Table 1.
Clinical characteristics of healthy non-smokers, healthy smokers, and individuals with confirmed COPD that underwent bronchial biopsies to obtain bronchial brushings.
Fig 1.
Co-infection of airway epithelial cells with human rhinovirus (HRV) and Pseudomonas aeruginosa (PA) results in a synergistic increase in human beta-defensin (HBD)-2 production.
Human bronchial epithelial (HBE) cells were infected with HRV-16 or PA, alone and in combination. HBD-2 mRNA (n = 6) (A) and HBD-2 protein (n = 9) (B) levels were measured 48 h post infection. Data are expressed as mean ±SEM (*p<0.05 between HRV+PA and the sum of the values from each treatment alone).
Fig 2.
Flagellin (Fla), but not lipoteichoic acid (LTA) or lipopolysaccharide (LPS), induce HBD-2 production from human bronchial epithelial (HBE) cells.
HBE cells were treated with medium control, HRV-16 (HRV), Fla (30 ng/mL), LTA (1 μg/mL) LPS (1 μg/mL), alone and in combination with HRV. HBD-2 protein was assessed after 48 h. Data are expressed as mean ±SEM (n = 5).
Fig 3.
Synergy in human beta-defensin (HBD)-2 expression due to the combination human rhinovirus (HRV)-16 and flagellin (Fla) stimulation mimics HRV and live Pseudomonas aeruginosa (PA) co-infection in airway epithelial cells.
A) HBD-2 mRNA (n = 8) and B) HBD-2 protein (n = 11) levels were determined after stimulation with HRV-16, 30 ng/ml of Fla, or the combination for 48 h in human bronchial epithelial (HBE) cells. Data are expressed as mean ± SEM (*p<0.05 between HRV+Fla and the sum of the values from each treatment alone).
Fig 4.
The synergistic response of human beta defensin (HBD)-2 is lost after co-infection with flagellin deficient Pseudomonas aeruginosa (fliC) and human rhinovirus (HRV).
HBD-2 mRNA (n = 5) and HBD-2 protein (n = 10) levels were determined following infection of HBE cells for 48 h with HRV-16, wild-type P. aeruginosa (PA), flagellin-deficient P. aeruginosa (fliC) or the combination of either bacterial strain with HRV. Data are expressed as mean ± SEM. Hashtag indicates significant difference compared to all other treatment groups (p<0.05). (ns = not significant).
Fig 5.
Human rhinovirus (HRV) and Pseudomonas aeruginosa (PA) induced human beta-defensin (HBD)-2 expression is reduced following the knockdown of toll-like receptor (TLR)-5.
A) TLR5 protein levels in human bronchial epithelial (HBE) cell lysates (48 h after infection) induced following HRV/PA co-infection were assessed via western blotting following lipid-mediated transfection (24 h) of 10 nM TLR5 short-interfering RNA (siRNA) duplexes A or B, compared with non-targeting control siRNA. B) Densitometric analysis of TLR5 protein expression of HRV and PA co-infected HBE cells transfected with TLR5 siRNA duplex A or B, or control siRNA. TLR5 expression levels were normalized to GAPDH and compared to siRNA control. C) HBD-2 protein (n = 6) levels were determined following 48 h infection with HRV-16, PA, alone and in combination, all in the presence or absence of TLR5 siRNA duplex A or B. Data are expressed as mean ± SEM (*p<0.05).
Fig 6.
Synergistic expression of HBD-2 is lost after co-infection with replication deficient UV-treated human rhinovirus (UV HRV) and P. aeruginosa (PA).
A) HBD-2 mRNA (n = 4) and B) protein (n = 8) expression from HBE cells after 48 h infection with HRV, PA, UV-HRV, or the combination of PA with either virus group. Data are expressed as mean ± SEM (#p<0.05 compared to all other treatment.
Fig 7.
Limited reduction in human beta-defensin (HBD)-2 protein is seen following knockdown of retinoic-acid inducible gene (RIG)-I or melanoma differentiation associated gene-5 (MDA5).
A) MDA5 and D) RIG-I knockdown was assessed via western blot analysis of HBE cells co-infected with human rhinovirus (HRV)-16 and P. aeruginosa (PA) following lipid-mediated transfection (24 h) of 10 nM short-interfering RNA (siRNA) duplex A or B specific for MDA5, compared with non-targeting siRNA control. Densitometric analysis of B) MDA5 and E) RIG-I knockdown. HBD-2 protein levels were determined 48 h post infection with HRV-16, PA or the combination in the presence or absence of C) MDA5 siRNA duplex A or B (n = 4) or F) RIG-I siRNA duplex A or B (n = 3). Data expressed as mean ± SEM.
Fig 8.
Cigarette smoke extract (CSE) exposure significantly attenuates HBD-2 expression following co-infection with human rhinovirus (HRV)-16 and P. aeruginosa (PA).
A) HBD-2 mRNA (n = 6) and B) protein (n = 8) expression from human bronchial epithelial (HBE) cells following 48 h stimulation with HRV-16, PA, or the combination, with and without addition of 50% CSE. Data expressed as mean ± SEM (#p<0.05 compared to all other treatment groups).
Fig 9.
Human beta-defensin (HBD)-2 expression following human rhinovirus (HRV)-16 and P. aeruginosa (PA) co-infection is significantly attenuated in healthy smokers and patients with COPD compared to normal non-smokers.
HBD-2 protein expression from epithelial cells obtained during bronchial biopsies of healthy non-smokers (n = 5), healthy smokers with normal lung function (n = 5), and patients with confirmed smoking-related COPD (n = 5) was measured following 48 h infection with HRV-16, PA, or the combination. Data expressed as mean ± SEM (*p<0.05).