Nicotinic Acetylcholine Receptor Subunit α7 Mediates Cigarette Smoke-Induced PD-L1 Expression in Human Bronchial Epithelial Cells

Abstract

Tobacco smoking is the top risk factor for lung cancer development. Nicotine in cigarettes can induce addiction, and its derivatives become potent carcinogens after metabolic activation and activate oncogenic signaling in lung epithelial cells through their expressed nicotinic acetylcholine receptors (nAChRs). However, the effects of smoking on the tumor immune microenvironment are under investigation. In the current study, we investigated whether nicotine activation of nicotinic acetylcholine receptor subunit α7 (nAChRα7, CHRNA7) would induce PD-L1 expression in lung epithelial cells. The expression levels of nAChRα7 and PD-L1 in eight human bronchial epithelial cell (HBEC) lines were measured after treatment with cigarette smoke extract (CSE) or nicotine derivatives. The results showed that PD-L1 expression levels increased in HBECs after exposure to CSE or nicotine derivatives. This induction of PD-L1 expression could be diminished by treatment with CHRNA7 small-interfering RNA, and the relevant signaling was mediated via STAT3 phosphorylation and NRF2 expression. In summary, this study demonstrated that the well-known nicotine derivative-activated nAChRα7 could induce STAT3/NRF2 pathways and subsequently promote PD-L1 expression in normal lung epithelial cells. This information provides mechanistic insight into cigarette smoke-induced immune evasion in lung epithelial cells.

Keywords: CHRNA7; PD-L1; bronchial epithelial cells; cigarette smoke; lung cancer.

Figure 1

CHRNA7 expression and clinicopathological correlations in TCGA-LUSC cohort. (A) Expression levels of CHRN subunit genes in normal lung from GTEx portal. (B) Boxplots of CHRNA7 gene expression comparing normal lung tissues against different stages of lung cancer (upper panel), or non-smoking patients against patients with different smoking habits (lower panel) in the TCGA-LUSC dataset; median of each bar is shown. (C) Kaplan–Meier analysis of lung squamous cell carcinoma patients in the TCGA-LUSC cohort (Oncolnc).

Figure 1

CHRNA7 expression and clinicopathological correlations in TCGA-LUSC cohort. (A) Expression levels of CHRN subunit genes in normal lung from GTEx portal. (B) Boxplots of CHRNA7 gene expression comparing normal lung tissues against different stages of lung cancer (upper panel), or non-smoking patients against patients with different smoking habits (lower panel) in the TCGA-LUSC dataset; median of each bar is shown. (C) Kaplan–Meier analysis of lung squamous cell carcinoma patients in the TCGA-LUSC cohort (Oncolnc).

Figure 2

Cigarette smoke and the carcinogen NNK induce PD-L1 expression on human bronchial epithelial cells (HBECs). HBECs derived from smokers (BS62N-KT, BS65.2N-KT, BS160N-KT, and BS189.2N-KT) or non-smokers (BS150N-KT, BS197N-KT, BS198N-KT, and BS202.5N-KT) were exposed to cigarette smoke extract (CSE, 5 or 10%) or NNK (5 or 10 µM) for 48 h, and CD274 and CHRNA7 (A) mRNA expression was measured by real-time RT-PCR; (B) PD-L1 and nAChRα7 protein expression was determined by Western blot analysis; (C) surface PD-L1 expression was measured by immunofluorescence staining. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.

Figure 2

Cigarette smoke and the carcinogen NNK induce PD-L1 expression on human bronchial epithelial cells (HBECs). HBECs derived from smokers (BS62N-KT, BS65.2N-KT, BS160N-KT, and BS189.2N-KT) or non-smokers (BS150N-KT, BS197N-KT, BS198N-KT, and BS202.5N-KT) were exposed to cigarette smoke extract (CSE, 5 or 10%) or NNK (5 or 10 µM) for 48 h, and CD274 and CHRNA7 (A) mRNA expression was measured by real-time RT-PCR; (B) PD-L1 and nAChRα7 protein expression was determined by Western blot analysis; (C) surface PD-L1 expression was measured by immunofluorescence staining. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.

Figure 3

nAChRα7-mediated NNK-induced PD-L1 expression. (A) HBECs were treated with non-targeting or CHRNA7-specific siRNA (50 nM) for 24 h, and the expression of CHRNA7 mRNA was measured by real-time RT-PCR. HBECs with or without knockdown of CHRNA7 by siRNA (50 nM) for 24 h were further treated with NNK (10 μM) for another 48 h; the expression of CD274 (PD-L1) (B) mRNA expression was measured by real-time RT-PCR; (C) protein expression was determined by Western blot analysis. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.

Figure 3

nAChRα7-mediated NNK-induced PD-L1 expression. (A) HBECs were treated with non-targeting or CHRNA7-specific siRNA (50 nM) for 24 h, and the expression of CHRNA7 mRNA was measured by real-time RT-PCR. HBECs with or without knockdown of CHRNA7 by siRNA (50 nM) for 24 h were further treated with NNK (10 μM) for another 48 h; the expression of CD274 (PD-L1) (B) mRNA expression was measured by real-time RT-PCR; (C) protein expression was determined by Western blot analysis. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.

Figure 4

nAChRα7-mediated NNK-induced PD-L1 expression through NRF2 and STAT3 signaling. (A) HBECs transfected with non-targeting or CHRNA7-targeting siRNA (50 nM) for 24 h were seeded on the assay plate of a Cignal 45-pathway reporter array and then reverse-transfected with reporter plasmids. Cells were then treated with NNK (10 µM) for another 24 h. Firefly and Renilla luciferase activities were measured. Normalized luciferase activities were compared with those of non-NNK-treated cells. Representative pathways are shown. (B) HBECs were treated with non-targeting or CHRNA7-targeting siRNA (50 nM) for 24 h and then treated with NNK (10 µM) for another 48 h. The expression of NRF2 and STAT3 phosphorylation was measured with Western blot analysis. (C) HBECs were co-treated with NNK (10 µM) and the STAT3 inhibitor C188 (1 µM) or the NRF2 inhibitor ML385 (5 µM) for 48 h. PD-L1 protein expression levels were determined by Western blot analysis. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.

Figure 4

nAChRα7-mediated NNK-induced PD-L1 expression through NRF2 and STAT3 signaling. (A) HBECs transfected with non-targeting or CHRNA7-targeting siRNA (50 nM) for 24 h were seeded on the assay plate of a Cignal 45-pathway reporter array and then reverse-transfected with reporter plasmids. Cells were then treated with NNK (10 µM) for another 24 h. Firefly and Renilla luciferase activities were measured. Normalized luciferase activities were compared with those of non-NNK-treated cells. Representative pathways are shown. (B) HBECs were treated with non-targeting or CHRNA7-targeting siRNA (50 nM) for 24 h and then treated with NNK (10 µM) for another 48 h. The expression of NRF2 and STAT3 phosphorylation was measured with Western blot analysis. (C) HBECs were co-treated with NNK (10 µM) and the STAT3 inhibitor C188 (1 µM) or the NRF2 inhibitor ML385 (5 µM) for 48 h. PD-L1 protein expression levels were determined by Western blot analysis. The values are presented as mean ± S.D. from technical triplicate experiments. *** p < 0.001.