Continuous and Discontinuous Cigarette Smoke Exposure Differentially Affects Protective Th1 Immunity against Pulmonary Tuberculosis

<div><p>Pulmonary tuberculosis (TB), caused by <i>Mycobacterium tuberculosis,</i> is the leading cause of death due to a bacterial pathogen. Emerging epidemiologic evidence suggests that the leading risk factor associated with TB mortality is cigarette smoke exposure. Despite this, it remains poorly understood what is the effect of cigarette smoke exposure on anti-TB immunity and whether its potential detrimental effect can be reversed by cigarette smoking cessation. In our current study, we have investigated the impact of both continuous and discontinuous cigarette smoke exposure on the development of anti-mycobacterial type 1 immunity in murine models. We find that while continuous cigarette smoke exposure severely impairs type 1 immunity in the lung, a short-term smoking cessation allows rapid restoration of anti-mycobacterial immunity. The ability of continuous cigarette smoke exposure to dampen type 1 protective immunity is attributed locally to its affects on innate immune cells in the lung. Continuous cigarette smoke exposure locally, by not systemically, impairs APC accumulation and their production of TNF, IL-12, and RANTES, blunts the recruitment of CD4+IFN-γ+ T cells to the lung, and weakens the formation of granuloma. On the other hand, smoking cessation was found to help restore type 1 immunity by rapidly improving the functionality of lung APCs, enhancing the recruitment of CD4+IFN-γ+ T cells to the lung, and promoting the formation of granuloma. Our study for the first time demonstrates that continuous, but not discontinuous, cigarette smoke exposure severely impedes the lung expression of anti-TB Th1 immunity via inhibiting innate immune activation and lung T cell recruitment. Our findings thus suggest cigarette smoking cessation to be beneficial to the control of pulmonary TB.</p> </div

Continuous, but not discontinuous smoke exposure, impairs the production of type 1 cytokines while enhancing the production of IL-4, and reducing the production of bactericidal nitric oxide by lung MNCs following mycobacterial infection.

<p>Following the exposure-challenge model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059185#pone-0059185-g002" target="_blank">Figure 2A</a>, we evaluated the impact of cigarette smoke exposure on the production of type 1 &2 cytokines and nitric oxide by mycobacteria infected lung MNCs. Following 48 hr lung MNC culture, the levels of TNF (A), IL-12p40 (B), IFN-γ (C), IL-4 (D) were evaluated by cytokine ELISA, and production of nitric oxide (E) by a modified Griess assay. Values represent the mean and standard error for 5 mice per exposure protocol. *p≤0.05; **p≤0.01; ***p≤0.001.</p

Continuous cigarette smoke exposure alters the balance of Th1 and Th2 CD4 T cells in the lung.

<p>Following the exposure-challenge model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059185#pone-0059185-g002" target="_blank">Figure 2A</a>, we evaluated the impact of cigarette smoke exposure on the frequency of CD4+IFN-γ+ (A) and CD4+IL-4+ (B) T cells in the airway lumen of mycobacterial infected mice. Values represent the mean and standard error for 5 mice per exposure protocol. *p≤0.05; **p≤0.01; ***p≤0.001.</p

Assessment of Histopathological Changes in the Lung following <i>M.tb</i> challenge.

<p>Granuloma size, granuloma number, and lung mononuclear cell infiltration were scored.</p><p>Results are representative of <i>n</i>  = 5/exposure/time point.</p><p>+++, moderate; ++++, marked; +++++, severe. (+ half point).</p

Continuous cigarette smoke exposure does not impair the generation of type 1 immunity in the in the spleen or MLN.

<p>Following the exposure-challenge model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059185#pone-0059185-g002" target="_blank">Figure 2A</a>, we evaluated the impact of cigarette smoke exposure on the establishment of type 1 immune response in the spleen and MLN of mycobacterial infected mice. The numbers of CD4+ and CD4+IFN-γ+ T cells were evaluated in the spleen (A&B), and the MLN (C&D). Values represent the mean and standard error for 5 mice per exposure protocol. *p≤0.05; **p≤0.01.</p

Prolonged cigarette smoke cessation enhances cellular infiltration, granuloma formation and bacterial control following mycobacterial challenge.

<p>Mice where either exposed to cigarette smoke or room air for a period of 6 wks, at which time both groups were subjected to challenge with <i>M.tb</i> H₃₇Rv (A). The bacterial burden was determined in the lung (B), and spleen (C) by colony formation assay of organ homogenates from infected mice at 4 and 6 wk post infection. The histological impact on lung pathology was determined by H&E staining of lung sections isolated 4 and 6 wks post infection with <i>M.tb</i> (D&E). CFU numbers represent the mean and standard error of 5 mice per exposure protocol. The 4 wk challenge data is representative of two independent experiments. Selected histological sections are representative of their exposure protocol. *p≤0.05; **p≤0.01.</p

Continuous cigarette smoke exposure alters the surface marker expression of lung, but not spleen or MLN APC populations following mycobacterial infection.

<p>Following the exposure-challenge model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059185#pone-0059185-g002" target="_blank">Figure 2A</a>, we evaluated the impact of cigarette smoke exposure on the expression of common APC markers, CD11b+ and CD11c+ in the lung of mycobacterial infected mice. The distribution of CD11b+ and CD11c+ by APC populations of the lung (A), MLN (B) and spleen (C) were evaluated. Panels are representative flow plots for lung mononuclear cells isolated from 5 mice for each exposure protocol.</p

Continuous cigarette smoke exposure alters lung pathology and decreases bacterial control following pulmonary mycobacterial infection.

<p>Following 6 wks of cigarette smoke (or room air) exposure, mice were subjected to Bacillus Calmette–Guérin - <i>M. bovis</i> challenge (A). At the time of challenge one group of previously cs exposed mice was discontinued from cigarette smoke exposure to determine the impact of cessation of mycobacterial immunity, while another continued exposure for the duration of infection. At 4 wks post-infection, the bacterial burden following the various exposure protocols was determined by colony formation assay in the lung and spleen of mycobacterial infected mice (B&C), and the histological impact on lung pathology by H&E staining of lung sections isolated from infected mice (D–F). CFU numbers represents the mean and standard error of 5 mice per exposure protocol. Selected histological sections are representative of their exposure protocol. *p≤0.05.</p