Title: A Novel Nonhuman Primate Model of Cigarette Smoke–Induced Airway Disease
Abstract: Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)–exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD. Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)–exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD. Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide.1Murray C.J. Lopez A.D. Measuring the global burden of disease.N Engl J Med. 2013; 369: 448-457Crossref PubMed Scopus (1256) Google Scholar, 2Murray C.J. Lopez A.D. Alternative projections of mortality and disability by cause 1990-2020: global Burden of Disease Study.Lancet. 1997; 349: 1498-1504Abstract Full Text Full Text PDF PubMed Scopus (5666) Google Scholar COPD is characterized by airflow limitation that is not fully reversible and associated with abnormal pulmonary inflammation induced by noxious particles and gases most commonly present in cigarette smoke (CS). Mice are widely used to investigate the biological pathways that contribute to lung pathologies occurring in CS-induced COPD and to test the efficacy of novel therapies for COPD.3Churg A. Cosio M. Wright J.L. Mechanisms of cigarette smoke-induced COPD: insights from animal models.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L612-L631Crossref PubMed Scopus (235) Google Scholar Mice exposed to CS for 6 months exhibit some features of human COPD, including chronic pulmonary inflammation, modest airspace enlargement, and mild small airway fibrosis.3Churg A. Cosio M. Wright J.L. Mechanisms of cigarette smoke-induced COPD: insights from animal models.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L612-L631Crossref PubMed Scopus (235) Google Scholar The use of mice to model COPD has several advantages, including the following: i) opportunities for genetic manipulation and the availability of molecular reagents to probe changes in pathways in vivo, ii) rapid breeding rate, and iii) small size, which is advantageous for dosing expensive drugs. However, murine COPD models have several limitations. In contrast to humans, mice lack bronchial submucosal glands, clearly defined respiratory bronchioles, and a distinct lobular architecture.4March T.H. Green F.H. Hahn F.F. Nikula K.J. Animal models of emphysema and their relevance to studies of particle-induced disease.Inhal Toxicol. 2000; 12: 155-187PubMed Google Scholar Mice also have a monopodial airway branching pattern, rather than the dichotomous pattern found in humans.5Plopper C.G. Hyde D.M. The non-human primate as a model for studying COPD and asthma.Pulm Pharmacol Ther. 2008; 21: 755-766Crossref PubMed Scopus (73) Google Scholar Moreover, there are differences in the innate and adaptive immune systems6Mestas J. Hughes C.C. Of mice and not men: differences between mouse and human immunology.J Immunol. 2004; 172: 2731-2738Crossref PubMed Scopus (2458) Google Scholar and in the expressed profiles of matrix metalloproteinases (MMPs) in humans versus mice.7Owen C.A. Proteinases and oxidants as targets in the treatment of chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2005; 2: 373-385Crossref PubMed Scopus (73) Google Scholar In mice, it is also challenging to perform serial sampling in blood or lungs to measure biomarkers of CS-induced lung injury or responses to therapies. Furthermore, mice do not develop robust airway pathologies, including mucus hypersecretion and small airway fibrosis, when exposed chronically to CS. Also, several therapies, including an anti-tumor necrosis factor α antibody,8Herfs M. Hubert P. Poirrier A.L. Vandevenne P. Renoux V. Habraken Y. Cataldo D. Boniver J. Delvenne P. Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers: implications for chronic obstructive pulmonary disease therapy.Am J Respir Cell Mol Biol. 2012; 47: 67-79Crossref PubMed Scopus (64) Google Scholar roflumilast,9Martorana P.A. Beume R. Lucattelli M. Wollin L. Lungarella G. Roflumilast fully prevents emphysema in mice chronically exposed to cigarette smoke.Am J Respir Crit Care Med. 2005; 172: 848-853Crossref PubMed Scopus (188) Google Scholar simvastatin,10Takahashi S. Nakamura H. Seki M. Shiraishi Y. Yamamoto M. Furuuchi M. Nakajima T. Tsujimura S. Shirahata T. Nakamura M. Minematsu N. Yamasaki M. Tateno H. Ishizaka A. Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L882-L890Crossref PubMed Scopus (76) Google Scholar and antioxidants11Nyunoya T. March T.H. Tesfaigzi Y. Seagrave J. Antioxidant diet protects against emphysema, but increases mortality in cigarette smoke-exposed mice.COPD. 2011; 8: 362-368Crossref PubMed Scopus (21) Google Scholar that have effectively treated emphysema in mice, were substantially less effective when tested in COPD patients,7Owen C.A. Proteinases and oxidants as targets in the treatment of chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2005; 2: 373-385Crossref PubMed Scopus (73) Google Scholar, 12Rennard S.I. Fogarty C. Kelsen S. Long W. Ramsdell J. Allison J. Mahler D. Saadeh C. Siler T. Snell P. Korenblat P. Smith W. Kaye M. Mandel M. Andrews C. Prabhu R. Donohue J.F. Watt R. Lo K.H. Schlenker-Herceg R. Barnathan E.S. Murray J. The safety and efficacy of infliximab in moderate to severe chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2007; 175: 926-934Crossref PubMed Scopus (375) Google Scholar, 13Calverley P.M. Sanchez-Toril F. McIvor A. Teichmann P. Bredenbroeker D. Fabbri L.M. Effect of 1-year treatment with roflumilast in severe chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2007; 176: 154-161Crossref PubMed Scopus (294) Google Scholar, 14Fabbri L.M. Calverley P.M. Izquierdo-Alonso J.L. Bundschuh D.S. Brose M. Martinez F.J. Rabe K.F. Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials.Lancet. 2009; 374: 695-703Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar raising the issue about the utility of smoke-exposed mice for testing novel therapeutics for the human disease. Smoke-exposed guinea pigs have been used as an alternative to mice as a small animal model of COPD but have several disadvantages, including the lack of availability of commercially available reagents for interrogating pathways in guinea pigs.15Wright J.L. Churg A. Cigarette smoke causes physiologic and morphologic changes of emphysema in the guinea pig.Am Rev Respir Dis. 1990; 142: 1422-1428Crossref PubMed Scopus (152) Google Scholar Thus, there is a need for alternative animal models that better recapitulate the physiological and pathological changes occurring in the lungs of human COPD patients. Other models for studying disease pathogenesis and preclinical testing of pharmaceuticals have been established in larger animals.5Plopper C.G. Hyde D.M. The non-human primate as a model for studying COPD and asthma.Pulm Pharmacol Ther. 2008; 21: 755-766Crossref PubMed Scopus (73) Google Scholar, 16Snider G.L. Lucey E.C. Stone P.J. Animal models of emphysema.Am Rev Respir Dis. 1986; 133: 149-169Crossref PubMed Scopus (250) Google Scholar Among these, nonhuman primates (NHPs) have great potential because their pulmonary anatomy and immune system are similar to those of humans. Also, NHP and human proteins have a high degree of homology, and molecular reagents used in studies of human samples often can be used to probe pathways in NHP samples.17Coffman R.L. Hessel E.M. Nonhuman primate models of asthma.J Exp Med. 2005; 201: 1875-1879Crossref PubMed Scopus (59) Google Scholar When challenged with allergens, NHPs develop allergic airway inflammation, hyperresponsiveness, and extensive airway remodeling pathologies resembling that which occurs in human asthmatics.5Plopper C.G. Hyde D.M. The non-human primate as a model for studying COPD and asthma.Pulm Pharmacol Ther. 2008; 21: 755-766Crossref PubMed Scopus (73) Google Scholar In addition, exposing NHPs to ozone produces a persistent chronic respiratory bronchiolitis18Tyler W.S. Tyler N.K. Last J.A. Gillespie M.J. Barstow T.J. Comparison of daily and seasonal exposures of young monkeys to ozone.Toxicology. 1988; 50: 131-144Crossref PubMed Scopus (65) Google Scholar similar to that occurring in human smokers.19Cosio M.G. Guerassimov A. Chronic obstructive pulmonary disease: inflammation of small airways and lung parenchyma.Am J Respir Crit Care Med. 1999; 160: S21-S25Crossref PubMed Scopus (135) Google Scholar However, to our knowledge, there have been no prior reports of the effects of CS exposure on the NHP lung. We hypothesized that when exposed to CS, NHPs would develop pathological and functional changes in their lungs similar to those occurring in the airways of COPD patients. To test this hypothesis, we evaluated the airway and alveolar pathologies and lung physiology in cynomolgus macaques (Macaca fascicularis) exposed to air or CS for up to 12 weeks. Some results have been presented in abstract form.20Polverino F. Doyle-Eisele M. McDonald J. Kelly E. Wilder J. Mauderly J. Divo M. Pinto-Plata V. Celli B. Tesfaigzi Y. Owen C.A. A novel non-human primate model of cigarette-smoke induced chronic obstructive pulmonary disease (abstract).Am J Respir Crit Care Med. 2014; (B45. COPD:pathogenesis. May 1, 2014, A2997)Google Scholar All cynomolgus macaque (M. fascicularis) NHPs were female, with an average age of 11 years ± SD 1 year and an average weight of 3.2 ± SD 0.37 kg. All animals studied were female because women who smoke have a higher risk of developing COPD than men regardless of smoking level or intensity.21Mucha L. Stephenson J. Morandi N. Dirani R. Meta-analysis of disease risk associated with smoking, by gender and intensity of smoking.Gend Med. 2006; 3: 279-291Abstract Full Text PDF PubMed Scopus (133) Google Scholar Also, female mice develop more severe airspace enlargement than male mice when exposed to the same dose of CS for the same duration.22March T.H. Wilder J.A. Esparza D.C. Cossey P.Y. Blair L.F. Herrera L.K. McDonald J.D. Campen M.J. Mauderly J.L. Seagrave J. Modulators of cigarette smoke-induced pulmonary emphysema in A/J mice.Toxicol Sci. 2006; 92: 545-559Crossref PubMed Scopus (107) Google Scholar Care of the animals complied with the regulations of the US Department of Agriculture guidelines on the protection of animals and the NIH's Guide for the Care and Use of Laboratory Animals23Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar used for scientific purposes. All experiments conducted on NHPs were approved by our Institutional Animal Care and Use Committee. The NHPs were socially housed (up to two animals per cage) in the Primate Facility at the Lovelace Respiratory Research Institute (Albuquerque, NM), in accordance with the Guide for Laboratory Animal Practice under the Association for the Assessment and Accreditation for Laboratory Animal Care International–approved animal environmental conditions. The animal facility maintained a 12-hour light cycle. NHPs were exposed to 100% freshly filtered air with 10 to 15 air changes per hour in the control and CS exposure group before initiating the exposures. Room temperature and relative humidity were maintained according to the Guide for the Care and Use of Laboratory Animals.23Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar NHPs were observed a minimum of twice daily for any sign of illness. NHPs were fed twice daily with water available at all times. Animals were weighed during each physical examination or collection period until necropsy. NHPs were placed into H2000 whole body exposure chambers and exposed to CS [250 mg/m3 total suspended particulate matter (TPM)] for 6 hours per day, 5 days per week, because this exposure protocol is most commonly used to model COPD in small animals, including mice.24Ganesan S. Comstock A.T. Kinker B. Mancuso P. Beck J.M. Sajjan U.S. Combined exposure to cigarette smoke and nontypeable Haemophilus influenzae drives development of a COPD phenotype in mice.Respir Res. 2014; 15: 11Crossref PubMed Scopus (29) Google Scholar, 25Lee K.M. Renne R.A. Harbo S.J. Clark M.L. Johnson R.E. Gideon K.M. 3-Week inhalation exposure to cigarette smoke and/or lipopolysaccharide in AKR/J mice.Inhal Toxicol. 2007; 19: 23-35Crossref PubMed Scopus (25) Google Scholar, 26Weissmann N. Lobo B. Pichl A. Parajuli N. Seimetz M. Puig-Pey R. Ferrer E. Peinado V.I. Dominguez-Fandos D. Fysikopoulos A. Stasch J.P. Ghofrani H.A. Coll-Bonfill N. Frey R. Schermuly R.T. Garcia-Lucio J. Blanco I. Bednorz M. Tura-Ceide O. Tadele E. Brandes R.P. Grimminger J. Klepetko W. Jaksch P. Rodriguez-Roisin R. Seeger W. Grimminger F. Barbera J.A. Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.Am J Respir Crit Care Med. 2014; 189: 1359-1373Crossref PubMed Scopus (67) Google Scholar We used an initial CS concentration of 100 mg/m3 TPM to acclimatize the animals to CS, and then we increased it to the target concentration of 250 mg/m3 TPM during the first 5 exposure days. These target CS exposure levels (100 and 250 mg/m3 TPM) were selected to simulate a heavy human smoking pattern. For a 3000-g primate inhaling an average volume of 800 mL/minute, a pulmonary TPM deposition of 20%, and a lung weight of 10 g, the weekly deposition of smoke particles would be approximately 2.9 or 7.2 TPM deposited per gram of lung per week, as was calculated previously.27Finch G.L. Lundgren D.L. Barr E.B. Chen B.T. Griffith W.C. Hobbs C.H. Hoover M.D. Nikula K.J. Mauderly J.L. Chronic cigarette smoke exposure increases the pulmonary retention and radiation dose of 239Pu inhaled as 239PuO2 by F344 rats.Health Phys. 1998; 75: 597-609Crossref PubMed Scopus (24) Google Scholar Assuming that a human smoker with 20 cigarettes per day over 1 week will have 1.8 mg TPM deposited per gram of lung per week, the 100 and 250 mg/m3 TPM CS dose for NHPs is similar to that of humans smoking approximately 1.8 or 4 packs per day, respectively. Two different studies were performed. In the first experiment, NHPs were exposed to CS (n = 8) or air (n = 4) for 4 weeks in H2000 whole-body inhalation chambers for 6 hours per day, 5 days per week, to determine whether this exposure results in significant increases in pulmonary inflammation and/or mucus metaplasia in airway epithelia. Physical examination and bronchoalveolar lavage (BAL) were performed at baseline before CS exposures were initiated and after 1 and 4 weeks, and lung tissue was obtained at necropsy (Table 1).Table 1Design of the 4- and 12-Week CS versus Air Exposure ExperimentExposure time pointsProcedures performed4-Week exposure BaselinePhysical examination, (BAL) on 12 NHPs 1 WeekAll animals: physical examination and BAL on eight CS-exposed and four air-exposed NHPs 4 WeeksEnd CS exposures on CS animalsAll animals: physical examination, BAL, and lung tissue collection on eight CS-exposed and four air-exposed NHPs12-Week exposure BaselinePhysical examination, BAL, and PFTs on 16 NHPs 4 WeeksAll animals: physical examination, BAL, and PFTs on eight CS-exposed and eight air-exposed NHPs 12 WeeksEnd CS exposures on CS animalsAll animals: physical examination, BAL, PFTs, and lung tissue collection on eight CS-exposed and eight air-exposed NHPsBAL, bronchoalveolar lavage; CS, cigarette smoke; NHP, nonhuman primate; PFT, pulmonary function test. Open table in a new tab BAL, bronchoalveolar lavage; CS, cigarette smoke; NHP, nonhuman primate; PFT, pulmonary function test. In the second experiment, NHPs were exposed to CS (n = 8) or air (n = 8) for 12 weeks in H2000 whole-body inhalation chambers for 6 hours per day, 5 days per week, to determine whether airway and/or airspace pathologies develop in the animals. Physical examination, pulmonary function tests (PFTs), and BAL were performed at baseline and after 4 and 12 weeks, and lung tissue was obtained at necropsy (Table 1). Exposures >12 weeks were not feasible because of the high costs associated with husbandry, performing the exposures, and sample collections. PFT was performed on NHPs exposed to air or CS for 12 weeks (n = 8 animals per group) using a whole-body flow plethysmography constructed and operated as described previously for dogs.28Mauderly J.L. Respiratory function responses of animals and man to oxidant gases and to pulmonary emphysema.J Toxicol Environ Health. 1984; 13: 345-361Crossref PubMed Scopus (24) Google Scholar The animals were anesthetized with 2% to 3% isoflurane and intubated, an esophageal catheter was inserted, and the animals were placed supine in the plethysmograph. The endotracheal tube was attached to an airway port, and the distal end of the esophageal catheter was passed through a small opening located near the airway port. The esophageal catheter was connected to a differential pressure transducer that was vented to the airway for measurement of transpulmonary pressure. A valve system allowed the airway to be connected to positive and negative pressure reservoirs for induction of inspiration and expiration. The data collection program was initiated, and the depth of the esophageal catheter was adjusted to maximize the transpulmonary pressure trace. Anesthesia was maintained at a light surgical plane for the duration of testing. During apnea induced by brief hyperventilation, the airway was connected to the positive pressure reservoir through a valve that provided a slow inspiration to total lung capacity, defined as a transpulmonary pressure of 30 cm H2O. The pressure-volume trace was recorded during quasistatic expiration (5 seconds), and the quasistatic chord compliance was measured as the slope between 10 cm H2O and functional residual capacity (relaxed lung volume). After a period of spontaneous respiration, the hyperventilation-inspiration sequence was repeated, followed by evacuation of the lung to the negative pressure reservoir without intentional limitation of flow. The forced vital capacity, the percentage of forced vital capacity exhaled in 0.1 seconds, and the peak expiratory flow were measured during this forced expiration. Bronchoscopy, followed by BAL, was performed at baseline and after 1, 4, and/or 12 weeks of exposure to CS or air (Table 1). Animals were sedated with 5 to 10 mg/kg ketamine (delivered by the i.m. route) and anesthetized with inhaled isoflurane delivered using a mask. An endotracheal tube of an appropriate size for the animal was placed into the animal's trachea. A bronchoscope (model BF-3C40; Olympus America Inc., Melville, NY) was maneuvered through the endotracheal tube and wedged in approximately a fourth- to sixth-generation airway in the right diaphragmatic lung lobe. Two 10-mL aliquots of sterile USP-grade endotoxin-free saline were instilled and aspirated in succession, followed by further aspiration with an empty syringe to recover as much BAL fluid (BALF) as possible, which was frozen to −80°C for subsequent analyses. All leukocytes, macrophages, polymorphonuclear neutrophils (PMNs), and lymphocytes were counted in BAL samples from air- and CS-exposed NHPs (four air- and eight CS-exposed NHPs in the 4-week exposure experiment and eight NHPs per group in the 12-week exposure experiment). IL-6, chemokine (C-X-C motif) ligand 8 (CXCL8), and chemokine (C-X-C motif) ligand 2 (CCL2) levels were measured in BALF from air- and CS-exposed NHPs (eight animals per group) using the Milliplex multi-analyte panels NHP cytokine Luminex multiplex assay (EMD Millipore, Billerica, MA). MMP-9 levels were measured in BALF samples using a human Duoset MMP-9 enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). We used Western blot analysis to measure MMP-12 levels in BALF samples from NHPs exposed to air (n = 3) or CS for 4 weeks (n = 3) and 12 weeks (n = 3), and in radioimmunoprecipitation assay extracts of BAL leukocytes from NHPs exposed to air (n = 7) or CS for 4 weeks (n = 4) or 12 weeks (n = 7). Briefly, equal amounts of total protein (30 μg) were reduced by adding Laemmli sample buffer containing dithiothreitol and heating to 90°C for 5 minutes. Proteins in the samples were separated on 12% SDS-polyacrylamide gels for 4 hours at 90 V. Proteins were then transferred to polyvinylidene difluoride membranes, blocked in phosphate-buffered saline (PBS) containing 3% nonfat milk and 0.1% Tween-20 for 2 hours at room temperature, and incubated overnight with rabbit anti–MMP-12 IgG (diluted 1:100; Abcam, Cambridge, MA) and rabbit anti-vinculin IgG (diluted 1:1000; Abcam) as a loading control. After washing the membranes with PBS containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (diluted 1:3000; BioRad, Hercules, CA) for 2 hours at room temperature, and developed using a chemiluminescence substrate (Thermo Scientific, Pittsburgh, PA) following the manufacturer's directions. The membranes were exposed to a charge-coupled device camera for 30 minutes (BioRad). Signals were quantified using densitometry with Scion Image software for Windows Beta 4.0.2 (Scion Corporation, Bethesda, MD), and MMP-12 signals were corrected for vinculin signals. To identify the inflammatory cell subsets that were recruited into the lung parenchyma of CS-exposed NHPs, we performed immunofluorescence staining for markers of macrophages and polymorphonuclear neutrophils in formalin-fixed lung sections from NHPs exposed to air or CS for 12 weeks (n = 5 per group). Briefly, the lung sections were deparaffinized, and antigen (Ag) retrieval was performed by heating the slides in a microwave in 0.01 mol/L sodium citrate and 2 mmol/L citrate buffer (pH 6.0). The sections were incubated for 1 hour at 37°C with either a murine IgG to human myeloperoxidase (diluted 1:20) as a marker of neutrophils or a rabbit anti-human CD68 IgG (diluted 1:20) as a marker of macrophages. Isotype-matched nonimmune murine and rabbit IgGs were used as controls. After washing the lung sections with PBS, the sections were incubated at 37°C for 1 hour with Alexa 488–conjugated goat anti-murine F(ab')2 diluted 1:100 or Alexa 488–conjugated goat anti-rabbit F(ab')2 diluted 1:100. Sections were then washed in PBS, and nuclei were counterstained with DAPI. Images of the stained lung sections were analyzed using a confocal microscope (Leica Microsystems, Buffalo Grove, IL). Confocal micrographs were recorded under a fluorescence imaging mode in which cells were exposed to 488-nm light attenuated by an acustotunable optical filter.29Knolle M.D. Nakajima T. Hergrueter A. Gupta K. Polverino F. Craig V.J. Fyfe S.E. Zahid M. Permaul P. Cernadas M. Montano G. Tesfaigzi Y. Sholl L. Kobzik L. Israel E. Owen C.A. Adam8 limits the development of allergic airway inflammation in mice.J Immunol. 2013; 190: 6434-6449Crossref PubMed Scopus (27) Google Scholar To quantify the number of lymphoid aggregates present in the airways and lung parenchyma, paraffin-embedded sections (5 μm thick) of lungs from NHPs exposed to air or CS for 12 weeks (seven animals per group) were stained with hematoxylin and eosin. For each NHP, 30 high-magnification fields were counted in a randomized manner using a Leica epifluorescence microscope (Leica Microsystems). We evaluated all lymphoid aggregates containing >40 contiguous mononuclear cells. Data were expressed as the number of parenchymal, peribronchial, and perivascular lymphoid aggregates/mm2 of tissue examined in 30 randomly acquired images per animal. To identify B- and T-lymphocyte subsets within the lymphoid aggregates, we performed triple-immunofluorescence staining of lung sections from NHPs exposed to air or CS for 12 weeks (n = 5 lung sections per group). Briefly, the lung sections were deparaffinized, and Ag retrieval was performed by heating the slides in a microwave in 0.01 mol/L sodium citrate and 2 mmol/L citrate buffer (pH 6.0). The sections were incubated for 2 hours at 37°C with murine anti-human CD4 IgG (diluted 1:50; Abcam), then for 1 hour at 37°C with rat anti-human CD8 IgG (diluted 1:100; Abcam), and then overnight at 4°C with rabbit anti-human CD20 IgG (diluted 1:100; Abcam). After washing the lung sections with PBS, the sections were incubated at 37°C for 1 hour with Alexa Cy5-conjugated goat anti-murine IgG (diluted 1:100), Alexa 546–conjugated goat anti-rat IgG (diluted 1:100), and Alexa 488–conjugated F(ab')2 fragment of goat anti-rabbit IgG (diluted 1:100). Sections were then washed in PBS, and nuclei were counterstained with DAPI. Images of the stained lung sections were analyzed using a confocal microscope (Leica Microsystems). Confocal micrographs were recorded under fluorescence imaging mode in which cells were exposed to 488-, 570-, and 670-nm light attenuated by an acustotunable optical filter.29Knolle M.D. Nakajima T. Hergrueter A. Gupta K. Polverino F. Craig V.J. Fyfe S.E. Zahid M. Permaul P. Cernadas M. Montano G. Tesfaigzi Y. Sholl L. Kobzik L. Israel E. Owen C.A. Adam8 limits the development of allergic airway inflammation in mice.J Immunol. 2013; 190: 6434-6449Crossref PubMed Scopus (27) Google Scholar Lung sections from seven NHPs exposed to air and eight NHPs exposed to CS for 4 or 12 weeks were stained with periodic acid–Schiff stain using commercial kits (Sigma-Aldrich, St. Louis, MO) following the manufacturer's instructions. We also immunostained lung sections from NHPs that were exposed to air (n = 6) or CS (n = 10) for 4 or 12 weeks for MUC5AC. Briefly, the lung sections were deparaffinized, and Ag retrieval was performed by heating the slides in a microwave in 0.01 mol/L sodium citrate and 2 mmol/L citric acid buffer citrate buffer (pH 6.0). Sections were incubated with blocking medium (PBS containing 1% normal donkey serum, 3% bovine serum albumin, 1% gelatin, 0.2% Triton X-100, and 0.2% saponin) at 37°C for 30 minutes and then washed in 1% bovine serum albumin, followed by 0.05% Brij-35, and then incubated overnight at 4°C with murine anti-human MUC5AC IgG1 (diluted 1:500; Chemicon, Billerica, MA) or nonimmune murine IgG to identify cells undergoing mucus metaplasia within the bronchial epithelium. After washing in PBS containing 1% bovine serum albumin followed by 0.05% Brij, the sections were incubated at 37°C for 1 hour with Alexa 556–conjugated donkey anti-murine IgG diluted 1:300. Sections were then washed in PBS, and nuclei were counterstained with DAPI. Immunofluorescence was imaged using Axioplan 2 (Carl Zeiss, Inc., Thornwood,