BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance

Abstract

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Keywords: mucin; mucociliary clearance; mucus; rheology; viscoelasticity.

Graphical abstract

Figure 1.

Decreased mucociliary clearance in Bpifb1 KO mice. A: percent bead clearance in tracheal mucociliary clearance assay in naive wild-type (WT) and Bpifb1 knockout (KO) mice. Means ± SE depicted; n = 6/genotype; male (square), female (triangle). **P < 0.01 from two-sided Student’s t test. B: ion transport measured in Ussing chambers. Amil, amiloride; Forsk, forskolin; Bumet, bumetanide. Means ± SE depicted; n = 6/genotype. No differences between genotypes by two-sided Student’s t test. C: ciliary beat frequency by in situ tracheal assay. Means ± SE depicted; n = 7 or 8/genotype; male (square), female (triangle). P = 0.1 for two-sided Welch’s t test by genotype.

Figure 2.

Decreased complex viscosity of apical wash samples from Bpifb1 knockout (KO) mouse tracheal epithelial cultures (mTECs). Complex viscosity of short (15 min) and long (3 h) apical washes of mTECs from wild-type (WT) (white) and Bpifb1 KO (gray) mice grown at air-liquid interface (ALI). Box plots depict median and interquartile ranges where each point represents an independent well of cells pooled from three mice. n = 17 or 18/genotype/wash group. Significance differences between groups by Wilcoxon rank sum test denoted as *P < 0.05 and #P < 0.1.

Figure 3.

Reduced percent solids in mucus from the apical surface of Bpifb1 knockout (KO) tracheal epithelial cultures (mTECs). Mass measurements of fluid (A) and organic solids (B) from mucus collected from the surface of wild-type (WT) and Bpifb1 KO mTECs. C: percentage of the total mass that is solids. Significant differences by genotype by two-sided Student’s t test denoted as *P < 0.05 and #P < 0.1. n = 6/genotype.

Figure 4.

Increased complex viscosity of mucus flakes in lavage fluid from Bpifb1 knockout (KO) mice. A: distribution of rheological signal in bronchoalveolar lavage (BAL) from a representative allergen exposed wild-type (WT) and Bpifb1 KO mouse. B: complex viscosity of BAL mucus fraction. Boxplots depict median and interquartile ranges. n = 13/genotype; male (square), female (triangle). *P < 0.05, significant difference between genotypes by ANCOVA modeling. MUC5B in pellet (C) and supernatant (D) fraction of BAL from naive WT and Bpifb1 KO mice by mucin immunoblotting. Means ± SEM depicted. n = 5/genotype; male (square), female (triangle). Significant difference between genotypes by two-sided Student’s t test denoted as *P < 0.001.

Figure 5.

BPIFB1 and MUC5B colocalize in murine airway secretory epithelial cells. A: BPIFB1 (green) and MUC5B (red) immunostaining in airway epithelial cells from allergen-challenged mice. Scale bar = 10 μm. B: percentage of cells positively stained for BPIFB1 only, MUC5B only, or both in allergen-challenged mice. n = 7 mice; square (male), female (circle). C: BPIFB1 (green) and MUC5B (red) immunostaining in large airways of allergen-challenged mice demonstrating MUC5B and BPIFB1 staining in luminal mucus. Scale bar = 50 μm.

Figure 6.

BPIFB1 is part of the mucus network in human bronchial epithelial cell cultures. Primary human bronchial epithelial cell cultures were established and differentiated at an air-liquid interface for 4 wk, then fixed and stained for MUC5B and BPIFB1. Immunogold-labeled scanned electron microscopy for negative control (rabbit IgG; A), MUC5B (B), and BPIFB1 (C) is shown. Scale bar = 1 μm.