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. 2019 Nov 3;199(2):230–243. doi: 10.1111/cei.13388

Nicotine modulates molecules of the innate immune response in epithelial cells and macrophages during infection with M. tuberculosis

C E Valdez‐Miramontes 1,2, L A Trejo Martínez 1, F Torres‐Juárez 1,2, A Rodríguez Carlos 1,2, S P Marin‐Luévano 1,2, J P de Haro‐Acosta 1, J A Enciso‐Moreno 1, B Rivas‐Santiago 1,✉
PMCID: PMC6954679  PMID: 31631328

Summary

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll‐like receptors (TLR)‐2, TLR‐4 and NOD‐2 in all three cell types, as well as the production of the SP‐D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)‐6 and C‐C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL‐8, IL‐6, tumor necrosis factor (TNF)‐α, IL‐10, CCL2, C‐X‐C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP‐D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.

Keywords: cigarette smoke, epithelial cells, macrophages, Mycobacterium tuberculosis, nicotine

Nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.

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Introduction

The tobacco epidemic is one of the biggest public health problems in the world, killing more than 8 million people a year. The World Health Organization (WHO) estimates that more than 7 million of those deaths are the result of direct tobacco consumption, whereas approximately 1·2 million are the result of non‐smokers being exposed to second‐hand smoke. Cigarette smoking is considered the single most important preventable risk to humans, as it has been associated with multiple diseases. Epidemiological studies have reported that cigarette consumption increases the possibility of developing tuberculosis; for instance, it has been reported that individuals who smoke more than 20 cigarettes a day have two to four times more risk to reactivate tuberculosis 1. Similarly, other studies have provided evidence that smokers are five times more prone to develop active tuberculosis than non‐smoking individuals2. Moreover, it has been reported that the risk of death due to tuberculosis is nine times higher for smokers than for never‐smokers 3. Indeed, the WHO estimates that 7·9% of tuberculosis cases worldwide are attributable to smoking 4.

Cigarette smoke contains more than 4500 chemical components, including both particulate and vapor phases. It has been observed that chronic exposure to these particles can affect the functioning of the immune system. Interestingly, the chronic exposure of the vapor phase of cigarette smoke does not suppress the immune system, which indicates that a component in the particulate phase is immunosuppressive 5. Nicotine is a potent immunomodulatory molecule found in the particulate phase of the cigarette and it is known that it can inhibit the expression of important cytokines; indeed, nicotine is considered the main immunomodulatory molecule of the cigarette. Several studies have reported that smokers show alterations in several components of the innate immune system, such as in pathogen recognition receptors. It has been reported that Toll‐like receptors (TLR)‐4 and TLR‐2, which recognize lipid components of the mycobacterial envelope, are decreased in smokers in nasal epithelium and alveolar macrophages 6, 7. In addition to the TLRs, the nucleotide‐binding oligomerization domain cytoplasmic receptors (NOD), which have an important role in Mycobacterium tuberculosis (Mtb) recognition, are decreased in intestinal epithelia in the presence of cigarette smoke components, thus affecting its ability to recognize and therefore respond to infection 8.

Other important components of the innate immunity are associated with the elimination of Mtb with either anti‐microbial or immunoregulatory properties. Such is the case of surfactant proteins (SP), which are a complex of lipoproteins, synthesized and secreted mainly by type II alveolar cells; although there are four SPs, only SP‐A and ‐D have been related to innate immunity. Both surfactant proteins have shown important functions, acting as opsonins for Mtb and facilitating its elimination by macrophages 9, 10. It has been reported that cigarette smoke alters the component and function of pulmonary surfactant proteins. Several studies have shown that smokers have lower levels of both SP‐A and SP‐D 11, 12, thus creating conditions that allow the development of tuberculosis.

Another critical component related to the innate immunity against Mtb concerns cytokines and chemokines, which are molecules known to regulate cell anti‐microbial activity, proliferation, differentiation and chemotaxis. In this context, tumor necrosis factor (TNF)‐α has been identified as a key cytokine released by macrophages immediately after exposure to Mtb antigens, activating cells such as macrophages and dendritic cells for mycobacteria killing and granuloma formation 13. In addition, IL‐1β, IL‐12 and IL‐8 can induce the production of anti‐microbial substances by other cells 14, 15, 16, while other cytokines such as IL‐10 and IL‐6 have an opposite role, as they can modulate and suppress immune responses creating a favorable environment for the persistence of microbes 17, 18. Additionally, chemokines that attract monocytes, lymphocytes and polymorphonuclear (PMN) cells to the site of infection, such as C‐C chemokine ligand (CCL)2, CCL5, C‐X‐C chemokine ligand (CXCL)9 and CXCL10, are critical for mycobacterial elimination 19, 20.

Although there are reports of the effect of cigarette smoke on key molecules involved in the defense against Mtb, it is unknown whether nicotine modulates innate immunity during Mtb infection, therefore contributing to the susceptibility for developing tuberculosis. In the present study we sought to determine the effect of nicotine on the expression of TLR‐2, TLR‐4 and NOD2 receptors as well as SP‐A and SP‐D, IL‐6, IL‐8, TNF‐α, IL‐10, IL‐8 and CCL‐2, CCL‐5, CXCL9 and CXCL10 and in lung epithelial cells and macrophages during infection with Mtb.

Materials and methods

Cell preparation and Mtb culture

The A549 cell line, further referred to as type II pneumocytes (A549 ATCC® CCL185™; American Type Culture Collection, Manassas, VA, USA) and NCI‐H292, further referred to as airway basal epithelial cells (NCI‐H292, ATCC® CRL1848™), were grown in 75‐cm2 culture flasks (Corning Cellgro, Manassas, VA, USA) with Roswell Park Memorial Institute medium (RPMI‐1640; Corning Cellgro) supplemented with 10% fetal bovine serum (FBS; Corning Cellgro) and 100 μg/ml of antibiotic penicillin (×100; Corning Cellgro), except for the cell line NCI‐H292 which, in addition to all the above, was supplemented with 2 mM of L‐glutamine (200 mM; Corning Cellgro). Both cell lines were incubated at 37°C 5% CO2 until they reached a confluence of between 80 and 85%, and thereafter were seeded in 24‐well plates (Thermo Fisher Scientific, Waltham, MA, USA) with 1% FBS and maintained for 24 h in the presence of 5% CO2 at 37°C until infection with Mtb.

Macrophages derived from human monocytes (MDM) were obtained according to the Declaration of Helsinki and approved by the National Committee of Ethics and National Commission of Scientific Research of the Mexican Institute of Social Security (IMSS). Briefly, after obtaining written informed consent, subjects underwent venipuncture, and heparinized blood was obtained from four purified protein derivative (PPD)‐negative healthy donors, none of whom had a history of prior exposure to tuberculosis patients. The isolation and differentiation to macrophages procedure was carried out according to previous reports 21. Briefly, peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (StemCell Technologies, Vancouver, Canada). PBMC (2·5 × 106) were cultured in RPMI medium using 24‐well ultra‐low adhesion plates (Costar, Corning, NY, USA). After 2 h, non‐adherent cells were removed and subsequently differentiated for 7 days with RPMI medium (Corning Cellgro) supplemented with 10% of decomplemented pool human AB serum (Biowest, Riverside, MO, USA). To assess macrophage phenotype, the CD68 differentiation marker was evaluated by flow cytometry, obtaining greater than 95% of positive cells (data not shown).

Infection and stimuli

Mtb H37Rv strain (ATCC 27294) was cultured in 25‐cm2 flasks with 20 ml Middlebrook 7H9 medium (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 10% oleic acid, albumin, dextrose and catalase (OADC enrichment medium; BBL, Becton Dickinson), 0·5% Tween 80 and 0·2% glycerol and incubated at 37°C with a 5% CO2 atmosphere. The strain was kept in culture until reaching the logarithmic phase, which was determined by spectrophotometry. The culture was then divided into working aliquots of 1 × 107 cells/ml. Suspension aliquots were centrifuged for 5 min at 6000 g, and the resulting Mtb pellets were declumped by vortexing (5 min) with five sterile 3‐mm glass beads in 1 m of RPMI medium enriched with 10% pooled human AB serum (Biowest, Mexico City, Mexico). The remaining Mtb clumps were removed with an additional centrifugation step at 350 g for 5 min. The Mtb suspension volumes required to obtain the desired multiplicity of infection (MOI = 5) were calculated based on the colony‐forming unit (CFU) numbers known to be present in the Mtb suspension supernatants. The actual CFU numbers used for in vitro infections were confirmed in each experiment.

Cells were infected for 2 h; the non‐phagocytosed mycobacteria were then removed through vigorous washing with phosphate‐buffered saline (PBS). Subsequently, stimulations were performed using RPMI medium at 1% for the epithelial cells (EpCs). Macrophages were infected in the presence of human serum AB (Biowest, Mexico) at 30%. Subsequently, stimulation with nicotine (Sigma‐Aldrich, St Louis, MO, USA) was performed using a 10 μM concentration, as it has been reported that this is the average plasma concentration found in smokers 22. Stimulation was carried out in RPMI with 10% of the decomplemented human serum for 18 and 24 h. In some cases, mecamylamine hydrochloride (Abcam Biochemicals, Bristol, UK) was used, which is a non‐competitive antagonist of the nicotinic acetylcholine receptor α7 23, and was added at the time of infection at a concentration of 5 μM, 2 h before the stimulation with nicotine, and was added again during stimulation. During all the experiments a control condition was added, which consisted in the cultured cells without any stimuli (none). Once the stimulation time had elapsed, the supernatant was removed and the cells disintegrated enzymatically for subsequent antibody labeling. To determinate cellular viability after the stimuli, a cell viability assay was performed using Guava ViaCount Assay (Millipore, Billerica, MA, USA), showing 95% viability.

TLR‐2, TLR‐4 and NOD‐2 evaluation using flow cytometry

After obtaining the epithelial cells (EpCs) or MDMs, the cells were placed in polystyrene tubes (Corning) to perform the corresponding immunolabeling. Cells were fixed and permeabilized using cytofix/cytoperm (BD Biosciences, San Diego, CA, USA) for 20 min. Subsequently, the corresponding antibodies were added: TLR‐2‐Alexa488 (BD Biosciences) and NOD‐2‐Alexa647 (Novus Biologicals, Centennial, CO, USA); TLR‐4‐phycoerythrin (PE) (BD Biosciences, San Diego, CA, USA) labeling was performed separately. For CD68+ analysis in macrophages, anti‐CD68‐peridinin chlorophyll (PerCP) was used (Santa Cruz Biotechnology, Dallas, TX, USA). All antibodies were incubated for 30 min at 4°C protected from light. Subsequently, the cells were washed twice and resuspended for subsequent analysis in a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using the FACSDiva software version 6.1.3.

Quantification of SP‐A and SP‐D by ELISA

Supernatants from each experimental condition in type II pneumocytes were collected in Eppendorf tubes with a protease inhibitor (Thermo Scientific) and samples were stored at –70°C until use. Measurement of the surfactant proteins was carried out using the enzyme‐linked immunosorbent assay (ELISA) kit, SP‐A (BioVendor Research, Asheville, NC, USA) and SP‐D (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

Cytokine determinations using cytometric bead arrays

Cytokine and chemokine concentrations for IL‐10, TNF‐α and IL‐1β (mature form), IL‐6, IL‐8 and IL‐12 and transforming growth factor (TGF‐β), as well as CCL2, CCL5, CXCL9 and CXCL10, were measured in cell supernatants using the BD cytometric bead array (CBA) system (BD Biosciences). For these determinations, 50 μl from each cell supernatant was used. Fluorescence intensity (FI) from the immunoassay was measured using a BD FACScanto II cytometry system and the FACSdiva version 6.1.3, and for data CBA analysis the BD FCAP Array software version 3.0.1 was used (BD Biosciences).

Statistical analysis

Statistical analyses were performed using the GraphPad Prism software for Mac (GraphPad Software version 6.01; GraphPad, San Diego, CA, USA). Normal distribution was assessed using the Kolmogorov–Smirnov test for each data set, together with a non‐parametric two‐group comparison Mann‐Whitney U‐test or the Kruskal–Wallis multiple comparison test to identify differences among the groups. When statistical significance (P < 0·05) was found, a Dunn’s post‐test was performed. Two‐sided P‐values of < 0·05 were considered statistically significant.

Results

Nicotine does not affect the viability of the EpCs and MDMs

Because it has been reported that the concentration of nicotine found in the blood of smokers is approximately 10 μM 22, whether this concentration affects the viability of the cells used for the present study was evaluated. Our results show that neither nicotine, even at higher concentrations (30 μM) nor the mecamylamine showed cytotoxicity in any of the tested cells (Fig. 1a–c).

Figure 1.

Figure 1

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Viability of the cells treated with nicotine. Viability was assessed in type II pneumocytes (a), airway basal epithelial cells and (c) macrophages derived from human monocytes (MDMs) treated with different nicotine concentrations, mecamylamine and dimethylsulfoxide (DMSO) as positive control. ***P < 0·001; **P < 0·005.

Nicotine decreases the expression of TLR‐2 in EpCs and MDMs

To explore whether nicotine affects the expression levels of TLR‐2, TLR‐4 and NOD‐2, the expression changes of these receptors were evaluated after 18 and 24 h of nicotine treatment with or without Mtb infection. The results, expressed as mean fluorescence intensity (MFI), showed that nicotine significantly decreases the expression of TLR‐2 at 18 h post‐stimulation in type II pneumocytes, compared to the control (P < 0·01) (Fig. 2a). Similarly, nicotine decreased the expression of this receptor in these cells (P < 0·05) after 24 h (Fig. 2b). At both times, a combination of the treatment with nicotine and infection decreased the expression of TLR‐2 even when compared with nicotine treatment alone (P < 0·01 and P < 0·001, respectively, for 18 and 24 h). Similarly, in airway epithelial basal cells, nicotine decreased TLR‐2 expression when compared with the control (P < 0·05) at both 18 and 24 h (Fig. 2c,d). This shift is more evident with a combination of nicotine treatment and infection (P < 0·01, P < 0·001). For MDMs, a decrease in receptor expression was observed only 18 h after nicotine treatment (P < 0·01) (Fig. 2e). Only infection with Mtb with or without the presence of nicotine showed a difference when compared with the control (Fig. 2f). Mecamylamine treatment did not restore the decreased receptor expression in any of the evaluated cells.

Figure 2.

Figure 2

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Effects of nicotine on Toll‐like receptor (TLR)‐2 expression during Mycobacterium tuberculosis (Mtb) infection. Pneumocytes type II (a,b), airway epithelial basal cells (c,d) and macrophages derived from human monocytes (MDMs) (e,f) were stimulated with nicotine for 24 h; subsequently, cells were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for 18 (a,c,e) and 24 h (b,d,f). TLR‐2 was assessed using mean fluorescence intensity (MFI). Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0·0001; n.s. = not significant.

Nicotine decreases the expression of TLR‐4 in EpCs and MDMs

TLR‐4 expression analysis showed that infection with Mtb with or without nicotine reduces the expression of this receptor mainly at 18 h post‐infection (P < 0·0001) (Fig. 3a). At 24 h post‐infection, nicotine decreases the expression of this receptor significantly in type II pneumocytes (P < 0·05) (Fig. 3b). Regarding the airway epithelial basal cells, nicotine decreased the TLR‐4 expression when compared with the control (P < 0·05) at both 18 and 24 h (P < 0·05) (Fig. 3c,d). This shift is more evident with the combination of nicotine treatment and infection (P < 0·01, P < 0·001). However, there was no difference between Mtb‐infected and Mtb‐infected + nicotine cells. Infection with Mtb with or without nicotine decreases the receptor expression in MDMs compared with the control condition after 18 h (P < 0·05) (Fig. 3e). However, at 24 h nicotine treatment can reduce the expression of the receptor on its own (P < 0·05) (Fig. 3f). Mecamylamine treatment did not restore the decreased receptor expression in any of the evaluated cells.

Figure 3.

Figure 3

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Effect of nicotine on Toll‐like receptor (TLR)‐4 expression during Mycobacterium tuberculosis (Mtb) infection. (a) Pneumocytes type II (a,b), airway epithelial basal cells (c,d) and macrophages derived from human monocytes (MDMs) (e,f) were stimulated with nicotine for 24 h; subsequently, they were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for 18 (a,c,e) and 24 h (b,d,f). TLR‐4 was assessed using mean fluorescence intensity (MFI). Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0·0001.

Nicotine decreases the expression of NOD‐2 in type II pneumocytes and MDMs

NOD‐2 expression analysis showed that nicotine treatment decreased the expression of this receptor at both 18 and 24 h (P < 0·05) (Fig. 4a,b). Infection with Mtb with or without the presence of nicotine decreased the receptor expression substantially (P < 0·01 and P < 0·001). Moreover, with regard to the airway epithelial basal cells, only infection with Mtb induced a reduction in NOD‐2 (P < 0·01 and P < 0·001, respectively, for 18 and 24 h) (Fig. 4c,d); similar results were observed in MDMs. Infection reduced receptor expression at 18 h. However, after 24 h post‐infection, the nicotine alone reduced the receptor expression (P < 0·01 and P < 0·001, respectively, for 18 and 24 h) (Fig. 4e,f).

Figure 4.

Figure 4

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Effect of nicotine on nucleotide‐binding oligomerization domain cytoplasmic receptor‐2 (NOD2) expression during Mycobacterium tuberculosis (Mtb) infection. Pneumocytes type II (a,b), airway epithelial basal cells (c,d) and macrophages derived from human monocytes (MDMs) (e,f) were stimulated with nicotine for 24 h; subsequently, they were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for 18 (a,c,e) and 24 h (b,d,f). NOD2 was assessed using mean fluorescence intensity (MFI). Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05; **P < 0·01; ***P < 0·001.

Nicotine decreases the expression surfactant A in type II pneumocytes

To assess whether nicotine affects the production of surfactant proteins A and D, the amount of these proteins was determined in the supernatant of type II pneumocytes after infection and/or nicotine stimulation. The results depicted in nanograms/milliliters showed that nicotine decreased the expression of SP‐A, and infection with Mtb in the presence of nicotine decreases the protein expression even more, compared with the none condition (P < 0·05) (Fig. 5a). Moreover, the SP‐D protein was not detected with any of the treatments evaluated (Fig. 5b).

Figure 5.

Figure 5

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Effect of nicotine on surfactant proteins expression during Mycobacterium tuberculosis (Mtb) infection. Type II pneumocytes were stimulated with 10 μM of nicotine for 24 h; subsequently, they were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for 24 h. Surfactant protein A and D (a,b, respectively) were measured in supernatants. Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05.

Nicotine affects the expression of cytokines in EpCs and MDMs

In order to determine the production of cytokines such as IL‐6, IL‐8, TNF‐α, IL‐10 and TGF‐β in EpCs and MDMs during Mtb infection, supernatants were collected after infection or nicotine stimulation; cytokine levels were then assessed using CBA. As depicted in Fig. 6, cytokine levels in type II pneumocytes showed that Mtb infection decreased IL‐6 (P < 0·05) (Fig. 6a). Regarding TGF‐β, nicotine alone decreases the expression of this cytokine (P < 0·001), a change that is inverted with the infection (P < 0·01) using mecamylamine (P < 0·001) (Fig. 6). IL‐8 expression remained unchanged in all conditions (Fig. 6b). In airway epithelial basal cells, IL‐6 expression decreased with a combination of infection and nicotine (P < 0·001) (Fig. 6d), while TGF‐β was found to be increased during Mtb infection, which becomes more evident with the presence of nicotine. However, the mecamylamine partially reversed this increment (P < 0·05) (Fig. 6f). No differences were found in these cells regarding the production of IL‐8 with any of the treatments used (Fig. 6e). Interestingly, the analysis of the production of cytokines in MDMs showed a different pattern. It was observed that IL‐6 levels increased with Mtb infection (P < 0·001), although this increase was partially reversed with the combination of nicotine (P < 0·01) (Fig. 6g). The same pattern was observed for IL‐8 (P < 0·001 and P < 0·01) (Fig. 6h). TGF‐β and IL‐10 levels were decreased in cells with nicotine treatment. However, no change was observed during Mtb infection when compared with non‐infected cells only with TGF‐β expression (P < 0·05) (Fig. 6i,k). Regarding TNF‐α levels, CBA analysis showed that infection increased the levels of this cytokine (P < 0·0001), while nicotine decreased TNF‐α levels during infection (P < 0·01) (Fig. 6j). TNF‐α and IL‐10 were identified in macrophages, but not in EpCs.

Figure 6.

Figure 6

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Effect of nicotine on cytokine expression during Mycobacterium tuberculosis (Mtb) infection. Type II pneumocytes (a–c), airway epithelial basal cells (d–f) and macrophages derived from human monocytes (MDMs) (j,k) were stimulated with 10 μM of nicotine for 24 h; subsequently, they were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for another 24 h. Cytokine levels were measured in supernatants. Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0·0001.

Nicotine affects the expression of chemokines in EpCs and MDMs

Chemokine levels were assessed using CBA in the different types of cells, as depicted in Fig. 7a, in type II pneumocytes; Mtb infection plus nicotine increased CCL2 levels (P < 0·05). Regarding CCL5 levels, results showed that nicotine decreases its expression, which is more evident during infection (P < 0·05) (Fig. 7b). It was observed that both nicotine and infection increased the expression of CCL5 in airway epithelial basal cells (Fig. 7c) (P < 0·01 and P < 0·05), although CXCL10 was found to be reduced with nicotine treatment (P < 0·05), which was more noticeable with infection and a combination of infection and nicotine (P < 0·01) (Fig. 7d). Nicotine reduces the expression of CCL2 in MDMs (P < 0·05) (Fig. 7e), whereas Mtb infection increased the production of this chemokine (P < 0·01). CCL5 only increases during Mtb infection with or without nicotine (P < 0·05) (Fig. 7f). Moreover, when the supernatant levels of CXCL9 and CXCL10 where analyzed, results showed that infection increases the levels of these chemokines, while the combination of infection and nicotine decreased these levels (P < 0·05) (Fig. 7g,h). CXCL9 and CXCL10 were detected in macrophages, but not in EpCs.

Figure 7.

Figure 7

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Effect of nicotine on chemokines expression during Mycobacterium tuberculosis (Mtb) infection. Type II pneumocytes (a,b), airway epithelial basal cells (c,d) and macrophages derived from human monocytes (MDMs) (e–h) were stimulated with 10 μM of nicotine for 24 h; subsequently, they were infected with Mtb [multiplicity of infection (MOI) = 5 : 1] for another 24 h. Representative chemokines for each type of cell type were measured in supernatants. Graphs are represented as mean ± standard deviation (s.d.). *P < 0·05; **P < 0·01.

Discussion

Nicotine is a lipophilic molecule that is found in the particulate phase of tobacco smoke and has been considered as an immunomodulator 5. Several studies have reported that exposure to nicotine dramatically reduces the production of antibodies and the proliferation of T lymphocytes with values ​​similar to those exposed to cigarette smoke 24, 25, 26, suggesting that this modulation of the immune system leads to an increased susceptibility to infectious diseases, such as tuberculosis 1, 2. Previous studies have demonstrated that both nicotine‐exposed monocytes and alveolar macrophages showed higher rates of Mtb intracellular growth in comparison to non‐nicotine‐exposed cells 27. In the present study, we evaluated whether nicotine affects key molecules of the innate immune response in epithelial cells or macrophages during infection with Mtb.

One cigarette contains an average of 8·4 mg of nicotine. When tobacco is burned, nicotine is aerosolized into tar droplets that deliver ∼1·6 mg of nicotine per cigarette. Inhaled tobacco smoke reaches the small airways and alveoli of the lungs, where 82–92% of nicotine is absorbed. Several studies have reported that the mean concentration of nicotine in smokers serum is approximately 1–2 μg/l (10–30 μM) 28. Thus, whether the use of these concentrations had a cytotoxic effect on the cells studied was evaluated. Using both 10 and 30 μM concentrations, it was determined that the effect observed was due to the stimuli instead of any possible cytotoxicity. In previous studies, it has been reported that the MOI used for the present study does not affect cellular viability 29. The selection criteria using 18 and 24 h to evaluate the expression of TLRs was based on previous studies, where it was demonstrated that stimulation of human macrophages with Mtb modulates the expression of TLR‐2 and TLR‐4, mainly at 24 h post‐infection 30. Other studies have shown the effect of cigarette smoke extract on the expression of TLRs in the human bronchial epithelial cell line (16‐HBE), where it was found that the expression of TLR‐4 decreased at 18 h of stimulation 31.

Toll‐like receptors are one of the main mechanisms that promote an effective immune response against Mtb. TLR‐2 is expressed throughout the human airway epithelium, predominantly in bronchial and alveolar epithelial cells, which respond to a wide variety of mycobacterial components 32. It has been observed that TLR‐2‐deficient mice infected with Mtb are more susceptible to developing progressive infection compared to wild‐type mice. Additionally, these mice showed defects in controlling chronic Mtb infection 33. Another key receptor identified in Mtb recognition is TLR‐4; this receptor is activated by the thermal shock protein p60/65, a protein secreted by several species of Mtb 34, 35, 36. TLR‐4–/– mice have shown an increased susceptibility to Mtb infection, similar to that observed in TLR‐2‐deficient mice 37. The NOD‐2 receptor has an important role in mounting an appropriate immunological activity against Mtb 38, 39. It has been reported that NOD‐2‐deficient mice have a deficient production of cytokines and nitric oxide when infected with Mtb which, in turn, makes them more prone to developing active tuberculosis 40. Due to the importance of these receptors in recognition of Mtb, we assessed whether or not nicotine can modulate its expression. The results showed that nicotine can reduce the expression of TLR‐2, TLR‐4 and NOD‐2 in EpCs and MDMs. These results are consistent with those reported previously, where A549 cells exposed to cigarette smoke extract decreased TLR‐4 expression substantially. Similar results were obtained regarding TLR‐4 expression in the nasal epithelium of smokers compared to non‐smokers 6. The present study is the first report, to our knowledge, of the effects of nicotine on the expression of TLRs in respiratory cells and MDMs. The results observed in TLR expression could suggest that nicotine suppresses recognition of Mtb through these receptors, inhibiting signaling pathways that lead to cytokine and anti‐microbial peptide expression important for mycobacteria elimination. It was observed that NOD‐2 expression is diminished in type II pneumocytes and MDMs after being stimulated with nicotine. This modulation would probably lead to weakening innate immunity, affecting the ability to detect and respond to mycobacterial components.

Furthermore, the present results showed that during Mtb infection, EpCs and MDMs decreased the expression of the three receptors evaluated. Analysis of these receptors did not show a synergistic effect when using a combination of infection and treatment with nicotine, which indicates that the observed decrease is an effect attributed mainly to Mtb. It has been reported elsewhere that Mtb lipid fractions could decrease TLR‐2, TLR‐4 and MHC class II expression as a Mtb evading mechanism 30.

Following the results obtained in the expression of Mtb recognition receptors, we investigated whether nicotine could modulate the expression of SP‐A and SP‐D, which are produced by type II pneumocytes and have essential functions for the elimination of Mtb 9, 10. It has been observed that both proteins can bind to mycobacterial‐wall lipoarabinomannan 41, 42, enhancing the Mtb uptake via mannose receptors 43. In the present study, SP‐A decreased in the presence of nicotine in Mtb‐infected EpCs. Previous studies have found that both proteins are decreased in bronchioalveolar lavage from smokers 44, which could imply that this deficiency increases the risk of mycobacterial infection by compromising the innate immune response. Interestingly, SP‐A is decreased in bronchoalveolar lavage of patients with Mtb, but these levels are restored after anti‐Mtb therapy 45. Taken together, this suggests that the nicotine contained in cigarette smoke down‐modulates the expression of SP‐A in smokers who, after exposure to Mtb, would not adequately opsonize the bacteria, and therefore could not mount an efficient recognition and phagocytosis process. Surfactant protein D was not detected in type II pneumocytes in any of the studied conditions; similar results have been previously reported in these cell types 46.

The key role of several cytokines, such as IL‐6, IL‐8, TNF‐α, TGF‐β and IL‐10 18, 47, 48, 49has been widely demonstrated in the response to tuberculosis. Moreover, an important function for chemokines such as CCL‐2, CCL‐5, CXCL9 and CXCL10 has been established in the defense against tuberculosis promoting the recruitment of leukocytes from the blood to lung 50.

In this study we observed that nicotine can decrease the production of IL‐6 during infection with Mtb in airway epithelial basal cells. In addition to IL‐6, there is also a decrease in IL‐8, TNF‐α and IL‐10 in MDMs when infected with Mtb. It has been widely reported that nicotine can induce an anti‐inflammatory effect, suppressing the production of proinflammatory cytokines in monocytes and macrophages in response to stimuli of exogenous or endogenous ligands through TLR‐2, TLR‐3, TLR‐4, TLR‐7 and TLR‐9 51. Hence, this modulation in cytokine expression could be related to the susceptibility of diseases related to tobacco consumption, such as tuberculosis. Moreover, infection with Mtb alone induces increased levels of IL‐6, IL‐8, IL‐10 and TNF‐α in MDMs, similar to previous findings 52. Interestingly, it was observed that when the EpCs were infected the nicotine increased TGF‐β levels. This finding coincides with previous studies, where it was observed that nicotine induced TGF‐β production by regulatory T cells (Treg) cells 53. As this cytokine is known to reduce macrophage antibacterial‐related molecules, these data suggest that nicotine promotes a TGF‐β‐rich microenvironment impairing Mtb growth.

It was identified that nicotine decreases CCL5 expression in chemokines, alone or in combination with Mtb in EpCs. This same pattern was observed for CXCL10, which was only detected in airway epithelial basal cells. It was observed that nicotine decreases the expression of CCL2, CXCL9 and CXCL10 in MDMs. Mtb infection induced over‐expression of most of the evaluated chemokines, which correlates with previous findings 54. This could indicate that nicotine deregulates chemokine production, thus inhibiting leukocyte recruitment during Mtb infection. Other studies have reported that nicotine reduced leukocyte infiltration in the lung, as well as the production of proinflammatory cytokines and chemokines, after treatment with lipopolysaccharide (LPS) 55.

An interesting finding in these results was that the blockade of the nicotinic acetylcholine receptor α7 using mecamylamine did not reverse the decrease of any of the studied receptors or the surfactant proteins, suggesting that modulation of the expression of these receptors induced by nicotine is probably mediated by another route, involving activation of another receptor or even an independent receptor pathway. Although this receptor has been described as the main receptor involved in the nicotine‐related immune response, some authors have proposed the participation of other nicotinic receptors 56 – for instance, the participation of the α4β2 nicotinic receptor. Nicotine binds to this receptor in glial cells and blocks the activity of the transcription nuclear factor kappa B (NF‐κB) through the activation of Janus kinase 2 and JAK2–signal transducer and activator of transcription 3 (STAT3). This pathway is shared with the α7 receptor to inhibit the production of proinflammatory cytokines 57. However, further research is needed to identify the mechanisms by which nicotine regulates the activation of these receptors in immune system cells. Taken together, the present results show that nicotine could modulate the expression of innate immunity molecules necessary to control Mtb growth.

Disclosures

Authors declare no conflicts of interest.

Author contributions

Original idea: B. R. S.; experimental procedures: L. A. T. M., F. T. J., C. E. V. M., S. P. M. L., A.R. C. and J. P. H. A.; writing, review and editing: B. R. S., C. E. V. M. and J. A. E. M.

Acknowledgements

We thank CONACyT for the grant CICB2015‐255236 and IMSS for the grant FIS/PROT/PRIO18/064. C. V. M. has a scholarship from CONACyT and B. R. S. has a scholarship from Fundación IMSS. We would also like to thank Lucca Stine for reviewing and correcting the manuscript.

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