Smoking, immunity, and DNA damage

Tobacco toxicology and teratogenic effects

The normal lung of non-smokers contains alveolar macrophages as guardian residents. However, when tobacco smoke enters the lungs it triggers a dramatic influx of macrophages and neutrophils in the bronchi and pulmonary epithelia. Smoking directly exposes the epithelial tissue to at least 60 powerful chemical carcinogens with the potential to cause DNA damage to larynx, bronchi, and lung epithelial cells. The most common compounds in tobacco smoke are nicotine, formaldehyde, ammonia, carbon monoxide, carbon dioxide, benzopyrenes, tar, acetone, hydroxyquinone, cadmium, and nitrogen oxides (1).

Tar and nicotine have immunosuppressive effects on the innate immune response and tobacco products containing high concentrations of tar and nicotine cause the greatest immunologic changes. For instance, cigarette smoke altogether suppresses or decreases neutrophils phagocytic activity and affects chemotaxis, kinesis, and cell signaling. It also inhibits the release of reactive oxygen species (ROS), thus compromising pathogen killing by neutrophils and other cells of the innate immunity (1).

In addition to the carcinogenic and epigenetic chemicals present in the smoke of cigarettes, cigars, pipes, etc., nicotine may also contribute to the genesis of lung cancer. The tobacco-related damage of the respiratory epithelia plus the suppressive role of nicotine on the programmed cell-death (apoptosis) mechanism may be an important factor for the survival of malignant cells and the formation of multiple epithelial lesions known as field-mutations. Apoptosis is the main protective cellular mechanism against proliferation of cells bearing irreversible DNA damage (2). Numerous field-mutations are found spread across the epithelia on the lungs of smokers and former smokers, each cell surrounded by an inflammatory response. Chronic inflammation is considered a major consequence of smoking that also leads to increased cell proliferation in the exposed pulmonary epithelia by enhancing progressive cell transformation and survival, which finally results in cancer cells (3). Eventually one of those field-mutations escapes from the inflammatory siege and starts promoting stroma formation, angiogenesis and rapid cell proliferation, resulting in epithelial tumor.

Nicotine is a toxic and highly addictive alkaloid with affinity to nicotinic acetylcholine receptors (nAChR), which are found on the surface of pulmonary epithelial cells as well as on mesotheliomas, SCLC, and NSCLC, showing a suppressive effect on the pro-apoptotic signaling pathways. Of course, nicotine is also rapidly absorbed from the lungs into the blood stream and reaches the cholinergic receptors on neurons of the reward centers in the CNS, causing strong addiction. It works also at the nicotinic receptors in the peripheral nervous system (4,5).

nAChR participate in the autocrine-proliferative network involved in the growth of cancer cells by upregulating growth factors, such as vascular endothelial growth factors (VEGF) that promotes angiogenesis, and fibroblastic growth factors (FGF) that promotes stromal tissue growth. Therefore, nicotine may play a pleiotropic role that includes cell proliferation and angiogenesis, besides down-regulating anti-inflammatory microRNAs in lung cells and stimulating the expression of inflammatory cytokines which contribute to tissue damage. These findings suggest that nicotine might play a direct role in the pathogenesis of human lung cancers and in the etiology of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), and emphysema (6-10).

According to toxicology studies, nicotine is highly toxic and the lethal dose for adult humans is around 1.0 mg/kg or ~60 mg in a human with average body mass. Oral ingestion of 4 to 8 mg of nicotine may cause vomiting, high blood pressure, dizziness, convulsions, and tachycardia—even leading to atrial fibrillation, depending on the body mass (11). The potential toxicity of nicotine in candies, chewing gums, and e-cigarettes should also not to be taken lightly, especially with children in the household. It suffices 1 g of nicotine to send a toddler to the emergency room (12).

Tobacco impact on immunity

As mentioned before, one of the first impacts on immunity caused by smoking is to trigger an inflammatory chronic response in the lungs, which increases over the years due to repeated exposure to smoke and the addition of new field-mutations across the pulmonary epithelia. Macrophages are considered the main players of chronic inflammation, which results in tissue destruction due to the increased release of high levels of pro-inflammatory cytokines and matrix metalloproteinases, as compared to non-smokers (13,14).

The augmented expression of adhesion molecules on activated macrophages enhances cell-to-cell interactions with T cells, which results in cytokine synthesis and release. Il-1 secreted by macrophages activates clonal proliferation of helper T lymphocytes (CD4+ Th). T helper cells secrete interleukin 2 (IL-2) that activates cytotoxic T lymphocyte effector T cells (CD8+ T cell). Alveolar macrophages are the main phagocytic cells in the lungs of non-smokers and those levels are increased in the bronchi-alveolar fluid (BAL) of smokers. Those macrophages also show altered phenotypes with the phagocytic activity significantly reduced, which decreases macrophages ability to eliminate inflammatory debris and apoptotic neutrophils from the lung (11).

Heguy et al. [2006] genotyped alveolar macrophages from smokers and non-smokers and found that 40 genes had increased expression whereas 35 others had decreased expression in smokers, when compared to non-smokers. Most of the altered genes played roles in the inflammatory response, cell adhesion, extracellular matrix, modulation of proteolysis, signal transduction, lysosomal function, antioxidant activity, and transcription factors (15).

Forsslund et al. [2014] investigated subpopulations of lymphocytes in 40 current smokers, 40 never smokers, and 40 former smokers, including patients with COPD (smokers and former smokers). They analyzed the BAL and the peripheral blood of all subjects and found that smokers (with or without COPD) had higher percentages of CD8+ T cells and natural killer (NK) T-like cells in the BAL than did never-smokers and ex-smokers with COPD. However, the percentages of CD4+ T cells were lower in smokers than in the nonsmoking subjects. In peripheral blood, the frequency of CD4+ T cells was increased in the two smoking groups (with and without COPD). Current smokers also had increased levels of activated naive and effector CD4+ T cells than nonsmokers, especially non-smoker patients with COPD. In male smokers with COPD, the percentage of CD8+ T cells in BAL fluid positively correlated with the daily number of smoked cigarettes (16).

NK cells (CD56+ CD3-) are also present in increased rates in the BAL fluid and peripheral blood of smokers, with and without COPD, while NK CD16+ levels are comparatively lower in the peripheral circulation of non-smokers without the disease (17,18). Other studies found that cigarette smoke have a suppressive activity on the expression of genes coding interferon gamma (IFN-γ) and tumor necrosis factor alfa (TNF-α) in human NKs CD56+ CD3-, in addition to reducing production of perforin, cytotoxicity, and cytolysis (19-21).

Cigarette smoke also contains high concentrations of ROS, and a recently published report showed that cigarette smoke extract (CSE) stimulates the synthesis of interleukin-17A (IL-17A) which, by its turn, induced the production of interleukine-8 (IL-8) via the receptors IL-17R on the surface of epithelial cells of the lungs and bronchi. IL-17 also activates pro-inflammatory signaling pathways by occupying receptors IL-17RA and IL-17RC on fibroblasts, endothelial and epithelial cells. IL-17 also recruits neutrophils to the respiratory tract by releasing IL-8 from the endothelial cells (22). Despite the amplification of inflammation, neutrophils exposed to tobacco smoke lose their ability to generate respiratory bursts and reduces phagocytosis, thus becoming less efficient in fighting pathogens in the respiratory tract (23,24).

The immune system always triggers, simultaneously, pro-inflammatory and anti-inflammatory responses, aiming at eliminating a pathogen (or a mutated protein) while preventing autoimmunity. Transforming growth factor beta (TGF-β) binds to receptors on the surface of cytotoxic T cells and transforms them into regulatory T cells (Tregs) which act by modulating the inflammatory process. Three subpopulations of Tregs were found in healthy smokers and patients with COPD: suppressive (rTregs) CD25++ CD45RA+; activated CD+++CD45RA- (aTregs), and pro-inflammatory CD25++CD45RA- (FrIII). FrIII secretes pro-inflammatory cytokines and when its population predominates over the other two, inflammation is enhanced and the risk of autoimmunity increases (25). It was found that smoking impairs the immunosuppressive function of Tregs and causes imbalance between Treg subpopulations by reducing the number of rTregs and favoring autoimmunity in COPD patients, when compared with smokers without the disease. In contrast, rTreg levels are detected abnormally high in some healthy smokers, which predisposes to respiratory infections due to its immune suppressive effect. In COPD patients it was found that lower levels of rTregs and aTregs are accompanied by larger populations of FrIII cells which enhance cytokine secretion and the maintenance of inflammation (26).

Final thoughts

There is a vast literature on the impact of smoking on the immune system, which corroborates the mentioned studies; and new findings are underway. Carcinogens present in the smoke of tobacco products have an important role in altering the genome of immune cells, whether by implanting chemical adducts in the cellular DNA or by inducing irreversible genetic damage (27). Nicotine-releasing non-smoking surrogates are not at all innocuous and although some studies have been considering nicotine administration as a potential immune modulator for Parkinson, Alzheimer and Crohn’s diseases, the matter remains highly controversial and requires deeper investigations (28).

All in all, tobacco smoking continues to be a huge public health challenge and a social and economic burden to nations, families, and individuals who precociously lose their health, productivity, and their lives under the yoke of nicotine addiction.

Acknowledgments

None.

Footnote

Conflicts of Interest: The author has no conflicts of interest to declare.

References

1. Hecht SS. Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer 2003;3:733-44. [Crossref] [PubMed]
2. Dasgupta P, Kinkade R, Joshi B, et al. Nicotine inhibits apoptosis induced by chemotherapeutic drugs by up-regulating XIAP and survivin. Proc Natl Acad Sci U S A 2006;103:6332-7. [Crossref] [PubMed]
3. Behrens C, Feng L, Kadara H, et al. Expression of interleukin-1 receptor-associated kinase-1 in non-small cell lung carcinoma and preneoplastic lesions. Clin Cancer Res 2010;16:34-44. [Crossref] [PubMed]
4. Catassi A, Servent D, Paleari L, et al. Multiple roles of nicotine on cell proliferation and inhibition of apoptosis: Implications on lung carcinogenesis. Mutation research 2008;659:221-31. [Crossref] [PubMed]
5. Jain R, Mukherjee K. Biological Basis of Nicotine Addiction. Indian Journal of Pharmacology 2003;35:281-9.
6. Ebrahimpour A, Shrestha S, Bonnen MD, et al. Nicotine modulates growth factors and microRNA to promote inflammatory and fibrotic processes. J Pharmacol Exp Ther 2019;368:169-78. [Crossref] [PubMed]
7. Carlisle DL, Hopkins TM, Gaither-Davis A, et al. Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts. Respir Res 2004;5:27. [Crossref] [PubMed]
8. Conklin BS, Zhao W, Zhong DS, et al. Nicotine and cotinine up-regulate vascular endothelial growth factor expression in endothelial cells. Am J Pathol 2002;160:413-8. [Crossref] [PubMed]
9. Cooke JP, Ghebremariam YT. Endothelial nicotinic acetylcholine receptors and angiogenesis. Trends Cardiovasc Med 2008;18:247-53. [Crossref] [PubMed]
10. Cucina A, Sapienza P, Corvino V, et al. Nicotine-induced smooth muscle cell proliferation is mediated through bFGF and TGF-beta 1. Surgery 2000;127:316-22. [Crossref] [PubMed]
11. Skold CM, Lundahl J, Hallden G, et al. Chronic smoke exposure alters the phenotype pattern and the metabolic response in human alveolar macrophages. Clin Exp Immunol 1996;106:108-13. [Crossref] [PubMed]
12. Connolly GN, Richter P, Aleguas A Jr, et al. Unintentional child poisonings through ingestion of conventional and novel tobacco products. Pediatrics 2010;125:896-9. [Crossref] [PubMed]
13. Kirkham PA, Spooner G, Rahman I, et al. Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix protein modified by cigarette smoke and lipid peroxidation products. Biochem Biophys Res Commun 2004;318:32-7. [Crossref] [PubMed]
14. Barnes PJ. Alveolar macrophages in chronic obstructive pulmonary disease (COPD). Cell Mol Biol (Noisy-le-grand) 2004;50 Online Pub:OL627-37.
15. Heguy A, O’Connor TP, Luettich K, et al. Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking. J Mol Med 2006;84:318-28. [Crossref] [PubMed]
16. Forsslund H, Mikko M, Karimi R. Distribution of T-cell subsets in BAL fluid of patients with mild to moderate COPD depends on current smoking status and not airway obstruction. Chest 2014;145:711-22. [Crossref] [PubMed]
17. Tollerud DJ, Clark JW, Brown LM, et al. Association of cigarette smoking with decreased numbers of circulating natural killer cells. Am Rev Respir Dis 1989;139:194-8. [Crossref] [PubMed]
18. Stolberg VR, Martin B, Mancuso P, et al. Role of CC Chemokine Receptor 4 in Natural Killer Cell Activation during Acute Cigarette Smoke Exposure. Am J Pathol 2014;184:454-63. [Crossref] [PubMed]
19. Mian MF, Lauzon NM, Stampfli MR, et al. Impairment of human NK cell cytotoxic activity and cytokine release by cigarette smoke. J Leukoc Biol 2008;83:774-84. [Crossref] [PubMed]
20. Arimilli S, Damratoski BE, Prasad GL. Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions. Toxicol In Vitro 2013;27:1992-2004. [Crossref] [PubMed]
21. Mian MF, Pek EA, Mossman KL, et al. Exposure to cigarette smoke suppresses IL-15 generation and its regulatory NK cell functions in poly I:C-augmented human PBMCs. Mol Immunol 2009;46:3108-16. [Crossref] [PubMed]
22. Lee KH, Lee CH, Woo J, et al. Cigarette Smoke Extract Enhances IL-17AInduced IL-8 Production via Up-Regulation of IL-17R in Human Bronchial Epithelial Cells. Mol. Cells 2018;41:282-9. [PubMed]
23. Ryder MI. Nicotine effects on neutrophil F-actin formation and calcium release: implications for tobacco use and pulmonary diseases. Exp Lung Res 1994;20:283-96. [Crossref] [PubMed]
24. Keast D, Taylor K. The effects of chronic tobacco smoke exposure from high-tar cigarettes on the phagocytic and killing capacity of polymorphonuclear cells of mice. Environ Res 1983;31:66-75. [Crossref] [PubMed]
25. Miyara M, Yoshioka Y, Kitoh A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 2009;30:899-911. [Crossref] [PubMed]
26. Hou J, Sun YC, Hao Y, et al. Imbalance between subpopulations of regulatory T cells in COPD. Thorax 2013;68:1131-9. [Crossref] [PubMed]
27. Alexandrov LB, Ju YS, Haase K, et al. Mutational signatures associated with tobacco smoking in human cancer. Science 2016;354:618-22. [Crossref] [PubMed]
28. Piao WH, Campagnolo D, Dayao C, et al. Nicotine and inflammatory neurological disorders. Acta Pharmacol Sin 2009;30:715-22. [Crossref] [PubMed]