Effects of CSE and MRs on the apoptosis of CD4⁺ T cells.

<p>T cells from healthy nonsmokers and patients with SCOPD were cultured in the presence of medium alone, CSE, mAChR agonist/antagonist or combinations of these factors (<i>top panels</i>) for 5 days. (<b>A</b>, <b>B</b>) The representative flow cytometric dot-plots show Annexin V/PI co-staining for the identification of apoptotic CD4⁺ T cells. (<b>C</b>) The apoptosis index was calculated by dividing the percentage of apoptotic CD4⁺ T cells in various groups by the percentage of apoptotic CD4⁺ T cells in the medium alone, and the proliferation index of the controls was 100. Comparing the apoptotic CD4⁺T (<i>excluding Tregs</i>) cells from healthy nonsmokers (<i>solid bars</i>) or patients with SCOPD (<i>open bars</i>) in each group, the results were reported as the mean ± SEM from 5 independent experiments. The comparisons were determined by the Kruskal-Wallis one-way analysis of variance on ranks or Mann-Whitney U test. *P<0.05 compared with control, **P<0.05 comparison between healthy nonsmokers and patients with SCOPD. # P<0.05 indicates CSE plus Atro compared with CSE alone and Muscarine plus Atro compared with Muscarine alone.</p

Effects of CSE and MRs on the proliferative response of Tregs.

<p>(<b>A</b>, <b>B</b>) The proliferation of Tregs was further analyzed by flow cytometry, and the representative flow cytometric dot-plots are shown. (<b>C</b>) The proliferation index was calculated by dividing the percentage of Ki-67⁺Tregs cells of various groups by the percentage of Ki-67⁺Tregs cells under the stimulation of PMA and PHA alone, and the proliferation index of controls was considered 100. Comparisons of the proliferation of Tregs from the healthy nonsmokers (<i>solid bars</i>) or patients with SCOPD (<i>open bars</i>) in various trial groups were made; the results were reported as the mean ± SEM from 5 independent experiments. The comparisons were determined by the Kruskal-Wallis one-way analysis of variance on ranks or the Mann-Whitney U test. *P<0.05 compared with control, **P<0.05 for the comparison between healthy nonsmokers and patients with SCOPD. # P<0.05 indicates CSE plus Atro compared with CSE; Muscarine plus Atro compared with Muscarine.</p

Effects of CSE and MRs on the apoptosis of Tregs.

<p>(<b>A</b>, <b>B</b>) The apoptosis of Tregs was further analyzed by flow cytometry, and the representative flow cytometric dot-plots were shown. (<b>C</b>) The apoptosis index was calculated by dividing the percentage of apoptotic Tregs from various groups by the percentage of apoptotic Treg cells in the medium alone, and the apoptosis index of controls was considered to be 100. Comparisons of the apoptosis of Tregs from the healthy nonsmokers (<i>solid bars</i>) or patients with SCOPD (<i>open bars</i>) in the various trial groups were made. The results are reported as the mean ± SEM from 5 independent experiments. The comparisons were determined by the Kruskal-Wallis one-way analysis of variance on ranks or Mann-Whitney U test. *P<0.05 compared with control, **P<0.05 comparison between healthy nonsmokers and patients with SCOPD. # P<0.05 indicates CSE plus Atro compared with CSE alone and Muscarine plus Atro compared with Muscarine alone.</p

MRs and CSE affect the expression of T cell subset nuclear transcription factors.

<p>PBT cells from healthy nonsmokers (<b>A</b>) or patients with SCOPD (<b>B</b>) were cultured and stimulated with anti-CD3 and anti-CD28 Abs in the presence of CSE, mAChR agonist/antagonist, or combinations of these factors (<i>as shown in bottom panels</i>). The agonist and antagonist of mAChRs were muscarine (Mus) and atropine (Atro), respectively. After 5 days in culture, the cells were harvested, and the total RNA was extracted. The expression levels of nuclear transcription factor mRNA, namely that of T-bet, GATA3, RORC, and FOXP3, were detected by qPCR, with GAPDH serving as an internal control. The data from 7 independent experiments are expressed as the mean ± SD of the mRNA in question relative to that in unstimulated T cells (controls), which was set as 1. The comparisons were determined by the Kruskal-Wallis one-way analysis of variance on ranks.*P<0.05 compared with the relevant control. ^# P<0.05 compared with CD3/CD28 plus CSE or CD3/CD28 plus Muscarine (Mus).</p

Effects of CSE and MRs on the proliferative response of CD4⁺ T cells.

<p>(<b>A</b>, <b>B</b>) T cells from healthy nonsmokers and patients with SCOPD were cultured in the presence of CSE, mAChR agonist/antagonist, or combinations of these factors (<i>top panels</i>) under stimulation with PMA and PHA for 5 days. Ki-67⁺CD4⁺T cells (<i>excluding Tregs</i>) were determined by flow cytometry, and the representative flow cytometric dot-plots are shown. (<b>C</b>) The proliferation index was calculated by dividing the percentage of Ki-67⁺CD4⁺ T cells of various groups by the percentage of Ki-67⁺CD4⁺T cells stimulated with PMA and PHA alone, and the proliferation index of control was considered to be 100. Comparisons of the proliferation of CD4⁺T cells from the healthy nonsmokers (<i>solid bars</i>) or patients with SCOPD (<i>open bars</i>) in various trial groups were made; the results are reported as the mean ± SEM from 5 independent experiments. The comparisons were determined by the Kruskal-Wallis one-way analysis of variance on ranks or Mann-Whitney U test. *P<0.05 compared with control, **P<0.05 in the comparison between healthy nonsmokers and patients with SCOPD. # P<0.05 indicates CSE plus Atro compared with CSE alone and Muscarine plus Atro compared with Muscarine alone.</p

The percentage of Tregs is increased in patients with AECOPD, and the percentages of Th10 and CD4⁺α7⁺ T cells are decreased in both patients with SCOPD and patients with AECOPD.

<p>(<b>A</b>) Representative flow cytometric dot-plots of Tregs, Th10 and CD4⁺α7⁺ T cells (<i>Tregs: CD3⁺CD8⁻CD25⁺FOXP3⁺ T cells, Th10: CD3⁺CD8⁻IL-10⁺T cells, and CD4⁺α7⁺ T: CD3⁺CD8⁻α7⁺ T cells</i>) in healthy nonsmokers, patients with SCOPD and patients with AECOPD are shown. (<b>B</b>) Comparisons of the percentages of Th1, Th2 and Th17 cells in healthy nonsmokers, patients with SCOPD and patients with AECOPD <i>(n = 14, 24, and 14, respectively</i>) are shown. Horizontal bars indicate the mean value. The comparisons were made using a Kruskal-Wallis one-way ANOVA on ranks.</p

Real-time RT-PCR primer sequences.

<p>All of these primers were synthesized by TaKaRa in Dalian.</p><p>Real-time RT-PCR primer sequences.</p

Demographic and spirometric values of healthy nonsmokers, patients with SCOPD and patients with AECOPD.

<p>All data are presented as the mean±SD. FEV1: Forced expiratory volume in one second, measured post bronchodilatation and FVC: Forced vital capacity. *P<0.05 compared with healthy nonsmokers; ^#P<0.05 compared with SCOPD patients.</p><p>Demographic and spirometric values of healthy nonsmokers, patients with SCOPD and patients with AECOPD.</p

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