Cigarette Smoke Disturbs the Survival of CD8⁺ Tc/Tregs Partially through Muscarinic Receptors-Dependent Mechanisms in Chronic Obstructive Pulmonary Disease

<div><p>Background</p><p>CD8⁺ T cells (Cytotoxic T cells, Tc) are known to play a critical role in the pathogenesis of smoking related airway inflammation including chronic obstructive pulmonary disease (COPD). However, how cigarette smoke directly impacts systematic CD8⁺ T cell and regulatory T cell (Treg) subsets, especially by modulating muscarinic acetylcholine receptors (MRs), has yet to be well elucidated.</p><p>Methods</p><p>Circulating CD8⁺ Tc/Tregs in healthy nonsmokers (n = 15), healthy smokers (n = 15) and COPD patients (n = 18) were evaluated by flow cytometry after incubating with anti-CD3, anti-CD8, anti-CD25, anti-Foxp3 antibodies. Peripheral blood T cells (PBT cells) from healthy nonsmokers were cultured in the presence of cigarette smoke extract (CSE) alone or combined with MRs agonist/antagonist for 5 days. Proliferation and apoptosis were evaluated by flow cytometry using Ki-67/Annexin-V antibodies to measure the effects of CSE on the survival of CD8⁺ Tc/Tregs.</p><p>Results</p><p>While COPD patients have elevated circulating percentage of CD8⁺ T cells, healthy smokers have higher frequency of CD8⁺ Tregs. Elevated percentages of CD8⁺ T cells correlated inversely with declined FEV1 in COPD. CSE promoted the proliferation and inhibited the apoptosis of CD8⁺ T cells, while facilitated both the proliferation and apoptosis of CD8⁺ Tregs. Notably, the effects of CSE on CD8⁺ Tc/Tregs can be mostly simulated or attenuated by muscarine and atropine, the MR agonist and antagonist, respectively. However, neither muscarine nor atropine influenced the apoptosis of CD8⁺ Tregs.</p><p>Conclusion</p><p>The results imply that cigarette smoking likely facilitates a proinflammatory state in smokers, which is partially mediated by MR dysfunction. The MR antagonist may be a beneficial drug candidate for cigarette smoke-induced chronic airway inflammation.</p></div

Imbalance of circulating CD8⁺ Tc/Tregs in patients with chronic obstructive pulmonary disease (COPD).

<p>Comparisons of percentages of CD8⁺ T cells (A) and CD8⁺ Tregs (B) in peripheral blood from healthy controls (HC, n = 15), healthy smokers (HS, n = 15) and COPD patients (n = 18). Correlation of CD8⁺ T cells (C) and CD8⁺ Tregs (D) with FEV1% predicted value in COPD patients (n = 18). * P<0.05.</p

Effects of CSE and muscarinic receptors on proliferation and apoptosis of CD8⁺ T cells.

<p>PBT cells from healthy nonsmokers were cultured for 5 days in the presence of CSE and MRs agonist/antagonist, either alone or in various combinations. (A) In the proliferation assays, medium combined with PHA and PMA was considered as control treated wells. Ki-67⁺CD8⁺ T cells (excluding CD8⁺ Tregs) were examined by flow cytometry, and the representative flow cytometric dot plots are shown. (B) The proliferation index of control wells was considered to be 100, and data are expressed as fold increase relative that in the control wells (n = 5). (C) In the apoptosis assays, medium alone was considered as control treated wells. Apoptotic CD8⁺ T cells (excluding CD8⁺ Tregs) were examined by flow cytometry, and the representative flow cytometric dot plots are shown. (D) The apoptosis index of control wells was considered to be 100, and data are expressed as fold increase relative that in the control wells (n = 6). The results are reported as the mean ± SEM. *P<0.05 compared with the control wells; #P<0.05 indicates CSE plus Atro compared with CSE or Mus plus Atro compared with Mus.</p

Representative dot plots show the gating strategy of the assays.

<p>In the proliferation assays, CD8⁺ Tregs were gated from CD8⁺ T cells (CD3⁺CD8⁺) based on their positive expression of Foxp3, and the expression of Ki-67 by CD8⁺ T cells and CD8⁺ Tregs was then further analyzed. However, in the apoptosis assays, CD8⁺ Tregs were gated from CD8⁺ T cells (CD3⁺CD8⁺) based on their high expression of CD25, and the apoptosis of CD8⁺ T cells and CD8⁺ Tregs gated on propidium iodide-negative and Annexin V-positive cells was then further analyzed.</p