Exposure to cigarette smoke inhibits the pulmonary T-cell response to influenza virus and Mycobacterium tuberculosis

Abstract

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-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ 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-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ 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.

FIG. 1.

Effects of cigarette smoke exposure on IFN-γ 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-γ 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.

FIG. 2.

Effects of cigarette smoke exposure on the number of IFN-γ-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-γ together with anti-CD3 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-γ 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.

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.

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-γ 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.

FIG. 6.

Effect of cigarette smoke exposure on T-cell production of IFN-γ 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-γ together with splenocytes from naïve and nonexposed mice, pulsed with the influenza A peptide (NP_366-374) 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.

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.