1 Cigarette smoke induces overexpression of active human cathepsin S in lungs 2 from current smokers with or without COPD 3 4 1,2,#,* 3* 1,2 5 Pierre-Marie Andrault , Andrea C. Schamberger , Thibault Chazeirat , Damien 1,4 1, † 3 3 6 Sizaret , Justine Renault , Claudia A. Staab-Weijnitz , Elisabeth Hennen , Agnès Petit- 7 Courty1,2, Mylène Wartenberg1,2, Ahlame Saidi1,2, Thomas Baranek1,2, Serge Guyetant1,4, 1,2 3, ‡ 1,2 1,2¶ 8 Yves Courty , Oliver Eickelberg , Gilles Lalmanach , Fabien Lecaille 9 10 1 Université de Tours, Tours, France 11 2 INSERM, UMR 1100, Centre d'Etude des Pathologies Respiratoires, Team Mécanismes 12 Protéolytiques dans l'Inflammation, Tours, France. 13 3 Comprehensive Pneumology Center, Institute of Lung Biology and Disease, University 14 Hospital, Ludwig-Maximilians-University and Helmholtz Zentrum München, Member of the 15 German Center for Lung Research (DZL), Munich, Germany. 16 4 Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Service d'Anatomie et 17 Cytologie Pathologique, Tours, France. 18 19 # Current address: Department of Oral Biological and Medical Sciences, Faculty of Dentistry, 20 University of British Columbia, Vancouver, Canada. 21 † Current address: Unité de Biologie Fonctionnelle et Adaptative, CNRS, UMR 8251, 22 Université Paris Diderot, Paris 7, France 23 ‡ Current address: Division of Pulmonary and Critical Care Medicine, University of Colorado 24 Anschutz School of Medicine, Aurora, USA. 25 26 ¶Corresponding author information : Fabien Lecaille, PhD, Université de Tours, INSERM, 27 UMR 1100, CEPR, 10 Boulevard Tonnellé, F-37032 Tours cedex, France. Tel: (+33) 28 247366047; e-mail: [email protected] 29 30 *These authors contributed equally to this manuscript. 31 32 Running title: Cigarette smoke increases human cathepsin S activity 33 34 35 36 37 38 39 1 Downloaded from www.physiology.org/journal/ajplung at Helmholtz Zentrum Muenchen (146.107.003.004) on September 27, 2019. 40 41 42 Abbreviations: 43 AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AMP, antimicrobial 44 peptide and protein; ASL, airway surface liquid ; BALF, bronchoalveolar lavage fluid ; BM, 45 basement membrane; CA-074, N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- 46 isoleucyl-L-proline; Cat, cathepsin; CF, cystic fibrosis; COPD, chronic obstructive pulmonary 47 disease; CS, current smoker; CSE, cigarette smoke extract; ECM, extracellular matrix ; FEV, 48 forced expiratory volume; FS, former smoker; GOLD, global initiative for chronic obstructive 49 lung disease; hCAP, human cathelicidin antimicrobial peptide ; LHVS, morpholinourea- 50 leucinyl-homophenylalanine-vinyl-sulfone; MMP, matrix metallo-proteinase; MMTS, S- 51 methyl thiomethanesulfonate; NS, never-smoker; NSP, neutrophil serine proteases; pHBECs, 52 primary human bronchial epithelial cells; PMSF, phenylmethylsulfonyl fluoride; ProCat, 53 procathepsin. 54 2 Downloaded from www.physiology.org/journal/ajplung at Helmholtz Zentrum Muenchen (146.107.003.004) on September 27, 2019. 55 Abstract: 56 Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, 57 inflammatory cell recruitment and a protease/anti-protease imbalance. These effects 58 contribute to tissue remodeling and destruction resulting in loss of lung function in chronic 59 obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that 60 is involved in the remodeling/degradation of connective tissue and basement membrane. 61 Aberrant expression or activity of CatS has been implicated in a variety of diseases, including 62 arthritis, cancer, cardiovascular and lung diseases. However, little is known about the effect of 63 cigarette smoking on both CatS expression and activity, as well as its role in smoking-related 64 lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues 65 from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing 66 environment, CatS expression and activity were significantly higher in current smokers (both 67 non-COPD and COPD) compared to never-smokers, and correlated positively with smoking 68 history. Moreover, we found that the exposure of primary human bronchial epithelial cells to 69 cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS 70 upregulation. The present data suggest that excessive CatS expression and activity contribute, 71 beside other proteases, to the deleterious effects of cigarette smoke on pulmonary 72 homeostasis. 73 74 Keywords: cigarette smoke, COPD, cysteine protease, oxidation, smokers 75 76 3 Downloaded from www.physiology.org/journal/ajplung at Helmholtz Zentrum Muenchen (146.107.003.004) on September 27, 2019. 77 Introduction: 78 Over 7 million deaths per year worldwide were recorded in 2017 that could be attributed to 79 past or current smoking, and trends indicate that this will increase to 8 million annually by 80 2030 (63). Besides longtime smokers having a shorter life-expectancy (10 years) compared 81 with never-smokers (www.cdc.gov/tobacco/data_statistics), long-term effects of cigarette 82 smoking include the risk of cardiovascular disease, lung cancer and chronic obstructive 83 pulmonary disease (COPD), which is characterized by progressive and irreversible airflow 84 obstruction. Conversely, cigarette smoking cessation improves substantially longevity in men 85 and women, irrespective of the age (27, 59). Cigarette smoke exposure contributes to chronic 86 airway inflammation and pathological lung tissue remodeling and destruction by increasing 87 the recruitment of inflammatory cells such as neutrophils and macrophages (23, 45). The 88 burden of oxidative stress associated with cigarette smoke exposure enhances recurrent 89 bacterial infections and leads to a protease/anti-protease imbalance (12, 36). 90 The principal classes of proteases expressed in human lungs are serine proteases, matrix 91 metalloproteases (MMP), and cysteine proteases (33). Lysosomal cysteine cathepsins are 92 produced by a wide variety of cell types such as fibroblasts, macrophages, and epithelial cells, 93 and these can be active outside lysosomes (secretory vesicles, cytosol, mitochondria, nucleus, 94 and extracellular medium). Among them, cathepsins B, K, L, and S play important roles in 95 diverse physiological events and are capable of degrading extracellular matrix (ECM) and 96 basement membrane (BM) constituents as well as antimicrobial peptides (AMPs) (17). 97 Besides MMPs and serine proteases, dysregulation of cysteine cathepsins expression may 98 disrupt these normal biological processes and contribute to a number of diseases such as 99 cancer, osteoporosis, arthritis, neurodegenerative and airway diseases (for review: (31, 43, 53, 100 54)). In particular, cathepsin S (CatS), which is predominantly expressed in dendritic cells and 101 macrophages (antigen-presenting cells, APCs), and epithelial cells, is thought to play a pivotal 102 role in chronic inflammatory lung diseases including cystic fibrosis (CF) and COPD (6, 28, 103 32, 56–58). Various studies have implicated CatS in extensive degradation of elastin fibers 104 and collagens as well as in the cleavage and the inactivation of key antimicrobials in CF 105 airways (for review: (17, 33)). Contrary to other related cathepsin family members that are 106 rapidly inactivated at neutral pH, CatS remains stable, a property that enables it to be active 107 extracellularly, particularly during ECM remodeling. Overexpression of CatS is observed in 108 mouse models of experimental emphysema induced by IL-13 or IFN, and CatS inhibition by 109 synthetic inhibitors reduces significantly the severity of emphysema and inflammation (18, 110 40, 55, 61). These data are supported by the observation that exposure of macrophages to 4 Downloaded from www.physiology.org/journal/ajplung at Helmholtz Zentrum Muenchen (146.107.003.004) on September 27, 2019. 111 bronchoalveolar lavage fluid (BALF) from patients with COPD markedly increased CatS 112 secretion, which was inhibited by IFN- neutralizing antibodies (19). This finding is in line 113 with the observation that IFN- stimulates CatS expression and activity in various cell types, 114 including keratinocytes and monocytes (20, 49). Interestingly, cigarette smoke enhanced the 115 production of IL-18 (an IFN- inducing factor), and induced the expression of immuno- 116 reactive CatS in lung macrophages of cigarette smoke-exposed mice as well as in lung tissues 117 of patients with COPD (28). 118 Despite increasing evidence for a role of CatS in COPD experimental animal models, very 119 little is known in human. Especially, the relationship between smoking status (current and 120 former smokers) and history (pack-years) and human CatS expression and activity in the 121 lungs of (ex)-smokers with or without COPD remains unsolved. Furthermore, the active site 122 cysteine residue (Cys25, papain numbering) of thiol-dependent cathepsins is highly sensitive 123 to oxidative reagents. The fact that CatS remains active in animal models and in COPD 124 patients, raises the question of its resistance to unfavorable conditions due to CS-induced 125 oxidative stress. 126 Therefore, in this report we compared both the protein levels and the enzymatic activity of 127 human CatS in lung tissue from never-smokers, and current or ex-smokers with normal lung 128 function or with various stages of COPD severity, according to the Global Initiative for 129