, co-culture models incorporating key host immune components (mucin, HA-binding antibodies, and surfactant protein D) will be implemented.
Task 2.3 In vivo assessment of pathophysiology hAM- or hAEC-derived influenza virions. If we find differences in pathophysiology between virions from different cell sources (Tasks 2.1 or 2.2), we will test supernatant of IAV-infected hAMs and hAECs in mice.
Work package 3. To characterize the macrophage-origin virion in the human context
Task 3.1. Exploration of IAV-infected AM in human lungs. Because the production of virions by macrophages is a terra incognita so far, we will explore the infection of hAM using human ex vivo model (human Precision Cut Lung Slices, hPCLS) in collaboration with the Thoracic Surgery Unit from Tours Academic Hospital. Infected hPCLS will be examined using confocal imaging to reveal viral nucleoprotein expression in hAMs. Supernatant of infected and non-infected hPCLS will be analyzed using a Malvern instrument for a quantitative analysis of viral shape and compared with results from Task 1.1.
Task 3.2. To investigate the macrophage-origin virions in IAV-infected patients.
The aim is to collect virions in IAV-infected patients for further characterisation. BAL fluid of IAV-infected patients from the intensive care unit of Tours Academic Hospital (IRB approved Tours Academic Hospital) will be processed as previously defined. Composition (protein and lipid) and shape of virions will be compared with results generated in Task 1.1, 1.2 and 3.1.

Excellentes compétences relationnelles, sens du travail en équipe et aptitude à évoluer dans un environnement multidisciplinaire (immunologie, virologie, bioinformatique). Communication claire à l'oral comme à l'écrit. Autonomie, sens de l'organisation et capacité d'adaptation dans un contexte scientifique présentant des défis expérimentaux.
Doctorant(e) faisant preuve d'une grande rigueur scientifique, d'intégrité et d'esprit critique. Curiosité intellectuelle, capacité d'analyse et persévérance.
Etre passionné(e) par la science :)