CD103-mediated regulation of airway hypersensitivity responses to bioaerosol-associated antigens

Les mécanismes immunitaires impliqués dans le maintien de l’homéostasie pulmonaire sont finement régulés étant donné l’exposition constante des voies aériennes aux bioaérosols. Plusieurs cellules participent au maintien de l’homéostasie pulmonaire, telles les cellules dendritiques. Un sous-type de cellules dendritiques pulmonaires attire particulièrement l’attention dans l’homéostasie pulmonaire, les cellules dendritiques CD103+, étant donné qu’il a été démontré qu’elles participent dans la tolérance immune. Toutefois, ce rôle reste controversé, car des études démontrent qu’elles participent plutôt au développement de réponses inflammatoires pulmonaires. De plus, le CD103 (une intégrine exprimée par des sous-types de cellules dendritiques et de lymphocytes T), est surtout utilisé comme marqueur cellulaire et le rôle spécifique joué par l’expression du CD103 sur ces cellules reste inconnu. L’homéostasie pulmonaire n’est pas toujours maintenue. Chez des individus susceptibles, l’exposition aux bioaérosols peut mener au développement de réponses inflammatoires. C’est le cas pour l’asthme et l’alvéolite allergique extrinsèque, deux réponses d’hypersensibilités pulmonaires, de type I et de type mixte III/IV respectivement. Récemment, des espèces d’archées, Methanosphaera stadtmanae (MSS) et Methanobrevibacter smithii (MBS), ont été retrouvées en grande concentration dans les bioaérosols d’environnements agricoles et il a été démontré que l’exposition pulmonaire à leur extrait mène au développement d’une réponse immune chez la souris. Toutefois, le type de réponse d’hypersensibilité pulmonaire qu’elles induisent reste méconnu, une information cruciale qui permettra la poursuite de la recherche sur leur potentiel d’induire une réponse pulmonaire chez l’humain. De plus, même si plusieurs thérapies contre les maladies d’hypersensibilité pulmonaires existent, ce ne sont pas tous les sous-groupes de patients qui répondent à la médication, menant à des conséquences socio-économiques importantes pour le système de santé et pour les patients. Ainsi, il demeure important de poursuivre la recherche sur de potentielles cibles thérapeutiques, telles les cellules impliquées dans le maintien de l’homéostasie pulmonaire. Cette thèse vise donc à évaluer le rôle de l’expression du CD103 dans le maintien de l’homéostasie pulmonaire dans le contexte de maladies d’hypersensibilité pulmonaires induites par des antigènes retrouvés dans les bioaérosols. Le rôle de l’expression du CD103 dans l’hypersensibilité de type I induite par l’ovalbumine ou l’extrait d’acariens (modèles d’asthme) a d’abord été a évalué via l’utilisation de souris Cd103-/-. Nous démontrons que l’expression du CD103 est cruciale pour le contrôle de la sévérité de l’inflammation pulmonaire et qu’elle pourrait être impliquée dans l’initiation de la phase de résolution de la réponse inflammatoire. De plus, l’expression du CD103 sur les cellules dendritiques joue un rôle dans leur migration aux ganglions lymphatiques. Ensuite, nous avons évalué le rôle de l’expression du CD103 dans la réponse d’hypersensibilité de type mixte III/IV en réponse à Saccharopolyspora rectivirgula (SR; modèle d’alvéolite allergique extrinsèque) en utilisant des souris Cd103-/-. De plus, en utilisant des modèles de transfert de cellules, nous avons évalué le rôle de l’expression du CD103 dans la réponse au SR lorsque seulement exprimé par les cellules dendritiques ou seulement par les lymphocytes T CD4. Nous démontrons que c’est l’expression du CD103 sur les cellules dendritiques spécifiquement qui est impliquée dans la régulation de l’initiation de la réponse inflammatoire. Après avoir déterminé le type de réponse d’hypersensibilité induite par l’extrait de MSS ou MBS, nous avons étudié le rôle de l’expression du CD103 en réponse à ces archées. Nous démontrons que l’exposition à MSS induit une réponse immune typique d’une hypersensibilité pulmonaire de type IV. Les résultats obtenus après l’exposition à MBS indiquent aussi que la réponse développée est une hypersensibilité de type IV, même si cela reste à confirmer. Finalement, étant donné une grande variabilité entre nos expériences chez les souris Cd103-/-, nous n’avons pu obtenir de conclusion sur le rôle de l’expression du CD103 dans les réponses d’hypersensibilités induites par les archées. Ces résultats démontrent que l’expression du CD103 sur les cellules dendritiques joue un rôle dans le contrôle de l’homéostasie pulmonaire en réponse à des bioaérosols spécifiques qui induisent une hypersensibilité pulmonaire. Les mécanismes exacts régulés par le CD103 sur les cellules dendritiques menant au maintien de l’homéostasie pulmonaire restent à être élucidé. De plus, nos résultats confirment que les espèces d’archées MSS et MBS induisent chacune une réponse d’hypersensibilité pulmonaire qui lui est spécifique, des résultats qui contribueront à déterminer si ces microorganismes induisent une pathologie chez l’Homme.As we breathe, the lungs are constantly exposed to bioaerosols that challenge the maintenance of airway homeostasis. Many cells are involved in the maintenance of lung homeostasis, such as airway dendritic cells (DCs). A subset of airway DCs has gained special interest in the past years for its role in immune tolerance: CD103+ DCs. Yet, this role remains controversial as there are also reports that they induce airway inflammatory responses. Furthermore, CD103 (an integrin expressed by subsets of DCs and T cells) is mostly used as a marker and whether CD103 expression on these cells plays a specific role remains unknown. Airway homeostasis is not always maintained. Exposure to bioaerosols can elicit an immune response in susceptible individuals, such as in asthma and hypersensitivity pneumonitis, two common airway hypersensitivity diseases of type I and mixed type III/IV hypersensitivity, respectively. Recently, archaea species Methanosphaera stadtmanae (MSS) and Methanobrevibacter smithii (MBS) were identified in high concentrations in bioaerosols from agricultural environments and their extracts were shown to induce an immune response in the airways of mice. However, the type of airway hypersensitivity response they induce remains unknown, a key information that is required if research is pursued on whether they elicit an airway hypersensitivity response in humans. Furthermore, although many therapies for airway hypersensitivity diseases exist, not all subsets of patients respond to the current medication, resulting in high social and economic impacts on the health system and patients. Therefore, research on potential therapy targets for airway hypersensitivity diseases, such as those involved in the maintenance of airway homeostasis, remains important. This thesis focuses on the role of CD103 expression in the maintenance of lung homeostasis in the context of airway hypersensitivity responses induced by antigens found in bioaerosols. We first assessed the role of CD103 expression in type I hypersensitivity in response to ovalbumin or house dust mite extract (models of experimental asthma) using Cd103-/- mice. We found that CD103 expression is crucial in controlling the severity of airway inflammation and could be involved in initiating the resolution of the inflammatory response. Furthermore, CD103 expression on DCs regulates DC trafficking to the draining lymph nodes. We then assessed the role for CD103 expression in mixed type III/IV hypersensitivity in response to Saccharopolyspora rectivirgula extract (SR; model of experimental hypersensitivity pneumonitis) using Cd103-/- mice. Furthermore, using models of cell transfers, we evaluated the role for CD103 expression in the response to SR when specifically expressed by dendritic cells or specifically by CD4 T cells. We demonstrate that CD103 expression on DCs specifically is involved in regulating the onset of the inflammatory response. We finally studied the role for CD103 expression in response to the airway exposure of MSS and MBS extracts, after elucidating the type of hypersensitivity response they induce. We demonstrate that exposure to MSS induces a typical type IV hypersensitivity response. The results obtained after exposure to MBS also indicate development of a type IV hypersensitivity response, although it remains to be confirmed. Finally, due to high variability in the results using Cd103-/- mice, we were unable to reach a conclusion on the role for CD103 expression in response to archaea species. These results demonstrate that CD103 expression by DCs is involved in the control of airway homeostasis to specific airway hypersensitivity-inducing bioaerosols. The exact mechanisms regulated by CD103 on DCs leading to the maintenance of airway homeostasis remain to be elucidated. Furthermore, our results confirm that archaea species MSS and MBS induce a specific type of hypersensitivity response, which will contribute to the elucidation of whether they induce an airway pathology in humans

Chorographie du Royaume de Léon des Provinces des Asturies et de Galice

Representación planimétrica de hidrografía, vales, pontes, vías de comunicacion, abadías, castelos e bispadosLiñas límite de reinos, iluminadas con técnica de aguadaO relevo está representado por perfís abatidos (pequenas montañas)Toponimia local en castelán aínda que con erros. Cartela, rosa dos ventos e escalas gráficas en francésOrixe de lonxitude na illa del HierroContén rosa dos ventos indicando a orientación, expresando cunha flor de lis a direccion N

Transfers of DCs and CD4⁺ T cells.

<p>WT and <i>Cd103</i>^<i>-/-</i> DCs were injected in a criss-cross manner in WT and <i>Cd103</i>^<i>-/-</i> mice. 24h after injection, recipient mice were submitted to either the acute or chronic model. <b>A)</b> Total BAL neutrophils (cells/mL) and <b>B)</b> total BAL cells (cells/mL) of SR-exposed mice in the acute model. <b>C)</b> Total BAL lymphocytes (cells/mL) of SR-exposed mice in the chronic model. WT or <i>Cd103</i>^<i>-/-</i> CD4⁺ T cells were injected in <i>Rag</i>^<i>-/-</i> mice. 1 month after the transfer, recipient mice were submitted to the chronic model. <b>D)</b> Total BAL cells (cells/mL) and <b>E)</b> total BAL lymphocytes (cells/mL) of saline and SR-exposed mice. Results were pooled from two experiments; n = 6–10 mice/group. * = p < 0.05 with multi-comparison test.</p

Characterization of the airway inflammatory response in the chronic model of HP: BAL and IgGs.

<p><b>A)</b> Timeline of the chronic and acute models of exposure to SR. Full line represents days of intranasal instillation of either saline or SR while dashed line represents day of euthanasia. B-E): WT and <i>Cd103</i>^<i>-/-</i> mice were exposed to SR for 3 weeks and the BAL content and serum immunoglobulins were measured. <b>B)</b> BAL total and differential cell numbers (cells/mL) in saline and SR-exposed mice. <b>C)</b> BAL differential cell % (MΦ: macrophages, Ly: lymphocytes, Ne: neutrophils) in saline and SR-exposed mice. SR-specific serum levels of <b>D)</b> IgG₁ and <b>E)</b> IgG_2a. For B and C, results were pooled between two experiments, and are representative of over 5 separate experiments; n = 8–12 mice/group. For D and E, results are representative of 2 different experiments; n = 4–6 mice/group. * = p < 0.05.</p

Modulation of CD103 expression on DCs in response to SR <i>in vitro</i>.

<p>DCs were isolated from lung and spleen of WT mice and stimulated with 0μg/mL (ctrl), 1μg/mL or 5μg/mL of SR extract for 18h. DCs were identified as autofluorescence^-/ CD11c⁺/ MHC-II^hi cells and CD103 and XCR1 expression was analyzed as shown in <b>A)</b>. <b>B)</b> Flow cytometry examples of CD103 and XCR1 expression on SR-stimulated spleen- and lung-isolated DCs. <b>C)</b> Viability, measured with trypan blue, of spleen- and lung-isolated DCs after stimulation with SR. <b>D)</b> % of spleen- and lung-isolated DCs expressing CD103 (CD103⁺XCR1^-) or XCR1 and CD103 (CD103⁺XCR1⁺). <b>E)</b> Mean Fluorescence Intensity (MFI) of CD103 from spleen- and lung-isolated CD103⁺XCR1⁺ DCs. <b>F)</b> Transcript level of CD103 relative to Rplp0 and GNB in spleen- and lung-isolated DCs. <b>G)</b> <i>In vivo</i> CD103⁺ or CD103⁺XCR1⁺ on lung CD11c⁺/ MHC-II^hi DCs (as analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179678#pone.0179678.g004" target="_blank">Fig 4A</a>) 18h after SR exposure. Results are representative of 2 separate experiments; n = 5–6 mice/group. * = p < 0.05. † = p < 0.05 with multi-comparison test.</p

Characterization of the airway inflammatory response in the chronic model of HP: Histology and cytokine.

<p>WT and <i>Cd103</i>^<i>-/-</i> mice were exposed to SR for 3 weeks and a section of the left lobe was used for histology while other lobes were used for analysis of cytokine production. <b>A)</b> Lung sections of WT and <i>Cd103</i>^<i>-/-</i> mice exposed to saline or SR. <b>B)</b> Histological score was obtained and compared between WT and <i>Cd103</i>^<i>-/-</i> mice. <b>C)</b> Flow cytometry gating strategy for the polarity of the effector lung response after <i>ex vivo</i> stimulation of lung leukocytes isolated from SR-exposed mice. CD4⁺ T cells were gated from total lung cells and cytokine-positive cells were analyzed using Fluorescence Minus One (FMO) controls. <b>D)</b> Number of cells and <b>E)</b> % of IFNg⁺, IL-13⁺ and IL-17A⁺CD4⁺ T cells in the lung of saline and SR-exposed mice. Results representative of 2 different experiments; n = 4–6 mice/group. * = p < 0.05.</p

Airway inflammatory response in WT and <i>Cd103</i>^<i>-/-</i> mice in the acute model of HP.

<p>WT and <i>Cd103</i>^<i>-/-</i> mice were exposed to SR for 3 days and the BAL content was evaluated: <b>A)</b> BAL total and differential cell numbers (cells/mL) in saline and SR-exposed mice; <b>B)</b> BAL differential cell % (MΦ: macrophages, Ly: lymphocytes, Ne: neutrophils, Eo: eosinophils) in saline and SR-exposed mice. C-E): WT and <i>Cd103</i>^<i>-/-</i> mice were exposed to SR and the BAL content was evaluated 2h, 12h and 18h after the exposure: <b>C)</b> BAL total numbers (cells/mL) in saline and SR-exposed mice; <b>D)</b> BAL differential numbers (cells/mL) and <b>E)</b> % 18h after exposure to saline or SR. For A and B, results from 2 experiments were pooled; n = 6–12 mice/group. For C and D, results are representative of 2 different experiments; n = 3–4 mice/group. * = p < 0.05.</p

Benchmarking bioinformatic tools for fast and accurate eDNA metabarcoding species identification

Bioinformatic analysis of eDNA metabarcoding data is crucial toward rigorously assessing biodiversity. Many programs are now available for each step of the required analyses, but their relative abilities at providing fast and accurate species lists have seldom been evaluated. We used simulated mock communities and real fish eDNA metabarcoding data to evaluate the performance of 13 bioinformatic programs and pipelines to retrieve fish occurrence and read abundance using the 12S mt rRNA gene marker. We used four indices to compare the outputs of each program with the simulated samples: sensitivity, F-measure, root-mean-square error (RMSE) on read relative abundances, and execution time. We found marked differences among programs only for the taxonomic assignment step, both in terms of sensitivity, F-measure and RMSE. Running time was highly different between programs for each step. The fastest programs with best indices for each step were assembled into a pipeline. We compare this pipeline to pipelines constructed from existing toolboxes (OBITools, Barque, and QIIME 2). Our pipeline and Barque obtained the best performance for all indices and appear to be better alternatives to highly used pipelines for analyzing fish eDNA metabarcoding data with a complete reference database. Real eDNA metabarcoding data also indicated differences for taxonomic assignment and execution time only. This study reveals major differences between programs during the taxonomic assignment step. The choice of algorithm for the taxonomic assignment can have a significant impact on diversity estimates and should be made according to the objectives of the study