Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung

Abstract

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and predisposes subjects to severe respiratory tract infections. Epidemiological studies have shown that cigarette smokers are seven times more likely to contract influenza infection than nonsmokers. The mechanisms underlying this increased susceptibility are poorly characterized. Retinoic acid-inducible gene (RIG)-I is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focused on how cigarette smoke extract (CSE) alters the influenza-induced proinflammatory response and suppresses host antiviral activity in the human lung using a unique lung organ culture model. We first determined that treatment with 2-20% CSE did not induce cytotoxicity as assessed by LDH release. However, CSE treatment inhibited influenza-induced IFN-inducible protein 10 protein and mRNA expression. Induction of the major antiviral cytokine IFN-β mRNA was also decreased by CSE. CSE also blunted viral-mediated RIG-I mRNA and protein expression. Inhibition of viral-mediated RIG-I induction by CSE was prevented by the antioxidants N-acetyl-cysteine and glutathione. These findings show that CSE suppresses antiviral and innate immune responses in influenza virus-infected human lungs through oxidative inhibition of viral-mediated induction of the pattern recognition receptor RIG-I. This immunosuppressive effect of CSE may play a role in the enhanced susceptibility of smokers to serious influenza infection in the lung.

Fig. 1.

Cigarette smoke extract (CSE) suppresses influenze A virus (IAV)-stimulated innate immune cytokine release in the human lung. Human lung slices were exposed for the times indicated to 6 × 10⁶ plaque-forming units (PFU)/ml of IAV PR8 in the presence or absence of 2% CSE. IFN-inducible protein-10 (IP-10; A) and monocyte chemoattractant protein-1 (MCP-1; B) cytokine protein levels were determined by ELISA in lung slice supernatants. Data are expressed as means ± SE from 3 separate lung slice donor experiments. Statistical significance was determined by ANOVA. Means were compared with data from the negative control group. *P < 0.05.

Fig. 2.

CSE suppresses IAV-stimulated cytokine and retanoic acid-inducible gene (RIG)-I mRNA expression in the human lung. Human lung slices were exposed to 6 × 10⁶ PFU/ml of IAV PR8 in the presence or absence of CSE for 24 h. Total RNA was then isolated from lung slices. Relative end-point RT-PCR products were separated by agarose gel electrophoresis, and mRNA expression was determined by densitometry of the appropriate bands on ethidium bromide-stained gels. Transcript levels of cytokines (A), IFNs (B), and RIG-I (C) were normalized relative to the constitutively expressed GAPDH gene. Data are representative of 3 separate experiments. *Lanes were not adjacent but came from the same original gel.

Fig. 3.

Effect of CSE on cell viability in human lung slices. Lung slices were incubated with 0%, 2%, 10%, 15%, 20%, 50%, and 100% CSE for 24 h. Staurosporine (10 μM)-treated lung slices were used as a positive control for cytotoxicity. CSE-free medium was used as a negative control. A: LDH activity of the supernatant and lung slices was measured using an LDH-Cytotoxicity Assay Kit (BioVision Research Products) according to the manufacturer's instructions. Cytotoxicity is expressed as the percentage of supernatant-released LDH to total (supernatant + tissue) LDH. B: the relative number of live cells was determined using Cell Counting Kit-8, which detects the activity of dehydrogenases in live cells. Results are shown as means ± SE from 4 separate experiments. Means were compared with data from the negative control group. *P < 0.05.

Fig. 4.

Antioxidants prevent CSE-mediated suppression of IAV induction of IP-10 in the human lung. For antioxidant treatment, lung slices were treated with increasing amounts of N-acetyl-cysteine (NAC; A) or 1,000 mU/ml catalase, 1 mM DTT, or 1 mM GSH (B) for 3 h before 2% CSE was added. All antioxidants were maintained throughout the experiment. In additional wells [C; reverse group (Rev)], there was no antioxidant treatment, and CSE was removed after 24 h of treatment and incubated for another 4 h in normal lung slice medium before treatment with virus. All indicated slices were exposed to 6 × 10⁶ PFU/ml of IAV PR8 for 24 h. IP-10 protein levels were determined by ELISA of lung slice supernatants. Data are expressed as means ± SE from 3 separate lung slice donor experiments. Statistical significance was determined by ANOVA. Means were compared with data from the PR8-treated group. NS, no significant difference. *P < 0.05.

Fig. 5.

Antioxidants prevent CSE-mediated suppression of IAV-induced cytokine and RIG-I expression in the human lung. Lung slices were treated with increasing amounts of NAC or GSH (1 mM) for 3 h before 2% CSE was added. For the reverse group, CSE was removed after 24 h of treatment and incubated for another 4 h in normal lung slice medium before treatment with virus. All indicated slices were exposed to 6 × 10⁶ PFU/ml of IAV PR8 for 24 h. A and B: relative end-point RT-PCR was used to determine mRNA expression. Transcript levels of cytokines and RIG-I were normalized relative to the constitutively expressed GAPDH gene. C: Western blot analysis was used to determine RIG-I protein expression in lung slices. Membranes were probed with anti-RIG-I or anti-GAPDH antibodies. Protein expression of RIG-I was normalized relative to GAPDH. Data are representative of 3 separate experiments.

Fig. 6.

Effects of CSE and antioxidants on induction of mRNA expression of other pattern recognition receptors by IAV in the human lung. Lung slices were treated with increasing amounts of NAC or GSH (1 mM) for 3 h before 2% CSE was added. For the reverse group, CSE was removed after 24 h of treatment and incubated for another 24 h in normal lung slice medium. Lung slices were exposed as indicated to 6 × 10⁶ PFU/ml of IAV PR8 for 24 h. Relative end-point RT-PCR was used to determine mRNA expression. Transcript levels of Toll-like receptor 3 (TLR3), nucleopeptide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3), and nucleotide-binding oligomerization domain 2 (NOD2) were normalized relative to GAPDH mRNA expression. Data are representative of 3 separate experiments.

Fig. 7.

CSE suppresses IP-10 induction by influenza virus in macrophages in the human lung. Lung slices were exposed to 6 × 10⁶ PFU/ml of influenza virus PR8 or virus diluents for 24 h in the presence of brefeldin A (BFA) to enhance the detection of cytokines. Slices were then processed for immunohistochemistry for the detection of the chemokine IP-10 using goat polyclonal antibodies, viral nucleoprotein (NP) using rabbit polyclonal antibody, and macrophages using anti-CD68 monoclonal antibody. Nuclei were stained with SYTOX green. Top: PR8 alone. Bottom: CSE + PR8. A–D: fluorescent images that demonstrate nuclei (A; blue), NP (B; red), IP-10 (C; green), and macrophages (D; cyan). E: bright-field images that demonstrate that lung architecture is preserved during the experiment. F: overlays of the fluorescent images that demonstrate that the primary cellular source of IP-10 is alveolar macrophages (arrows). Bars = 100 μm.

Fig. 8.

CSE suppresses IFN-β induction by influenza virus in epithelial cells in the human lung. Lung slices were exposed to 6 × 10⁶ PFU/ml of influenza virus PR8 or virus diluents for 24 h in the presence of BFA to enhance the detection of cytokines. Slices were then processed for immunohistochemistry for the detection of IFN-β using goat polyclonal antibodies, viral NP using rabbit polyclonal antibody, and epithelial cells using anti-cytokeratin monoclonal antibody. Top: PR8 alone. Bottom: CSE + PR8. A–D: fluorescent images that demonstrate nuclei (A; blue), NP (B; red), IFN-β (C; green), and epithelial cells (D; cyan). E: bright-field images. F: overlays of the fluorescent images that demonstrate that the primary cellular sources of the cytokines are epithelial cells (arrows). Bars = 100 μm.