Cigarette smoke extract induces differential expression levels of beta-defensin peptides in human alveolar epithelial cells
More details
Hide details
School of Systems Biology, George Mason University, Manassas, USA
National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, USA
Discovery Hall, George Mason University, Manassas, USA
Publish date: 2013-04-29
Tobacco Induced Diseases 2013;11(April):10
The damaging effects of cigarette smoke on the lungs are well known in terms of cancer risks. Additional molecular changes within the lung tissue can also occur as a result of exposure to cigarette smoke. The human β-defensin (hBD) class of antimicrobial peptides is the focus of our research. In addition to antimicrobial activity, β-defensins also have immunomodulatory functions. Over 30 previously unrecognized β-defensin genes have recently been identified in the human genome, many with yet to be determined functions. We postulated that altered β-defensin production may play a role in the pathogenesis observed in the lungs of smokers. Our hypothesis is that cigarette smoke exposure will affect the expression of β-defensins in human lung alveolar epithelial cells (A549).

We exposed A549 cells to cigarette smoke extract (CSE) and measured the changes in mRNA levels of several antimicrobial peptides by quantitative real-time PCR, and directly observed peptide expression in cells by immunofluorescence (IF) microscopy.

We found that hBD3, hBD5, and hBD9 gene expression was upregulated in A549 cells exposed to CSE. HBD1, hBD8, hBD18 and LL-37 gene expression did not significantly change upon exposure to CSE. Expression of hBD3 and hBD4 peptides was visualized by IF.

This differential expression suggests that hBD3, hBD5, and hBD9 may play a role in the changes to the lung tissue observed in smokers. Establishing differential β-defensin expression following CSE treatment will add to our understanding of the molecular response of the lung alveolar epithelium to cigarette smoke exposure.

Monique L van Hoek   
School of Systems Biology, George Mason University, Manassas, VA, USA
1. Doll R: Uncovering the effects of smoking: historical perspective. Stat Methods Med Res. 1998, 7 (2): 87-117. 10.1191/096228098668199908.
2. Sangodkar J: Lung adenocarcinoma: lessons in translation from bench to bedside. Mt Sinai J Med. 2010, 77 (6): 597-605. 10.1002/msj.20226.
3. Brusselle GG, Joos GF, Bracke KR: New insights into the immunology of chronic obstructive pulmonary disease. Lancet. 2011, 378 (9795): 1015-1026. 10.1016/S0140-6736(11)60988-4.
4. McMaster SK: Cigarette smoke inhibits macrophage sensing of Gram-negative bacteria and lipopolysaccharide: relative roles of nicotine and oxidant stress. Br J Pharmacol. 2008, 153 (3): 536-543. 10.1038/sj.bjp.0707595.
5. Kier LD, Yamasaki E, Ames BN: Detection of mutagenic activity in cigarette smoke condensates. Proc Natl Acad Sci U S A. 1974, 71 (10): 4159-4163. 10.1073/pnas.71.10.4159.
6. Smith CJ: IARC carcinogens reported in cigarette mainstream smoke and their calculated log P values. Food Chem Toxicol. 2003, 41 (6): 807-817. 10.1016/S0278-6915(03)00021-8.
7. Shin HJ: Effect of cigarette filters on the chemical composition and in vitro biological activity of cigarette mainstream smoke. Food Chem Toxicol. 2009, 47 (1): 192-197. 10.1016/j.fct.2008.10.028.
8. Nikota JK, Stampfli MR: Cigarette smoke-induced inflammation and respiratory host defense: Insights from animal models. Pulm Pharmacol Ther. 2012, 25 (4): 257-262. 10.1016/j.pupt.2012.05.005.
9. Dwyer TM: Cigarette smoke-induced airway inflammation as sampled by the expired breath condensate. Am J Med Sci. 2003, 326 (4): 174-178. 10.1097/00000441-200310000-00004.
10. Doss M: Human defensins and LL-37 in mucosal immunity. J Leukoc Biol. 2010, 87 (1): 79-92. 10.1189/jlb.0609382.
11. Andresen E: Increased expression of beta-defensin 1 (DEFB1) in chronic obstructive pulmonary disease. PLoS One. 2011, 6 (7): e21898-10.1371/journal.pone.0021898.
12. Liao Z: Enhanced expression of human beta-defensin 2 in peripheral lungs of patients with chronic obstructive pulmonary disease. Peptides. 2012, 38 (2): 350-356. 10.1016/j.peptides.2012.09.013.
13. Janssens W: Genomic copy number determines functional expression of beta-defensin 2 in airway epithelial cells and associates with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010, 182 (2): 163-169. 10.1164/rccm.200905-0767OC.
14. Matsushita I: Genetic variants of human beta-defensin-1 and chronic obstructive pulmonary disease. Biochem Biophys Res Commun. 2002, 291 (1): 17-22. 10.1006/bbrc.2002.6395.
15. Han S, Bishop BM, van Hoek ML: Antimicrobial activity of human beta-defensins and induction by Francisella. Biochem Biophys Res Commun. 2008, 371 (4): 670-674. 10.1016/j.bbrc.2008.04.092.
16. Swain RJ: Assessment of cell line models of primary human cells by Raman spectral phenotyping. Biophys J. 2010, 98 (8): 1703-1711. 10.1016/j.bpj.2009.12.4289.
17. Hiemstra PS: Defensins and cathelicidins in inflammatory lung disease: beyond antimicrobial activity. Biochem Soc Trans. 2006, 34 (Pt 2): 276-278.
18. Schutte BC: Discovery of five conserved beta -defensin gene clusters using a computational search strategy. Proc Natl Acad Sci U S A. 2002, 99 (4): 2129-2133. 10.1073/pnas.042692699.
19. Rodriguez-Jimenez FJ: Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis. Genomics. 2003, 81 (2): 175-183. 10.1016/S0888-7543(02)00034-4.
20. Yamaguchi Y, Ouchi Y: Antimicrobial peptide defensin: identification of novel isoforms and the characterization of their physiological roles and their significance in the pathogenesis of diseases. Proc Jpn Acad Ser B Phys Biol Sci. 2012, 88 (4): 152-166. 10.2183/pjab.88.152.
21. Garcia JR: Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res. 2001, 306 (2): 257-264. 10.1007/s004410100433.
22. Garcia JR: Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J. 2001, 15 (10): 1819-1821.
23. Vareille M: The airway epithelium: soldier in the fight against respiratory viruses. Clin Microbiol Rev. 2011, 24 (1): 210-229. 10.1128/CMR.00014-10.
24. Kao CY: ORFeome-based search of airway epithelial cell-specific novel human beta-defensin genes. Am J Respir Cell Mol Biol. 2003, 29 (1): 71-80. 10.1165/rcmb.2002-0205OC.
25. Weinberg A: The yin and yang of human Beta-defensins in health and disease. Front Immunol. 2012, 3: 294.
26. Domagala-Kulawik J: Effects of cigarette smoke on the lung and systemic immunity. J Physiol Pharmacol. 2008, 59 (Suppl 6): 19-34.
27. Chen X: Antimicrobial peptides human beta-defensin (hBD)-3 and hBD-4 activate mast cells and increase skin vascular permeability. Eur J Immunol. 2007, 37 (2): 434-444. 10.1002/eji.200636379.
28. Chen L: Cigarette smoke enhances beta-defensin 2 expression in rat airways via nuclear factor-kappaB activation. Eur Respir J. 2010, 36 (3): 638-645. 10.1183/09031936.00029409.
29. Mahanonda R: Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells. J Periodontal Res. 2009, 44 (4): 557-564. 10.1111/j.1600-0765.2008.01153.x.
30. Semlali A: Whole cigarette smoke increased the expression of TLRs, HBDs, and proinflammory cytokines by human gingival epithelial cells through different signaling pathways. PLoS One. 2012, 7 (12): e52614-10.1371/journal.pone.0052614.
31. Kulkarni R: Cigarette smoke inhibits airway epithelial cell innate immune responses to bacteria. Infect Immun. 2010, 78 (5): 2146-2152. 10.1128/IAI.01410-09.
32. Amer LS, Bishop BM, van Hoek ML: Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella. Biochem Biophys Res Commun. 2010, 396 (2): 246-251. 10.1016/j.bbrc.2010.04.073.
33. Alekseeva L: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms. BMC Microbiol. 2009, 9: 33-10.1186/1471-2180-9-33.
34. Pierson T: Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine. J Proteome Res. 2011, 10 (3): 954-967. 10.1021/pr1009756.
35. Herr C: Suppression of pulmonary innate host defence in smokers. Thorax. 2009, 64 (2): 144-149. 10.1136/thx.2008.102681.
36. O'Neil DA: Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium. J Immunol. 1999, 163 (12): 6718-6724.
37. Eckmann L: Defence molecules in intestinal innate immunity against bacterial infections. Curr Opin Gastroenterol. 2005, 21 (2): 147-151. 10.1097/01.mog.0000153311.97832.8c.
38. Andreeva AV, Kutuzov MA, Voyno-Yasenetskaya TA: Regulation of surfactant secretion in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol. 2007, 293 (2): L259-L271. 10.1152/ajplung.00112.2007.
39. Fehrenbach H: Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res. 2001, 2 (1): 33-46. 10.1186/rr36.
40. Hsieh SJ: Biomarkers increase detection of active smoking and secondhand smoke exposure in critically ill patients. Crit Care Med. 2011, 39 (1): 40-45. 10.1097/CCM.0b013e3181fa4196.
41. Liu J: Relationship between biomarkers of cigarette smoke exposure and biomarkers of inflammation, oxidative stress, and platelet activation in adult cigarette smokers. Cancer Epidemiol Biomarkers Prev. 2011, 20 (8): 1760-1769. 10.1158/1055-9965.EPI-10-0987.
42. Morin A: Estimation and correlation of cigarette smoke exposure in Canadian smokers as determined by filter analysis and biomarkers of exposure. Regul Toxicol Pharmacol. 2011, 61 (3 Suppl): S3-S12.
43. Naufal ZS: Differential exposure biomarker levels among cigarette smokers and smokeless tobacco consumers in the National Health and Nutrition Examination Survey 1999-2008. Biomarkers. 2011, 16 (3): 222-235. 10.3109/1354750X.2010.546013.
44. Sexton K: Proteomic profiling of human respiratory epithelia by iTRAQ reveals biomarkers of exposure and harm by tobacco smoke components. Biomarkers. 2011, 16 (7): 567-576. 10.3109/1354750X.2011.608855.
45. Yuchuan H: Circulating biomarkers of hazard effects from cigarette smoking. Toxicol Ind Health. 2011, 27 (6): 531-535. 10.1177/0748233710393393.
46. Bernert JT: Increases in tobacco exposure biomarkers measured in non-smokers exposed to sidestream cigarette smoke under controlled conditions. Biomarkers. 2009, 14 (2): 82-93. 10.1080/13547500902774613.
47. Otri AM: Variable expression of human Beta defensins 3 and 9 at the human ocular surface in infectious keratitis. Invest Ophthalmol Vis Sci. 2012, 53 (2): 757-761. 10.1167/iovs.11-8467.
48. Scharf S: Legionella pneumophila induces human beta defensin-3 in pulmonary cells. Respir Res. 2010, 11: 93-10.1186/1465-9921-11-93.
49. Gross CA: Beta2-agonists promote host defense against bacterial infection in primary human bronchial epithelial cells. BMC Pulm Med. 2010, 10: 30-10.1186/1471-2466-10-30.
50. Liao Z: Enhanced expression of human beta-defensin 2 in peripheral lungs of patients with chronic obstructive pulmonary disease. Peptides. 2012, 38: 350-356. 10.1016/j.peptides.2012.09.013.
51. Pace E: Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients. PLoS One. 2012, 7 (3): e33601-10.1371/journal.pone.0033601.
52. Zhang W: Cigarette smoke modulates PGE(2) and host defence against Moraxella catarrhalis infection in human airway epithelial cells. Respirology. 2011, 16 (3): 508-516. 10.1111/j.1440-1843.2010.01920.x.
53. Shibata Y: Altered expression of antimicrobial molecules in cigarette smoke-exposed emphysematous mice lungs. Respirology. 2008, 13 (7): 1061-1065.
54. Yamin M: Cigarette smoke combined with Toll-like receptor 3 signaling triggers exaggerated epithelial regulated upon activation, normal T-cell expressed and secreted/CCL5 expression in chronic rhinosinusitis. J Allergy Clin Immunol. 2008, 122 (6): 1145-1153 e3. 10.1016/j.jaci.2008.09.033.
55. Nakamura S: Nicotine induces upregulated expression of beta defensin-2 via the p38MAPK pathway in the HaCaT human keratinocyte cell line. Med Mol Morphol. 2010, 43 (4): 204-210. 10.1007/s00795-010-0493-4.
56. Lee J, Taneja V, Vassallo R: Cigarette smoking and inflammation: cellular and molecular mechanisms. J Dent Res. 2012, 91 (2): 142-149. 10.1177/0022034511421200.
57. Xu J, Xu F, Lin Y: Cigarette smoke synergizes lipopolysaccharide-induced interleukin-1beta and tumor necrosis factor-alpha secretion from macrophages via substance P-mediated nuclear factor-kappaB activation. Am J Respir Cell Mol Biol. 2011, 44 (3): 302-308. 10.1165/rcmb.2009-0288OC.
58. Kanda N: Human beta-defensin-2 enhances IFN-gamma and IL-10 production and suppresses IL-17 production in T cells. J Leukoc Biol. 2011, 89 (6): 935-944. 10.1189/jlb.0111004.
59. Winter J: Human beta-defensin-1, -2, and -3 exhibit opposite effects on oral squamous cell carcinoma cell proliferation. Cancer Invest. 29 (3): 196-201.
60. Kreuter A: Expression of antimicrobial peptides in different subtypes of cutaneous lupus erythematosus. J Am Acad Dermatol. 2011, 65 (1): 125-133. 10.1016/j.jaad.2010.12.012.
61. Shestakova T: Immunohistochemical analysis of beta-defensin-2 expression in human lung tumors. Exp Oncol. 2010, 32 (4): 273-276.
1. Cigarette smoke-induced inflammation: NLRP10-mediated mechanisms
Gagandeep Kaur, Prathyusha Bagam, Rakeysha Pinkston, Dhirendra P. Singh, Sanjay Batra
2. Comparative analysis of cigarette smoke induced cellular proteome distributions on bovine aortic endothelial cells
Jiho Min, Ji-Young Ahn, Simranjeet Singh Sekhon, Yoon Mi Jin, Hyun-Ju Um, Inho Jo, Yang-Hoon Kim
Molecular & Cellular Toxicology
3. Monocyte-derived exosomes upon exposure to cigarette smoke condensate alter their characteristics and show protective effect against cytotoxicity and HIV-1 replication
Sanjana Haque, Namita Sinha, Sabina Ranjit, Narasimha M. Midde, Fatah Kashanchi, Santosh Kumar
Scientific Reports
4. Airwayβ-Defensin-1 Protein Is Elevated in COPD and Severe Asthma
Katherine J. Baines, Thomas K. Wright, Jodie L. Simpson, Vanessa M. McDonald, Lisa G. Wood, Kristy S. Parsons, Peter A. Wark, Peter G. Gibson
Mediators of Inflammation
5. Membrane microdomains regulate NLRP10- and NLRP12-dependent signalling in A549 cells challenged with cigarette smoke extract
Dhirendra P. Singh, Gagandeep Kaur, Prathyusha Bagam, Rakeysha Pinkston, Sanjay Batra
Archives of Toxicology
6. Effects of whole cigarette smoke on human beta defensins expression and secretion by oral mucosal epithelial cells
Wen-mei Wang, Pei Ye, Ya-jie Qian, Ya-fan Gao, Jing-jing Li, Fang-fang Sun, Wei-yun Zhang, Xiang Wang
Tobacco Induced Diseases
7. Human β-defensin 3 increases the TLR9-dependent response to bacterial DNA
Sarah L. McGlasson, Fiona Semple, Heather MacPherson, Mohini Gray, Donald J. Davidson, Julia R. Dorin
European Journal of Immunology
8. LL-37 secreted by epithelium promotes fibroblast collagen production: a potential mechanism of small airway remodeling in chronic obstructive pulmonary disease
Congcong Sun, Maoxiang Zhu, Zhihua Yang, Xiujie Pan, Yuke Zhang, Qin Wang, Wei Xiao
Laboratory Investigation
9. Antimicrobial peptides as a possible interlink between periodontal diseases and its risk factors: A systematic review
S. Li, G. Schmalz, J. Schmidt, F. Krause, R. Haak, D. Ziebolz
Journal of Periodontal Research
10. Azithromycin attenuates cigarette smoke extract-induced oxidative stress injury in human alveolar epithelial cells
Molecular Medicine Reports
11. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells
PSS Rao, Anusha Ande, Namita Sinha, Anil Kumar, Santosh Kumar, Srikumar P Chellappan