Deficits in inflammasomes, a key element of innate immunity, confer increased susceptibility to infection. We report that sputum cells from asthmatics have decreased expression of inflammasome factors, consistent with reports of increased infection risk in asthmatics

Alexis, N.E.

Brickey, W.J.

Hernandez, M.L.

Peden, D.B.

Reed, W.

Ting, J.P.Y.

Carolina Digital Repository

Sputum Inflammatory Cells from Persons with Allergic Rhinitisand Asthma Have Decreased Inflammasome Gene ExpressionWillie June Brickey, PhDa, Neil E. Alexis, PhDb,c, Michelle L. Hernandez, MDb,c, WilliamReed, PhDb,c, Jenny PY. Ting, PhDa,d,e, and David B. Peden, MDa,b,c,faDepartment of Microbiology and Immunology, The School of Medicine, The University of NorthCarolina at Chapel Hill, Chapel Hill, North CarolinabDepartment of Pediatrics, The School of Medicine, The University of North Carolina at ChapelHill, Chapel Hill, North CarolinacCenter for Environmental Medicine, Asthma and Lung Biology, The School of Medicine, TheUniversity of North Carolina at Chapel Hill, Chapel Hill, North CarolinadCenter for Translational Immunology, The School of Medicine, The University of North Carolinaat Chapel Hill, Chapel Hill, North CarolinaeLineberger Comprehensive Cancer Center, The School of Medicine, The University of NorthCarolina at Chapel Hill, Chapel Hill, North CarolinafDepartment of Medicine, The School of Medicine, The University of North Carolina at Chapel Hill,Chapel Hill, North CarolinaCapsule SummaryDeficits in inflammasomes, a key element of innate immunity, confer increased susceptibility toinfection. We report that sputum cells from asthmatics have decreased expression ofinflammasome factors, consistent with reports of increased infection risk in asthmatics.KeywordsInnate Immunity; Asthma; Atopy; Inflammasome; IL-1βTo the EditorThere are numerous reports supporting the hypothesis that decreased exposure to microbesas well as genetic factors that attenuate innate immune responsiveness increase risk fordeveloping asthma and atopy. A growing body of evidence also suggests that adverse healthoutcomes due to bacterial infection or microbial colonization in the airways may beincreasing in asthmatics and other atopic persons. Numerous investigators have shown that© 2011 American Academy of Allergy, Asthma and Immunology. Published by Mosby, Inc. All rights reserved.Corresponding Author: David B. Peden, MD, Center for Environmental Medicine, Asthma and Lung Biology, 104 Mason Farm Road,UNC School of Medicine, Chapel Hill, NC 27516, peden@med.unc.edu, phone 919-966-0768, fax 919-966-9863.Disclosures: N. E. Alexis has contracts with Centocor Pharmacueticals, GlaxoSmithKline and MedImmune. D. B. Peden has been aconsultant to GlaxoSmithKline and Aquinox Pharmaceuticals and a contract with MedImmune. J. PY. Ting had a one-year contractwith GlaxoSmithKline (Philadelphia). The rest of the authors have declared that they have no conflicts of interest.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptJ Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.Published in final edited form as:J Allergy Clin Immunol. 2011 October ; 128(4): 900–903. doi:10.1016/j.jaci.2011.08.012.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptasthmatics have increased risk of invasive pneumococcal disease (1;2). Additionally, personswith poorly controlled asthma have increased bacterial colonization of the airway, indicatedby either increased airway endotoxin levels(3) or elevated 16S rRNA load in bronchialsamples(4). Asthmatics also have increased lower respiratory symptoms, greater impairmentof lung function and bronchial hyperreactivity, diminished circulating CD8+ lymphocytes,and deficient IFN-γ and IL-10 responses following experimental rhinoviral infection,consistent with other studies which suggest that the severity of viral infection is increased,and that viral mechanisms to evade immune response have evolved(5). Taken together, theseobservations suggest that increased disease severity in asthmatics may be associated withdiminished innate immunity and increased susceptibility to viral or bacterial airwayinfection.Inflammasomes are newly appreciated innate immune regulatory complexes which processIL-1β and Il-18 when activated by a number of pathogen, environmental or host-deriveddanger signals. We have made seminal observations demonstrating the important role ofinflammasomes in mediating host defense against influenza and bacterial pathogens, as wellas modulating responses to alum and contact dermatitis(6). The mounting evidence thatpersons with asthma may have impaired innate immune responses propelled us tohypothesize that inflammasome function may be reduced in asthmatics compared to healthynon-atopic normal volunteers.To test this hypothesis, we examined sputum samples in our sample repository from normalvolunteers (NV), asthmatics (Asth, 5 with atopy), and volunteers with allergic rhinitis (AR,all with positive skin tests) recruited using protocols approved by the UNC IRB aspreviously described(7) to assess IL-1β and inflammasome-regulated mechanisms that alterimmune responsiveness. All volunteers had normal lung function, with allergy statusconfirmed by history and allergen skin tests, and asthma status confirmed by clinical historyand methacholine challenge (Table I). Sputum cell pellets contained predominantlymacrophages (Mac) and neutrophils (PMN) with less than 10% squamous epithelial cells(Table I). While cell counts were not significantly different between the groups, trends ofdouble PMNs in Asth compared to the other two groups and double macrophage in ARcompared to NV were evident. The sputum supernatants from asthmatic (Asth) and normalvolunteers (NV) were assessed for protein levels of IL-1α, IL-1β, IL-6 and TNFα bymultiplex immunoassay (RayBiotech). Asthmatics showed decreased levels of IL-1α andIL-1β, while no significant differences in IL-6 or TNFα were observed between these groups(Figure 1A).Messenger RNA from sputum cell pellets was assayed for gene expression ofinflammasome-associated genes (ASC, CARD8, CASP1, NLRP1, NLRP3, and NLRC4); non-inflammasome Nucleotide-Binding Leucine-Rich Repeat factors (NLRP12, NOD1, NOD2,BIRC2); cytokines IL-1β, IL-18, IL-6 and TNFα; and HLA molecules. To assess geneexpression, two-color cDNA microarray hybridization was performed on labeled sputumRNA probes (EpiCentre) with signal intensity ratios normalized to housekeeping genes (β-actin and 60S ribosomal RNA). Secondly, total sputum cell RNA was transcribed to cDNAusing MMLV RT and amplified by quantitative PCR using gene-specific probes sets(Applied Biosystems) to assess gene expression relative to housekeeping gene β-actin.Gene expression for the MHC molecules HLA-DPA1, HLA-A and HLA-C, and theinflammasome molecules ASC, CARD8 and NLRC4 were significantly decreased in Asthand AR compared to NV, with CASP1 trending toward reduced levels in Asth compared toNV (Figure 1B). Additionally, NOD2 and BIRC2 expression was lower in Asth compared toNV, while expression of NLRP1, NLRP3, NLRP12, and NOD1 did not vary significantlybetween groups. Lowered gene expression for IL-1β, IL-18 and TNFα was evident in AsthBrickey et al. Page 2J Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptand AR. In comparison, real-time PCR results showed significantly diminished expressionof ASC, CARD8, NLRC4, CASP1 and TNFα in Asth and AR versus NV, with trends oflowered IL-1β, HLA-DPA1 and HLA-C expression (Figure 1C). While the results betweenmicroarray and PCR analyses mostly align, there are discrepancies that might be attributedto technical issues such as dissimilar oligonucleotide targets between microarray and PCRmethods used for transcript detection and/or disparate efficiencies in measuring lowabundant or degraded transcripts. Gene-specific PCR amplification may provide a moreaccurate representation of sputum cell expression due to sensitive detection.Our results are consistent with the hypothesis that relative to NV, Asth and AR volunteersdisplay reduced innate immune capability. Specifically, we found diminished airway cellexpression of inflammasome components ASC, CARD8, and NLRC4 as well as IL-1β, thekey mediator released following inflammasome activation, in both Asth and AR groups.However, we were unable to determine gene expression of specific cell types in sputum, orwhich cell type might account for decreased gene expression in Asth and AR groups.Eosinophils, which are more typical in sputum atopic persons, have less expression ofinflammasome genes than dendritic cells, neutrophils or macrophages (8). Also, we wereunable to differentiate M1 from M2 macrophages in this study. Macrophages can bepolarized to the M2 phenotype by the TH2 cytokines IL-4 and IL-13, and have decreasedinflammasome activity with decreased IL-1β secretion compared to M1 (9). Sincemacrophages were the most abundant cell in sputum from each group, and M2 macrophagesare more likely to be present in atopic persons, we hypothesize that M2 macrophages in theairways of Asth and AR volunteers may account for the observed diminished sputum cellinflammasome gene expression. Finally, it is not known if upon appropriate stimulation,inflammasome expression would increase in cells from asthmatic and atopic patients.In summary, our pilot data support the hypothesis that development of asthma and atopicdiseases is associated with decreased inflammasome function. Future studies will focus ondetermining inflammasome gene expression and function in isolated sputum macrophagesand granulocytes from asthmatics and atopic persons, and the relationship between TH2cytokines, the expression and function of inflammasome complexes, and asthma diseaseseverity.Sincerely,Willie June Brickey, PhDa, Neil E. Alexis, PhDb,c, Michelle L. Hernandez, MDb,c, WilliamReed, PhDb,c, Jenny PY. Ting, PhDa,d,e, and David B. Peden, MDa,b,c,fa. Department of Microbiology and Immunologyb. Department of Pediatricsc. Center for Environmental Medicine, Asthma and Lung Biologyd. Center for Translational Immunologye. Lineberger Comprehensive Cancer Centerf. Department of MedicineThe School of MedicineThe University of North Carolina at Chapel HillChapel Hill, North CarolinaBrickey et al. Page 3J Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcknowledgmentsFunding Sources: W.J.B., N.E.A., W.R., J.PY.T., D.B.P. are supported by NIAID U19AI077437. MLH issupported by NIH KL2RR025746AbbreviationsFEV1 Forced Expiratory Volume in One SecondPD20 provocative dose causing 20% reduction in airflowAsth asthmaticAR allergic rhiniticNV normal volunteerPMN neutrophilMac macrophageLym lymphocyteEos eosinophilBEC basal endothelial cellReferences1. Juhn YJ, Kita H, Yawn BP, Boyce TG, Yoo KH, McGree ME, et al. Increased risk of seriouspneumococcal disease in patients with asthma. J Allergy Clin Immunol. 2008; 122(4):719–23.[PubMed: 18790525]2. Talbot TR, Hartert TV, Mitchel E, Halasa NB, Arbogast PG, Poehling KA, et al. Asthma as a riskfactor for invasive pneumococcal disease. N Engl J Med. 2005; 352(20):2082–90. [PubMed:15901861]3. Goleva E, Hauk PJ, Hall CF, Liu AH, Riches DW, Martin RJ, et al. Corticosteroid-resistant asthmais associated with classical antimicrobial activation of airway macrophages. J Allergy ClinImmunol. 2008; 122(3):550–9. [PubMed: 18774390]4. Huang YJ, Nelson CE, Brodie EL, Desantis TZ, Baek MS, Liu J, et al. Airway microbiota andbronchial hyperresponsiveness in patients with suboptimally controlled asthma. J Allergy ClinImmunol. 2011; 127(2):372–81. [PubMed: 21194740]5. Newcomb DC, Peebles RS Jr. Bugs and asthma: a different disease? Proc Am Thorac Soc. 2009;6(3):266–71. [PubMed: 19387028]6. Davis BK, Wen H, Ting JP. The Inflammasome NLRs in Immunity, Inflammation, and AssociatedDiseases. Annu Rev Immunol. 20107. Alexis NE, Brickey WJ, Lay JC, Wang Y, Roubey RA, Ting JP, et al. Development of an inhaledendotoxin challenge protocol for characterizing evoked cell surface phenotype and genomicresponses of airway cells in allergic individuals. Ann Allergy Asthma Immunol. 2008; 100(3):206–15. [PubMed: 18426139]8. Guarda G, Zenger M, Yazdi AS, Schroder K, Ferrero I, Menu P, et al. Differential expression ofNLRP3 among hematopoietic cells. J Immunol. 2011; 186(4):2529–34. [PubMed: 21257968]9. Pelegrin P, Surprenant A. Dynamics of macrophage polarization reveal new mechanism to inhibitIL-1beta release through pyrophosphates. EMBO J. 2009; 28(14):2114–27. [PubMed: 19536133]Brickey et al. Page 4J Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Assessment of innate immune factors in sputum airway cells. (A) Sputum supernatantprotein for NV and Asth subjects are analyzed by multiplex ELISA with mean levelsindicated by bar. Gene expression is assessed by oligonucleotide microarray hybridization(B) and PCR analyses (C). Mean normalized microarray signal intensity ratio ofexperimental to universal reference RNA ± SEM in NV, Asth and AR are plotted (n=8–10/group). Mean PCR-amplified transcripts relative to ACTB ± SEM are plotted (n=5/group).Statistical tests include (A) Unpaired t test and (B, C) One-way Analysis of Variance forthree groups (with overall p values depicted over the line) with Dunnett’s MultipleComparison Test to NV (stars) with p values indicated (*p<0.05, **p<0.01, ***p<0.001).Brickey et al. Page 5J Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptBrickey et al. Page 6Table IDemographic Data of Subjects and Sputum Cell Composition.SubjectPhysiologyCell Number (×1000)TypenFEV1 (%)Methacholine Challenge PD20(mg)MedicationsAllergiesPMNMacLymEosBECNV985–105negativenonenone79±33150±302.0±0.51.3±0.96.5±2.9Asth783–1241.0 – 10.0albuterolcat, cockroach, dust, grass, mite, shellfish, tree, weed160±110250±904.9±1.95.8±5.614±8AR9100–120negativenone, OCP, aldactonecockroach, grass, mite, rodent, tree, weed85±28350±2202.8±1.62.0±1.611±5NV, normal volunteer; Asth, asthmatic; AR, allergic rhinitic; FEV1, forced expiratory volume in one second; PD20, provocative dose causing 20% reduction in airflow; OCP, over the counter prescription;PMN, neutrophil; Mac, macrophage; Lym, lymphocyte; Eos, eosinophil; BEC, basal endothelial cellJ Allergy Clin Immunol. Author manuscript; available in PMC 2012 October 1.

Sputum inflammatory cells from patients with allergic rhinitis and asthma have decreased inflammasome gene expression

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Sputum inflammatory cells from patients with allergic rhinitis and asthma have decreased inflammasome gene expression

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