Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 is online at: average * The Journal of Immunology , 10 of which you can access for free at: 2012; 188:832-843; Prepublished online 14 from submission to initial decision 4 weeks from acceptance to publication December 2011; doi: 10.4049/jimmunol.1102349 http://www.jimmunol.org/content/188/2/832 Shifting of Immune Responsiveness to House Dust Mite by Influenza A Infection: Genomic Insights Amal Al-Garawi, Mainul Husain, Dora Ilieva, Alison A. Humbles, Roland Kolbeck, Martin R. Stampfli, Paul M. O'Byrne, Anthony J. Coyle and Manel Jordana J Immunol cites 38 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2011/12/14/jimmunol.110234 9.DC1 This article http://www.jimmunol.org/content/188/2/832.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 29, 2021. The Journal of Immunology Shifting of Immune Responsiveness to House Dust Mite by Influenza A Infection: Genomic Insights Amal Al-Garawi,*,†,1 Mainul Husain,*,†,1 Dora Ilieva,*,† Alison A. Humbles,‡ Roland Kolbeck,‡ Martin R. Stampfli,*,† Paul M. O’Byrne,x Anthony J. Coyle,*,†,{ and Manel Jordana*,† Respiratory viral infections have been associated with an increased incidence of allergic asthma. However, the mechanisms by which respiratory infections facilitate allergic airway disease are incompletely understood. We previously showed that exposure to a low dose of house dust mite (HDM) resulted in enhanced HDM-mediated allergic airway inflammation, and, importantly, marked airway hyperreactivity only when allergen exposure occurred during an acute influenza A infection. In this study, we evaluated the impact of concurrent influenza infection and allergen exposure at the genomic level, using whole-genome micro- array. Our data showed that, in contrast to exposure to a low dose of HDM, influenza A infection led to a dramatic increase in gene Downloaded from expression, particularly of TLRs, C-type lectin receptors, several complement components, as well as Fc«R1. Additionally, we observed increased expression of a number of genes encoding chemokines and cytokines associated with the recruitment of proinflammatory cells. Moreover, HDM exposure in the context of an influenza A infection resulted in the induction of unique genes, including calgranulin A (S100a8), an endogenous damage-associated molecular pattern and TLR4 agonist. In addition, we observed significantly increased expression of serum amyloid A (Saa3) and serine protease inhibitor 3n (Serpina3n). This study showed that influenza infection markedly increased the expression of multiple gene classes capable of sensing allergens and http://www.jimmunol.org/ amplifying the ensuing immune-inflammatory response. We propose that influenza A infection primes the lung environment in such a way as to lower the threshold of allergen responsiveness, thus facilitating the emergence of a clinically significant allergic phenotype. The Journal of Immunology, 2012, 188: 832–843. llergic asthma is a chronic immune-inflammatory disease is immunologic homeostasis and that additional factors contribute of the airways that occurs following sensitization to to triggering aberrant immune responses to these allergens (2). In A common aeroallergens, such as house dust mite (HDM), addition to a genetic predisposition, environmental factors, such as by guest on September 29, 2021 the most ubiquitous indoor aeroallergen worldwide. However, respiratory viral infections, have been implicated with the clinical despite universal exposure, only ∼20% of the population develops expression of allergic airway inflammation (3, 4). In this regard, the disease (1). This suggests that the natural response to allergens there is abundant evidence in humans of an association between viral infections and the expression of asthma (5, 6). In mice, we previously reported that exposure to a concentration of HDM, *Division of Respiratory Diseases and Allergy, Center for Gene Therapeutics, McMas- ter University, Hamilton, Ontario L8S 4K1, Canada; †Department of Pathology and which by itself elicits negligible airway inflammation and no Molecular Medicine, McMaster University, Hamilton, Ontario L8S 4K1, Canada; changes in lung function, results in a phenotype characterized by ‡Department of Respiratory, Inflammation and Autoimmunity, MedImmune LLC, Gai- thersburg, MD 20878; xDepartment of Medicine, McMaster University, Hamilton, robust allergic airway inflammation, enhanced mucus production, Ontario L8S 4K1, Canada; and {Centers for Therapeutic Innovation, Pfizer Inc., Cam- and marked lung dysfunction in the context of an acute influenza bridge, MA 02138 A virus (Flu) infection (7). 1 A.A.-G. and M.H. contributed equally to this work. In this study, we used genome-wide transcriptional profiling to Received for publication August 12, 2011. Accepted for publication November 4, investigate the nature of the Flu-induced environment in the lung, by 2011. examining global gene expression during the early phase (EP; 4 d) This work was supported in part by the Canadian Institute of Health Research and MedImmune LLC. A.A.-G. holds a scholar award from the King Abdullah University and late phase (LP; 7 d) of HDM exposure. We found that exposure to of Science and Technology, and M.J. holds a Senior Canada Research Chair in a low concentration of HDM alone elicited minimal alterations in the Immunobiology of Respiratory Disease and Allergy. gene profile, whereas Flu infection led to a pervasive upregulation The sequences presented in this article have been submitted to the European Molec- of genes associated with the general response to stimuli and stress. ular Biology Laboratory-European Bioinformatics Institute ArrayExpress database (http://www.ebi.ac.uk/microarray-as/ae) under accession number E-MEXP-3325. In particular, Flu infection dramatically increased the expression Address correspondence and reprint requests to Dr. Manel Jordana, Division of of a number of cell surface receptors, notably TLRs, C-type lectin Respiratory Diseases and Allergy and Department of Pathology and Molecular Med- receptors (CLRs), and FcRs, and a prolific number of chemokines and icine, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada. E-mail address: [email protected] chemokine receptors. Interestingly, exposure to HDM in the context of this Flu-induced environment led to the increased expression of The online version of this article contains supplemental material. several hundred genes, which were not expressed in mice exposed to Abbreviations used in this article: CLR, C-type lectin receptor; DC, dendritic cell; EP, early phase; Flu, influenza A virus; Flu+HDM, house dust mite in the context of allergen alone. These data suggested that Flu lowered the threshold influenza A virus infection; GO, gene ontology; HDM, house dust mite; LP, late of HDM responsiveness by establishing a global, heightened state of phase; qPCR, quantitative real-time PCR. immune sensing in the lung and launching multiple pathways in- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 volved in inflammatory responses to exogenous Ags. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102349 The Journal of Immunology 833 Materials and Methods corrected; p , 0.05) were considered significantly enriched. Microarray Animals data files from this study are available at European Molecular Biology Laboratory-European Bioinformatics Institute ArrayExpress database Female BALB/c mice (6–8 wk old) were purchased from Charles River (http://www.ebi.ac.uk/microarray-as/ae). ArrayExpress accession number Laboratories (Saint-Constant, QC, Canada). The mice were housed under for these files is E-MEXP-3325. specific pathogen-free conditions and maintained on a 12-h light-dark cycle, with food and water ad libitum. All experiments described in this Quantitative real-time PCR for gene validation study were approved by the Animal Research Ethics Board of McMaster RNA was quantified and normalized, and RNA integrity was assessed by University. Agilent Bioanalyzer. cDNA was generated using the Super Script III Re- verse Transcriptase kit (Life Technologies, Carlsbad, CA), according to Flu and infection protocol the manufacturer’s instructions. Relative transcript expression assay was Flu strain A/PR/8/34 (H1N1) was prepared, as described previously (8), conducted, as described previously (13), using the Fluidigm Biomark and kindly provided by MedImmune. The viral stock suspension (109 system (Fluidigm, San Francisco, CA). BestKeeper (version 1) (14) was PFU/ml) was diluted, and 10 PFU was administered intranasally to used to identify the stably expressed housekeeping gene to be used as an isoflurane-anesthetized BALB/c mice in 35 ml sterile PBS solution. internal reference. Among the