Exposure to Cigarette Smoke Inhibits the Pulmonary T-Cell Response ...

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INFECTION AND IMMUNITY, Jan. 2011, p. 229–237 0019-9567/11/$12.00 doi:10.1128/IAI.00709-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

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Exposure to Cigarette Smoke Inhibits the Pulmonary T-Cell Response to Influenza Virus and Mycobacterium tuberculosis䌤 Yan Feng,1† Ying Kong,1†‡ Peter F. Barnes,1,2 Fang-Fang Huang,1 Peter Klucar,1§ Xisheng Wang,1 Buka Samten,1,2 Mayami Sengupta,1¶ Bruce Machona,1储 Ruben Donis,3 Amy R. Tvinnereim,1 and Homayoun Shams1,2* Center for Pulmonary and Infectious Disease Control,1 Departments of Microbiology and Immunology,2 The University of Texas Health Science Center at Tyler, Tyler, Texas 75708, and Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, Georgia 303333 Received 1 July 2010/Returned for modification 31 August 2010/Accepted 19 October 2010

Smoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected with Mycobacterium tuberculosis by aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenic M. tuberculosis protein. Gamma interferon (IFN-␥) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-␥ during stimulation in vitro with anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis and influenza A virus in vivo. Reduced IFN-␥ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-␥ expression. Cigarette smoke exposure increased the bacterial burden in mice infected with M. tuberculosis and increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response to M. tuberculosis and influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens. derscores the potential for this infection to spread rapidly and cause substantial morbidity and mortality. Given the prevalence of exposure to cigarette smoke, it is critical to determine if cigarette smoke increases susceptibility to tuberculosis and influenza or if it affects the efficacy of vaccination against these diseases. T cells contribute significantly to host defenses against both Mycobacterium tuberculosis and influenza virus. CD4⫹ cells produce gamma interferon (IFN-␥), which activates macrophages to kill intracellular M. tuberculosis, in part through the induction of nitric oxide (3, 5, 39). CD8⫹ T cells are critical for the clearance of influenza virus infection (9, 10, 44), and recent evidence suggests that CD4⫹ cells also contribute to immunity against influenza (4, 7, 42). Limited information is available on the effects of cigarette smoke on T-cell responses to M. tuberculosis and influenza infection. Most studies of the effects of smoking on the immune response have used in vitro assays, such as the addition of tobacco extracts to cell cultures or nicotine exposure through subcutaneous pumps, which impairs T-cell proliferation and T-cell receptor-mediated signaling (14). We evaluated the effect of cigarette smoke exposure on the lung immune response to M. tuberculosis and influenza A virus, using a physiologically relevant model in which animals were exposed to environmental cigarette smoke. Using this model, we also investigated how cigarette smoke exposure affected the mucosal response to vaccination against M. tuberculosis.

It is estimated that 5.5 trillion cigarettes are produced globally each year and are smoked by more than 1.1 billion people (http://www.who.int/tobacco/en/atlas8.pdf). An even greater number of people are exposed to second-hand cigarette smoke, when smoke exhaled by the smoker is inhaled by others. Epidemiological studies have shown that tuberculosis and influenza are both more common in smokers than nonsmokers (1, 12, 19, 20, 27, 30). However, it is unclear how cigarette smoke exposure increases susceptibility to these infections. Candidate vaccines against both tuberculosis and influenza are being tested in clinical trials, but it is uncertain if exposure to cigarette smoke reduces the efficacy of these vaccines. Tuberculosis claims 1.9 million lives annually world-wide, and influenza kills an estimated 40,000 persons annually in the United States. Pandemic influenza has killed up to 50 million persons (45), and the recent pandemic of H1N1 swine influenza un-

* Corresponding author. Mailing address: Center for Pulmonary and Infectious Disease Control, The University of Texas Health Science Center at Tyler, Tyler, TX 75708. Phone: (903) 877-2837. Fax: (903) 877-7989. E-mail: [email protected] † These authors contributed equally. ‡ Present address: Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University, 407 Reynolds Medical Bldg., College Station, TX 77843-1114. § Present address: Baylor Institute for Immunology Research, 3434 Live Oak St., Dallas, TX 75204. ¶ Present address: Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461. 储 Present address: Department of Biological Sciences, CW 405, Biological Sciences Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2E9. 䌤 Published ahead of print on 25 October 2010.

MATERIALS AND METHODS Animals. Specific-pathogen-free 6- to 8-week-old female DR4 transgenic mice expressing the human HLA-DRB1*0401 allele (DR4) or their wild-type

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C57BL/6 counterparts were purchased from Taconic (Albany, NY) or bred at the University of Texas Health Science Center at Tyler. The Institutional Animal Care and Use Committee approved all of the protocols for the animal experiments. Exposure to second-hand cigarette smoke. Mice were randomly divided into unexposed (control) and cigarette smoke-exposed groups. A smoke exposure chamber (Teague Enterprises, Davis, CA) was used to expose animals to a combination of fresh air and a defined level of cigarette smoke, mimicking natural exposure to environmental cigarette smoke in daily life. We exposed the mice to cigarette smoke from a 1R4F cigarette (University of Kentucky Reference Cigarette) for 120 min, twice (with a 2-h break in between) daily, 5 days a week for 6 weeks. The total smoke particulate concentration during exposure was approximately 80 mg/m3. Control animals were kept in an adjacent room. Mice that were infected with M. tuberculosis were housed in the biosafety level 3 laboratory, where a smoke exposure chamber is not available. Therefore, mice were not exposed to cigarette smoke after infection. Mice that were infected with influenza A virus continued to be exposed to cigarette smoke after infection. Isolation of T cells. Cell suspensions generated from lungs and spleens were centrifuged, red blood cells were lysed, and the cells were washed twice with complete RPMI medium. T cells were isolated by immunomagnetic separation according to the manufacturer’s recommendations (Miltenyi Biotec). Cell purity was confirmed by flow cytometry. T-cell stimulation. Cells were stimulated with suboptimal concentrations of anti-CD3/anti-CD28. Cells were added to 96-well plates, precoated with 3 ␮g/ml anti-mouse CD3 and 1 ␮g/ml anti-mouse CD28, and incubated at 37°C, 95% relative humidity, and 5% CO2. The supernatants were collected 72 h later and stored at ⫺70°C for the measurement of IFN-␥ levels by enzyme-linked immunoassay (ELISA). For the enzyme-linked immunospot (ELISPOT) assay, plates were coated with anti-CD3, anti-CD28, and anti-IFN-␥. Cells then were added and incubated for 24 to 48 h, as noted below. ELISPOT assay to determine the frequency of IFN-␥-producing T cells. The ELISPOT assay was performed as previously described (21). To evaluate stimulated cells, plates were coated with 3 ␮g/ml anti-CD3, 1 ␮g/ml anti-CD28, and 10 ␮g/ml anti-IFN-␥. After overnight incubation and blocking, cells were added to the plates and incubated for 24 to 48 h. After being washed, the detection antibody (Ab) R4-6A2-Biotin (Mabtech) was added, followed by streptavidinalkaline phosphatase (Mabtech) and a substrate solution (5-bromo-4-chloro-3indolylphosphate-nitroblue tetrazolium [BCIP-NBT]; BD Bioscience). Plates were rinsed with tap water and air dried, and spots were counted with a stereomicroscope (Olympus SZ2-LGB). ELISA to measure IFN-␥ concentrations. The ELISA was performed as previously described (21), using coating and detection monoclonal Abs (MAbs) to mouse IFN-␥ (Mabtech). The sensitivity of the assay was 50 pg/ml. DNA immunization. The construction of the pcDNA3.1(⫹) eukaryotic expression vector to express the M. tuberculosis 10-kDa culture filtrate protein (CFP10) and lysosomal integral membrane protein II and vaccination with this construct were performed as described previously (21). Briefly, endotoxin-free plasmid was diluted in 5% glucose to 1 mg/ml, and 100 ␮g of plasmid DNA was mixed with linear polyethylenimine (PEI; Bridge Bioscience) solution according to the manufacturer’s instructions, and 200 ␮l of the mixture containing 100 ␮g of plasmid DNA was administered once intravenously (i.v.) via the retro-orbital route, as described elsewhere (21). Animal infections. (i) Tuberculosis. Mice were infected with M. tuberculosis H37Rv via aerosol, using an exposure chamber made to order by the University of Wisconsin. To confirm the accuracy of infection, 24 h after infection, 5 mice were sacrificed, their lungs were homogenized and plated on 7H10 agar, and the number of CFU was determined. We reproducibly infected mice with 10 to 25 CFU. (ii) Influenza. Mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus was grown in the allantoic cavity of 10-day-old embryonated hen eggs and stored at ⫺70°C. Uninfected allantoic fluid also was collected and stored at ⫺70°C as a control inoculum. In all experiments, mice were anesthetized and intranasally inoculated with 50 ␮l of influenza virus or uninfected allantoic fluid. Infected mice were monitored daily, and clinical signs of disease and weight were recorded. Pilot studies with 2-fold serial dilutions of virus stocks were carried out to determine the 50% lethal infectious dose (LD50) of the PR8 virus in C57BL/6 mice. Influenza virus titers. Virus titers were measured by infecting Madin-Darby canine kidney cells with serially diluted supernatants from lung homogenates. After incubation at 37°C for 24 h, 0.02% tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) was added and incubated for an additional 48 h. Virus titers were determined based on the presence or absence of cytopathic effect, and the median tissue culture infectious dose was calculated.

INFECT. IMMUN. Flow cytometry. Lung cells and splenocytes were stained with allophycocyanin anti-CD3, fluorescein isothiocyanate anti-CD4, and phycoerythrin anti-CD8 (all from BD Pharmingen) and then analyzed with a FACSCalibur flow cytometer and CellQuest pro software (BD Biosciences). At least 10,000 gated events were analyzed in each experiment. Evaluation of transcription factors by Western blotting. Whole-cell protein extracts of T cells from lungs and spleens were prepared as described previously (36–38) and were quantified by bicinchoninic acid assay (Pierce Biotechnology). SDS-PAGE and Western blotting were performed as previously described (36). The blot then was stripped and reblotted with anti-glyceraldehyde-3-phosphate dehydrogenase. All antibodies were obtained from Santa Cruz Biotechnology. Statistical analysis. All of the data were processed in Excel and Prism. Student’s t test or the Mann-Whitney t test was used to test for statistical significance. P ⬍ 0.05 was considered statistically significant.

RESULTS Cigarette smoke exposure reduces IFN-␥ production in response to stimulation through the T-cell receptor. To determine if cigarette smoke reduces the cell-mediated immune response, we evaluated its effects on the production of IFN-␥, which is a key cytokine in mediating protective immunity against intracellular pathogens. T cells from control or cigarette smoke-exposed C57BL/6 mice were stimulated with suboptimal concentrations of anti-CD3 and anti-CD28. Cigarette smoke exposure markedly reduced IFN-␥ production by lung T cells by ⬎80% (P ⬍ 0.0001) (Fig. 1A), with similar effects on CD4⫹ and CD8⫹ T cells (Fig. 1B and C). Exposure to cigarette smoke did not significantly reduce IFN-␥ production in antiCD3-stimulated splenocytes (Fig. 1D to F). We next used ELISPOT assays to measure the frequency of IFN-␥-producing cells upon stimulation with anti-CD3 and anti-CD28. Cigarette smoke exposure significantly reduced the number of IFN-␥-producing total lung and CD4⫹ T cells (P ⫽ 0.025 for both comparisons) (Fig. 2). However, spleen cells were not affected. Using flow cytometry, we found that cigarette smoke exposure did not affect the total number of CD3⫹ cells from the lungs and spleens or the percentages of CD4⫹ and CD8⫹ T cells (data not shown), indicating that cigarette smoke did not reduce IFN-␥ production by decreasing T-cell numbers. Cigarette smoke exposure reduces expression of transcription factors that control IFN-␥ production. We have shown previously that the transcription factors cyclic AMP response element binding protein (CREB), activating transcription factor w (ATF-2), and c-Jun bind to the IFN-␥ proximal promoter and upregulate IFN-␥ production by T cells (37, 38). The phosphorylation of these transcription factors increases their binding to the transcriptional complex. To evaluate the effect of cigarette smoke exposure on these transcription factors, we isolated T cells from the lungs and spleens of cigarette smokeexposed and control mice, stimulated them with anti-CD3 and anti-CD28, and performed Western blotting on cellular protein extracts. The expression of phosphorylated CREB, ATF-2, and c-Jun in the lung T cells of cigarette smoke-exposed mice was markedly reduced compared to that of nonexposed control mice (Fig. 3A and C). Similarly, cigarette smoke exposure reduced expression in lung T cells of T-bet, which controls IFN-␥ production. The effect of cigarette smoke exposure was specific, as it did not affect NF-␬B expression (Fig. 3A). In contrast to the effects on lung T cells, cigarette smoke exposure

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FIG. 1. Effects of cigarette smoke exposure on IFN-␥ production by T cells stimulated through the T-cell receptor. C57BL/6 mice (5 to 10 mice per group) were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks. CD4⫹ or CD8⫹ cells were isolated from lungs (A to C) and spleens (D to F) by positive immunomagnetic selection and cultured in plates coated with anti-CD3 and anti-CD28 or with medium alone. Forty-eight to 72 h later, supernatants were collected and IFN-␥ concentrations were measured by ELISA. A total of 106 cells/ml were cultured in 48-well plates. For lung CD4⫹ and CD8⫹ T cells, 2 ⫻ 105 cells/ml were cultured. A representative result is shown of three independent experiments. ns, not significant.

did not reduce the splenic T-cell expression of any transcription factors that control IFN-␥ production (Fig. 3B). Cigarette smoke exposure reduces T-cell responses to DNA vaccination against M. tuberculosis. The studies described

FIG. 2. Effects of cigarette smoke exposure on the number of IFN-␥producing T cells. C57BL/6 mice (5 to 10 mice per group) were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks. CD4⫹ T cells were isolated from lungs (top) and spleens (bottom) by positive immunomagnetic selection. CD4⫹ and total lung and spleen cells were cultured in ELISPOT plates coated with anti-IFN-␥ together with antiCD3 and anti-CD28 or with medium alone. After overnight incubation, the plates were developed and the number of spots counted. A representative result is shown of three independent experiments.

FIG. 3. Effects of cigarette smoke exposure on expression of transcription factors that regulate IFN-␥ production. C57BL/6 mice (5 to 10 mice per group) were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks. T cells were isolated from lungs (A) or spleens (B) by positive immunomagnetic selection and cultured in plates coated with anti-CD3 and anti-CD28. Thirty minutes later, cells were collected and pooled. Cellular protein extracts were prepared and resolved by SDS-PAGE, and Western blotting was performed with the antibodies shown. A representative result of three experiments is shown. (C) Densitometric results were obtained for all experiments and normalized for glyceraldehyde 3-phosphate dehydrogenase, and mean values and standard errors are shown.

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FIG. 4. Effects of cigarette smoke exposure on the response to vaccination with a DNA vaccine expressing an immunogenic M. tuberculosis protein. Mice transgenic for HLA-DRB1*0401 were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks and immunized either with an empty pcDNA3.1 plasmid or with a pcDNA3.1 plasmid expressing CFP10 and lysosomal integral membrane protein II, which targets antigens for presentation by the MHC class II pathway. Two weeks after immunization, CD4⫹ T cells from lungs and spleens were isolated by positive immunomagnetic selection and incubated on an ELISPOT plate together with splenocytes as antigen-presenting cells, either unpulsed (No Ag) or pulsed with CFP10. Five mice per group were used. Mean values and standard errors are shown.

above indicate that cigarette smoke reduces the capacity of T cells to produce IFN-␥ in response to T-cell receptor activation. To determine if cigarette smoke also affects the T-cell response to a specific antigen, we used C57BL/6 mice transgenic for human HLA-DRB1*0401 (DR4). We have demonstrated previously that the M. tuberculosis 10-kDa culture filtrate protein (CFP10) contains several epitopes for human CD4⫹ T cells that are recognized in the context of DRB1*0401 (41), and that DR4 mice recognize human epitopes of CFP10 (21). Cigarette smoke-exposed and control DR4 mice were immunized with a pcDNA3.1 plasmid expressing CFP10 and the lysosomal integral membrane protein II, which targets antigens for presentation by the major histocompatibility complex (MHC) class II pathway (35). Mice also were vaccinated with an empty pcDNA3.1 plasmid as controls. Two weeks after immunization, CD4⫹ T cells from lungs and spleens were isolated and incubated on an ELISPOT plate together with antigen-presenting cells (naïve splenocytes) and either medium alone or CFP10. Cigarette smoke-exposed mice had markedly reduced numbers of CFP10-responsive IFN-␥⫹ lung cells compared to those of unexposed mice (P ⫽ 0.0005) (Fig. 4A). Cigarette smoke exposure caused a more-modest reduction of approximately 30% in the number of splenic CFP10-responsive IFN-␥⫹ cells (P ⫽ 0.007) (Fig. 4B). These findings indicate that cigarette smoke exposure reduces the pulmonary mucosal and systemic T-cell response to vaccination, with the greatest effect being on the pulmonary response. Cigarette smoke exposure impairs T-cell immunity to M. tuberculosis. To determine if cigarette smoke affects IFN-␥ production in response to an infection in vivo, cigarette smokeexposed and unexposed control DR4 mice were infected with the M. tuberculosis strain H37Rv by aerosol. Four weeks after infection, mice were sacrificed and the frequency of IFN-␥producing cells in their lungs and spleens were quantified by ELISPOT assay. To evaluate the effects of cigarette smoke on

IFN-␥ production in vivo, we used an ex vivo assay and seeded cells immediately after isolation on ELISPOT plates precoated with anti-IFN-␥ without stimulation in vitro. The number of IFN-␥⫹ cells from lungs and spleens of cigarette smoke-exposed mice were reduced by more than 90% (Fig. 5A and B) (P ⬍ 0.05 for both comparisons). Cigarette smoke also reduced the number of IFN-␥⫹ CD4⫹ splenic T cells (Fig. 5C). Because IFN-␥ is central to immunity against M. tuberculosis (16, 47), we next studied if cigarette smoke exposure affected the bacterial burden after infection. Mice were exposed to cigarette smoke for 6 weeks and then infected with M. tuberculosis H37Rv by aerosol (10 to 25 CFU/mouse). Ten weeks after infection, CFU in the lungs of cigarette smoke-exposed mice were significantly higher than those in control mice (P ⫽ 0.002) (Fig. 5D). The bacterial burdens in the spleens of both groups were only 3 to 10% of those in the lungs and were comparable in both groups (data not shown). Cigarette smoke exposure inhibits T-cell IFN-␥ production in response to influenza virus infection. The data described above demonstrate that cigarette smoke exposure reduces the T-cell response to M. tuberculosis. To determine if this effect extended to another pulmonary pathogen, cigarette smokeexposed and unexposed C57BL/6 mice were immunized with a sublethal dose (0.1 LD50) of live influenza A virus (PR8) intranasally. One week later, lung and spleen T cells were isolated by positive immunomagnetic selection and incubated with antigen-presenting cells (irradiated splenocytes from naïve mice) on ELISPOT plates precoated with anti-mouse IFN-␥. ELISPOT plates were developed 24 h later, and the frequency of antigen-specific IFN-␥-producing T cells ex vivo was determined. Cigarette smoke exposure reduced the number of IFN-␥⫹ T cells in both the lungs and the spleen (P ⬍ 0.03 for all comparisons) (Fig. 6A). We next evaluated the effect of cigarette smoke exposure on viral replication in the lungs of infected mice. Three days after

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FIG. 5. Effects of cigarette smoke exposure on the T cell response and bacterial burden after M. tuberculosis infection. Mice were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks and then infected with 10 to 25 CFU of M. tuberculosis H37Rv by aerosol (five mice per group). Four weeks after infection, T cells were isolated from lungs (A) and T cells and CD4⫹ cells were isolated from spleens (B and C, respectively) by positive immunomagnetic selection. Without any further stimulation they were placed on ELISPOT plates coated with anti-IFN-␥ antibodies. Twenty-four hours later, the number of spots was counted with a stereomicroscope. Mean values and standard errors are shown for one experiment, which is representative of two performed. In comparisons of cigarette smoke-exposed and control mice, P values were 0.01, 0.05, and 0.07 for panels A, B, and C, respectively. (D) Mice were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks and then infected with M. tuberculosis H37Rv by aerosol (nine mice per group). Ten weeks after infection, the lungs were homogenized and plated on 7H10 agar, and CFU were determined. The horizontal line shows the median value for each group.

infection with 0.1 LD50, lungs from cigarette smoke-exposed mice had slightly higher viral loads than their control counterparts, but this difference was not statistically significant (P ⫽ 0.14) (Fig. 6B). By 6 days after infection, viral burdens were essentially identical in control and cigarette smoke-exposed mice. These findings indicate that the reduced number of IFN␥-producing T cells due to cigarette smoke exposure was not due to a reduction in the viral burden and antigen load. Cigarette smoke exposure increases weight loss and mortality from influenza virus infection. We next examined the effect of cigarette smoke on weight loss due to IAV infection as a clinical indicator of the efficacy of the immune response. Cigarette smoke-exposed and unexposed C57BL/6 mice were immunized with a sublethal dose (0.1 LD50) of IAV PR8 intranasally and weighed daily. Ten days after immunization, the control group stopped losing weight and soon started to gain weight, with some reaching their preinfection weight 2 weeks after infection (Fig. 7A). Cigarette smoke-exposed animals also lost weight during the first 10 days after infection, but most failed to regain weight by 2 weeks postinfection (Fig. 7A). At 2 weeks postinfection, cigarette smoke-exposed mice lost sig-

nificantly more weight than their control counterparts (P ⫽ 0.006). Unexpectedly, sublethal infection resulted in the death of 10 to 20% of cigarette smoke-exposed mice but no unexposed mice (data not shown). The studies described above evaluated the effects of cigarette smoke exposure on sublethal infection with influenza virus. To determine if cigarette smoke exposure affects more severe infection, we infected cigarette smoke-exposed and control mice with a lethal dose of IAV PR8 virus (⬃1 LD50). All cigarette smoke-exposed mice succumbed to infection, whereas approximately one-third of control mice survived (P ⫽ 0.008) (Fig. 7B). Mice that survived until day 15 all recovered completely (data not shown). DISCUSSION In this report, we provide the first evidence that exposure to cigarette smoke directly inhibits the lung T-cell production of IFN-␥ during stimulation in vitro with anti-CD3/CD28, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis

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FIG. 6. Effect of cigarette smoke exposure on T-cell production of IFN-␥ in response to influenza A virus (A) and viral burden (B). Cigarette smoke-exposed (CS) and unexposed mice were infected intranasally with 0.1 LD50 of the influenza A virus PR8 strain. (A) One week later, CD4⫹ and CD8⫹ T cells were isolated from the lungs and spleens and incubated on an ELISPOT plate coated with anti-IFN-␥ together with splenocytes from naïve and nonexposed mice, pulsed with the influenza A peptide (NP366-374) that contains an epitope for CD8⫹ T cells, or infected with live virus for CD4⫹ T cells. Eighteen hours later, the plate was developed and the number of spots was counted with a stereomicroscope. Five mice per group were used. Mean values and standard errors of the means (SEM) are shown. (B) Three and 6 days after infection, lung tissue was aseptically removed at the bronchial level, snap-frozen, and kept at ⫺80°C. The lungs then were thawed and homogenized, after which 10-fold dilutions of lung homogenates were applied to confluent MDCK cells. Plates were monitored daily for 72 h, after which the cytopathic effect of the virus was recorded and the TCID50 was calculated using the Reed and Muench formula. Five mice per group were used. Representative results of two independent experiments with similar results are shown. Error bars show the SEM.

and influenza A virus in vivo. Reduced IFN-␥ production was mediated through the decreased phosphorylation of CREB, ATF-2, and c-Jun, which positively regulate IFN-␥ transcription. The effects of cigarette smoke exposure on the T-cell production of IFN-␥ were associated with reduced immunity, as manifested by increased bacterial burden in the case of tuberculosis and increased weight loss and mortality in the case of influenza virus infection. These findings provide the first demonstration that cigarette smoke exposure reduces resistance to tuberculosis and influenza in an animal model, and they suggest that increased susceptibility is mediated in part through direct inhibitory effects on T cells. The effects of smoking on the immune response are multifaceted and complex. On the one hand, smoking favors the development of pulmonary inflammation and chronic obstructive pulmonary disease, and cigarette smoke exposure in animals increases the expression of the proinflammatory cytokines

IL-18 (18) and IL-1 (11) and activates natural killer cells (26). Furthermore, smoking in humans is associated with the increased expression of STAT4 and IFN-␥ by lymphocytes in bronchial biopsy specimens and bronchoalveolar lavage fluid (8). In contrast to its proinflammatory effects, cigarette smoke can inhibit dendritic cell maturation and the production of IL-12 in animal models (22, 33), suggesting the potential to inhibit Th1 responses that depend on IL-12. The effects of cigarette smoke on T-cell responses have varied in different studies. Some authors found no effect of mainstream cigarette smoke on splenic T-cell production of IFN-␥ (46), whereas others found the marked inhibition of splenocyte proliferation and the ability to mount a Ca2⫹ flux in response to T-cell receptor ligation (17). The few studies of the effects of cigarette smoke on pulmonary T-cell responses have focused on Th2 responses that contribute to allergic inflammation and have yielded contradictory results. Mainstream

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FIG. 7. Effect of cigarette smoke exposure on weight loss and on mortality after influenza A virus infection. (A) Mice were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks (eight mice exposed, nine unexposed) and then infected intranasally with 0.1 LD50 of the influenza A virus PR8 strain. Mice were monitored and weighed daily after infection, and one cigarette smoke-exposed mice died 9 days after infection. Weights were plotted as a percentage of the baseline weight prior to infection and are depicted as means and standard errors of the means. (B) Mice were left unexposed or were exposed to cigarette smoke (CS) for 6 weeks and then infected intranasally with 1 LD50 of the influenza A virus PR8 strain. The survival curves for cigarette smoke-exposed and control groups (18 mice per group) are shown. Results were pooled from three separate experiments. Mice that survived to day 15 recovered from infection and remained well for an additional 2 weeks.

cigarette smoke has been reported to reduce (24, 34) and enhance (25, 40) T-cell-mediated allergic airway inflammation. These differences may reflect differing levels of smoke exposure, as one study showed that higher levels inhibited T-cell cytokine production, whereas lower levels did not (43). Previous studies have evaluated the effects of mainstream cigarette smoke on T-cell function, but our current work provides the first evaluation of the effects of environmental cigarette smoke on T cells. Cigarette smoke exposure inhibited both the lung and splenic CD4⫹ and CD8⫹ T-cell production of IFN-␥ in response to M. tuberculosis and influenza (Fig. 4, 5, and 6A), suggesting that both local and systemic T-cell function are reduced during infection. However, exposure to cigarette smoke inhibited the capacity of lung but not splenic T cells to produce IFN-␥ in response to stimulation through the T-cell receptor independently of antigen-presenting cells (Fig. 1). Anti-CD3 and anti-CD28 provide a stronger T-cell stimulus than bacterial and viral antigens and may overcome the inhibitory effects of cigarette smoke on splenic but not lung cell responses, since the concentrations of immunosuppressive components of cigarette smoke are highest in the lung and are likely to have the greatest effect on local T cells. Although cigarette smoke exposure affected antigen-specific responses, this was not due to global effects on T-cell recruitment, as exposure did not reduce the trafficking of CD4⫹ or CD8⫹ T cells to the lungs or mediastinal lymph nodes, either before or after influenza infection (H. Shams, unpublished data). Furthermore, cigarette smoke exposure did not significantly affect the recruitment of naïve or effector memory T cells, or T cells expressing the integrins LFA-1 and VLA-1, to the lungs after influenza infection (H. Shams, unpublished data). Smoking and tuberculosis are strongly epidemiologically linked. One recent study of 17,700 Taiwanese persons showed a 2-fold increase in the risk of tuberculosis among current smokers, with a significant dose-response relationship with the number of cigarettes smoked per day and the number of packyears of smoking (23). Cigarette smoke exposure also was independently associated with a 70% increase in the likelihood of the development of culture-proven tuberculosis among

15,500 nonsmoking women (2). Despite these epidemiologic data, no published information is available on the mechanisms through which smoking increases susceptibility to tuberculosis. IFN-␥ plays a central role in immunity against tuberculosis, as mice with a deleted IFN-␥ gene rapidly succumb to tuberculosis (6, 13), and T cells from tuberculosis patients with ineffective immunity produce low concentrations of IFN-␥ compared to that of healthy tuberculin reactors with protective immunity (16, 47). In this report, we immunized mice with a plasmid DNA vaccine that has been shown previously to deliver an immunogenic mycobacterial protein to the lung and to elicit strong T-cell responses (21). Cigarette smoke exposure inhibited the lung and splenic T-cell production of IFN-␥ in response to vaccination (Fig. 4). Furthermore, when mice were infected with M. tuberculosis, cigarette smoke exposure reduced IFN-␥ production (Fig. 5A to C) and the bacillary burden was greater in cigarette smoke-exposed animals (Fig. 5D), suggesting that the inhibition of the T-cell response significantly impacted the capacity of the immune system to control bacterial infection. Smoking predisposes healthy adults to the development of influenza, which is more severe in smokers (20, 28). Of three studies of smoking and influenza in mice in vivo, one used miniosmotic pumps to administer nicotine and another administered extremely high smoke exposure for only 4 days prior to influenza infection (15, 31), situations that are unlikely to mimic conditions in humans. The third study found that mainstream cigarette smoke reduced the local airway inflammatory response after infection with a low dose of influenza virus but enhanced inflammation and increased mortality after highdose infection (32). Our study is the first to evaluate the effects of cigarette smoke on the T cell response to influenza in animals. Similarly to our findings for tuberculosis, cigarette smoke reduced the local and systemic T-cell production of IFN-␥ in mice infected with influenza A virus (Fig. 6A), and this effect was associated with increased weight loss and mortality (Fig. 7A and B). IFN-␥ is critical for human defenses against M. tuberculosis and other bacterial, fungal, and viral intracellular pathogens,

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and the proximal promoter of IFN-␥ is necessary and sufficient for its transcription in activated T cells (29). In the present report, we demonstrated that cigarette smoke exposure reduced the phosphorylation of CREB, ATF-2, and c-Jun (Fig. 3), which are known to bind to and positively regulate the proximal promoter of IFN-␥ in primary human T cells in response to mycobacterial antigen (38). It will be important to delineate the upstream mechanisms through which cigarette smoke exposure reduces the expression of these transcription factors. In summary, we provide the first evidence that cigarette smoke exposure directly inhibits the pulmonary and systemic T-cell production of IFN-␥ during infection with M. tuberculosis and influenza A virus in vivo, at least in part through the decreased phosphorylation of CREB, ATF-2, and c-Jun. The inhibition of IFN-␥ production was associated with an increased bacterial burden in the case of tuberculosis and increased weight loss and mortality in the case of influenza virus infection. Given the enormous numbers of people exposed to cigarette smoke and the tremendous morbidity and mortality attributable to tuberculosis and influenza world-wide, future studies will be critical to fully understanding the molecular mechanisms of these effects of cigarette smoke exposure, so that immunomodulatory strategies can be developed to correct these defects. ACKNOWLEDGMENTS We thank Barry Starcher for his helpful discussions on cigarette smoke exposure protocols and assistance with the cigarette smoke exposure chamber. This work was supported by a grant from the Flight Attendant Medical Research Institute (052338 and 092015 Clinical Innovator Awards to H.S.) and the Margaret E. Byers Cain Chair for Tuberculosis Research and James Byers Cain Research Endowment (both to P.F.B.). REFERENCES 1. Arcavi, L., and N. L. Benowitz. 2004. Cigarette smoking and infection. Arch. Intern. Med. 164:2206–2216. 2. Benowitz, N. L. 2010. Secondhand smoke and infectious disease in adults: a global women’s health concern: comment on “passive smoking and tuberculosis.” Arch. Intern. Med. 170:292–293. 3. Boom, W. H. 1996. The role of T-cell subsets in Mycobacterium tuberculosis infection. Infect. Agents Dis. 5:73–81. 4. Brown, D. M., A. M. Dilzer, D. L. Meents, and S. L. Swain. 2006. CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch. J. Immunol. 177:2888–2898. 5. Caruso, A. M., N. Serbina, E. Klein, K. Triebold, B. R. Bloom, and J. L. Flynn. 1999. Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis. J. Immunol. 162:5407– 5416. 6. Cooper, A. M., D. K. Dalton, T. A. Stewart, J. P. Griffin, D. G. Russell, and I. M. Orme. 1993. Disseminated tuberculosis in interferon gamma genedisrupted mice. J. Exp. Med. 178:2243–2247. 7. Crowe, S. R., S. C. Miller, D. M. Brown, P. S. Adams, R. W. Dutton, A. G. Harmsen, F. E. Lund, T. D. Randall, S. L. Swain, and D. L. Woodland. 2006. Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine 24:457–467. 8. Di Stefano, A., G. Caramori, A. Capelli, I. Gnemmi, F. L. Ricciardolo, T. Oates, C. F. Donner, K. F. Chung, P. J. Barnes, and I. M. Adcock. 2004. STAT4 activation in smokers and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 24:78–85. 9. Doherty, P. C., L. E. Brown, A. Kelso, and P. G. Thomas. 2009. Immunity to avian influenza A viruses. Rev. Sci. Tech. 28:175–185. 10. Doherty, P. C., D. J. Topham, R. A. Tripp, R. D. Cardin, J. W. Brooks, and P. G. Stevenson. 1997. Effector CD4⫹ and CD8⫹ T-cell mechanisms in the control of respiratory virus infections. Immunol. Rev. 159:105–117. 11. Doz, E., N. Noulin, E. Boichot, I. Guenon, L. Fick, M. Le Bert, V. Lagente, B. Ryffel, B. Schnyder, V. F. Quesniaux, and I. Couillin. 2008. Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/ MyD88 signaling dependent. J. Immunol. 180:1169–1178.

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