Sustained Club Cell Injury in Mice Induces Histopathologic Features of Deployment-Related Constrictive Bronchiolitis

Abstract

Histopathologic evidence of deployment-related constrictive bronchiolitis (DRCB) has been identified in soldiers deployed to Southwest Asia. While inhalational injury to the airway epithelium is suspected, relatively little is known about the pathogenesis underlying this disabling disorder. Club cells are local progenitors critical for repairing the airway epithelium after exposure to various airborne toxins, and a prior study using an inducible transgenic murine model reported that 10 days of sustained targeted club cell injury causes constrictive bronchiolitis. To further understand the mechanisms leading to small airway fibrosis, a murine model was employed to show that sustained club cell injury elicited acute weight loss, caused increased local production of proinflammatory cytokines, and promoted accumulation of numerous myeloid cell subsets in the lung. Transition to a chronic phase was characterized by up-regulated expression of oxidative stress-associated genes, increased activation of transforming growth factor-β, accumulation of alternatively activated macrophages, and enhanced peribronchiolar collagen deposition. Comparative histopathologic analysis demonstrated that sustained club cell injury was sufficient to induce epithelial metaplasia, airway wall thickening, peribronchiolar infiltrates, and clusters of intraluminal airway macrophages that recapitulated key abnormalities observed in DRCB. Depletion of alveolar macrophages in mice decreased activation of transforming growth factor-β and ameliorated constrictive bronchiolitis. Collectively, these findings implicate sustained club cell injury in the development of DRCB and delineate pathways that may yield biomarkers and treatment targets for this disorder.

Figure 1

Exposure of CC-DTA mice to doxycycline for 10 consecutive days leads to sustained club cell injury. A: Schematic representation of exposure protocol. Cohorts of CC-DTA and control mice were exposed to doxycycline on days 0 to 9 of the protocol to induce club cell injury. B: Representative lung sections of control (top panels) and CC-DTA (bottom panels) mice at the indicated protocol time points were stained using fluorescent immunohistochemistry for club cell secretory protein (CCSP; green), α-smooth muscle actin (α-SMA; red), and DAPI (blue). C:Scgb1a1 (CCSP; left panel) and Foxa2 (right panel) relative gene expression, as measured by quantitative RT-PCR analysis in total lung homogenates at the indicated time points. Expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase and expressed relative to the mean expression of control mice at day 10. D: CCSP protein concentrations measured in bronchoalveolar lavage fluid (BALF; left panel) and serum (right panel) at the indicated time points. C and D: Cumulative data shown are from two independent experiments and present data from 8 to 10 mice. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (C and D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test corrected for multiple comparisons using the Holm-Sidak method). Scale bars = 50 μm (B). AW, airway; V, blood vessel.

Figure 2

Exposure of CC-DTA mice to doxycycline for 10 consecutive days modulates body weight, oxidative stress, and cytokine production. A: Percentage change in body weight from baseline (day 0) in CC-DTA (black squares) and control (black circles) mice at the indicated time points. Data represent at least three independent experiments. B: Relative gene expression measured by quantitative RT-PCR analysis in total lung homogenates for genes associated with oxidative stress: Hmox1 (heme oxygenase 1), Cat (catalase), Gpx1 (glutathione peroxidase), and Sod1 (superoxide dismutase) at the indicated time points. C: Cytokine concentrations were measured by a cytometric bead array in bronchoalveolar lavage fluid for tumor necrosis factor (TNF)-α, IL-6, B-cell activating factor (BAFF), and free active transforming growth factor (TGF)-β1 at the indicated time points. B and C: Cumulative data are from two independent experiments and are presented as data from 8 to 10 mice. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (B and C). n = 5 (A). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test corrected for multiple comparisons using the Holm-Sidak method).

Figure 3

Exposure of CC-DTA mice to doxycycline for 10 consecutive days induces chronic accumulation of alternatively activated lung macrophages. A: Total numbers of CD45⁺ leukocytes and each myeloid subset per mouse lung were calculated at the indicated time points. B: Representative cell surface CD206 expression on alveolar macrophages (left panel) and exudate macrophages (right panel) obtained at protocol day 20 from a CC-DTA (thick black line; open histogram) and control mouse (thin black line; gray histogram) relative to isotype-matched control staining (thin orange line; open histogram). C: CD206 cell surface expression [Δ geometric mean fluorescence intensity (GMFI) relative to isotype-matched control antibody staining] on alveolar macrophages (AMs) and exudate macrophages (ExMs) at protocol day 20. D: Relative gene expression measured by quantitative RT-PCR analysis in total lung homogenates for genes associated with alternative macrophage activation: Lgals3 (galectin-3), Retnla (resistin-like α; Fizz1), Chil3 (chitinase-like 3; YM1), and Mrc1 (mannose receptor C type 1; CD206) at protocol day 20. A, C, and D: Cumulative data are from two independent experiments and presented as data from 10 mice. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (A, C, and D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 (t-test corrected for multiple comparisons using the Holm-Sidak method). cDC, conventional dendritic cell; max, maximum; moDC, monocyte-derived dendritic cell.

Figure 4

Exposure of CC-DTA mice to doxycycline for 10 consecutive days induces chronic peribronchiolar fibrosis. A: Representative lung sections stained using picrosirius red (to identify collagen) from control and CC-DTA mice at protocol day 20. B: Relative gene expression measured by quantitative RT-PCR analysis in total lung homogenates for Col1a1 at the indicated time points. C: Hydroxyproline assay for total lung collagen content quantification at the indicated time points. B and C: Cumulative data are from two independent experiments and are presented as data from 8 to 10 mice. C: The two graphs represent separate, independent experiments. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (B and C). ∗∗P < 0.01, ∗∗∗P < 0.001 (t-test corrected for multiple comparisons using the Holm-Sidak method). Scale bars: 100 μm (A, left panels); 10 μm (A, right panels). AW, airway; V, blood vessel.

Figure 5

Airway wall thickening and epithelial metaplasia in murine and deployment-related constrictive bronchiolitis (DRCB). Representative images of lung sections (hematoxylin and eosin stained) obtained from a CC-DTA mouse (A and B) at protocol day 20 and a veteran diagnosed with DRCB (C and D). Note thickening of the airway wall with subepithelial matrix deposition (blue brackets; B and D) and squamous metaplasia (red arrows; B and D) present in both specimens. Scale bars: 100 μm (A and C); 40 μm (B and D). AW, airway; CB, constrictive bronchiolitis.

Figure 6

Chronic airway inflammation in murine and deployment-related constrictive bronchiolitis (DRCB). Representative images of lung sections (hematoxylin and eosin stained) obtained from a CC-DTA mouse (A–D) at protocol day 20 and a veteran diagnosed with DRCB (E–H). Note the peribronchiolar mononuclear cell infiltrates (orange brackets; B and F) and collections of intraluminal cells with the appearance of foamy macrophages (blue arrows; D and H) present in both specimens. Scale bars: 100 μm (A, C, E, and G); 40 μm (B, D, F, and H). AW, airway; CB, constrictive bronchiolitis; MCI, mononuclear cell infiltrate.

Figure 7

Peribronchiolar myeloid cell infiltrates and large intraluminal macrophages characterize both murine and deployment-related constrictive bronchiolitis (DRCB). Representative images of lung sections obtained from a CC-DTA mouse (A–F) at protocol day 20 and a veteran diagnosed with DRCB (G–J). Murine frozen sections (A–J) were stained with isotype control antibody (A and B) or anti-CD68 (C–F) using an aminoethyl carbazole peroxidase reaction (red staining), whereas sections from the veteran with DRCB were paraffin embedded and stained with anti-CD68 (G–J) using a 3,3′-diaminobenzidine peroxidase reaction (brown staining). Both specimens were counterstained with hematoxylin. Note the presence of smaller CD68⁺ mononuclear cells localized to the subepithelial infiltrates (red arrows; D, F, and H) and the larger CD68⁺ cells with macrophage morphology residing within airway lumens (blue arrows; F and J) present in both specimens. Scale bars: 100 μm (A, C, E, and G–J); 40 μm (B, D, and F). AW, airway; CB, constrictive bronchiolitis.

Figure 8

Depletion of alveolar macrophages ameliorates murine constrictive bronchiolitis in CC-DTA mice exposed to doxycycline for 10 consecutive days. A: Schematic representation of macrophage depletion protocol. Cohorts of CC-DTA and control mice were exposed to doxycycline on days 0 to 9 of the protocol to induce club cell injury. Mice also received either clodronate liposomes (CLs) or empty liposomes (ELs) via oropharyngeal aspiration on protocol days 0, 3, 7, 10, 13, and 17. B: Percentage change in body weight from baseline (day 0) in doxycycline-exposed CC-DTA mice treated with CLs (red squares) or ELs (black circles) relative to doxycycline-exposed control mice treated with ELs (black triangles) at the indicated time points. Data are representative of one of three experiments. C: Representative lung sections from doxycycline-exposed control or CC-DTA mice given the indicated treatments stained using picrosirius red (top panels) or trichrome (bottom panels) to identify peribronchiolar collagen deposition at protocol day 20. Right panels: Note the reduction in collagen staining in doxycycline-exposed CC-DTA mice treated with CLs. D: Hydroxyproline assay for total lung collagen content quantification at protocol day 20. E: Free active transforming growth factor (TGF)-β1 concentrations measured by a cytometric bead array in bronchoalveolar lavage fluid at protocol day 20. D and E: Cumulative data shown are from two independent experiments and presented as data from 9 to 10 mice. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (D and E). n = 5 mice per experimental condition (B). ∗P < 0.05, ∗∗P < 0.01 (one-way analysis of variance followed by the Tukey multiple comparisons test). Scale bars = 100 μm (C).

Supplemental Figure S1

Identification of lung myeloid cells in murine constrictive bronchiolitis using flow cytometric analysis. Gating strategy used in flow cytometric analysis to identify CD45⁺ leukocytes (CD45⁺), neutrophils, eosinophils (Eos), CD103⁺ conventional dendritic cells (CD103⁺ cDCs), CD11b⁺ conventional dendritic cells (CD11b⁺ cDCs), monocyte-derived dendritic cells (moDCs), Ly6C⁺ monocytes (Ly6C⁺ monos), alveolar macrophages (AMs), and exudate macrophages (ExMs) within lung-derived single-cell suspensions obtained from CC-DTA and control mice at the indicated time points (representative plots from a CC-DTA mouse at protocol day 20 are shown; initial gates set to eliminate doublets, debris, and nonviable cells are not shown). FSC, forward scatter; MHC, major histocompatibility complex; SSC, side scatter.

Supplemental Figure S2

Monocyte and macrophage accumulation in murine constrictive bronchiolitis (CB). Representative images of lung sections obtained from a CC-DTA mouse at protocol day 20. Murine frozen sections were stained with isotype control antibody (A and B), anti-F4/80 (C and D), and anti-CD11c (E and F) using an aminoethyl carbazole peroxidase reaction (red staining). Both specimens were counterstained with hematoxylin. Note the presence of smaller F4/80⁺ mononuclear cells localized to the subepithelial infiltrates (red arrows; C and D) and the larger CD11c⁺ cells with macrophage morphology residing within airway lumens (blue arrows; F). Scale bars: 100 μm (A); 40 μm (B–F). AW, airway.

Supplemental Figure S3

Alveolar macrophage depletion using clodronate liposomes. A: Protocol day 5 enumeration of alveolar and exudate macrophages (by flow cytometric analysis) in the lungs of CC-DTA mice exposed to doxycycline and administered clodronate liposomes (CLs) or empty liposomes (ELs) on days 0 and 3. Data are from one experiment and presented as data from three mice. B: Protocol day 20 enumeration of alveolar and exudate macrophages in the lungs of CC-DTA and control mice exposed to doxycycline and administered clodronate or empty liposomes on protocol days 0, 3, 7, 10, 13, and 17. Cumulative data from two independent experiments are shown and presented as data from 10 mice. CC-DTA mice, open bars; control mice, closed bars. Data are presented as means ± SEM (A and B). ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 [unpaired t-test (A) or one-way analysis of variance, followed by the Tukey multiple comparisons test (B)].