In this report, we provide the first evidence that exposure to cigarette smoke directly inhibits the lung T-cell production of IFN-γ during stimulation in vitro with anti-CD3/CD28, 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 CREB, ATF-2, and c-Jun, which positively regulate IFN-γ transcription. The effects of cigarette smoke exposure on the T-cell production of IFN-γ 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-γ 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
), 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-γ (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 cigarette smoke has been reported to reduce (24
) and enhance (25
) 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-γ in response to M. tuberculosis and influenza (Fig. , , and ), 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-γ in response to stimulation through the T-cell receptor independently of antigen-presenting cells (Fig. ). 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 pack-years 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-γ plays a central role in immunity against tuberculosis, as mice with a deleted IFN-γ gene rapidly succumb to tuberculosis (6
), and T cells from tuberculosis patients with ineffective immunity produce low concentrations of IFN-γ compared to that of healthy tuberculin reactors with protective immunity (16
). 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-γ in response to vaccination (Fig. ). Furthermore, when mice were infected with M. tuberculosis
, cigarette smoke exposure reduced IFN-γ production (Fig. ) and the bacillary burden was greater in cigarette smoke-exposed animals (Fig. ), 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
). 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
), 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 high-dose 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-γ in mice infected with influenza A virus (Fig. ), and this effect was associated with increased weight loss and mortality (Fig. ).
IFN-γ is critical for human defenses against M. tuberculosis
and other bacterial, fungal, and viral intracellular pathogens, and the proximal promoter of IFN-γ 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. ), which are known to bind to and positively regulate the proximal promoter of IFN-γ 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-γ 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-γ 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.