Tobacco exposure inhibits SPLUNC1-dependent antimicrobial activity

Abstract

Background: Tobacco smoke exposure impairs the lung's innate immune response, leading to an increased risk of chronic infections. SPLUNC1 is a secreted, multifunctional innate defense protein that has antimicrobial activity against Gram negative organisms. We hypothesize that tobacco smoke-induced SPLUNC1 dysfunction contributes to the observed defect in innate immunity in tobacco smokers and that this dysfunction can be used as a potential biomarker of harm.

Methods: We collected sputum from never-smokers and otherwise healthy smokers. We performed Western blotting to determine SPLUNC1 levels and determined antimicrobial activity against nontypeable Haemophilus influenzae. An in vitro exposure model was utilized to measure the effect of tobacco exposure on human bronchial epithelial culture (HBEC) antimicrobial activity against H. influenzae. The direct effects of cigarette and little cigar smoke exposure on SPLUNC1 function was determined using 24 h growth measurements and LPS binding assays.

Results: H. influenzae growth in cigarette smoker's sputum was significantly greater compared to never-smokers sputum over 24 h. HBEC supernatants and lysates contained significantly higher numbers of H. influenzae following chronic cigarette and little cigar smoke exposure compared to air-exposed controls. Furthermore, SPLUNC1's antimicrobial activity and LPS-binding capability against both H. influenzae and P. aeruginosa was attenuated following cigarette and little cigar exposure.

Conclusions: These data suggest that cigarette and little cigar exposure impairs SPLUNC1's antimicrobial ability and that this inhibition may serve as a novel biomarker of harm that can be used to assess the toxicity of commercial tobacco products.

Keywords: BPIFA1; COPD; Little cigars; Sputum.

Fig. 1

SPLUNC1 protein levels are similar in smokers’ and never-smokers’ sputum. Representative Western blots showing endogenous expression of SPLUNC1 (a) and neutrophil elastase (c) in never smoker and smokers sputum from n = 6 donors per group. Membranes were probed for SPLUNC1 prior to stripping and re-probing for neutrophil elastase. Bar graphs showing mean densitometry of SPLUNC1 (b) and neutrophil elastase (d) protein abundance in never smokers and smokers sputum

Fig. 2

Cigarette smoker’s sputum has increased H. influenzae growth. Sputum was obtained from never-smokers and smokers (n = 6 per group). 10⁵ CFU H. influenzae were added to 20 μl of Sputum and CFUs were measured 24 h later. **p < 0.001 different to never-smokers (Mann-Whitney U-test)

Fig. 3

Tobacco smoke exposure leads to increased H. influenzae colonization. HBECs obtained from normal donors were exposed to air or tobacco smoke from Marlboro cigarettes, Cheyenne little cigars and Swisher Sweets Strawberry little cigars, once a day for 5 days. 20 μl of H. influenzae at 10⁶ CFU/ml were added apically to HBECs for 2 h at 37 °C/5% CO₂. a Apical supernatants and (b) lysates were collected, serially diluted, plated on LB agar plates and incubated at 37 °C for 48 h to determine colony-forming units (CFU) for bacterial load (all n = 9). Statistically significant differences were measured using the Kruskal-Wallis test. **p < 0.001, ***p < 0.0001 different to air control

Fig. 4

Tobacco smoke exposure reduces SPLUNC1 bacteriostatic activity. a, c 10 μM of SPLUNC1 or Kentucky cigarette-exposed SPLUNC1 dissolved in Ringer’s solution were co-incubated with 10⁶ of H. influenzae or P. aeruginosa for 24 h (all n = 3 per group). b, d H. influenzae or P. aeruginosa were co-incubated with 10 μM of SPLUNC1 or tobacco-exposed SPLUNC1 for 24 h; Camel, Marlboro, Djarum, Cheyenne, Swisher Sweets, Swisher Sweets Strawberry, and Captain Black (all n = 3 per group). Statistically significant differences were measured using Kruskal-Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001 different to control

Fig. 5

Tobacco smoke reduces SPLUNC1’s ability to bind LPS. Graphs show Non-linear regression fit generated from one site specific binding of LPS derived from either H. influenzae (a) or P. aeruginosa (b) with 2 fold dilutions of SPLUNC1, cigarette and tobacco smoke exposed SPLUNC1 dissolved in Ringer’s solution to commercial cigarettes and several little cigars (all n = 9 per group). Statistically significant differences were measured using Kruskal-Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 different to Air + SPLUNC1