Mucus Hypersecretion and Ciliary Impairment in Conducting Airway Contribute to Alveolar Mucus Plugging in Idiopathic Pulmonary Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease attributed to the complex interplay of genetic and environmental risks. The muco-ciliary clearance (MCC) system plays a critical role in maintaining the conduit for air to and from the alveoli, but it remains poorly understood whether the MCC abnormalities in conducting airway are involved in IPF pathogenesis. In this study, we obtained the surgically resected bronchi and peripheral lung tissues from 31 IPF patients and 39 control subjects, and we sought to explore the morphologic characteristics of MCC in conducting airway by using immunostaining and scanning and transmission electron microscopy. In the submucosal regions of the bronchi, we found that the areas of mucus glands (MUC5B⁺) were significantly larger in IPF patients as compared with control subjects (p < 0.05). In the surface epithelium of three airway regions (bronchi, proximal bronchioles, and distal bronchioles), increased MUC5B and MUC5AC expression of secretory cells, decreased number of ciliated cells, and increased ciliary length were observed in IPF patients than control subjects (all p < 0.05). In addition, the mRNA expression levels of MUC5B were up-regulated in both the bronchi and peripheral lung of IPF patients than those of control subjects (p < 0.05), accompanied with 93.55% IPF subjects who had obvious MUC5B⁺ mucus plugs in alveolar regions. No MUC5B rs35705950 single-nucleotide polymorphism allele was detected in both IPF patients and control subjects. Our study shows that mucus hypersecretion and ciliary impairment in conducting airway are major causes of mucus plugs in alveolar regions and may be closely related to the alveolar injuries in IPF patients.

Keywords: MUC5B; conducting airway mucosa; idiopathic pulmonary fibrosis; muco-ciliary clearance; mucus plugs.

FIGURE 1

Quantification of glands and MUC5B expression in bronchial submucosal region. Representative H&E staining images in the bronchi from control subjects (A) and IPF patients (B), and the median SMG area (expressed as the proportion of SMG region over the total mucosal region) was significantly higher in IPF patients than that in control subjects (C). The IF staining shows that the median areas of mucus component (MUC5B-positive) in SMGs were significantly increased in IPF patients compared to those in control subjects (D–F). DAPI labeling of nuclei (blue). DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; MUC5B, mucin 5B; SMG, submucosal glands; IF, immunofluorescence; IPF, idiopathic pulmonary fibrosis.

FIGURE 2

Morphology and expression of secretory proteins in the surface epithelium of the conducting airways. Representative images of H&E staining, TEM, and IF staining showing the morphology and immunopositive secretory cells (A–C); H&E staining and TEM revealed hyperplasia of secretory cells in the epithelium of bronchi and proximal and distal bronchioles from IPF patients, respectively. Quantitation of positive expression areas of MUC5B (red), MUC5AC (green), and CC10 (white) in different airway compartments between control subjects and IPF patients (D–F). DAPI labeling of nuclei (blue). CC10, club cell 10 kDa protein; DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; IPF, idiopathic pulmonary fibrosis; MUC5AC, mucin 5AC; MUC5B, mucin 5B; TEM, transmission electron microscope; ns, not significant.

FIGURE 3

Quantification of ciliated cells and ciliary length in conducting airways. IF staining for α-tubulin (stained in green) in different compartments of conducting airway in control subjects (A–C) and IPF patients (D–F). The median number of ciliated cells was significantly reduced in all three airway compartments (G), along with the significant increase of ciliary length (H) in IPF patients as compared to the controls, respectively. DAPI labeling of nuclei (blue). DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; IPF, idiopathic pulmonary fibrosis.

FIGURE 4

Characteristics of surface epithelium revealed by SEM. SEM was performed to show the distribution of ciliated cells and secretory cells in different regions of conducting airway from control subjects (A–D) and IPF patients (E–H). IPF, idiopathic pulmonary fibrosis; SEM, scanning electron microscope. EPs, epitheliums; SG, submucosal gland.

FIGURE 5

MUC5B⁺ mucus plugging in conducting airway lumens and alveolar regions. The IF staining showing obvious MUC5B⁺ mucus (red) secreted in conducting airway lumens in IPF patients (A). A representative H&E staining of peripheral lung tissue from a control subject, showing that both the distal bronchioles and alveoli have normal size and architecture, without fibrosis or inflammation around (B). A representative H&E staining of honeycomb fibrosis (characterized by cystic fibrotic airspaces that are frequently lined by bronchiolar epithelium and filled with mucus and inflammatory cells) in peripheral lung tissue from an IPF patient (C). Compared with control subjects (B), we found obvious MUC5B⁺ mucus plugging in alveolar regions (SPC-positive for alveolar type 2 cell) (C). As compared to the controls, the MUC5B mRNA levels were significantly higher in both the bronchi (D) and peripheral lung (E) tissues of IPF patients, respectively. DAPI labeling of nuclei (blue). DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; IPF, idiopathic pulmonary fibrosis; MUC5B, mucin 5B; SPC, surfactant protein C.