Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis

Abstract

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Keywords: alveolar type cells; mucus; nontuberculous mycobacteria; secretoglobin family 3A member 2; surfactant protein B.

Figure 1.

Morphologic characterization of control and non–cystic fibrosis bronchiectasis (NCFB) lungs. (A) Representative images of block sections from control lungs stained with hematoxylin and eosin (H&E) and alcian blue and periodic acid–Schiff (AB-PAS). Larger (Aii) and smaller (Aiii) bronchioles were defined only by relative size. (B) Representative images of NCFB lung. Note, dilated and larger bronchiole (Bii) and clusters of smaller bronchioles (Biii) lined by increased numbers of AB-PAS epithelial cells and filled with AB-PAS–positive material and surrounded by mononuclear cell and lymphatic infiltration. Arrows point to the region enlarged in the inset (Aii, Aiii, Bii, and Biii). Scale bars, 5 mm (Ai and Bi); 200 μm (Aii, Aiii, Bii, and Biii). (C–E) Morphometric quantitation of airways in H&E-stained lung block sections from control (n = 5) and Japanese NCFB lungs (n = 8). (C) Total airway lumen perimeter (Ci) or total airway lumen area (Cii) per block section area. (D) Total airway lumen perimeter per block section area of airways classified by presence or absence of SMG or cartilage as bronchi and bronchioles, respectively. (E) Total lumen perimeter per block section area in NCFB-ectatic (NCFB-E) versus nonectatic (NCFB-NE) block sections as measures of disease heterogeneity (see supplemental methods). (F) Percentage of bronchioles within each lung specimen obstructed by mucus for control, NCFB-NE, and NCFB-E sections as measured in AB-PAS–stained sections (see supplemental methods). Histogram bars and error bars represent mean ± SEM. Each unique color represents one donor for NCFB (in E and F). **P < 0.01, Mann-Whitney test (C, D, and F comparing control vs. others), paired t test (E and F comparing NCFB-NE vs. NCFB-E). SMG = submucosal gland.

Figure 2.

SFTPB and SFTPC RNA expression in distal regions of control and non–cystic fibrosis bronchiectasis (NCFB) lungs. (A) Representative images of a control lung probed for SFTPB (blue) and SFTPC (red) by RNA in situ hybridization (RNA-ISH). Proximal bronchioles are defined as SFTPB-negative airways without cartilage or submucosal glands. (B) Representative images of an NCFB-ectatic (NCFB-E) lung similarly probed for SFTPB and SFTPC expression. Note increased numbers of SFTPB+ distal bronchioles and cellular infiltrates in surrounding areas. Scale bars, 500 μm (low magnification); scale bars, 20 μm (high magnification). (C–F) Morphometric quantification of RNA-ISH SFTPB in bronchiolar airway structures within control (n = 5), NCFB-nonectatic (NCFB-NE) (n = 6, all from Japanese specimens), and NCFB-E (n = 11, from Japanese and U.S. specimens) block sections. All bronchioles in the five control lung block sections were quantified (total of 41 bronchioles from five donors). To maintain similar sampling frequencies, 8 bronchioles per block section were randomly selected for quantification in NCFB-NE (48 bronchioles from six donors) and NCFB-E (88 bronchioles from 11 donors). Each unique color in C and D represents one donor of NCFB. (C) Number of SFTPB+ bronchioles per block section area for control, NCFB-NE, and NCFB-E donors. (D) Fraction of SFTPB-stained epithelial area per epithelial surface area as a measure of intensity of SFTPB expression. (E) Fractions of SFTPB-stained area per epithelial surface area versus airway size as a measure of regional expression of SFTPB. (F) Percentage of total SFTPB+ bronchioles in the 1–2 mm and >2 mm size range for control, NCFB-NE, and NCFB-E lungs. (G) Percentage of mucus-obstructed SFTPB+ bronchioles in control (n = 5), NCFB-NE (n = 6), and NCFB-E (n = 11) lungs. (H) Quantitation of SFTPB protein in induced sputum as measured by peptide intensity (total precursor ion intensity) mass spectrometry (control, n = 18; NCFB, n = 10). Each unique color represents one donor of NCFB or control lungs. Histogram bars and error bars represent mean ± SEM. *P < 0.05 and **P < 0.01, Mann-Whitney test (C, G, and H), mixed effect model (D and E).

Figure 3.

SCGB3A2 RNA expression and characterization of alveolar type 0 cells in distal regions of control and non–cystic fibrosis bronchiectasis (NCFB) lungs. (A) Representative images of a control lungs probed for SCGB3A2 (red) by RNA in situ hybridization (RNA-ISH). Note selective expression of SCGB3A2 in distal bronchioles. (B) Representative images of NCFB-ectatic (NCFB-E) probed as in A. Note the increased number of SCGB3A2+ distal bronchioles. Scale bars, 500 μm (low magnification); 20 μm (high magnification). (C–E) Morphometric quantification of RNA-ISH SCGB3A2 in bronchiolar structures within control (n = 5, 49 bronchioles), NCFB-nonectatic (NCFB-NE) (n = 6, 48 bronchioles), and NCFB-E (n = 11, 110 bronchioles) lungs. (C) Number of SCGB3A2+ bronchioles per block section area for control, NCFB-NE, and NCFB-E lungs. Each unique color represents one control or NCFB donor lung. (D) Fraction of SCGB3A2-stained area per epithelial surface area in each group, as measure of intensity of SCGB3A2 expression, versus airway size. (E) Percentage of total SCGB3A2 bronchioles in the 1–2 mm and >2 mm size range for each group. (F) Quantitation of SCGB3A2 protein in induced sputum by peptide intensity (total precursor ion activity) as determined by mass spectrometry (control, n = 18; NCFB, n = 10). (G) Representative images of NCFB-E block sections stained with H&E or immunofluorescence for SCGB3A2 (green), SFTPB (white), and SFTPC (red) in DAPI-stained sections. Scale bars, 100 μm. (H and I) Quantification of percentages of SCGB3A2+ SFTPC+ cells in bronchioles (H) and alveoli (I) for control, NCFB-NE, and NCFB-E lungs. Histogram bars and error bars depict mean ± SEM. *P < 0.05 and **P < 0.01. Mann-Whitney test (C, F, H, and I in control vs. others), paired t test (H and I in NCFB-NE vs. NCFB-E), mixed effect model (D). AT2 = alveolar type 2; H&E = hematoxylin and eosin.

Figure 4.

MUC5B and MUC5AC RNA and protein expression in distal regions of control and non–cystic fibrosis bronchiectasis (NCFB) lungs. (A) Representative images of control (upper) and NCFB-ectatic (NCFB-E) tissue sections (lower) probed for MUC5B (blue) and MUC5AC (red) by RNA in situ hybridization (RNA-ISH) and IF for MUC5B (green) and MUC5AC (red) protein staining. Scale bars, 100 μm. (B and C) Morphometric quantification of RNA-ISH MUC5B (B) and MUC5AC (C) in bronchioles within control (n = 5, 41 bronchioles), NCFB-NE (n = 6, 48 bronchioles), NCFB-E (n = 11, 110 bronchioles) lungs. Each unique color in Bi and Ci represents one control or NCFB donor. (Bi) Fraction of MUC5B-stained surface area per epithelial surface area. (Bii) Correlation between fraction of MUC5B-stained area per surface area and airway size per group (note, y-axis reflects cubic transformation of RNA-ISH data; see supplemental methods). The method for airway size calculation is described in the online supplement. (Ci) Fraction of MUC5AC surface area per epithelial surface area per group. (Cii) Correlations between fraction of MUC5AC-stained area per airway size per group. (D) Representative images of control and NCFB-E tissue sections probed for AGR2 and XBP1S. Scale bars, 20 μm. Mixed effect model and Pearson’s correlation tests (B and C). ^†The area fraction was cubic root transformed (Bii and Cii). IF = immunofluorescence; NCFB-NE = NCFB-nonectatic.

Figure 5.

Characterization of ectasia and MUC5B and MUC5AC mucin expression in proximal bronchioles within non–cystic fibrosis bronchiectasis (NCFB)-ectatic (NCFB-E) lungs. (A) Representative images from NCFB-E lung stained with AB-PAS and probed for SFTPB (blue), SFTPC (red), and UGT2A1 (red) by RNA in situ hybridization (RNA-ISH). Note absence of SFTPB and UGT2A1 signals in proximal bronchioles. Scale bars, 5 mm (low magnification); 200 μm (high magnification). (B) Quantification of total lumen perimeter (Bi) or lumen area (Bii) of SFTPB-negative bronchioles per block section area and percentage of mucus-obstructed SFTPB-negative bronchioles (Biii) in control (n = 5), NCFB-NE (n = 6), and NCFB-E (n = 11) lungs. Each unique color represents one donor. (C) Representative images of fluorescent RNA-ISH for SFTPB (white), MUC5AC (red), and MUC5B (green) in control and NCFB-E lungs. Scale bars, 500 μm. (D) Quantitation of (Di) MUC5B- and (Dii) MUC5AC-stained area per epithelial surface area in SFTPB-negative bronchioles from control (n = 4) and NCFB-E (n = 5) lungs. Histogram bars and error bars represent mean ± SEM. *P < 0.05 and ***P < 0.001. Mann-Whitney test (B and D). AB-PAS = alcian blue and periodic acid–Schiff; NCFB-NE = NCFB-nonectatic.

Figure 6.

Effect of IL-1 receptor deletion on secretory mucin RNA expression in human bronchial epithelial (HBE) cell cultures exposed to sterile sputum supernatants (SSS) from subjects with non–cystic fibrosis bronchiectasis (NCFB) and detection of IL-1 expression in NCFB-E lungs. (A) NCFB subject sputum concentrations of IL-1α and IL-1β measured by ELISA. Note, normal control sputum values for IL-1β were 0.07 (0.004–0.081) ng/ml; n = 51. Normal sputum IL-1α concentrations reported as ∼0.030 ng/ml (48). (B) Comparisons of SSS administration to CRISPR/Cas9-mediated IL-1 receptor knockout (KO) versus negative–guide RNA control (NC) HBE cells with respect to relative expression of MUC5B, MUC5AC, XBP1s, and AGR2 RNA normalized to TBP 3 days after exposure. Data were derived from HBE cells from n = 3 donors. Each unique color represents one donor. (C) Representative images of CRISPR/Cas9-mediated IL-1 receptor KO versus NC HBE cells 3 days after SSS administration stained with hematoxylin and eosin (H&E) and AB-PAS and immunohistochemically for MUC5B and MUC5AC protein. Scale bar, 10 μm. (D) Representative images of control and NCFB-ectatic (NCFB-E) block sections probed for IL1A (blue) and IL1B (red) by RNA-ISH in epithelial and luminal cells in a distal bronchus and bronchiole. Scale bars, 100 μm. (E) A representative image of NCFB-E bronchiole stained with H&E (scale bars, 50 μm) (Ei), with quantitation of macrophages and neutrophils in mucus plugs from five NCFB-E donor lungs (Eii). (F) Immunohistochemical staining for MPO (green), CD68 (white), and IL-1β (red) protein in bronchiolar epithelia and lumens. (G) Percentage of IL-1β colocalization with CD68+, MPO+, or double-positive cells in NCFB-E lungs (n = 5). Scale bars, 50 μm. A total of 100 randomly selected IL-1+β+ cells in submucosa and lumen were counted (Fi, Fii, and G). Each unique color represents one donor of NCFB (E and G). Histogram bars and error bars represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, mixed effect model (B). AB-PAS = alcian blue and periodic acid–Schiff; PBS = phosphate-buffered saline; RNA-ISH = RNA in situ hybridization.

Figure 7.

Transcriptional digital spatial profiler (DSP) studies reveal distinct transcriptional pathway changes in distal bronchioles from control, non–cystic fibrosis bronchiectasis (NCFB)-nonectatic (NCFB-NE), and NCFB-ectatic (NCFB-E) lungs. (A) Principal component analysis plot for control, NCFB-E, and NCFB-NE distal bronchioles. Symbols depict all regions of interest (ROIs) denoted by unique color representing each group. (B) Volcano plots of DSP differentially expressed genes are shown for NCFB-E versus control distal bronchioles (Bi) and NCFB-E versus NCFB-NE distal bronchioles (Bii). (C) Normalized enrichment scores (ES) and adjusted P values obtained from DSP pathway enrichment analyses for NCFB-NE versus controls, NCFB-E versus controls, and NCFB-E versus NCFB-NE bronchioles. Statistical analyses are detailed in the supplemental methods. NS = not significant. (D) Representative images of control and NCFB-E distal bronchioles stained with immunohistochemistry for acetylated α-tubulin (cilia) (Di). Quantitation of proportion of cilia-positive length in bronchiole from control and NCFB-E lungs (both n = 3, nine bronchioles) (Dii). (E) DSP Q3-normalized counts of MMP7 (upper) and PIGR (lower) expression from ROIs of distal bronchioles in control, NCFB-NE, and NCFB-E sections. Details of Q3-nomalized count are described in the online supplement. Graphs represent all ROIs selected, with each unique color representing one donor. (F) Representative images of control and NCFB-E distal bronchioles probed for MMP7 and PIGR by RNA-ISH. Arrows point to the region enlarged in the inset. (G) Principal component analysis plot of NCFB-E distal bronchioles and plugs. (H) DSP Q3-normalized counts depicting CD68, IL1B, and IL1RN expression across ROIs in distal bronchiolar epithelium in control, NCFB-NE, and NCFB-E lungs and NCFB-E mucus plugs. Scale bars, 100 μm (D and F). Each unique color in Dii, E, and H represents one donor. Histogram bars and error bars represent mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, mixed effect model (Dii, E, and H). RNA-ISH = RNA in situ hybridization.

Figure 8.

Graphical summary of the morphological/gene data describing proximal and distal bronchioles in normal, non–cystic fibrosis bronchiectasis (NCFB) (nonectatic) and NCFB (bronchiolectatic) lungs. Bronchiolectasis was observed in the proximal, but not distal, bronchioles of NCFB (bronchiolectasis) lungs. Both MUC5B and MUC5AC were increased in proximal bronchioles, but only MUC5B was increased in distal bronchioles of NCFB lungs. SFTPB was upregulated, whereas cilia-associated genes were downregulated in distal bronchioles of NCFB (bronchiolectatic) lungs. Spatial transcriptomic analyses of distal bronchioles identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β–producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. eNOS = endothelial nitric oxide synthase; MMP = matrix metalloproteinases; SASP = senescence-associated secretory phenotype; UPR = unfolded protein response.