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1. The gut microbiota and its metabolite butyrate shape metabolism and antiviral immunity along the gut-lung axis in the chicken

Author

Vincent Saint-Martin, Vanaique Guillory, Mélanie Chollot, Isabelle Fleurot, Emmanuel Kut, Ferdinand Roesch, Ignacio Caballero, Emmanuelle Helloin, Emilie Chambellon, Brian Ferguson, Philippe Velge, Florent Kempf, Sascha Trapp, and Rodrigo Guabiraba

Subjects
Biology (General), QH301-705.5
Abstract

Abstract The gut microbiota exerts profound influence on poultry immunity and metabolism through mechanisms that yet need to be elucidated. Here we used conventional and germ-free chickens to explore the influence of the gut microbiota on transcriptomic and metabolic signatures along the gut-lung axis in poultry. Our results demonstrated a differential regulation of certain metabolites and genes associated with innate immunity and metabolism in peripheral tissues of germ-free birds. Furthermore, we evidenced the gut microbiota’s capacity to regulate mucosal immunity in the chicken lung during avian influenza virus infection. Finally, by fine-analysing the antiviral pathways triggered by the short-chain fatty acid (SCFA) butyrate in chicken respiratory epithelial cells, we found that it regulates interferon-stimulated genes (ISGs), notably OASL, via the transcription factor Sp1. These findings emphasize the pivotal role of the gut microbiota and its metabolites in shaping homeostasis and immunity in poultry, offering crucial insights into the mechanisms governing the communication between the gut and lungs in birds.

Published
2024
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2. Exploring type I interferon pathway: virulent vs. attenuated strain of African swine fever virus revealing a novel function carried by MGF505-4R

Author

Juliette Dupré, Mireille Le Dimna, Evelyne Hutet, Pascal Dujardin, Aurore Fablet, Aurélien Leroy, Isabelle Fleurot, Grégory Karadjian, Ferdinand Roesch, Ignacio Caballero, Olivier Bourry, Damien Vitour, Marie-Frédérique Le Potier, and Grégory Caignard

Subjects
African Swine Fever Virus, IFN, viral immune evasion, virulence factor, TRAF3, Immunologic diseases. Allergy, RC581-607
Abstract

African swine fever virus represents a significant reemerging threat to livestock populations, as its incidence and geographic distribution have surged over the past decade in Europe, Asia, and Caribbean, resulting in substantial socio-economic burdens and adverse effects on animal health and welfare. In a previous report, we described the protective properties of our newly thermo-attenuated strain (ASFV-989) in pigs against an experimental infection of its parental Georgia 2007/1 virulent strain. In this new study, our objective was to characterize the molecular mechanisms underlying the attenuation of ASFV-989. We first compared the activation of type I interferon pathway in response to ASFV-989 and Georgia 2007/1 infections, employing both in vivo and in vitro models. Expression of IFN-α was significantly increased in porcine alveolar macrophages infected with ASFV-989 while pigs infected with Georgia 2007/1 showed higher IFN-α than those infected by ASFV-989. We also used a medium-throughput transcriptomic approach to study the expression of viral genes by both strains, and identified several patterns of gene expression. Subsequently, we investigated whether proteins encoded by the eight genes deleted in ASFV-989 contribute to the modulation of the type I interferon signaling pathway. Using different strategies, we showed that MGF505-4R interfered with the induction of IFN-α/β pathway, likely through interaction with TRAF3. Altogether, our data reveal key differences between ASFV-989 and Georgia 2007/1 in their ability to control IFN-α/β signaling and provide molecular mechanisms underlying the role of MGF505-4R as a virulence factor.

Published
2024
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3. TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

Author

Isabelle, Fleurot, Raquel, López-Gálvez, Pascal, Barbry, Antoine, Guillon, Mustapha, Si-Tahar, Andrea, Bähr, Nikolai, Klymiuk, Jean-Claude, Sirard, Ignacio, Caballero, Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Gene Center and Center for Innovative Medical Models (CIMM), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Association Vaincre la Mucoviscidose (grants RF20150501357, RF20160501644 and RF20170502036), ANR-18-CE20-0024,PIGIMMUNITY,PIGIMMUNITY: une approche de biologie des systèmes pour stimuler la réponse innée chez le porc(2018), Infectiologie et Santé Publique [UMR ISP], Institut de pharmacologie moléculaire et cellulaire [IPMC], Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 [CEPR], Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Ludwig Maximilian University [Munich] (LMU), CHU Lille, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), brea, deborah, APPEL À PROJETS GÉNÉRIQUE 2018 - PIGIMMUNITY: une approche de biologie des systèmes pour stimuler la réponse innée chez le porc - - PIGIMMUNITY2018 - ANR-18-CE20-0024 - AAPG2018 - VALID, Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Cystic Fibrosis, Swine, education, Cystic Fibrosis Transmembrane Conductance Regulator, respiratory system, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Epithelium, respiratory tract diseases, Toll-Like Receptor 5, Animals, Humans, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Lung, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Flagellin
Abstract

International audience; Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.

Published
2021
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4. Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa

Author

Raquel López-Gálvez, Isabelle Fleurot, Pablo Chamero, Sascha Trapp, Michel Olivier, Claire Chevaleyre, Céline Barc, Mickael Riou, Christelle Rossignol, Antoine Guillon, Mustapha Si-Tahar, Tobias May, Pascal Barbry, Andrea Bähr, Nikolai Klymiuk, Jean-Claude Sirard, Ignacio Caballero

Abstract

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity–mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as toPseudomonas aeruginosainfection. Moreover,invivoadministration of flagellin decreased the severity ofP. aeruginosa–induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by usingex vivoprecision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Published
2021

5. Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets

Author

Catherine Robbe-Masselot, Mustapha Si-Tahar, Ignacio S. Caballero, Renaud Léonard, Nicolas Pons, Andrea Bähr, Pascal Barbry, Antoine Guillon, Agnès Paquet, Claire Chevaleyre, Nikolai Klymiuk, Isabelle Fleurot, Mustapha Berri, Bélinda Ringot-Destrez, Kevin Lebrigand, Carole Baron, Céline Barc, Reuben Ramphal, Isabelle Lantier, Centre National de la Recherche Scientifique (CNRS), Université Côte d'Azur (UCA), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Université Côte d'Azur (UCA), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Service de Médecine Intensive et Réanimation [Tours], Plateforme d'Infectiologie Expérimentale (PFIE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ludwig Maximilian University [Munich] (LMU), Association Vaincre la Mucoviscidose (Grant No. RF20150 501357), Association Grégory Lemarchal (Grant No. RIF20160501690), Fondation pour la Recherche Médicale (DEQ20180339158), ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Chanteloup, Nathalie Katy, Centres d'excellences - Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie - - SIGNALIFE2011 - ANR-11-LABX-0028 - LABX - VALID, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Université de Tours (UT), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours (UT), Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Gene Center and Center for Innovation Medical Models (CiMM), Ludwig Maximilians University of Munich, Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)

Subjects
0301 basic medicine, Pulmonary and Respiratory Medicine, Male, Glycosylation, Mucociliary clearance, Swine, [SDV]Life Sciences [q-bio], Sus scrofa, Cystic Fibrosis Transmembrane Conductance Regulator, Context (language use), Inflammation, Respiratory Mucosa, Mucociliary transport, medicine.disease_cause, Cystic fibrosis, Microbiology, 03 medical and health sciences, 0302 clinical medicine, Medicine, Animals, CFTR, ComputingMilieux_MISCELLANEOUS, Lung, medicine.diagnostic_test, business.industry, Pseudomonas aeruginosa, Mucin, Mucins, respiratory system, medicine.disease, 3. Good health, respiratory tract diseases, Trachea, [SDV] Life Sciences [q-bio], Mucin glycosylation, 030104 developmental biology, medicine.anatomical_structure, Bronchoalveolar lavage, 030228 respiratory system, Animals, Newborn, Mucociliary Clearance, Pediatrics, Perinatology and Child Health, Female, medicine.symptom, MESH: CFTR, business
Abstract

International audience; Background: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process.Methods: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa.Results: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation.Conclusions: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.

Published
2020
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6. Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets

Author

Mustapha Berri, Isabelle Fleurot, Sandrine Melo, Laura Soler, Ignacio S. Caballero, Ana-Paula Teixeira-Gomes, Valérie Labas, Nicolas Cenac, Mustapha Si-Tahar, Julien Pujo, Andrea Bähr, Déborah Bréa, Claire Chevaleyre, Nikolai Klymiuk, Antoine Guillon, SEGUIN, Nathalie, Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biosynthèse & Toxicité des Mycotoxines (ToxAlim-BioToMyc), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Infectiologie et Santé Publique (UMR ISP), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire d'Analyses des Interactions Moléculaires, Plate-forme d'Analyses intégrative des Biomarqueurs cellulaires et moléculaires (PAIB), UMR6175, Institut National de la Recherche Agronomique (INRA), Institut de Recherche en Santé Digestive (IRSD ), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Gene Center and Center for Innovation Medical Models (CiMM), Ludwig-Maximilians University [Munich] (LMU), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Vaincre la Mucoviscidose [grant numbers RF20130500925 and RF2015050357, Region Centre (France) [grant number 320 0 0604 ]., Metatoul- Lipidomique Core Facility-MetaboHub, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ludwig Maximilians University of Munich, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), and Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0301 basic medicine, Pulmonary and Respiratory Medicine, Male, Proteomics, Swine, Neutrophils, [SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], [SDV]Life Sciences [q-bio], Inflammation, medicine.disease_cause, Cystic fibrosis, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Immunophenotyping, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, CFTR, chemistry.chemical_classification, Reactive oxygen species, Proteomic Profile, Lung, medicine.diagnostic_test, Pseudomonas aeruginosa, business.industry, Models, Theoretical, medicine.disease, Pig model, Molecular biology, 3. Good health, [SDV] Life Sciences [q-bio], Disease Models, Animal, 030104 developmental biology, medicine.anatomical_structure, 030228 respiratory system, chemistry, Animals, Newborn, ICM-MS, Pediatrics, Perinatology and Child Health, Fatty Acids, Unsaturated, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, medicine.symptom, business, Reactive Oxygen Species
Abstract

International audience; Background: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process.Methods: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated.Results: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed.Conclusions: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.

Published
2020
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7. Novel approaches boosting innate immunity against Pseudomonas aeruginosa

Author

Isabelle Fleurot, Mickael Riou, Claire Chevaleyre, Sandrine Melo, Michel Olivier, Christelle Rossignol, Céline Barc, Jérémy Pezant, Alexis Pleau, Alain Deslis, Nikolai Klymiuk, Jean Claude Sirard, Ignacio Caballero, Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Plateforme d'Infectiologie Expérimentale (PFIE), Institut National de la Recherche Agronomique (INRA), Ludwig Maximilians University of Munich, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP), Société Française de Microbiologie (SFM). FRA., and Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT)

Subjects
[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Cystic Fibrosis, [SDV.BA]Life Sciences [q-bio]/Animal biology, Pseudomonas aeruginosa, bacteria, TLR5, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, Flagellin
Abstract

National audience; Pseudomonas aeruginosa, an opportunistic gram-negative bacterium that rarely infects human lungs unless the host immune system has been impaired, is one of the main pathogens found in cystic fibrosis (CF) patients. P. aeruginosa infections in CF patients are difficult to treat, becoming chronic and contributing to exacerbated lung inflammation and respiratory failure. Modulation of innate immunity has been proposed as an alternative to improve defence against infections. This approach is particularly attractive in CF since exacerbated immune response is central to the pathogenesis of CF lung disease. Innate immunity in epithelial and immune cells can be stimulated through activation of Toll-like receptors (TLRs), the main family of pattern recognition receptors. Stimulation of TLR5 through flagellin-based interventions have demonstrated protective activity against several gram-negative bacteria (Salmonella sp., Burkholderia cepacia, Yersinia pseudotuberculosis), restoring immune-competence and promoting tissue repair processes. Here, we aimed to determine the effect of flagellin stimulation on the innate immune response against P. aeruginosa using an experimental pig model of lung infection. The pig model presents several advantages since swine and human lungs are similar in terms of anatomical, histological, biochemical, and physiological features. This is especially true in CF, where pigs lacking CFTR present a similar phenotype to what is typically observed in human patients. P. aeruginosa-infected pigs showed an acute neutrophilic response with an exacerbated release of neutrophil serine proteases that peaked 3-6 h post-infection (p.i.) leading to lung destruction and tissue hepatisation 24h p.i. When animals were pre-treated with flagellin 24h before the experimental infections, we observed a significant decrease in the expression of pro-inflammatory markers. In addition, a better lung status was observed on infected animals that had been pre-treated with flagellin compared to infected controls. The effect of flagellin pre-treatment on immune response to P. aeruginosa infection was confirmed using ex-vivo and in vitro models of lung epithelium from CFTR-/- pigs. In conclusion, our data point to a modulatory role of flagellin pre-treatment on the immune response to P. aeruginosa. This approach may have a therapeutic potential to improve inflammatory manifestations of CF.

Published
2019

8. Neutrophils in cystic fibrosis. Are they intrinsically defective?

Author

Deborah Brea, Laura Soler Vasco, Antoine Guillon, Claire Chevaleyre, Céline Barc, Mustapha Berri, Michel Olivier, Sandrine Melo, Isabelle Fleurot, Jérémy Pezant, Valérie Labas, Ana Paula Teixeira, Pierre Sarradin, Mickaël Riou, Nikolai Klymiuk, Mustapha Si-Tahar, Ignacio Caballero-Posadas, Institut National de la Santé et de la Recherche Médicale (INSERM), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), Plateforme d'Infectiologie Expérimentale (PFIE), Institut National de la Recherche Agronomique (INRA), Gene Center and Center for Innovative Medical Models (CIMM), ProdInra, Migration, Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Université de Tours

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], [INFO]Computer Science [cs], [INFO] Computer Science [cs]
Abstract

National audience; Cystic fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF lungs are characterized by chronic colonization by opportunistic pathogens and a neutrophilic infiltration that results in excessive neutrophil protease release and lung tissue destruction. Previous studies have reported neutrophil dysfunction in CF. However, the exact etiology of the altered functionality of CF neutrophils (i.e.a result of the genetic defect or a consequence of chronic infection, remains unclear. Recently, a CFTR-/- knockout pig model has been developed that displays most of the features of the human CF disease1. We and others have shown that at birth these animals do not show early signs of systemic or lung infection. This fact and the similarities between human and pig neutrophils3 make them a suitable model to develop a research project aimed to answer the following key question: are CF neutrophils intrinsically defective? Blood neutrophils will be collected from CFTR-/- and wild-type (WT) newborn piglets and differences in their proteomic profiling will be evaluated using Intact Cell MALDI-TOF MS (ICM-MS). MS spectra will be further processed and analyzed with ClinProTools v3.0. Our preliminary data shows that ICM-MS-based analysis discriminates CFTR-/- and WT neutrophils suggesting differences in their protein composition. Further analysis will include the characterization of the molecular composition of the spectra through high-throughput top-down protein by nanoLC-MS/MS on a LTQ Orbitrap Velos. Functional assays will be performed on neutrophils in order to establish a link between differentially-expressed proteins and neutrophil activity. The expected data could provide a molecular target for CF therapies modulating neutrophil function.

Published
2015

9. Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

Author

Jacques-Antoine Hennekinne, Alexandra Gruss, Renaud Fleurot, Elise Borezée-Durant, Claire Darrigo, Marina Aigle, A. Delacroix-Buchet, Isabelle Fleurot, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Unité Staphylocoques, Bacillus, Clostridies, Lait (Unité SBCL), Laboratoire de sécurité des aliments de Maisons-Alfort, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Ile-de-France, Biologie du développement et reproduction (BDR), Centre National de la Recherche Scientifique (CNRS)-École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Laboratoire de sécurité des aliments de Maisons-Alfort (LSAl), and Fleurot, Isabelle

Subjects
Staphylococcus aureus, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], milk, bacteria, United-States, identification, dairy-product, in-situ hybridization, microstructure, foodborne illness, enterotoxin production, lactococcus-lactis, Ingénierie des aliments, Food Contamination, medicine.disease_cause, Staphylococcal infections, Applied Microbiology and Biotechnology, Microbiology, Enterotoxins, Bacterial Proteins, Cheese, [SDV.IDA]Life Sciences [q-bio]/Food engineering, medicine, Food microbiology, Food engineering, Humans, Pathogen, Ecosystem, 2. Zero hunger, Food poisoning, Ecology, biology, Lactococcus lactis, Staphylococcal Infections, medicine.disease, biology.organism_classification, Food Microbiology, Bacteria, Food Science, Biotechnology, Food contaminant, Autre (Sciences du Vivant)
Abstract

Human intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on the in situ distribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate of Staphylococcus aureus , an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring of S. aureus populations and targeted gene expression. The combination of complementary techniques revealed that S. aureus localizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.

Published
2014
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