α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

Figures & data

Table 1 Characteristics of human study subjects

	Age (years)	Sex (M/F)	Smoking (pack-years)	FEV1, % predicted	FVC, % predicted	FEV1/FVC%
Normal (n=4)	53.0±3.2	1/3	1 (0) 3 (34.3±16.7)	93.7±12.8	88.0±11.6	83.0±2.5
COPD (n=4)	61.5±8.1	1/3	4 (55.3±21.3)	41.0±13.4a	62.5±21.9	41.7±6.2a

Notes: Data are presented as mean ± SEM.

a Indicates that P<0.05 compared to normal subjects.

Abbreviations: M, male; F, female; FEV₁, forced expiratory volume in the first second; FVC, forced vital capacity; SEM, standard error of the mean.

Figure 1 Rhinovirus infection increased expression of antiviral genes in human bronchial epithelial cells.

Notes: Brushed bronchial epithelial cells cultured in ALI from normal subjects (n=4) and patients diagnosed with COPD (n=4) were exposed to air for 10 minutes and then infected with HRV-16 for 24 hours or treated with cell culture medium (−) as a control. HRV-16 significantly increased the mRNA expression of antiviral genes IFN-λ1, OAS1, and MX1. Relative levels of the three genes were normalized to the housekeeping gene GAPDH. The red horizontal bars represent the medians of the eight subjects. The Wilcoxon test was used to compare the difference between (−) and HRV-infected groups. Blue represents normal subjects, while black represents patients diagnosed with COPD.

Abbreviations: ALI, air–liquid interface; HRV, human rhinovirus; mRNA, messenger RNA; IFN-λ1, interferon-λ1.

Figure 2 WCS increased viral load and decreased antiviral gene expression in rhinovirus-infected human bronchial epithelial cells.

Notes: Brushed bronchial epithelial cells cultured in ALI from normal subjects (n=4) and patients diagnosed with COPD (n=4) were exposed to air for 10 minutes and then infected with HRV-16 for 24 hours or treated with cell culture medium as a control. WCS significantly increased viral load (A) but reduced the mRNA expression of antiviral genes (B). Relative levels of the three genes were normalized to the housekeeping gene GAPDH. The red horizontal bars represent the medians of the eight subjects. The Wilcoxon test was used to compare the difference between air and WCS groups. Blue represents normal subjects, while black represents patients diagnosed with COPD.
Abbreviations: WCS, whole cigarette smoke; ALI, air–liquid interface; HRV, human rhinovirus; mRNA, messenger RNA; IFN-λ1, interferon-λ1.

Figure 3 A1AT decreased viral load in WCS-exposed human bronchial epithelial cells.

Notes: Brushed bronchial epithelial cells cultured in ALI from normal subjects (n=4) and patients diagnosed with COPD (n=4) were exposed to air or WCS for 10 minutes. Cells were treated with BSA as a control or A1AT (1 mg/mL) in the presence of HRV-16 for 24 hours. A1AT decreased viral load in cells exposed to WCS (A), and expression of the antiviral genes IFN-λ1, OAS1, or MX1 in cells exposed to WCS (B) or air (C). Relative levels of the three genes were normalized to the housekeeping gene GAPDH. The red horizontal bars represent the medians of the eight subjects. The Wilcoxon test was used to compare the difference between BSA and AIAT groups. Blue represents normal subjects, while black represents patients diagnosed with COPD.
Abbreviations: A1AT, α1-antitrypsin; WCS, whole cigarette smoke; ALI, air–liquid interface; BSA, bovine serum albumin; HRV, human rhinovirus; IFN-λ1, interferon-λ1.

Figure 4 A1AT did not affect HRV-3C protease activity.

Notes: A1AT (1 mg/mL) was added to HRV-3C protease and its substrate, GST-Syk fusion protein, incubated overnight, electrophoresed on an SDS-PAGE gel, and then stained with Coomassie blue. The image is from one of the three independent experiments, all showing similar results. (−) indicates no HRV-3C.
Abbreviations: A1AT, α1-antitrypsin; HRV, human rhinovirus; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; BSA, bovine serum albumin.

Figure 5 A1AT treatment decreased ICAM-1 mRNA expression in human bronchial epithelial cells.

Notes: Brushed bronchial epithelial cells cultured in ALI from normal subjects (n=4) and patients diagnosed with COPD (n=4) were exposed to air or WCS for 10 minutes. Cells were treated with BSA as a control or A1AT (1 mg/mL) in the presence of HRV-16 for 24 hours. In air-exposed and HRV-16-infected cells, A1AT significantly reduced ICAM-1 mRNA (A). In WCS-exposed and HRV-16-infected cells, A1AT tended to decrease ICAM-1 mRNA (B). Relative ICAM-1 mRNA levels were normalized to the housekeeping gene GAPDH. An ANOVA was used to compare all the groups, followed by a paired t-test to compare the two groups. Data are presented as mean ± SEM.
Abbreviations: A1AT, α1-antitrypsin; ICAM-1, intercellular adhesion molecule-1; mRNA, messenger RNA; ALI, air–liquid interface; WCS, whole cigarette smoke; BSA, bovine serum albumin; HRV, human rhinovirus; ANOVA, analysis of variance; SEM, standard error of the mean.