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Clinical Infectious Diseases DPP4 in smoker s lungs CID 2018 66 1 January 45 DPP4 the Middle East Respiratory Syndrome Coronavirus Receptor is Upregulated in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients Leen J M Seys 1 2 W Widagdo 3 Fien M Verhamme 1 Alex Kleinjan 4 Wim Janssens 5 Guy F Joos 1 Ken R Bracke 1 Bart L Haagmans 3 a and Guy G Brusselle 1 a 1 Laboratory for Translational Research in Obstructive Pulmonary Diseases Department of Respiratory Medicine Ghent University Hospital and 2 Laboratory of Immunoregulation and Mucosal Immunology VIB UGent Center for Inflammation Research Ghent Belgium Departments of 3 Viroscience and 4 Pulmonary Medicine Erasmus MC Rotterdam The Netherlands and 5 University Hospital Leuven Respiratory Division and Rehabilitation Leuven Belgium Background Middle East respiratory syndrome coronavirus MERS CoV causes pneumonia with a relatively high case fatality rate in humans Smokers and chronic obstructive pulmonary disease COPD patients have been reported to be more susceptible to MERS CoV infection Here we determined the expression of MERS CoV receptor dipeptidyl peptidase IV DPP4 in lung tissues of smokers without airflow limitation and COPD patients in comparison to nonsmoking individuals never smokers Methods DPP4 expression was measured in lung tissue of lung resection specimens of never smokers smokers without air flow limitation COPD GOLD stage II patients and in lung explants of end stage COPD patients Both control subjects and COPD patients were well phenotyped and age matched The mRNA expression was determined using qRT PCR and protein expression was quantified using immunohistochemistry Results In smokers and subjects with COPD both DPP4 mRNA and protein expression were significantly higher compared to never smokers Additionally we found that both DPP4 mRNA and protein expression were inversely correlated with lung function and diffusing capacity parameters Conclusions We provide evidence that DPP4 is upregulated in the lungs of smokers and COPD patients which could partially explain why these individuals are more susceptible to MERS CoV infection These data also highlight a possible role of DPP4 in COPD pathogenesis Keywords Middle East respiratory syndrome coronavirus MERS CoV dipeptidyl peptidase 4 DPP4 smoking chronic obstructive pulmonary disease COPD Middle East Respiratory Syndrome coronavirus MERS CoV is a newly emerging pathogen that mainly causes pneu monia with a relatively high case fatality rate 1 2 Since 2012 1900 laboratory confirmed cases have been reported to the World Health Organization WHO 2 The major ity of cases occurred in familial or hospital related clusters through human to human transmission 3 5 The clinical course of MERS CoV infection ranges from asymptomatic to acute respiratory distress syndrome with need for ventila tory support 3 5 7 To infect its host MERS CoV attaches to its receptor an exopeptidase called dipeptidyl peptidase 4 DPP4 also known as CD26 8 DPP4 is a type II transmembrane glycoprotein that is expressed in many cell types and organs in the body It serves multiple functions among which post translational cleavage of hormones and chemokines T cell activation cell adhesion and apoptosis 9 11 In lungs however DPP4 is expressed at a minimum level 12 mainly in alveolar epithelial cells and endo thelial cells and to a lesser extent in bronchiolar epithelial cells airway submucosal glands alveolar macrophages lymphocytes and plasmacytoid dendritic cells 13 16 Importantly the alveo lar epithelial cells are the main target for MERS CoV 13 17 Several underlying comorbidities including chronic lung diseases have been reported to increase the risk of acquiring MERS CoV infection 18 Chronic obstructive pulmonary dis ease COPD is a highly prevalent chronic lung disease in older subjects and is currently the leading cause of death worldwide 19 20 The most common cause of COPD is chronic cigar ette smoking The inflammatory response to cigarette smoke results in an excessive release of chemokines and cytokines with a subsequent high influx of immune cells 20 Because smok ing has also been reported to increase susceptibility to MERS CoV infection 18 we aimed to investigate the expression of the MERS CoV receptor DPP4 in a large well phenotyped cohort of smokers with and without airflow limitation in comparison to age matched individuals that never smoked never smokers MAJOR ARTICLE The Author s 2017 Published by Oxford University Press for the Infectious Diseases Society of America All rights reserved For permissions e mail journals permissions DOI 10 1093 cid cix741 Received 3 May 2017 editorial decision 7 August 2017 accepted 12 August 2017 published online August 17 2017 a B L H and G G B contributed equally to this manuscript Correspondence Prof Dr G G Brusselle Department of Respiratory Medicine Ghent University Hospital De Pintelaan 185 9000 Ghent Belgium Guy Brusselle UGent be Clinical Infectious Diseases 2018 66 1 45 53 STANDARD XX XXXX Downloaded from by University of Washington user on 19 January 201846 CID 2018 66 1 January Seys et al METHODS Human Lung Tissue Samples Lung resection specimens were obtained from patients diag nosed with solitary pulmonary tumors at Ghent University Hospital Ghent Belgium or from explant lungs from end stage COPD patients UZ Gasthuisberg Leuven Belgium Based on preoperative spirometry diffusion capacity tests and question naires patients were categorized as never smokers with normal lung function smokers without airflow limitation or patients with COPD COPD severity was defined according to the Global Initiative for Chronic Obstructive Lung Disease GOLD classifi cation 19 None of the patients were treated with neo adjuvants chemotherapy Lung tissue of patients diagnosed with solitary pulmonary tumor was obtained at a maximum distance from the pulmonary lesions and without signs of retro obstructive pneu monia or tumor invasion and collected by a pathologist Lung tis sue of patients with COPD GOLD III IV was obtained from lung explants of end stage COPD patients undergoing lung transplan tation Written informed consent was obtained from all subjects This study was approved by the medical ethical committees of the Ghent University Hospital 2011 114 and the University Hospital Gasthuisberg Leuven S51577 Patient characteristics are listed in Table 1 Detailed patient characteristics per read out are provided in supplementary Tables S1 and S2 Human Proximal Bronchi Samples Biopsy samples of proximal bronchi were obtained from 21 patients 17 male and 4 female with moderate to severe COPD previously recruited for a separate study 21 Inclusion criteria were the following chronic productive cough age between 40 and 70 years current smokers negative skin tests for inhalation aller gens FEV 1 70 of predicted normal value or FEV 1 VC 0 70 reversibility of FEV 1 10 pred after 750 g terbutaline inhala tion and suffering from moderate to severe bronchial hyper re sponsiveness as determined by PC 20 value upon challenge with histamine and methacholine Exclusion criteria were a history of asthma complaints of wheezing recent respiratory tract infec tion and recent or concurrent usage of anti inflammatory drugs Oral anti inflammatory medication was discontinued for at least 3 months and inhaled glucocorticoids at least 6 weeks before the start of the study Bronchoscopy was performed with an Olympus BF 1T10 At least 6 biopsies were taken from the bronchi of the right and the left upper and lower lobes using a fenestrated forceps FB 18C or FB 20C All was according to published guidelines 22 The study was approved by the Medical Ethics Committee of the Erasmus University Medical Center Rotterdam and writ ten informed consent was obtained from all participants Patient characteristics are listed in Table 2 Proximal bronchi biopsy sam ples of 16 healthy individuals 8 male and 8 female previously described in the earlier study 23 were used as negative control Table 3 provides an overview of the different cohorts and samples used in this study Purification of Human Lung Dendritic Cell subsets Lung dendritic cells DC were isolated from single cell suspen sions of lung tissue of 3 patients as described previously 24 Lung tissues were rinsed cut into small fragments and incu bated in digestion medium Next the samples were resuspended Table 1 Characteristics of study population n 117 Never smokers Smokers a COPD II b COPD III IV c Number 21 32 37 27 Sex M F 6 15 d 23 9 d 34 3 d 12 14 d Age years 65 58 71 64 5 55 71 65 58 69 56 5 54 60 e f g Current ex smoker 19 13 d 24 13 d 0 27 d Smoking history PY 0 0 0 33 14 51 e 45 40 60 e f 30 25 36 e g FEV 1 post L 2 4 2 1 3 2 7 2 3 3 3 2 1 1 8 2 4 e f 0 7 0 5 1 e f g FEV 1 post predicted 103 92 117 95 92 112 69 61 75 e f 27 21 33 e f g FEV 1 FVC post 78 74 83 76 72 79 56 51 61 e f 30 27 35 e f g DLCO predicted 88 81 103 83 65 104 67 51 86 e f 34 32 37 e f g KCO predicted 95 86 121 93 78 106 85 65 107 e 52 46 59 e f g ICS yes no 1 19 d 2 30 d 16 21 d 25 1 d Abbreviations COPD chronic obstructive pulmonary disease DLCO diffusing capacity of the lung for carbon monoxide FEV 1 forced expiratory volume in 1 second FVC forced vital capac ity IQR interquartile range KCO transfer of carbon monoxide coefficient ICS inhaled corticosteroids PY pack years a Smokers without airflow limitation b subjects with COPD stage II as defined by the Global initiative for Obstructive Lung Disease GOLD Data are presented as median IQR Mann Whitney U test c Subjects with COPD stage III IV as defined by GOLD Fisher s exact test d P 001 e P 05 versus never smokers f P 05 versus smokers without COPD g P 05 versus COPD GOLD I II Downloaded from by University of Washington user on 19 January 2018DPP4 in smoker s lungs CID 2018 66 1 January 47 in Ca2 and Mg2 free PBS containing 10 mM EDTA and passed through a 40 m filter Subsequently pulmonary mon onuclear cells were separated on a Ficoll density gradient The cells were labeled with anti CD3 FITC anti CD19 FITC anti CD207 PE anti CD209 PerCp Cy5 and anti BDCA2 APC and sorted on a FACSAria BD Biosciences RNA Extraction and Real Time Polymerase Chain Reaction Analysis RNA extraction and polymerase chain reaction PCR analysis of lung tissue were performed as described previously 25 RNA extraction from lung tissue blocks of 92 subjects 18 never smok ers 26 smokers without airflow limitation 34 patients with COPD GOLD II 14 patients with COPD GOLD IV was per formed with the miRNeasy Mini kit Qiagen Hilden Germany following manufacturer s instructions Next complementary DNA cDNA was prepared with the iScript Advanced cDNA Synthesis Kit for RT qPCR Bio Rad Hercules California Taqman Gene Expression Assays Applied Biosystems Forster City California were used to measure the expression of DPP4 and the reference genes Glyceraldehyde 3 phosphate dehydro genase GAPDH Hypoxanthine phosphoribosyltransferase 1 HPRT 1 and Succinate Dehydrogenase complex flavoprotein subunit A SDHA Data were analyzed using the standard curve method and expression of DPP4 was calculated relative to the expression of the 3 reference genes using the geNorm applet according to the guidelines and theoretical framework previously described 25 26 For human lung DC subsets RNA extraction was performed with miRNeasy Mini kit Qiagen Hilden Germany whereas RNA amplification was with the Qiagen QuantiTect Whole Transcriptome kit both following manufacturer s instructions DPP4 expression in the DC subsets was calculated relative to the expression of GAPDH HPRT1 and peptidylprolyl isomer ase A PPIA as described previously 25 DPP4 Immunohistochemistry and Analyses Sections obtained from formalin fixed paraffin embedded lung tissue blocks of 98 subjects 19 never smokers 30 smokers without COPD 36 subjects with COPD GOLD II and 13 sub jects with COPD GOLD III IV were incubated with anti DPP4 antibody polyclonal goat anti human R SD standard deviation PC 20 provocative concentration causing a 20 fall in FEV 1 Table 3 Overview of the Cohorts and Samples Used in This Study Overview of cohorts and samples used 90 patients 21 never smokers 32 smokers without airflow limitation and 37 patients with COPD GOLD stage II who underwent lobectomia or pneu mectomia due to lung cancer 73 90 patients samples for both qRT PCR and IHC analyses 5 90 patients samples only for qRT PCR analysis 12 90 patients samples only for IHC analysis 27 patients with COPD GOLD stage III IV who underwent lung transplanta tion due to end stage COPD 14 27 patients samples for qRT PCR analysis 13 27 patients samples for IHC analysis 37 patients who underwent bronchial biopsies 21 37 patients with moderate to severe COPD ref samples used for IHC staining 16 37 control patients with airflow limitation ref samples used for IHC staining In this study we used resection lung tissue of 90 patients who underwent lobectomia pneumectomia due to lung cancer explant lung tissue of 27 end stage COPD patients and bronchial biopsy tissue of 37 patients Abbreviations COPD chronic obstructive pulmonary disease GOLD global initiative for chronic obstructive lung disease IHC immunohistochemistry qRT PCR quantitative reverse transcription polymerase chain reaction Downloaded from by University of Washington user on 19 January 201848 CID 2018 66 1 January Seys et al DPP4 detection in the frozen samples of proximal bronchi was performed with 1 g mL mouse anti DPP4 monoclonal anti body Santa Cruz Biotechnology Dallas Texas 15 after pre viously fixed with acetone and incubated with 10 normal goat serum Dako Glostrup Denmark for 1 hour at room temper ature These slides were subsequently stained with biotinylated goat antimouse Ig serum 1 50 in PBS BSA plus 10 human serum and with streptavidin alkaline phosphatase 1 50 in PBS BSA plus 10 human serum Biogenex Klinipath Duiven The Netherlands for 30 minutes each A positive signal was revealed with New Fuchsin substrate Chroma Kongen Germany Counterstaining was performed with Gill s hematoxylin Negative control staining was performed by the substitution of the primary monoclonal antibody with an isotype antibody Statistical Analysis Statistical analysis was performed with Sigma Stat software SPSS 23 0 Chicago Illinois using Kruskal W allis Mann Whitney U Fisher exact test and Spearman correlation analysis In addition one way analysis of variance ANOVA and T tests were used for statistical analyses of the DC subsets Characteristics of the study population are presented as a median and interquartile range Differences at P values 05 were considered to be signif icant P 05 P 01 and P 001 RESULTS DPP4 mRNA Expression is Upregulated in Lungs of Smokers and COPD Patients Messenger RNA mRNA expression of DPP4 was analyzed in lung tissue of 92 subjects Lung tissue was derived from either resection tissue of lobectomy never smokers smokers without airflow lim itation and patients with COPD GOLD stage II or explant lungs of lung transplantation patients with COPD GOLD stage III IV Patient characteristics are described in supplementary Table 1 Compared to never smokers mRNA expression of DPP4 in lung tissue of smokers without airflow limitation and patients with COPD was significantly increased Figure 1A Moreover DPP4 mRNA expression in lung tissue of patients with COPD GOLD stage III IV was significantly higher than in lung tissue of smokers without airflow limitation and patients with COPD GOLD stage II Figure 1A Quantification according to smok ing status ex vs current smokers is shown in Supplementary Figure S1 Furthermore DPP4 mRNA expression was inversely correlated with the severity of airflow limitation FEV 1 R 0 376 P 001 and FEV 1 FVC ratio R 0 527 P 001 Figure 1B C In addition the mRNA expression of DPP4 was also correlated inversely with the diffusing capacity of the lung DLCO R 0 402 P 001 and KCO R 0 408 P 001 Figure 1D E Linear regression analysis revealed that the asso ciation of DPP4 mRNA expression with the presence of COPD was significant even when corrected for age sex pack years and use of inhaled corticosteroids Supplementary Table S3 Additionally because dendritic cells DCs play a crucial role in antiviral immunity we investigated whether DPP4 mRNA expression differs between DC subsets Three DC subsets were sorted langerin positive DCs DC SIGN positive DCs and plasmacytoid DCs pDCs DPP4 mRNA was merely detected in pDCs Supplementary Figure S2 DPP4 Protein Expression is Upregulated in Lungs of Smokers and COPD Patients DPP4 protein expression was studied in lung tissue of nev er smokers smokers without airflow limitation and COPD patients by using immunohistochemistry IHC staining DPP4 was detected on the apical surface of bronchiolar epithelium and in the alveolar epithelial cells In the alveoli we observed that DPP4 protein was gradually increased from never smok ers to COPD GOLD stage III IV Figure 2 Additionally we performed immunohistochemical staining of DPP4 with both aquaporin 5 marker of type I alveolar epithelial cells and pro surfactant C marker of type II alveolar epithelial cells confirming that the upregulation of DPP4 protein can mainly be contributed to the alveolar epithelial cells Figure 2I L In contrast this increment was not observed in the bronchiolar epithelium Figure 2A as well as in the proximal bronchial epithelium Figure 3 Furthermore DPP4 was also detected in the endothelial cells alveolar macrophages immune cells in the submucosal region of airway epithelium and lymphoid aggregates Supplementary Figure S3 We further quanti fied DPP4 signals in the lung tissues of 98 subjects using the Axiovision software Zeiss Characteristics of these patients are presented in supplementary Table 2 Compared to never smokers DPP4 protein expression was sig nificantly increased in the alveolar epithelial cells of smokers and patients with COPD DPP4 protein expression was the highest in patients with COPD GOLD stage III IV Figure 4A Quantification of DPP4 protein expression according to smoking status ex vs current smoking is shown in Supplementary Figure S4 Similar to DPP4 mRNA expression DPP4 protein was also inversely corre lated with lung function parameters FEV 1 R 0 567 P 001 and FEV 1 FVC ratio R 0 689 P 001 as well as diffusing capacity parameters DLCO R 0 603 P 001 and KCO R 0 563 P 001 Linear regression analysis revealed that the association of alveolar DPP4 expression with the presence of COPD was signif icant even when corrected for age gender pack years and use of inhaled corticosteroids Supplementary Table S4 DISCUSSION Our study investigated the expression of the MERS CoV receptor DPP4 in lung tissues of smokers without airflow limitation and COPD patients in comparison to never smokers As previously reported DPP4 is mainly detected in the alveolar epithelial cells of the lungs the main target of MERS CoV infection 13 15 Downloaded from by University of Washington user on 19 January 2018DPP4 in smoker s lungs CID 2018 66 1 January 49 Among the dendritic cells we found that DPP4 mRNA is mainly expressed in pDCs confirming in vitro data showing that among the antigen presenting cells pDCs produce large amounts of type I and III interferon upon contact with MERS CoV 14 Most importantly we provide evidence that DPP4 is upregulated in the lungs both at mRNA and protein level not only in COPD patients but also in that of smokers This indicates that these individuals may be more susceptible to MERS CoV supporting both smoking and COPD as risk factors for MERS CoV infection 18 These results are in line with a recent study describing a higher DPP4 expression in lungs of 4 COPD patients compared to 16 control subjects of different ages 13 In this study we did not find any evidence of DPP4 upreg ulation in the bronchial and bronchiolar epithelium in the lungs of smokers and COPD patients suggesting that DPP4 upregulation in pulmonary epithelia is restricted to the alveolar Figure 1 DPP4 mRNA e