GILES F. FILLEY LECTURE Cigarette Smoke Effects on Innate Immune Mechanisms in the Nasal Mucosa Potential Effects on the Microbiome Ilona Jaspers1 1
Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina
Abstract It is well established that exposure to cigarette smoke (CS), through active smoking and through exposure to secondhand smoke, has immunosuppressive effects, yet how this might affect the microbiome is not known. In this manuscript we focus on the effects of CS on innate host defense response, with particular emphasis on the role of epithelial cells and mucosal immune responses in the nose and the potential effects on the microbiome. The studies described here briefly summarize the effects of CS on specific innate immune cells, such as neutrophils, macrophages/ monocytes, natural killer cells, and dendritic cells. A detailed description of how CS affects epithelial cells and why we consider this to be a central defect in the overall immunosuppressive effects of CS in the lung is provided. We
summarize data on the role of the “epimmunome” in the context of CS exposure, including the effects on soluble mediator production, such as cytokines, chemokines, and antimicrobial defense mediators. Separate emphasis is put on the expression of ligands on epithelial cells, which directly interact with receptors on immune cells, and the effects of CS on these interactions. We introduce the nose and nasal mucosa as a model to study the effects of CS exposure on host defense responses and changes in the microbiome in humans in vivo. Understanding the dynamics of a healthy microbiome and how CS affects this balance is important to uncovering the mechanisms of CS-induced disease. Keywords: cigarette smoke; innate host defense responses; nasal mucosa
(Received in original form June 12, 2013; accepted in final form July 8, 2013 ) Correspondence and requests for reprints should be addressed to Ilona Jaspers, University of North Carolina, Department of Pediatrics, 104 Mason Farm Rd CB#7310, Chapel Hill, NC 27599-7310. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S38–S42, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-154MG Internet address: www.atsjournals.org
Individuals chronically exposed to cigarette smoke (CS), either as active smokers or through exposure to secondhand smoke, experience increased risk of developing of a variety of pulmonary diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and infections (1). Smoking not only induces cellular damage and inflammation but also serves as an immunosuppressor (1). This article evaluates the role of epithelial cells during innate immune defense responses, identifies mechanisms by which CS modulates innate immune responses, and develops hypotheses of immune mechanisms affecting CS-induced changes in the nasal microbiome. S38
Components of CS CS is a multicomponent mixture of gasphase and particle-phase toxins with known immunosuppressive effects. Many of the individual components of CS have known immunomodulatory function, carcinogenic activity, or addictive activities (2). They include metals, volatile hydrocarbons, and gas-phase components, such as carbon monoxide (2). Among the components of CS, polycyclic aromatic hydrocarbons (PAHs), acrolein, and reactive oxygen species have been individually studied for their effects on respiratory immune responses. Acrolein is a highly reactive unsaturated aldehyde and a known
respiratory irritant (3, 4). Acrolein is electrophilic with high reactivity toward biological nucleophiles, resulting in protein and DNA adducts, which is thought to mediate the adverse health effects of this inhaled toxin (4). PAHs are generated during incomplete combustion of organic materials, such as fossil fuels, wood, and tobacco, and are linked to adverse health effects induced by inhalation of CS and other combustion emissions, such as diesel exhaust and wood smoke. Thus, it is conceivable that exposure to wood smoke, diesel exhaust, and other emissions caused by incomplete combustion of organic matter poses similar hazards to public health as those induced by exposure to CS.
AnnalsATS Volume 11 Supplement 1 | January 2014
GILES F. FILLEY LECTURE General Effects of Smoking on Mucosal Immune Responses Exposure to CS affects the respiratory immune system on several levels. Innate immune and inflammatory cells, such as neutrophils and macrophages, become activated and expand, resulting in increased levels of proinflammatory cytokines/ chemokines, such as IL-8, TNF-a, MCP-1, and many more (2). CS-induced neutrophilia is marked by the infiltration of immature neutrophils, which have a highly proinflammatory phenotype, as evidenced by increased production of reactive oxygen species and enhanced myeloperoxidase levels (5), and therefore are more likely to cause tissue damage. Despite the expansion of neutrophils and macrophages, these cells demonstrate impaired phagocytic capabilities. For example, exposure of macrophages to CS decreases clearance of bacteria or apoptotic cells, such as apoptotic PMNs (6, 7). Similarly, exposure of PMNs to CS extract (CSE) increases spontaneous production of superoxide radicals but decreases bacterial-stimulated production of superoxide (8), which is necessary for the efficient elimination of the pathogen. Other macrophage functions associated with bacterial clearance, such as surface receptor expression, are affected by CS exposure. For example, expression of TLR2, which is important for the recognition and response to gram-positive bacteria, is reduced in macrophages from smokers (9). Similarly, expression of MARCO, a member of the class A scavenger receptor family important for the binding of gram-positive and gramnegative bacteria, is decreased in macrophages exposed to CSE (10). The effects of CS on dendritic cell (DC) function are less defined. Exposure of peripheral blood monocyte–derived myeloid DCs to CSE interferes with antigen presentation by these cells (11). Production of IFN-g, which is critical to direct a Th1-type immune response to the immune challenge, and expression of CCR7, which is important for migration of DCs to the lymph nodes, are decreased in bronchoalveolar lavage fluid samples from smokers (12). CSE exposure of plasmacytoid DCs, which are important in the context of viral infections, inhibits antiviral activities, as marked by a decreased release of type I interferons and
other cytokines involved in antiviral defense responses (13, 14). Our studies have demonstrated that viral infection of cocultures composed of nasal epithelial cells from smokers with DCs leads to reduced expression of antiviral signaling molecules in these cells as compared with cocultures composed of nasal epithelial cells from nonsmokers and DCs (15), suggesting that smoking affects the ability of epithelial cells to communicate and activate DCs. CS-induced alterations of natural killer (NK) cell function have also been demonstrated. NK cells play important roles during viral infections and in eliminating tumor cells via cell-mediated cytotoxicity. This function is mediated by the release of cytotoxins, such as perforin and granzyme B. In addition to targeted cytotoxicity, NK cells are important sources of cytokines, such as IFN-g in the context of microbial infections. We have previously demonstrated that the number and function of cytotoxic NK cells is decreased in smokers challenged with the live attenuated influenza virus (16). Similarly, in vitro exposure of peripheral blood mononuclear cells to CS-conditioned medium decreases RNA-induced cytotoxic function and IFN-b production by NK cells (17–19). Other innate host defense mechanisms, such as mucociliary escalator and antimicrobial peptide release, are affected by CS exposure. The mucus layer covering the airway epithelium traps pathogens and, in concert with coordinated beating of the ciliated epithelium, propels the mucus to the larynx. CS exposure affects the mucocililary escalator at several levels, including decreased ciliary beating, mucus hypersecretion, and squamous cell metaplasia (20). In addition, the epithelial integrity can be compromised by CS exposure through the disruption of tight junctions, leading to increased epithelial permeability (20, 21). The direct effects of CS exposure on epithelial cells is discussed in more detail below.
Epithelial Cells: A Central Target for CS Exposure Epithelial cells are among the first targets for inhaled toxins and are a primary site for infection with respiratory pathogens. Thus, interactions affecting host defense responses in the context of CS exposure are likely mediated by epithelial cells, at least during
Jaspers: CS Effects on Innate Immune Mechanisms in the Nasal Mucosa
the initial stages of the infection. The term “epimmunome” was introduced by Swamy and colleagues in 2010 and describes the concept that epithelial cells regulate communication with different sets of immune cells through the release or expression of cytokines, chemokines, or adhesion molecules and therefore are important regulators of immune responses (22). To simplify the concept introduced by Swamy and colleagues, one can divide the “epimmunome” into (1) soluble mediators directly protecting the host, (2) soluble mediators orchestrating the immune response, and (3) ligand–receptor interactions to activate and mature immune cells. In the context of CS exposure, all of these functions are altered in epithelial cells lining the respiratory tract. For example, epithelial cells show altered production and release of soluble antimicrobial peptides, such as human b-defensin 2 (hBD-2). However, the CS-induced alterations in hBD-2 may be site specific because hBD-2 was decreased in the central but not distal airways from smokers (23, 24). Other soluble factors released by epithelial cells that are important for orchestrating the recruitment, activation, and maturation of immune cells are cytokines or chemokines, which are altered in the context of CS exposure. In particular, expression of neutrophil chemokines (e.g., IL-8 and GROa) and macrophage chemokines (e.g., MCP-1) are enhanced in CS-exposed epithelial cells (25), thus contributing to the enhanced recruitment of these cells into the airways. Other means of communication between epithelial cells and resident immune cells include the expression of receptor–ligand interactions. For example, several MHC class I–related molecules are strong activating ligands for NKG2D, which is a surface receptor expressed on lymphocytes, including NK cells, cytotoxic T cells, and g/d T cells (26). UL16 binding proteins, which were originally identified as ligands for the UL16 glycoprotein of the human cytomegalovirus (27), are expressed on stressed epithelial cells (28), and we have recently shown that their expression can be altered in nasal epithelial cells by oxidants (29). Other potential NKG2D ligands expressed on epithelial cells that are altered in the context of CS exposure include retinoic acid early transcript 1 and MHC class 1 chain–related molecules A and B (30, 31). Altered expression of these ligands results in S39
GILES F. FILLEY LECTURE modified activation of NK cells or other cytotoxic lymphocytes (30, 32). Thus, the role of the “epimmunome” in the context of CS exposure is diverse and plays a key role in the manifestations of altered immune responses. A schematic illustrating CS-induced changes in mucosal immune responses is shown in Figure 1.
The Nasal Mucosa as a Tool to Study the Effects of Smoking on Innate Immune Responses Although exposure of animal models and in vitro exposure of cell culture models are important tools to uncover mechanisms of CS-induced immune dysfunction, translating these findings into humans in vivo is of great clinical importance. Over the years we have developed tools to study the effects of CS exposure on immune defense responses by examining the nasal mucosa. Using the nose as a model to study mucosal immune responses has several advantages: (1) the nose is accessible and easy to sample in a relatively noninvasive way; (2) the nasal epithelium resembles the airway epithelium morphologically, and smoking-related gene expression and histological changes (e.g., mucus production) are similar in nasal and airway epithelial cells (33–35); and (3) the nasal mucosa is the primary target for inhaled pathogens and has a robust microbial flora that can be studied for potential CS-induced changes in the microbiome. CS-induced alterations in the nasal mucosal immune responses in humans can be studied through several different experimental tools. Nasal lavage fluid, which
is noninvasively obtained through saline irrigation of the nostrils followed by expelling the sample into a specimen cup, can be analyzed for changes in soluble mediators, including cytokines/chemokines, proteases/antiproteases, and antimicrobial peptides (16, 36–39). The cellular components of the nasal lavage can be used for gene expression analysis or immunophenotyping using flow cytometry (16, 38, 40). Superficial scrape biopsies obtained from the human subjects can be analyzed by routine biochemical analysis tools such as qRT-PCR or flow cytometry to reveal important information regarding intraepithelial cell immune cell populations and changes in epithelial cell function (29, 35, 41). Thus, sampling the nose represents an important tool to understand potential effects of CS exposure on innate immune responses and potentially to link these changes with alterations in the nasal microbiome.
The Nasal Microbiome and Changes Induced by CS Exposure The nasopharyngeal flora are quite diverse and can be sampled using nasal lavage, swabs, and scrape biopsies. Unlike the lower airways, where smokers (without any significant comorbidities) and nonsmokers do not differ in the microbiota measurable in alveolar lavage fluid (42), studies have shown that the nasopharyngeal flora of smokers and nonsmokers differ (43). Specifically, the nasal microbiota in smokers contain fewer commensal aerobic and anaerobic organisms and show greater
Figure 1. Schematic outlining cigarette smoke–induced changes in mucosal immune responses. NK = natural killer.
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numbers of potential pathogens (43). Subsequent studies by the same group demonstrate that smoking cessation reverts the nasopharyngeal microbiota to normal (44) and that the altered nasal microbiome in parents who smoke can be transferred to their children (45). However, smokers with significant comorbidities, such as COPD, differ in their lung and lower airway microbiome (46, 47), suggesting that disease progression could be associated with changes in the microbial flora populating the lower airways. Potential mechanisms mediating CS-induced changes in the microbiome include inhalation of pathogens contained in tobacco, impaired antimicrobial defenses, and increased adherence of pathogens to the epithelium. Studies have shown that cigarettes contain bacteria that could potentially be inhaled (48). Among the bacteria identified in cigarettes are Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia (49). Thus, inhalation of mainstream CS could introduce bacteria contained in cigarettes into the lower airways. The impaired antimicrobial defenses induced by CS exposure include impaired phagocytic activity of macrophages and neutrophils, leading to delayed and/or reduced clearance of bacteria and the increased possibility of colonization. Dysfunction of the mucociliary escalatory also contributes to impaired clearance of bacteria in smokers. In addition, CS exposure can increase the adherence of bacteria to the epithelium. This can be achieved through increased expression of platelet-activating factor receptor (PAFR) on epithelial cells. Specifically, exposure to CSE increases PAFR expression on respiratory epithelial cells and enhances pneumococcal adhesion to these cells in a PAFR-dependent manner (50). In addition, CS can directly affect bacteria and enhance their virulence. For example, exposure of Staphylococcus aureus to CS increases biofilm formation and adherence to epithelial cells, which is mediated by oxidative stress and through the enhanced expression of fibronectin binding protein A (51). These observations suggest that, in addition to compromising antimicrobial defense responses, exposure to CS could affect the microbiome by increasing adherence and virulence of certain species.
AnnalsATS Volume 11 Supplement 1 | January 2014
GILES F. FILLEY LECTURE Conclusions and Future Directions Existing and emerging data point to a relationship between exposure to CS, changes in the microbiome, and adverse
health effects. Understanding the dynamics of a healthy microbiome and how CS affects this balance are important to uncovering the mechanisms of CS-induced disease. The nasal mucosa and its microbial flora present an important tool to investigate the effects of
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GILES F. FILLEY LECTURE 35 Jaspers I, Horvath KM, Zhang W, Brighton LE, Carson JL, Noah TL. Reduced expression of IRF7 in nasal epithelial cells from smokers after infection with influenza. Am J Respir Cell Mol Biol 2010;43: 368–375. 36 Kesic MJ, Hernandez M, Jaspers I. Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased influenza A virus cleavage and replication. Respir Res 2012;13:82. 37 Meyer M, Kesic MJ, Clarke J, Ho E, Simmen RC, Diaz-Sanchez D, Noah TL, Jaspers I. Sulforaphane induces SLPI secretion in the nasal mucosa. Respir Med 2013;107:472–475. 38 Noah TL, Zhou H, Jaspers I. Alteration of the nasal responses to influenza virus by tobacco smoke. Curr Opin Allergy Clin Immunol 2012;12:24–31. 39 Noah TL, Zhou H, Zhang H, Horvath K, Robinette C, Kesic M, Meyer M, Diaz-Sanchez D, Jaspers I. Diesel exhaust exposure and nasal response to attenuated influenza in normal and allergic volunteers. Am J Respir Crit Care Med 2012;185:179–185. 40 Noah TL, Zhou H, Monaco J, Horvath K, Herbst M, Jaspers I. Tobacco smoke exposure and altered nasal responses to live attenuated influenza virus. Environ Health Perspect 2011;119:78–83. 41 Horvath KM, Brighton LE, Herbst M, Noah TL, Jaspers I. Live attenuated influenza virus (LAIV) induces different mucosal T cell function in nonsmokers and smokers. Clin Immunol 2012;142: 232–236. 42 Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, Flores SC, Fontenot AP, Ghedin E, Huang L, et al.; Lung HIV Microbiome Project. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013; 187:1067–1075.
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AnnalsATS Volume 11 Supplement 1 | January 2014
Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina
Abstract It is well established that exposure to cigarette smoke (CS), through active smoking and through exposure to secondhand smoke, has immunosuppressive effects, yet how this might affect the microbiome is not known. In this manuscript we focus on the effects of CS on innate host defense response, with particular emphasis on the role of epithelial cells and mucosal immune responses in the nose and the potential effects on the microbiome. The studies described here briefly summarize the effects of CS on specific innate immune cells, such as neutrophils, macrophages/ monocytes, natural killer cells, and dendritic cells. A detailed description of how CS affects epithelial cells and why we consider this to be a central defect in the overall immunosuppressive effects of CS in the lung is provided. We
summarize data on the role of the “epimmunome” in the context of CS exposure, including the effects on soluble mediator production, such as cytokines, chemokines, and antimicrobial defense mediators. Separate emphasis is put on the expression of ligands on epithelial cells, which directly interact with receptors on immune cells, and the effects of CS on these interactions. We introduce the nose and nasal mucosa as a model to study the effects of CS exposure on host defense responses and changes in the microbiome in humans in vivo. Understanding the dynamics of a healthy microbiome and how CS affects this balance is important to uncovering the mechanisms of CS-induced disease. Keywords: cigarette smoke; innate host defense responses; nasal mucosa
(Received in original form June 12, 2013; accepted in final form July 8, 2013 ) Correspondence and requests for reprints should be addressed to Ilona Jaspers, University of North Carolina, Department of Pediatrics, 104 Mason Farm Rd CB#7310, Chapel Hill, NC 27599-7310. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S38–S42, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-154MG Internet address: www.atsjournals.org
Individuals chronically exposed to cigarette smoke (CS), either as active smokers or through exposure to secondhand smoke, experience increased risk of developing of a variety of pulmonary diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and infections (1). Smoking not only induces cellular damage and inflammation but also serves as an immunosuppressor (1). This article evaluates the role of epithelial cells during innate immune defense responses, identifies mechanisms by which CS modulates innate immune responses, and develops hypotheses of immune mechanisms affecting CS-induced changes in the nasal microbiome. S38
Components of CS CS is a multicomponent mixture of gasphase and particle-phase toxins with known immunosuppressive effects. Many of the individual components of CS have known immunomodulatory function, carcinogenic activity, or addictive activities (2). They include metals, volatile hydrocarbons, and gas-phase components, such as carbon monoxide (2). Among the components of CS, polycyclic aromatic hydrocarbons (PAHs), acrolein, and reactive oxygen species have been individually studied for their effects on respiratory immune responses. Acrolein is a highly reactive unsaturated aldehyde and a known
respiratory irritant (3, 4). Acrolein is electrophilic with high reactivity toward biological nucleophiles, resulting in protein and DNA adducts, which is thought to mediate the adverse health effects of this inhaled toxin (4). PAHs are generated during incomplete combustion of organic materials, such as fossil fuels, wood, and tobacco, and are linked to adverse health effects induced by inhalation of CS and other combustion emissions, such as diesel exhaust and wood smoke. Thus, it is conceivable that exposure to wood smoke, diesel exhaust, and other emissions caused by incomplete combustion of organic matter poses similar hazards to public health as those induced by exposure to CS.
AnnalsATS Volume 11 Supplement 1 | January 2014
GILES F. FILLEY LECTURE General Effects of Smoking on Mucosal Immune Responses Exposure to CS affects the respiratory immune system on several levels. Innate immune and inflammatory cells, such as neutrophils and macrophages, become activated and expand, resulting in increased levels of proinflammatory cytokines/ chemokines, such as IL-8, TNF-a, MCP-1, and many more (2). CS-induced neutrophilia is marked by the infiltration of immature neutrophils, which have a highly proinflammatory phenotype, as evidenced by increased production of reactive oxygen species and enhanced myeloperoxidase levels (5), and therefore are more likely to cause tissue damage. Despite the expansion of neutrophils and macrophages, these cells demonstrate impaired phagocytic capabilities. For example, exposure of macrophages to CS decreases clearance of bacteria or apoptotic cells, such as apoptotic PMNs (6, 7). Similarly, exposure of PMNs to CS extract (CSE) increases spontaneous production of superoxide radicals but decreases bacterial-stimulated production of superoxide (8), which is necessary for the efficient elimination of the pathogen. Other macrophage functions associated with bacterial clearance, such as surface receptor expression, are affected by CS exposure. For example, expression of TLR2, which is important for the recognition and response to gram-positive bacteria, is reduced in macrophages from smokers (9). Similarly, expression of MARCO, a member of the class A scavenger receptor family important for the binding of gram-positive and gramnegative bacteria, is decreased in macrophages exposed to CSE (10). The effects of CS on dendritic cell (DC) function are less defined. Exposure of peripheral blood monocyte–derived myeloid DCs to CSE interferes with antigen presentation by these cells (11). Production of IFN-g, which is critical to direct a Th1-type immune response to the immune challenge, and expression of CCR7, which is important for migration of DCs to the lymph nodes, are decreased in bronchoalveolar lavage fluid samples from smokers (12). CSE exposure of plasmacytoid DCs, which are important in the context of viral infections, inhibits antiviral activities, as marked by a decreased release of type I interferons and
other cytokines involved in antiviral defense responses (13, 14). Our studies have demonstrated that viral infection of cocultures composed of nasal epithelial cells from smokers with DCs leads to reduced expression of antiviral signaling molecules in these cells as compared with cocultures composed of nasal epithelial cells from nonsmokers and DCs (15), suggesting that smoking affects the ability of epithelial cells to communicate and activate DCs. CS-induced alterations of natural killer (NK) cell function have also been demonstrated. NK cells play important roles during viral infections and in eliminating tumor cells via cell-mediated cytotoxicity. This function is mediated by the release of cytotoxins, such as perforin and granzyme B. In addition to targeted cytotoxicity, NK cells are important sources of cytokines, such as IFN-g in the context of microbial infections. We have previously demonstrated that the number and function of cytotoxic NK cells is decreased in smokers challenged with the live attenuated influenza virus (16). Similarly, in vitro exposure of peripheral blood mononuclear cells to CS-conditioned medium decreases RNA-induced cytotoxic function and IFN-b production by NK cells (17–19). Other innate host defense mechanisms, such as mucociliary escalator and antimicrobial peptide release, are affected by CS exposure. The mucus layer covering the airway epithelium traps pathogens and, in concert with coordinated beating of the ciliated epithelium, propels the mucus to the larynx. CS exposure affects the mucocililary escalator at several levels, including decreased ciliary beating, mucus hypersecretion, and squamous cell metaplasia (20). In addition, the epithelial integrity can be compromised by CS exposure through the disruption of tight junctions, leading to increased epithelial permeability (20, 21). The direct effects of CS exposure on epithelial cells is discussed in more detail below.
Epithelial Cells: A Central Target for CS Exposure Epithelial cells are among the first targets for inhaled toxins and are a primary site for infection with respiratory pathogens. Thus, interactions affecting host defense responses in the context of CS exposure are likely mediated by epithelial cells, at least during
Jaspers: CS Effects on Innate Immune Mechanisms in the Nasal Mucosa
the initial stages of the infection. The term “epimmunome” was introduced by Swamy and colleagues in 2010 and describes the concept that epithelial cells regulate communication with different sets of immune cells through the release or expression of cytokines, chemokines, or adhesion molecules and therefore are important regulators of immune responses (22). To simplify the concept introduced by Swamy and colleagues, one can divide the “epimmunome” into (1) soluble mediators directly protecting the host, (2) soluble mediators orchestrating the immune response, and (3) ligand–receptor interactions to activate and mature immune cells. In the context of CS exposure, all of these functions are altered in epithelial cells lining the respiratory tract. For example, epithelial cells show altered production and release of soluble antimicrobial peptides, such as human b-defensin 2 (hBD-2). However, the CS-induced alterations in hBD-2 may be site specific because hBD-2 was decreased in the central but not distal airways from smokers (23, 24). Other soluble factors released by epithelial cells that are important for orchestrating the recruitment, activation, and maturation of immune cells are cytokines or chemokines, which are altered in the context of CS exposure. In particular, expression of neutrophil chemokines (e.g., IL-8 and GROa) and macrophage chemokines (e.g., MCP-1) are enhanced in CS-exposed epithelial cells (25), thus contributing to the enhanced recruitment of these cells into the airways. Other means of communication between epithelial cells and resident immune cells include the expression of receptor–ligand interactions. For example, several MHC class I–related molecules are strong activating ligands for NKG2D, which is a surface receptor expressed on lymphocytes, including NK cells, cytotoxic T cells, and g/d T cells (26). UL16 binding proteins, which were originally identified as ligands for the UL16 glycoprotein of the human cytomegalovirus (27), are expressed on stressed epithelial cells (28), and we have recently shown that their expression can be altered in nasal epithelial cells by oxidants (29). Other potential NKG2D ligands expressed on epithelial cells that are altered in the context of CS exposure include retinoic acid early transcript 1 and MHC class 1 chain–related molecules A and B (30, 31). Altered expression of these ligands results in S39
GILES F. FILLEY LECTURE modified activation of NK cells or other cytotoxic lymphocytes (30, 32). Thus, the role of the “epimmunome” in the context of CS exposure is diverse and plays a key role in the manifestations of altered immune responses. A schematic illustrating CS-induced changes in mucosal immune responses is shown in Figure 1.
The Nasal Mucosa as a Tool to Study the Effects of Smoking on Innate Immune Responses Although exposure of animal models and in vitro exposure of cell culture models are important tools to uncover mechanisms of CS-induced immune dysfunction, translating these findings into humans in vivo is of great clinical importance. Over the years we have developed tools to study the effects of CS exposure on immune defense responses by examining the nasal mucosa. Using the nose as a model to study mucosal immune responses has several advantages: (1) the nose is accessible and easy to sample in a relatively noninvasive way; (2) the nasal epithelium resembles the airway epithelium morphologically, and smoking-related gene expression and histological changes (e.g., mucus production) are similar in nasal and airway epithelial cells (33–35); and (3) the nasal mucosa is the primary target for inhaled pathogens and has a robust microbial flora that can be studied for potential CS-induced changes in the microbiome. CS-induced alterations in the nasal mucosal immune responses in humans can be studied through several different experimental tools. Nasal lavage fluid, which
is noninvasively obtained through saline irrigation of the nostrils followed by expelling the sample into a specimen cup, can be analyzed for changes in soluble mediators, including cytokines/chemokines, proteases/antiproteases, and antimicrobial peptides (16, 36–39). The cellular components of the nasal lavage can be used for gene expression analysis or immunophenotyping using flow cytometry (16, 38, 40). Superficial scrape biopsies obtained from the human subjects can be analyzed by routine biochemical analysis tools such as qRT-PCR or flow cytometry to reveal important information regarding intraepithelial cell immune cell populations and changes in epithelial cell function (29, 35, 41). Thus, sampling the nose represents an important tool to understand potential effects of CS exposure on innate immune responses and potentially to link these changes with alterations in the nasal microbiome.
The Nasal Microbiome and Changes Induced by CS Exposure The nasopharyngeal flora are quite diverse and can be sampled using nasal lavage, swabs, and scrape biopsies. Unlike the lower airways, where smokers (without any significant comorbidities) and nonsmokers do not differ in the microbiota measurable in alveolar lavage fluid (42), studies have shown that the nasopharyngeal flora of smokers and nonsmokers differ (43). Specifically, the nasal microbiota in smokers contain fewer commensal aerobic and anaerobic organisms and show greater
Figure 1. Schematic outlining cigarette smoke–induced changes in mucosal immune responses. NK = natural killer.
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numbers of potential pathogens (43). Subsequent studies by the same group demonstrate that smoking cessation reverts the nasopharyngeal microbiota to normal (44) and that the altered nasal microbiome in parents who smoke can be transferred to their children (45). However, smokers with significant comorbidities, such as COPD, differ in their lung and lower airway microbiome (46, 47), suggesting that disease progression could be associated with changes in the microbial flora populating the lower airways. Potential mechanisms mediating CS-induced changes in the microbiome include inhalation of pathogens contained in tobacco, impaired antimicrobial defenses, and increased adherence of pathogens to the epithelium. Studies have shown that cigarettes contain bacteria that could potentially be inhaled (48). Among the bacteria identified in cigarettes are Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia (49). Thus, inhalation of mainstream CS could introduce bacteria contained in cigarettes into the lower airways. The impaired antimicrobial defenses induced by CS exposure include impaired phagocytic activity of macrophages and neutrophils, leading to delayed and/or reduced clearance of bacteria and the increased possibility of colonization. Dysfunction of the mucociliary escalatory also contributes to impaired clearance of bacteria in smokers. In addition, CS exposure can increase the adherence of bacteria to the epithelium. This can be achieved through increased expression of platelet-activating factor receptor (PAFR) on epithelial cells. Specifically, exposure to CSE increases PAFR expression on respiratory epithelial cells and enhances pneumococcal adhesion to these cells in a PAFR-dependent manner (50). In addition, CS can directly affect bacteria and enhance their virulence. For example, exposure of Staphylococcus aureus to CS increases biofilm formation and adherence to epithelial cells, which is mediated by oxidative stress and through the enhanced expression of fibronectin binding protein A (51). These observations suggest that, in addition to compromising antimicrobial defense responses, exposure to CS could affect the microbiome by increasing adherence and virulence of certain species.
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GILES F. FILLEY LECTURE Conclusions and Future Directions Existing and emerging data point to a relationship between exposure to CS, changes in the microbiome, and adverse
health effects. Understanding the dynamics of a healthy microbiome and how CS affects this balance are important to uncovering the mechanisms of CS-induced disease. The nasal mucosa and its microbial flora present an important tool to investigate the effects of
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