Sputum Leucine-Rich Alpha-2 Glycoprotein as a Marker of Airway Inflammation in Asthma

<div><p>Background</p><p>Asthma is a chronic inflammatory disease of airways, but an ideal biomarker that accurately reflects ongoing airway inflammation has not yet been established. The aim of this study was to examine the potential of sputum leucine-rich alpha-2 glycoprotein (LRG) as a new biomarker for airway inflammation in asthma.</p><p>Methods</p><p>We obtained induced sputum samples from patients with asthma (N = 64) and healthy volunteers (N = 22) and measured LRG concentration by sandwich enzyme-linked immunosorbent assay (ELISA). Ovalbumin (OVA)-induced asthma model mice were used to investigate the mechanism of LRG production during airway inflammation. The LRG concentrations in the bronchoalveolar lavage fluid (BALF) obtained from mice were determined by ELISA and mouse lung sections were stained with anti-LRG antibody and periodic acid-Schiff (PAS) reagent.</p><p>Results</p><p>Sputum LRG concentrations were significantly higher in patients with asthma than in healthy volunteers (p = 0.00686). Consistent with patients’ data, BALF LRG levels in asthma model mice were significantly higher than in control mice (p = 0.00013). Immunohistochemistry of lung sections from asthma model mice revealed that LRG was intensely expressed in a subpopulation of bronchial epithelial cells, which corresponded with PAS-positive mucus producing cells.</p><p>Conclusion</p><p>These findings suggest that sputum LRG is a promising biomarker of local inflammation in asthma.</p></div

Up-regulation of LRG in asthmatic patients.

<p>Levels of LRG in sputum obtained from healthy volunteers and patients with asthma. Concentrations of sputum LRG were determined by ELISA. The Mann-Whitney U-test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s004" target="_blank">S2 File</a>.</p

Detection of LRG in BALF, serum and lung section in a murine model of asthma.

<p>A) Levels of LRG in BALF and serum in a mouse model of asthma. Concentrations of BALF and serum LRG were determined by ELISA. The Student’s t-test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s005" target="_blank">S3B–S3E File</a>) Localization of LRG in mouse lung. Paraffin sections of the lung from control (B and D) and OVA-treated (C and E) mouse were stained with anti-LRG antibody (B and C) and PAS (D and E). Scale bar, 100 μm. F and G) Immunohistochemisry of MUC5AC (F) and LRG (G) of the lung from OVA-treated mouse. Arrows show MUC5AC (F) or LRG (G) positive cells. Scale bar, 20 μm</p

Induction of LRG in primary bronchial epithelial cells.

<p>A) LRG secretion in culture supernatant by primary bronchial epithelial cells. Cells were incubated with IL-13 for 5 days and then treated with indicated cytokines for 24 h. Control cells were incubated without IL-13 for 5 days and further stimulated by cytokines. LRG protein in culture supernatant was detected by western blot. B) LRG gene expression in primary bronchial epithelial cells treated with IL-13 for 5 days. Cells were stimulated by indicated cytokines for 6 h. LRG mRNA expression was analyzed by quantitative PCR. Dunnett’s test was used for statistical analysis. The individual values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162672#pone.0162672.s006" target="_blank">S4 File</a>.</p

Clinical characteristics of the study subjects.

<p>Clinical characteristics of the study subjects.</p

Additional file 5 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 5. Figure S4

Additional file 3 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 3. Table S1. Top 30 differentially expressed genes between nestin-expressing and -nonexpressing endothelial cells in the lung

Additional file 1 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 1. Figure S1

Additional file 6 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 6. Figure S5

Additional file 4 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 4. Figure S3