Cigarette smoke inhibits airway epithelial cell innate immune responses to bacteria

Abstract

The human upper respiratory tract, including the nasopharynx, is colonized by a diverse array of microorganisms. While the host generally exists in harmony with the commensal microflora, under certain conditions, these organisms may cause local or systemic disease. Respiratory epithelial cells act as local sentinels of the innate immune system, responding to conserved microbial patterns through activation of signal transduction pathways and cytokine production. In addition to colonizing microbes, these cells may also be influenced by environmental agents, including cigarette smoke (CS). Because of the strong relationship among secondhand smoke exposure, bacterial infection, and sinusitis, we hypothesized that components in CS might alter epithelial cell innate immune responses to pathogenic bacteria. We examined the effect of CS condensate (CSC) or extract (CSE) on signal transduction and cytokine production in primary and immortalized epithelial cells of human or murine origin in response to nontypeable Haemophilus influenzae and Staphylococcus aureus. We observed that epithelial production of interleukin-8 (IL-8) and IL-6 in response to bacterial stimulation was significantly inhibited in the presence of CS (P < 0.001 for inhibition by either CSC or CSE). In contrast, epithelial production of beta interferon (IFN-beta) was not inhibited. CSC decreased NF-kappaB activation (P < 0.05) and altered the kinetics of mitogen-activated protein kinase phosphorylation in cells exposed to bacteria. Treatment of CSC with antioxidants abrogated CSC-mediated reduction of epithelial IL-8 responses to bacteria (P > 0.05 compared to cells without CSC treatment). These results identify a novel oxidant-mediated immunosuppressive role for CS in epithelial cells.

FIG. 1.

CSC inhibits pathogen-induced epithelial cell innate immune responses. A549 human airway epithelial cells were exposed to H. influenzae (Hi) strain H233 or S. aureus (Sa) strain RN6390 in the presence of CSC, CSE, or vehicle control (DMSO for CSC; medium [VC] for CSE) for 18 h, and levels of IL-8 in the supernatants were determined by ELISA. CSC induced dose-dependent inhibition of IL-8 responses to HK NT H. influenzae bacteria (A), HK S. aureus (B), or live NT H. influenzae (C). Similar effects were observed when cells were treated with CSC and proinflammatory cytokine TNF-α (D) or with CSE and HK NT H. influenzae (E). P values for this and subsequent figures are as follows: *, <0.05, **, <0.01, and ***, <0.001, while NS (not significant) refers to a P value of >0.05. ND, not detected.

FIG. 2.

CSC decreases IL-6 and IL-8 mRNA transcription in response to bacteria. Immortalized A549 (A) or differentiated primary murine respiratory epithelial (B and C) cells were concurrently treated with CSC (200 μg/ml) or DMSO and 10⁸ CFU/ml of HK NT H. influenzae. The amount of IL-6 (A and B), IL-8 (A), or IFN-β (C) mRNA was determined using real-time PCR with normalization to GAPDH (A and B) or actin (C) transcript levels. RQ values obtained from comparative C_T analysis (ΔΔC_T) are shown. The RQ value for DMSO alone was standardized to 1. Comparison of C_T values representing GAPDH transcript levels in A549 cells treated with DMSO or CSC (200 μg/ml) for 2 or 4 h (D) demonstrates that CSC does not affect transcription globally.

FIG. 3.

MAPK activation in response to bacteria with or without CSC exposure. A549 cells were treated with 200 μg/ml CSC or DMSO with or without HK NT H. influenzae for 30 or 60 min, and cell lysates were separated on SDS-PAGE and probed for phosphorylated and total forms of p38, JNK, and ERK MAP kinases. Results for total MAPK are shown as loading controls.

FIG. 4.

CSC decreases NT H. influenzae-mediated NF-κB activation. (A) A549 monolayers were treated with 50 μM TPCK or DMSO for 30 min, washed with fresh medium, and treated with HK NT H. influenzae for 18 h prior to determination of IL-8 concentrations in cell-free supernatants. (B) Luciferase-based assay of NF-κB activity in A549 cells treated with 200 μg/ml CSC with or without HK NT H. influenzae for 6 h prior to cell lysis.

FIG. 5.

Antioxidant treatment abrogates CSC-mediated IL-8 suppression. A549 monolayers were treated with CSC and/or HK NT H. influenzae for 18 h. CSC or DMSO was pretreated with 25 mM NAC for 30 min. IL-8 concentrations in supernatants were assayed by ELISA. Similar results were obtained with H. influenzae at a concentration of 10⁸ CFU/ml (not shown).