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Lung Gene Expression in a Rhesus Allergic Asthma Model Correlates with Physiologic Parameters of Disease and Exhibits Common and Distinct Pathways with Human Asthma and a Mouse Asthma Model

2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2011.06.009

1525-2191

Alexander R. Abbas, Janet Jackman, Sherron Bullens, Sarah M. Davis, David F. Choy, Grazyna Fedorowicz, Martha Tan, Bao-Tran Truong, Y. Gloria Meng, Lauri Diehl, Lisa A. Miller, Edward S. Schelegle, Dallas M. Hyde, Hilary Clark, Zora Modrušan, Joseph R. Arron, Lawren C. Wu,

Respiratory and Cough-Related Research

Experimental nonhuman primate models of asthma exhibit multiple features that are characteristic of an eosinophilic/T helper 2 (Th2)-high asthma subtype, characterized by the increased expression of Th2 cytokines and responsive genes, in humans. Here, we determine the molecular pathways that are present in a house dust mite–induced rhesus asthma model by analyzing the genomewide lung gene expression profile of the rhesus model and comparing it with that of human Th2-high asthma. We find that a prespecified human Th2 inflammation gene set from human Th2-high asthma is also present in rhesus asthma and that the expression of the genes comprising this gene set is positively correlated in human and rhesus asthma. In addition, as in human Th2-high asthma, the Th2 gene set correlates with physiologic markers of allergic inflammation and disease in rhesus asthma. Comparison of lung gene expression profiles from human Th2-high asthma, the rhesus asthma model, and a common mouse asthma model indicates that genes associated with Th2 inflammation are shared by all three species. However, some pathophysiologic aspects of human asthma (ie, subepithelial fibrosis, angiogenesis, neural biology, and immune host defense biology) are better represented in the gene expression profile of the rhesus model than in the mouse model. Further study of the rhesus asthma model may yield novel insights into the pathogenesis of human Th2-high asthma. Experimental nonhuman primate models of asthma exhibit multiple features that are characteristic of an eosinophilic/T helper 2 (Th2)-high asthma subtype, characterized by the increased expression of Th2 cytokines and responsive genes, in humans. Here, we determine the molecular pathways that are present in a house dust mite–induced rhesus asthma model by analyzing the genomewide lung gene expression profile of the rhesus model and comparing it with that of human Th2-high asthma. We find that a prespecified human Th2 inflammation gene set from human Th2-high asthma is also present in rhesus asthma and that the expression of the genes comprising this gene set is positively correlated in human and rhesus asthma. In addition, as in human Th2-high asthma, the Th2 gene set correlates with physiologic markers of allergic inflammation and disease in rhesus asthma. Comparison of lung gene expression profiles from human Th2-high asthma, the rhesus asthma model, and a common mouse asthma model indicates that genes associated with Th2 inflammation are shared by all three species. However, some pathophysiologic aspects of human asthma (ie, subepithelial fibrosis, angiogenesis, neural biology, and immune host defense biology) are better represented in the gene expression profile of the rhesus model than in the mouse model. Further study of the rhesus asthma model may yield novel insights into the pathogenesis of human Th2-high asthma. Asthma is characterized by variable airflow obstruction, airway hyperreactivity, and chronic airway inflammation. Despite these common clinical features, asthma is a heterogeneous disease that can be subclassified by a number of different measures, including the nature of the airway inflammation.1Green R.H. Brightling C.E. Bradding P. The reclassification of asthma based on subphenotypes.Curr Opin Allergy Clin Immunol. 2007; 7: 43-50Crossref PubMed Scopus (88) Google Scholar, 2Wenzel S.E. Asthma: defining of the persistent adult phenotypes.Lancet. 2006; 368: 804-813Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar Airway eosinophilia, a hallmark of T helper 2 (Th2) inflammation, defines a major subtype of severe asthma, with other major subtypes defined by neutrophilic and paucigranulocytic inflammation.1Green R.H. Brightling C.E. Bradding P. The reclassification of asthma based on subphenotypes.Curr Opin Allergy Clin Immunol. 2007; 7: 43-50Crossref PubMed Scopus (88) Google Scholar, 2Wenzel S.E. Asthma: defining of the persistent adult phenotypes.Lancet. 2006; 368: 804-813Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar, 3Douwes J. Gibson P. Pekkanen J. Pearce N. Non-eosinophilic asthma: importance and possible mechanisms.Thorax. 2002; 57: 643-648Crossref PubMed Scopus (513) Google Scholar, 4Haldar P. Pavord I.D. Noneosinophilic asthma: a distinct clinical and pathologic phenotype.J Allergy Clin Immunol. 2007; 119 (quiz 1053–1044): 1043-1052Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar Recent analyses of airway gene expression in mild-to-moderate asthmatics not being treated with corticosteroids defines a molecular signature of Th2 inflammation that correlates with airway and peripheral eosinophilia, airway hyperreactivity, subepithelial fibrosis, distinct patterns of mucin expression, and elevated IgE.5Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. Fedorowicz G. Modrusan Z. Fahy J.V. Woodruff P.G. Arron J.R. Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (135) Google Scholar, 6Woodruff P.G. Modrek B. Choy D.F. Jia G. Abbas A.R. Ellwanger A. Koth L.L. Arron J.R. Fahy J.V. T-helper type 2-driven inflammation defines major subphenotypes of asthma.Am J Respir Crit Care Med. 2009; 180: 388-395Crossref PubMed Scopus (1393) Google Scholar The Th2-high subtype of asthma in mild-to-moderate asthmatics is associated with the cytokines IL-5 and IL-13 and is correlated with a clinical response to inhaled corticosteroid treatment. Nonhuman primate models of asthma have been used to study pathogenic mechanisms and the efficacy of therapies, given the close similarities between monkeys and humans.7Avdalovic M.V. Putney L.F. Schelegle E.S. Miller L. Usachenko J.L. Tyler N.K. Plopper C.G. Gershwin L.J. Hyde D.M. Vascular remodeling is airway generation-specific in a primate model of chronic asthma.Am J Respir Crit Care Med. 2006; 174: 1069-1076Crossref PubMed Scopus (35) Google Scholar, 8Coffman R.L. Hessel E.M. Nonhuman primate models of asthma.J Exp Med. 2005; 201: 1875-1879Crossref PubMed Scopus (61) Google Scholar, 9Fanucchi M.V. Schelegle E.S. Baker G.L. Evans M.J. McDonald R.J. Gershwin L.J. Raz E. Hyde D.M. Plopper C.G. Miller L.A. Immunostimulatory oligonucleotides attenuate airways remodeling in allergic monkeys.Am J Respir Crit Care Med. 2004; 170: 1153-1157Crossref PubMed Scopus (70) Google Scholar, 10Miller L.A. Hurst S.D. Coffman R.L. Tyler N.K. Stovall M.Y. Chou D.L. Putney L.F. Gershwin L.J. Schelegle E.S. Plopper C.G. Hyde D.M. Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.Clin Exp Allergy. 2005; 35: 894-906Crossref PubMed Scopus (38) Google Scholar, 11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12Seshasayee D. Lee W.P. Zhou M. Shu J. Suto E. Zhang J. Diehl L. Austin C.D. Meng Y.G. Tan M. Bullens S.L. Seeber S. Fuentes M.E. Labrijn A.F. Graus Y.M. Miller L.A. Schelegle E.S. Hyde D.M. Wu L.C. Hymowitz S.G. Martin F. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.J Clin Invest. 2007; 117: 3868-3878Crossref PubMed Scopus (183) Google Scholar, 13Van Scott M.R. Hooker J.L. Ehrmann D. Shibata Y. Kukoly C. Salleng K. Westergaard G. Sandrasagra A. Nyce J. Dust mite-induced asthma in cynomolgus monkeys.J Appl Physiol. 2004; 96: 1433-1444Crossref PubMed Scopus (35) Google Scholar Several features of lung and immune biology in nonhuman primates more accurately model human biology than the mouse, which is the most commonly used animal species for preclinical asthma studies.8Coffman R.L. Hessel E.M. Nonhuman primate models of asthma.J Exp Med. 2005; 201: 1875-1879Crossref PubMed Scopus (61) Google Scholar For example, in both humans and rhesus monkeys the primary distal airway is the respiratory bronchiole, whereas in rodents it is the nonalveolarized bronchiole. Basal cells are found throughout the tracheobronchial airways of both humans and rhesus monkeys but only in the tracheas of mice. In addition, Clara cells are found only in the bronchioles of both humans and rhesus monkeys but throughout the tracheobronchial airways of mice. Some unique features of asthma found in humans and rhesus monkeys but not mice include intrinsic airway hyperreactivity (except for the A/J mouse strain), smooth muscle hypertrophy in the more distal bronchi and respiratory bronchioles, exfoliation of epithelial sheets, and mast cell infiltration of airway smooth muscle.11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 14Boyce J.A. Austen K.F. No audible wheezing: nuggets and conundrums from mouse asthma models.J Exp Med. 2005; 201: 1869-1873Crossref PubMed Scopus (80) Google Scholar, 15Van Winkle L.S. Baker G.L. Chan J.K. Schelegle E.S. Plopper C.G. Airway mast cells in a rhesus model of childhood allergic airways disease.Toxicol Sci. 2010; 116: 313-322Crossref PubMed Scopus (7) Google Scholar Thus, the study of nonhuman primate models of asthma is an important complement and supplement to studies of mouse asthma models in preclinical asthma research. Gene expression profiling of disease tissues enables the genomewide assessment of molecular pathways that are dysregulated in disease. Although several groups have performed genomewide transcriptional profiling of preclinical mouse asthma models,16Di Valentin E. Crahay C. Garbacki N. Hennuy B. Gueders M. Noel A. Foidart J.M. Grooten J. Colige A. Piette J. Cataldo D. New asthma biomarkers: lessons from murine models of acute and chronic asthma.Am J Physiol Lung Cell Mol Physiol. 2009; 296: L185-L197Crossref PubMed Scopus (96) Google Scholar, 17Fulkerson P.C. Zimmermann N. Hassman L.M. Finkelman F.D. Rothenberg M.E. Pulmonary chemokine expression is coordinately regulated by STAT1. STAT6, and IFN-gamma.J Immunol. 2004; 173: 7565-7574Crossref PubMed Scopus (91) Google Scholar, 18Kelada S.N. Wilson M.S. Tavarez U. Kubalanza K. Borate B. Whitehead G. Maruoka S. Roy M.G. Olive M. Carpenter D.E. Brass D.M. Wynn T.A. Cook D.A. Evans C.M. Schwartz D.A. Collins F.S. Strain-dependent genomic factors affect allergen-induced airway hyper-responsiveness in mice.Am J Respir Cell Mol Biol. 2011; ([Epub ahead of press])https://doi.org/10.1165/rcmb.2010-0315OCCrossref Scopus (48) Google Scholar, 19Kuperman D.A. Lewis C.C. Woodruff P.G. Rodriguez M.W. Yang Y.H. Dolganov G.M. Fahy J.V. Erle D.J. Dissecting asthma using focused transgenic modeling and functional genomics.J Allergy Clin Immunol. 2005; 116: 305-311Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 20Lewis C.C. Aronow B. Hutton J. Santeliz J. Dienger K. Herman N. Finkelman F.D. Wills-Karp M. Unique and overlapping gene expression patterns driven by IL-4 and IL-13 in the mouse lung.J Allergy Clin Immunol. 2009; 123 (795–804e798)Abstract Full Text Full Text PDF Scopus (45) Google Scholar, 21Lewis C.C. Yang J.Y. Huang X. Banerjee S.K. Blackburn M.R. Baluk P. McDonald D.M. Blackwell T.S. Nagabhushanam V. Peters W. Voehringer D. Erle D.J. Disease-specific gene expression profiling in multiple models of lung disease.Am J Respir Crit Care Med. 2008; 177: 376-387Crossref PubMed Scopus (96) Google Scholar, 22Park S.G. Choi J.W. Kim H. Roh G.S. Bok J. Go M.J. Kwack K. Oh B. Kim Y. Genome-wide profiling of antigen-induced time course expression using murine models for acute and chronic asthma.Int Arch Allergy Immunol. 2008; 146: 44-56Crossref PubMed Scopus (19) Google Scholar, 23Rolph M.S. Sisavanh M. Liu S.M. Mackay C.R. Clues to asthma pathogenesis from microarray expression studies.Pharmacol Ther. 2006; 109: 284-294Crossref PubMed Scopus (31) Google Scholar, 24Zimmermann N. King N.E. Laporte J. Yang M. Mishra A. Pope S.M. Muntel E.E. Witte D.P. Pegg A.A. Foster P.S. Hamid Q. Rothenberg M.E. Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis.J Clin Invest. 2003; 111: 1863-1874Crossref PubMed Scopus (449) Google Scholar and a few publications have assessed gene expression in nonhuman primate asthma models,10Miller L.A. Hurst S.D. Coffman R.L. Tyler N.K. Stovall M.Y. Chou D.L. Putney L.F. Gershwin L.J. Schelegle E.S. Plopper C.G. Hyde D.M. Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.Clin Exp Allergy. 2005; 35: 894-906Crossref PubMed Scopus (38) Google Scholar, 25Ayanoglu G. Desai B. Fick Jr, R.B. Grein J. de Waal Malefyt R. Mattson J. McClanahan T. Olmstead S. Reece S.P. Van Scott M.R. Wardle R.L. Modelling asthma in macaques: longitudinal changes in cellular and molecular markers.Eur Respir J. 2011; 37: 541-552Crossref PubMed Scopus (12) Google Scholar, 26Zou J. Young S. Zhu F. Gheyas F. Skeans S. Wan Y. Wang L. Ding W. Billah M. McClanahan T. Coffman R.L. Egan R. Umland S. Microarray profile of differentially expressed genes in a monkey model of allergic asthma.Genome Biol. 2002; 3 (research0020)Crossref Google Scholar data are limited on genomewide gene expression in nonhuman primate asthma models, and we are not aware of any publications that have directly compared the genomewide transcriptional profiles of human asthma with those of any preclinical asthma model, regardless of species. A model of allergic asthma induced in rhesus monkeys by house dust mites (HDMs) exhibits multiple pathophysiologic features of human allergic asthma and has been used to assess the efficacy of immunomodulatory therapies.9Fanucchi M.V. Schelegle E.S. Baker G.L. Evans M.J. McDonald R.J. Gershwin L.J. Raz E. Hyde D.M. Plopper C.G. Miller L.A. Immunostimulatory oligonucleotides attenuate airways remodeling in allergic monkeys.Am J Respir Crit Care Med. 2004; 170: 1153-1157Crossref PubMed Scopus (70) Google Scholar, 10Miller L.A. Hurst S.D. Coffman R.L. Tyler N.K. Stovall M.Y. Chou D.L. Putney L.F. Gershwin L.J. Schelegle E.S. Plopper C.G. Hyde D.M. Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.Clin Exp Allergy. 2005; 35: 894-906Crossref PubMed Scopus (38) Google Scholar, 11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12Seshasayee D. Lee W.P. Zhou M. Shu J. Suto E. Zhang J. Diehl L. Austin C.D. Meng Y.G. Tan M. Bullens S.L. Seeber S. Fuentes M.E. Labrijn A.F. Graus Y.M. Miller L.A. Schelegle E.S. Hyde D.M. Wu L.C. Hymowitz S.G. Martin F. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.J Clin Invest. 2007; 117: 3868-3878Crossref PubMed Scopus (183) Google Scholar Here, we have conducted gene expression profiling of lung airway samples from this rhesus asthma model. We assess the relation between rhesus lung gene expression and physiologic parameters of inflammation and disease. In addition, we compare lung gene expression of human Th2-high asthma with that of the rhesus asthma model, as well as that of a commonly used mouse asthma model, to assess similarities and differences in lung gene expression between human Th2-high asthma and these two animal models of asthma. Bronchial biopsy RNA from 27 nonsmoking patients with mild-to-moderate asthma and healthy nonsmoking subjects were obtained from the University of California San Francisco Airway Tissue Bank, a specimen biorepository approved by the University of California San Francisco Committee on Human Research. Endobronchial biopsies had been collected from a subset of patients for whom we have previously described gene expression profiles of bronchial epithelial brushings.6Woodruff P.G. Modrek B. Choy D.F. Jia G. Abbas A.R. Ellwanger A. Koth L.L. Arron J.R. Fahy J.V. T-helper type 2-driven inflammation defines major subphenotypes of asthma.Am J Respir Crit Care Med. 2009; 180: 388-395Crossref PubMed Scopus (1393) Google Scholar, 27Woodruff P.G. Boushey H.A. Dolganov G.M. Barker C.S. Yang Y.H. Donnelly S. Ellwanger A. Sidhu S.S. Dao-Pick T.P. Pantoja C. Erle D.J. Yamamoto K.R. Fahy J.V. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids.Proc Natl Acad Sci U S A. 2007; 104: 15858-15863Crossref PubMed Scopus (679) Google Scholar Three to six endobronchial biopsies were collected from the carinae of the second- to fourth-order bronchi. Informed consent was obtained from all human subjects. An HDM-induced allergic asthma model in young adult rhesus monkeys (Macaca mulatta) has been described previously.12Seshasayee D. Lee W.P. Zhou M. Shu J. Suto E. Zhang J. Diehl L. Austin C.D. Meng Y.G. Tan M. Bullens S.L. Seeber S. Fuentes M.E. Labrijn A.F. Graus Y.M. Miller L.A. Schelegle E.S. Hyde D.M. Wu L.C. Hymowitz S.G. Martin F. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.J Clin Invest. 2007; 117: 3868-3878Crossref PubMed Scopus (183) Google Scholar The model consisted of an 18-month disease development phase, followed by a treatment phase. Data from the treatment phase was published previously.12Seshasayee D. Lee W.P. Zhou M. Shu J. Suto E. Zhang J. Diehl L. Austin C.D. Meng Y.G. Tan M. Bullens S.L. Seeber S. Fuentes M.E. Labrijn A.F. Graus Y.M. Miller L.A. Schelegle E.S. Hyde D.M. Wu L.C. Hymowitz S.G. Martin F. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.J Clin Invest. 2007; 117: 3868-3878Crossref PubMed Scopus (183) Google Scholar Data from the 18-month disease development phase is presented in this article. The animal protocol was approved by the Institutional Animal Care and Use Committee Ethical Review Board at the University of California Davis. All monkeys were selected from the California National Primate Research Center's breeding colony on the basis of social rank, treated with ivermectin subcutaneously at 0.2 mg/kg for potential parasites, and isolated indoors for 1 month. Briefly, 12 adult rhesus monkeys were sensitized with a subcutaneous injection of HDM allergen extract followed by 3 biweekly intranasal instillations of HDM and 6 weekly aerosol challenges of HDM. Ten adult rhesus monkeys were subjected to control treatment with PBS injections and mock aerosol challenges. After the sensitization procedure, sensitized monkeys were regularly exposed to aerosolized HDM for 2 to 3 hours twice a week, for a total of 18 months, and also received additional subcutaneous and intranasal HDM boosts at weeks 56, 71, and 75. Although all 12 sensitized animals were enrolled and characterized for the duration of the study, only 4 of the 10 control animals were kept and characterized beyond the postsensitization time point, because of cost constraints. The six control animals that were dropped from the study were randomly selected for exclusion such that there were no differences in the mean group values of measured parameters in the control group between the 4-animal subset and the entire group of 10 control animals. Data and samples were collected over a 2-week period, starting at weeks −4 to −8 for the presensitization time point, week 15 for the postsensitization time point, and week 75 for the 18-month time point, whereby week 0 denotes the beginning of the sensitization protocol. In the first week of data and sample collection, peripheral blood and serum samples were obtained just before an aerosol challenge for determination of complete blood counts, flow cytometric analysis, and serum ELISAs. Blood leukocyte values and differential counts were determined as described previously.11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Forty-eight hours after the aerosol challenge, pulmonary mechanics and airway hyperreactivity to methacholine were determined as described previously.11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar In the second week of data and sample collection, bronchoalveolar lavage fluid (BALF) samples were collected 48 hours after an aerosol challenge for determination of leukocyte differentials and flow cytometric analysis, as described previously.11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Immediately after the BALF collection, five lung biopsies were obtained from the subcarina at the lower or middle lobes of the lung by flexible bronchoscopy with the use of a 1-mm biopsy forceps, after intubation with an appropriate-sized, cuffed endotracheal tube. Animals were deprived of food 8 hours before the bronchoscopy to minimize the risk of aspiration during anesthesia. During the bronchoscopy procedure, oxygen saturation and heart rate monitoring were provided continuously, and supplemental oxygen was routinely given. The bronchoscope (2.7 to 3.6 mm in diameter; Olympus, Center Valley, PA) was passed through the nose, 5 mg of lidocaine was instilled on the larynx for topical anesthesia, and the bronchoscope was directed into the trachea. A second dose of 5 mg of lidocaine was administered through the bronchoscope for topical anesthesia and to reduce the cough reflex, and the bronchoscope was directed to the thin shelf of tissues dividing segmental or subsegmental airways. Three biopsies were preserved in RNAlater RNA stabilization reagent (Qiagen, Valencia, CA) for RNA extraction and gene expression profiling, and two biopsies were reserved for histopathology analyses. Intradermal skin testing for reactivity to HDM antigen was performed as described previously11Schelegle E.S. Gershwin L.J. Miller L.A. Fanucchi M.V. Van Winkle L.S. Gerriets J.P. Walby W.F. Omlor A.M. Buckpitt A.R. Tarkington B.K. Wong V.J. Joad J.P. Pinkerton K.B. Wu R. Evans M.J. Hyde D.M. Plopper C.G. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).Am J Pathol. 2001; 158: 333-341Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar at approximately 5 months before the start of sensitization for the presensitization time point and at week 5 for the postsensitization time point, whereby week 0 denotes the beginning of the sensitization protocol. During all data and sample collection, animals were sedated with ketamine (10 mg/kg, i.m.) and then anesthetized with propofol (0.1 to 0.2 mg/kg/min, i.v.), with the dose adjusted as deemed necessary by the attending veterinarian. Peripheral blood mononuclear cells and BALF cells were prepared for immunofluoresence staining and analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Total IgE in rhesus monkey sera was measured by ELISA with the use of monoclonal anti-IgE MAE11 (Genentech, South San Francisco, CA) for capture and peroxidase-conjugated goat anti-human IgE antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for detection. The standard range was 0.098 to 12.5 ng/mL for human IgE. The minimum sample dilution was 1:10 to avoid any interference from sera in the assay. Rhesus monkey IgE concentrations were calculated by dividing the concentrations obtained on the basis of a human IgE standard curve by a correlation factor of 0.029, which was determined with purified cynomolgus monkey IgE (Genentech). HDM-specific IgE titers were measured by ELISA with the use of monoclonal anti-IgE MAE11 for capture and biotinylated HDM allergen Der f1 (Indoor Biotechnologies, Charlottesville, VA) for detection, followed by horseradish peroxidase–conjugated streptavidin (GE Healthcare, Little Chalfont, Buckinghamshire, UK). For calculation of titers, a cut point was set at twice the absorbance of a 1:100 diluted blank rhesus monkey serum (Bioreclamation, Westbury, NY). The dilution factor at which an absorbance value equaled the cut point was calculated from a linear interpolation of absorbance values obtained from serial dilutions of samples. Titer is reported as the log10 of the dilution factor. Titers for negative samples are reported as <1.52 because a minimum sample dilution factor of 33.3 was used. Biopsies were placed individually in cryomolds and submerged in optimal cutting temperature compound media (Sakura Finetek, Torrance, CA). Cryomolds were then chilled to set optimal cutting temperature compound and stored at −80°C. All sections were cut 5-μm thick and stained with H&E for histologic analysis. All biopsies were scored in a blinded manner at the time of collection. Human lung airway biopsy RNA was isolated from homogenized bronchial biopsies as described previously.5Choy D.F. Modrek B. Abbas A.R. Kummerfeld S. Clark H.F. Wu L.C. Fedorowicz G. Modrusan Z. Fahy J.V. Woodruff P.G. Arron J.R. Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways.J Immunol. 2011; 186: 1861-1869Crossref PubMed Scopus (135) Google Scholar, 6Woodruff P.G. Modrek B. Choy D.F. Jia G. Abbas A.R. Ellwanger A. Koth L.L. Arron J.R. Fahy J.V. T-helper type 2-driven inflammation defines major subphenotypes of asthma.Am J Respir Crit Care Med. 2009; 180: 388-395Crossref PubMed Scopus (1393) Google Scholar Total RNA was extracted from individual biopsy samples with the use of RNeasy Mini Kits (Qiagen), following the manufacturer's guidelines, and all biopsy RNA from each individual subject was pooled for further processing and analysis. RNA samples were quantified with a Nanodrop ND-1000 UV-spectrophotometer (Thermo Scientific, West Palm Beach, FL), and RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The quantity of total RNA used in a two-round amplification protocol ranged from 10 ng to 50 ng per sample. First-round amplification and second-round cDNA syntheses were done with the Message Amp II aRNA Amplification Kit (Applied Biosystems, Foster City, CA). Cyanine-5 dye was incorporated with the Quick Amp Labeling kit (Agilent Technologies). Each cyanine-5–labeled test sample (750 ng) was pooled with cyanine-3–labeled Universal Human Reference RNA (Stratagene, La Jolla, CA) and hybridized onto Agilent Whole Human Genome 4 × 44K arrays as described in the manufacturer's protocol. Arrays were washed, dried, and scanned on the Agilent scanner according to the manufacturer's protocol. Microarray image files were analyzed with Feature Extraction software 9.5 (Agilent Technologies). Human lung airway biopsy microarray data have been deposited in Gene Expression Omnibus with the accession code GSE23611. Rhesus lung airway biopsies were recovered from RNAlater and homogenized in RLT buffer (Qiagen) with the use of an MM300 mixer mill (Retsch, Haan, Germany). RNA was isolated from homogenized tissue with the use of RNeasy Micro Kits (Qiagen) with on-column DNase treatment, following the manufacturer's guidelines, and concentrated by ethanol precipitation. RNA samples were quantified with a Nanodrop ND-1000 UV-spectrophotometer (Thermo Scientific), and RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, RNA was amplified with the Low RNA Input Fluorescent Linear Amplification protocol (Agilent Technologies). A T7 RNA polymerase single round of linear amplification was performed to incorporate Cyanine-3 and Cyanine-5 label into cRNA. Each Universal Human Reference (Stratagene) cRNA labeled with Cyanine-3 and test sample cRNA labeled with Cyanine-5 (750 ng) was fragmented for 30 minutes at 60°C before loading onto Agilent Whole Human Genome microarrays (Agilent Technologies). Samples were hybridized for 18 hours at 60°C with constant rotation. Microarrays were washed, dried, and scanned on the Agilent scanner according to the manufacturer's protocol. Microarray image files were analyzed with Feature Extraction software version 7.5 with default parameters and Lowess normalization to yield summary ratio data (Agilent Technologies). Rhesus lung airway biopsy microarray data have been deposited in Gene Expression Omnibus with the accession code GSE23327. Rhesus model data were expressed as mean ± SD. P va