Controlled and uncontrolled asthma display distinct alveolar tissue matrix compositions

Abstract

Objective: Whether distal inflammation in asthmatics also leads to structural changes in the alveolar parenchyma remains poorly examined, especially in patients with uncontrolled asthma. We hypothesized that patients who do not respond to conventional inhaled corticosteroid therapy have a distinct tissue composition, not only in central, but also in distal lung.

Methods: Bronchial and transbronchial biopsies from healthy controls, patients with controlled atopic and patients with uncontrolled atopic asthma were processed for immunohistochemical analysis of fibroblasts and extracellular matrix molecules: collagen, versican, biglycan, decorin, fibronectin, EDA-fibronectin, matrix metalloproteinase (MMP)-9 and tissue-inhibitor of matrix metalloproteinase (TIMP)-3.

Results: In central airways we found increased percentage areas of versican and decorin in patients with uncontrolled asthma compared to both healthy controls and patients with controlled asthma. Percentage area of biglycan was significantly higher in both central airways and alveolar parenchyma of patients with uncontrolled compared to controlled asthma. Ratios of MMP-9/TIMP-3 were decreased in both uncontrolled and controlled asthma compared to healthy controls. In the alveolar parenchyma, patients with uncontrolled asthma had increased percentage areas of collagen, versican and decorin compared to patients with controlled asthma. Patients with uncontrolled asthma had significantly higher numbers of myofibroblasts in both central airways and alveolar parenchyma compared to patients with controlled asthma.

Conclusions: Tissue composition differs, in both central and distal airways, between patients with uncontrolled and controlled asthma on equivalent doses of ICS. This altered structure and possible change in tissue elasticity may lead to abnormal mechanical properties, which could be a factor in the persistent symptoms for patients with uncontrolled asthma.

Figure 1

Percentage area of collagen (trichrome staining, % positively stained area) in bronchial and transbronchial biopsies from controls and patients with controlled and uncontrolled asthma (A-B). Data are presented as scatter dot plots where horizontal line denotes median value. Representative micrographs of tissue stained with trichrome staining (collagen: blue) from controls (C, F), controlled asthma (D, G) and uncontrolled asthma (E, H). Scale bars: C-E, G and H = 100 μm and F = 200 μm.

Figure 2

Percentage area of versican (A, B) and decorin (C, D) (% positively stained area) in bronchial and transbronchial biopsies from controls and patients with controlled and uncontrolled asthma. Data are presented as scatter dot plots where horizontal line denotes median value. Representative micrographs of double staining of versican (brown) and decorin (red) from controls (E, H) and patients with controlled asthma (F, I) and uncontrolled asthma (G, J) in bronchial (E-G) and transbronchial (H-J) biopsies. Scale bars: F, G = 50 μm, E, I and J = 100 μm, H = 200 μm.

Figure 3

Percentage area of biglycan (% positively stained area) in bronchial (A) and transbronchial biopsies (B) from controls and patients with controlled and uncontrolled asthma. Representative micrographs of staining of biglycan (brown) from controls (C, F) and patients with controlled asthma (D, G) and uncontrolled asthma (E, H) in bronchial (C-E) and transbronchial (F-H) biopsies. Scale bars: C-H = 50 μm.

Figure 4

Percentage area of MMP-9 (% positively stained area) in bronchial (A) and transbronchial biopsies (B) in controls and patients with controlled and uncontrolled asthma. Representative micrographs of staining of MMP-9 (brown) from controls (C, F) and patients with controlled asthma (D, G) and uncontrolled asthma (E, H) in bronchial (C-E) and transbronchial (F-H) biopsies. Scale bars: C-E and G = 100 μm, F and H = 200 μm.

Figure 5

Percentage area of TIMP-3 (% positively stained area) in bronchial (A) and transbronchial biopsies (B) in controls and patients with controlled and uncontrolled asthma. Representative micrographs of staining of TIMP-3 (brown) from controls (C, F) and patients with controlled asthma (D, G) and uncontrolled asthma (E, H) in bronchial (C-E) and transbronchial (F-H) biopsies. Scale bars: C-E and G = 100 μm, F and H = 200 μm.

Figure 6

Density of myofibroblasts per tissue area (triple positive cells/mm²) in central airways (A) and alveolar parenchyma (B) from controls and patients with controlled and uncontrolled asthma. Representative micrographs of triple staining of myofibroblasts: (C) nuclei: Hoechts 33342, blue, (D) prolyl-4OH: Alexa F488, green, (E) vimentin: AlexaF 647, deep red, (F) α-SMA: AlexaF 555, red. Merged image are shown in (G) where * denotes triple positive cells. High magnification image of fibroblast with protrusions are shown in (H). rbm: reticular basement membrane and ep: airway epithelium

Figure 7

Percentage area of EDA-fibronectin (% positively stained area) in controls and patients with uncontrolled asthma (F). Representative micrographs of staining of EDA-fibronectin (brown) from controls (C, F) and patients with controlled asthma (D, G) and uncontrolled asthma (E, H) in bronchial (C-E) and transbronchial (F-H) biopsies. Scale bars: C-H = 100 μm.