Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein
2022; Springer Nature; Volume: 41; Issue: 12 Linguagem: Inglês
10.15252/embj.2021108306
ISSN1460-2075
AutoresAntoine Guillon, Deborah Bréa-Diakite, Adeline Cezard, Alan Wacquiez, Thomas Baranek, Jérôme Bourgeais, Frédéric Picou, Virginie Vasseur, Léa Meyer, Christophe Chevalier, Adrien Auvet, José M. Carballido, Lydie Nadal‐Desbarats, Florent Dingli, Andrei Turtoï, Audrey Le Gouëllec, Florence Fauvelle, Amélie Donchet, Thibaut Crépin, Pieter S. Hiemstra, Christophe Paget, Damarys Loew, Olivier Hérault, Nadia Naffakh, Ronan Le Goffic, Mustapha Si‐Tahar,
Tópico(s)Immune Response and Inflammation
ResumoArticle4 May 2022Open Access Transparent process Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein Antoine Guillon Antoine Guillon INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Service de Médecine Intensive Réanimation, CHRU de Tours, Tours, France Contribution: Conceptualization, Formal analysis, Supervision, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Deborah Brea-Diakite Deborah Brea-Diakite INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Adeline Cezard Adeline Cezard orcid.org/0000-0001-8608-701X INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Alan Wacquiez Alan Wacquiez orcid.org/0000-0001-5266-5328 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Thomas Baranek Thomas Baranek orcid.org/0000-0002-5651-0937 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Jérôme Bourgeais Jérôme Bourgeais Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Frédéric Picou Frédéric Picou orcid.org/0000-0002-2066-2146 Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Virginie Vasseur Virginie Vasseur INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Investigation, Methodology Search for more papers by this author Léa Meyer Léa Meyer Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Investigation, Methodology Search for more papers by this author Christophe Chevalier Christophe Chevalier orcid.org/0000-0003-3231-9027 Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Resources, Methodology, Writing - review & editing Search for more papers by this author Adrien Auvet Adrien Auvet INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Service de Médecine Intensive Réanimation, CHRU de Tours, Tours, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author José M Carballido José M Carballido orcid.org/0000-0001-8465-0665 Novartis Institutes for BioMedical Research, Basel, Switzerland Contribution: Resources Search for more papers by this author Lydie Nadal Desbarats Lydie Nadal Desbarats UMR 1253, iBrain, Inserm, Université de Tours, Tours, France Search for more papers by this author Florent Dingli Florent Dingli orcid.org/0000-0002-7715-2446 Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, PSL Research University, Paris, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Andrei Turtoi Andrei Turtoi orcid.org/0000-0003-3813-6635 Tumor Microenvironment Laboratory, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Montpellier, France Institut du Cancer de Montpellier, Montpellier, France Université de Montpellier, Montpellier, France Contribution: Resources, Investigation, Writing - review & editing Search for more papers by this author Audrey Le Gouellec Audrey Le Gouellec orcid.org/0000-0002-4905-3854 CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, University Grenoble Alpes, Grenoble, France Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Florence Fauvelle Florence Fauvelle UGA/INSERM U1216, Grenoble Institute of Neurosciences, Grenoble, France UGA/INSERM US17, Grenoble MRI Facility IRMaGe, Grenoble, France Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Amélie Donchet Amélie Donchet Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France Contribution: Investigation, Methodology Search for more papers by this author Thibaut Crépin Thibaut Crépin orcid.org/0000-0003-2663-1794 Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France Contribution: Resources, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Pieter S Hiemstra Pieter S Hiemstra orcid.org/0000-0002-0238-5982 Department of Pulmonology, Leiden University Medical Center, Leiden, Netherlands Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Christophe Paget Christophe Paget orcid.org/0000-0002-5374-5407 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Damarys Loew Damarys Loew orcid.org/0000-0002-9111-8842 Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, PSL Research University, Paris, France Contribution: Resources, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Olivier Herault Olivier Herault Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Nadia Naffakh Nadia Naffakh orcid.org/0000-0002-0424-0277 Institut Pasteur, Unité Biologie des ARN et Virus Influenza, CNRS UMR3569, Paris, France Contribution: Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author Ronan Le Goffic Ronan Le Goffic orcid.org/0000-0002-2012-0064 Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author Mustapha Si-Tahar Corresponding Author Mustapha Si-Tahar [email protected] orcid.org/0000-0002-5792-7742 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Antoine Guillon Antoine Guillon INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Service de Médecine Intensive Réanimation, CHRU de Tours, Tours, France Contribution: Conceptualization, Formal analysis, Supervision, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Deborah Brea-Diakite Deborah Brea-Diakite INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology Search for more papers by this author Adeline Cezard Adeline Cezard orcid.org/0000-0001-8608-701X INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Alan Wacquiez Alan Wacquiez orcid.org/0000-0001-5266-5328 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Thomas Baranek Thomas Baranek orcid.org/0000-0002-5651-0937 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Jérôme Bourgeais Jérôme Bourgeais Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Frédéric Picou Frédéric Picou orcid.org/0000-0002-2066-2146 Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Virginie Vasseur Virginie Vasseur INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Investigation, Methodology Search for more papers by this author Léa Meyer Léa Meyer Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Investigation, Methodology Search for more papers by this author Christophe Chevalier Christophe Chevalier orcid.org/0000-0003-3231-9027 Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Resources, Methodology, Writing - review & editing Search for more papers by this author Adrien Auvet Adrien Auvet INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Service de Médecine Intensive Réanimation, CHRU de Tours, Tours, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author José M Carballido José M Carballido orcid.org/0000-0001-8465-0665 Novartis Institutes for BioMedical Research, Basel, Switzerland Contribution: Resources Search for more papers by this author Lydie Nadal Desbarats Lydie Nadal Desbarats UMR 1253, iBrain, Inserm, Université de Tours, Tours, France Search for more papers by this author Florent Dingli Florent Dingli orcid.org/0000-0002-7715-2446 Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, PSL Research University, Paris, France Contribution: Validation, Investigation, Methodology Search for more papers by this author Andrei Turtoi Andrei Turtoi orcid.org/0000-0003-3813-6635 Tumor Microenvironment Laboratory, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Montpellier, France Institut du Cancer de Montpellier, Montpellier, France Université de Montpellier, Montpellier, France Contribution: Resources, Investigation, Writing - review & editing Search for more papers by this author Audrey Le Gouellec Audrey Le Gouellec orcid.org/0000-0002-4905-3854 CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, University Grenoble Alpes, Grenoble, France Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Florence Fauvelle Florence Fauvelle UGA/INSERM U1216, Grenoble Institute of Neurosciences, Grenoble, France UGA/INSERM US17, Grenoble MRI Facility IRMaGe, Grenoble, France Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Amélie Donchet Amélie Donchet Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France Contribution: Investigation, Methodology Search for more papers by this author Thibaut Crépin Thibaut Crépin orcid.org/0000-0003-2663-1794 Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France Contribution: Resources, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Pieter S Hiemstra Pieter S Hiemstra orcid.org/0000-0002-0238-5982 Department of Pulmonology, Leiden University Medical Center, Leiden, Netherlands Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Christophe Paget Christophe Paget orcid.org/0000-0002-5374-5407 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Damarys Loew Damarys Loew orcid.org/0000-0002-9111-8842 Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, PSL Research University, Paris, France Contribution: Resources, Validation, Investigation, Methodology, Writing - review & editing Search for more papers by this author Olivier Herault Olivier Herault Université de Tours, Tours, France CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France Service d'Hématologie Biologique, CHRU de Tours, Tours, France Contribution: Resources, Validation, Writing - review & editing Search for more papers by this author Nadia Naffakh Nadia Naffakh orcid.org/0000-0002-0424-0277 Institut Pasteur, Unité Biologie des ARN et Virus Influenza, CNRS UMR3569, Paris, France Contribution: Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author Ronan Le Goffic Ronan Le Goffic orcid.org/0000-0002-2012-0064 Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France Contribution: Resources, Formal analysis, Validation, Writing - review & editing Search for more papers by this author Mustapha Si-Tahar Corresponding Author Mustapha Si-Tahar [email protected] orcid.org/0000-0002-5792-7742 INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France Université de Tours, Tours, France Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Antoine Guillon1,2,3,†, Deborah Brea-Diakite1,2,†, Adeline Cezard1,2,†, Alan Wacquiez1,2, Thomas Baranek1,2, Jérôme Bourgeais2,4,5, Frédéric Picou2,4,5, Virginie Vasseur1,2, Léa Meyer6, Christophe Chevalier6, Adrien Auvet1,2,3, José M Carballido7, Lydie Nadal Desbarats8, Florent Dingli9, Andrei Turtoi10,11,12, Audrey Le Gouellec13, Florence Fauvelle14,15, Amélie Donchet16, Thibaut Crépin16, Pieter S Hiemstra17, Christophe Paget1,2, Damarys Loew9, Olivier Herault2,4,5,‡, Nadia Naffakh18,‡, Ronan Le Goffic6,‡ and Mustapha Si-Tahar *,1,2 1INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France 2Université de Tours, Tours, France 3Service de Médecine Intensive Réanimation, CHRU de Tours, Tours, France 4CNRS ERL 7001 LNOx "Leukemic niche and redox metabolism", Tours, France 5Service d'Hématologie Biologique, CHRU de Tours, Tours, France 6Virologie et Immunologie Moléculaires, INRAe, Université Paris-Saclay, Jouy-en-Josas, France 7Novartis Institutes for BioMedical Research, Basel, Switzerland 8UMR 1253, iBrain, Inserm, Université de Tours, Tours, France 9Centre de Recherche, Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, PSL Research University, Paris, France 10Tumor Microenvironment Laboratory, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Montpellier, France 11Institut du Cancer de Montpellier, Montpellier, France 12Université de Montpellier, Montpellier, France 13CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, University Grenoble Alpes, Grenoble, France 14UGA/INSERM U1216, Grenoble Institute of Neurosciences, Grenoble, France 15UGA/INSERM US17, Grenoble MRI Facility IRMaGe, Grenoble, France 16Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France 17Department of Pulmonology, Leiden University Medical Center, Leiden, Netherlands 18Institut Pasteur, Unité Biologie des ARN et Virus Influenza, CNRS UMR3569, Paris, France † These authors contributed equally to this work ‡ These authors contributed equally to this work *Corresponding author. Tel: +33 247 36 60 45; Fax: +33 247 36 60 49; E-mail: [email protected] The EMBO Journal (2022)41:e108306https://doi.org/10.15252/embj.2021108306 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Influenza virus infection causes considerable morbidity and mortality, but current therapies have limited efficacy. We hypothesized that investigating the metabolic signaling during infection may help to design innovative antiviral approaches. Using bronchoalveolar lavages of infected mice, we here demonstrate that influenza virus induces a major reprogramming of lung metabolism. We focused on mitochondria-derived succinate that accumulated both in the respiratory fluids of virus-challenged mice and of patients with influenza pneumonia. Notably, succinate displays a potent antiviral activity in vitro as it inhibits the multiplication of influenza A/H1N1 and A/H3N2 strains and strongly decreases virus-triggered metabolic perturbations and inflammatory responses. Moreover, mice receiving succinate intranasally showed reduced viral loads in lungs and increased survival compared to control animals. The antiviral mechanism involves a succinate-dependent posttranslational modification, that is, succinylation, of the viral nucleoprotein at the highly conserved K87 residue. Succinylation of viral nucleoprotein altered its electrostatic interactions with viral RNA and further impaired the trafficking of viral ribonucleoprotein complexes. The finding that succinate efficiently disrupts the influenza replication cycle opens up new avenues for improved treatment of influenza pneumonia. Synopsis Metabolic defense mechanisms of lung epithelial cells exposed to influenza virus infection remain poorly understood. Here, combined metabolomics, in vitro and in vivo infection assays reveal a surprising anti-viral role of energy production metabolite succinate in the airways, suggesting new avenues for improved treatment of influenza pneumonia. Influenza A virus (IAV) infection of mice increases succinate levels in the airways. IAV-infected patients show elevated succinate levels in tracheal aspirates. Succinate inhibits IAV infection through succinylation and nuclear retention of the viral nucleoprotein. Succinate restores metabolic dysregulation in IAV-infected lung epithelial cells and impairs acute influenza pneumonia in vivo. Video Synopsis The energy-production metabolite succinate protects from pulmonary viral infection via post-translational modification-dependent interruption of the influenza replication cycle. Introduction In 1918, more than 50 million people died from severe influenza viral pneumonia. One century later, and despite considerable progress in medicine, annual epidemics are still estimated to result in 3–5 million cases of severe illness worldwide and up to 650,000 deaths (Taubenberger et al, 2019; Lampejo, 2020). Influenza viruses are the etiological agents and are classified into four types (A, B, C, and D) of which influenza A virus (IAV) is clinically the most significant. Vaccination against influenza constitutes the most effective preventive strategy. However, the short duration of vaccine-induced immunity coupled with the intrinsic antigenic drift of IAV negatively affects vaccine efficiency (Jang & Seong, 2019). Besides, drugs targeting IAV neuraminidase (NA) are currently recommended by the WHO but their efficacy is largely disputed (WHO, 2009; Jefferson et al, 2014; Duwe, 2017). Hence, the development of innovative antiviral drugs is required to better treat IAV pneumonia (Pizzorno et al, 2019). To this end, we need a better understanding of the mechanisms of IAV––host cell interactions that may lead to IAV-triggered lung hyper-inflammation. Indeed, several studies suggest that such an excessive and deleterious inflammatory response is responsible for the life-threatening "flu" syndrome (Le Goffic et al, 2006; Taubenberger et al, 2019). Interestingly, accumulating data support the view that metabolic pathways may promote or inhibit immune and inflammatory responses (Rambold & Pearce, 2018; Williams & O'Neill, 2018; Infantino et al, 2019), in addition to their role in energy production and macromolecular biosynthesis. Succinate is of special interest in this context. It is an intermediate of the tricarboxylic acid (TCA) cycle in mitochondria. Succinate is mainly produced from succinyl coenzyme A by succinyl coenzyme A synthetase (Grimolizzi & Arranz, 2018). It can also connect intracellular metabolic status and intercellular communication, as it can be released into the extracellular space through plasma membrane transporters of the SLC13 family (Grimolizzi & Arranz, 2018; Murphy & O'Neill, 2018). As a result, succinate can accumulate extracellularly in certain pathophysiological situations such as chronic inflammatory diseases (Ryan et al, 2019). Thus, the relationship between succinate and inflammatory signaling in leukocytes is increasingly studied (Rubic et al, 2008; Tannahill et al, 2013; Jha et al, 2015; Littlewood-Evans et al, 2016; Mills et al, 2016; Zasłona & O'Neill, 2020). In contrast, such effects of succinate on nonimmune cells such as the epithelial cells that line the airway mucosa have not been explored to a similar extent. This is important since airway epithelial cells are the main site of infection for respiratory pathogens such as IAV, and their role in controlling immune and inflammatory responses is pivotal. Furthermore, we and others have previously demonstrated that these cells are the source of major inflammatory mediators contributing to IAV-related lung injury (Simmons & Farrar, 2008; Si-Tahar et al, 2009; Gregory & Kobzik, 2015; Denney & Ho, 2018). In view of the pro-inflammatory properties of succinate (Rubic et al, 2008; Tannahill et al, 2013; Jha et al, 2015; Littlewood-Evans et al, 2016; Mills et al, 2016; Zasłona & O'Neill, 2020), we initially speculated that succinate could contribute to the deleterious immunopathological response associated with severe IAV pneumonia. Strikingly, the opposite situation was observed. Using in vitro and in vivo approaches, we reveal that succinate restricts IAV replication and downstream inflammatory signaling. The underlying mechanism involves a specific posttranslational modification and nuclear retention of the IAV nucleoprotein (NP). Hence, we demonstrate that succinate is a key player in the antiviral defense of the lung mucosa. Results Influenza infection increases succinate levels in airways Despite the growing interest in immunometabolism (Pearce & Pearce, 2018), little is known about metabolic reprogramming upon IAV infection (Smallwood et al, 2017; Tian et al, 2019; Bahadoran et al, 2020; Keshavarz et al, 2020; Wendt et al, 2021). Here, we conducted an unbiased quantification of the lung metabolome of mice four days after the infection by a sublethal dose of IAV (A/Scotland/20/74 (H3N2)). From the nuclear magnetic resonance (NMR) spectra of the bronchoalveolar lavage (BAL) fluids, we focused on succinate for two main reasons: (i) it was among the top ten most altered metabolites in the BAL fluids of IAV-infected mice, compared to control animals (fold change = 2.2, FDR-adjusted P-value = 8 × 10-7, Fig 1A) and (ii) succinate is increasingly recognized as a potent immunoregulatory mediator (Rubic et al, 2008; Tannahill et al, 2013; Jha et al, 2015; Littlewood-Evans et al, 2016; Mills et al, 2016; Zasłona & O'Neill, 2020). Figure 1. Influenza virus infection increases succinate levels in airways C57Bl/6 mice were infected (n = 9) or not (n = 8) intranasally with 150 pfu of influenza A virus (IAV; A/Scotland/20/74 (H3N2)). Bronchoalveolar lavage fluids (BALs) were collected 4 days postinfection. Samples were further processed for metabolomic analysis by NMR. Left panel shows a volcano-plot representing the comparison of "IAV-infected" versus "control, non-infected" animals. The graph plots the −log (P-value) against the fold change (log2 (ratio)) for individual metabolites. Vertical dashed blue lines mark the two-fold change and horizontal dashed blue line marks the cut-off P-value of 0.0001. Metabolites in the upper right square in red are significantly more abundant in "IAV-infected" compared to "control, non-infected" samples. Succinate (annotated by a red arrow) is one of these compounds with a FC = 2.2 and a FDR-adjusted P-value = 8 × 10−7. The boxplots whiskers correspond to the minimum and maximum values, while the central band is the mean. The first and third quartiles (boxes) are merely the average values between the median and the extrema. Shows succinate quantification by NMR from tracheal aspirates collected in mechanically ventilated patients with (pink bar, n = 9) or without (orange bar, n = 7) diagnostic of IAV pneumonia. *P < 0.05. IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates. Main baseline characteristics of the patients included in the study. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann–Whitney U-test. Download figure Download PowerPoint In agreement with the data from the mouse experiments (Fig 1A), succinate level in respiratory fluids (i.e., tracheal aspirates) was significantly higher in critically ill patients with influenza infection than in patients not infected by IAV but with similar severity and lung inflammation (Fig 1B). This second group of patients included critically ill individuals ventilated for coma (hypoxic-ischemic coma, stroke, or drug overdose) with similar comorbidities and severity score, as assessed by IL-6 and IL-8/CXCL8 measurements in the respiratory fluids (Fig 1C), the leucocyte count in respiratory fluids and the regular presence of positive bacterial culture (Fig 1D). The heterogeneity of succinate levels in the fluids of IAV-infected patients can partly be ascribed to the variability at the time of hospital admission and the subsequent symptoms-to-sampling time (median [IQR] = 9 [7; 18] days). Overall, we demonstrated that IAV infection was associated with high levels of succinate in lungs of mice and humans. Succinate limits the secretion of inflammatory mediators and restores metabolic dysregulation in IAV-infected lung epithelial cells To decipher the potential role of succinate in IAV-infected lungs, we first compared at 20 h postinfection (pi) the gene expression profiles of IAV-infected human bronchial epithelial cells, exposed or not to succinate. The volcano plot in Fig 2A (left panel) shows that IAV infection increased the expression of multiple gene pathways, including canonical immune and inflammatory pathways. The volcano plot in the right panel indicates that succinate treatment of IAV-infected cells results in a drastic downregulation of those pathways. As illustrated in Fig 2A and in Appendix Fig S1, those altered pathways included, but were not limited to, "inflammasome signaling," "TREM1 signaling," "acute phase response signaling," "role of PRR in recognition of bacteria and viruses," "iNOS signaling,", etc.). Of note is that succinate does not induce any cell cytotoxicity at the doses we used (Appendix Fig S2A and B). Figure 2. Succinate reverses the inflammatory response and the metabolic modifications induced by influenza virus Bronchial epithelial (BEAS-2B) cells were infected or not with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI = 1 for 4 h and treated or not with succinate (Suc; 4 mg/ml/24.7 mM) for 20 h. A. Volcano-plot showing the most significantly regulated canonical pathways determined by microarray analysis compared with mock-treated cells. Each dot represents a specific canonical pathway as determined by GSEA. Pathway representations result from the magnitude of regulation (z-score, x-axis) and significance (−log10 adjusted P-value, y-axis). The dashed horizontal line indicates the statistical significance threshold (P ≤ 0.01 after adjustment with the Bonferroni correction). The two vertical dashed lines show the z-score threshold (−2: repressed; 2: induced). Colored spots characterize the most highly regulated canonical pathways endowed with statistical significance. B. Representative inflammatory protein-array blots obtained from the supernatants of control- or IAV-infected- or IAV-infected and succinate-treated cells. The table on the right side indicates the 8 most regulated mediators b
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