Article Figures & Data
Figures
- Figure 1. Serum and lung SP-D levels during BMS-induced disease progression.
(A) Timeline after i.t. instillation of bleomycin (1.28 mg/kg body weight) with microbubbles followed by sonoporation (BMS) in D1CC×D1BC mice, showing initiation, acute, intermediate, and chronic phases of IP progression. (B) Serum SP-D levels over 14 wk after BMS (closed circle), bleomycin alone (closed triangle), or vehicle (open circle) in D1CC×D1BC mice evaluated by ELISA. The dotted line in inset graph indicates serum SP-D cutoff value (53.9 ng/ml). Data are means ± SE of seven mice at each time point. Asterisks show *P < 0.05, compared with vehicle. (C, D) Body weight variability ratio and (D) total lung weight after BMS administration in D1CC×D1BC mice. Data are means ± SE from nine mice per group. Asterisks show *P < 0.05 and ****P < 0.0001. (E, F, G) SP-D (E), SP-A (F), and SP-C (G) in whole lung extracts at weeks 0, 2, 8, and 14 measured by WB. The relative signal intensity from densitometrical analysis was calculated using ImageJ Fiji, with tubulin-α used as a loading control. Data are means ± SE from three mice per group. Asterisks show **P < 0.01, and ***P < 0.001, compared with 0 wk. (H) Kaplan–Meier analysis of mouse survival post-BMS from weeks 0–14 of 14 mice.
- Figure S1. Incidence of IP with different concentration of bleomycin in BMS method.
The incidence of IP was determined by whether the concentration of serum SP-D exceeded the cutoff value (53.9 ng/ml). Bleomycin was administrated intra-tracheally at 0.96 (closed triangle), 1.28 (closed square), and 1.60 mg/kg body weight (closed circle), respectively. 10 mice were used for each group.
- Figure S2. Absence of IP with sonoporation and/or microbubbles without bleomycin, or with lower doses of BMS.
Sonoporation or microbubbles, alone and in combination, or lower doses of BMS (bleomycin 0.008, 0.8, and 0.2 mg/kg body weight) are not sufficient to induce IP in D1CC×D1BC mouse. Serum SP-D was measured by ELISA in samples collected at week 2 after BMS administration, corresponding to peak levels with BMS (1.28 mg/kg body weight) in Fig 1. The results represent mean ± SE of eight mice per group.
- Figure S3. TUNEL assay with conventional bleomycin method.
Intra-tracheal instillation of bleomycin (40 μl/mouse, 3.84 mg/kg body weight, three times higher than in BMS method) was performed in D1CC×D1BC mice. Mice were euthanized at 6 or 24 h after bleomycin administration. Scale bars = 100 μm (yellow) and 10 μm (white).
- Figure 2. DNA damage in AEC1, AEC2, and bronchioles, but not in fibroblasts.
(A) TUNEL assays in mouse lungs at 0, 6, and 24 h, 3 and 7 days, and 2, 8, and 14 weeks after BMS administration. Arrows indicate cell death. (B) Percentage of γH2AX-positive cells at days 0, 3, 7 and 14 in mice treated with BMS. Images were captured and analyzed from five randomly selected fields on each slide at a magnification of 200×. Data are means ± SE from five mice per group. Asterisks show **P < 0.01, ****P < 0.0001, compared with 0 wk. (C) Percentage of γH2AX-positive cells in AEC1 (podoplanin), AEC2 (SP-C), bronchiolar epithelium (E-cadherin, Ecad), and fibroblasts (S100A4) at day 3. Images were captured and analyzed from 10 randomly selected fields on each slide at a magnification of 200×. Data are means ± SE from three mice per group. Asterisks show ****P < 0.0001, compared with the percentage of γH2AX-positive cell in different cell type. (D) Immunohistochemical staining of γH2AX (green), podoplanin (Pdpn, red), SP-C (white), E-cadherin (E-cad, red), S100A4 (white), F4/80 (red), CD45R (red), and CD3 (yellow). Scale bar = 100 μm (yellow) and 10 μm (white).
- Figure S4. Infiltration of lymphoid cells by day 7 post-BMS, without systemic tissue damage.
Histochemical staining with hematoxylin and eosin after BMS administration. (A) Lung sections at 0, 6, and 24 h, and 3 and 7 d with magnified image. (B) Liver and skin sections from control mice (left) and at week 2 after BMS (right). There were no overt phenotypic changes in the liver or skin after bleomycin treatment. Scale bars = 100 μm.
- Figure S5. Honeycomb structure in the chronic phase of BMS model.
Representative macrographs of Masson’s trichrome staining at days 0, 3, 7, and weeks 2, 4, 6, 8, and 14 after BMS administration in whole lung sections from D1CC×D1BC mice. L, left lobe; R, right lobe. Scale bar = 5 mm. Arrows indicate honeycombing at weeks 10 and 14.
- Figure 3. Bimodal fibrotic stages in IP progression.
(A) Representative histopathological micrographs of Masson’s trichrome–stained sections from BMS-induced D1CC×D1BC mice. In acute phase at week 2, many infiltrated cells were observed. In the chronic phase at week 14, honeycombing was indicated by yellow arrows. (B) Quantitative analysis of fibrosis from Masson’s trichrome stained sections as ratios of ECM area (blue) to total lung area, as calculated by ImageJ, Fiji. n = 6–7 per each group. (C) Quantitative analysis of Ashcroft score (averaged from scores by two individuals, Y Miura and S Kanazawa, performed in a blinded manner). n = 3 per group. (D) Representative images at weeks 0, 4, and 14 captured with HistoIndex show the fibrotic area (mostly collagen I and III, green) and the entire lung area (cell structure, red). (E, F) Collagen deposition and (F) collagen fiber density in 2-μm-thick unstained specimens captured by HistoIndex and analyzed using FibroIndex software. n = 3 per each group. (G) Quantitative analysis of total soluble collagen using sirius red. n = 5 per each group. (H) Collagen I expression determined by WB and ImageJ, Fiji. n = 3 per each group. (I) Immunohistochemical staining of Col1 (green) and S100A4 (red) at weeks 0, 2, 8, and 14. Asterisks indicates subpleural hyperplasia. (J) TGF-β expression determined by WB and ImageJ, Fiji. (A, D, I) Scale bars are 100 μm in (A) and (I), and 5 mm in (D). (G, H, J) n = 3 per each group. Data are presented means ± SE. Asterisks show *P < 0.05, **P < 0.01, and ***P < 0.001, compared with week 0.
- Figure S6. Severe inflammation in acute but not chronic phases of BMS model.
(A, B, C, D) Percentages of lymphoid cells in whole lung images, determined by immunohistochemical staining for (A) Macrophage (F4/80), (B) T cell (CD3), (C) neutrophil (PAD4), and (D) B cell (CD45R) at weeks 0, 2, 8, and 14 and calculated by ImageJ, Fiji. All results are represented as the means ± SE from lung sections from three mice for each time point. Asterisk shows P < 0.05, compared with 0 wk. (E, F) Each image represents immunohistochemical staining of (E) macrophages (F4/80, green), T cells (CD3, red), (F) neutrophils (PAD4, green), and B cells (CD45R, red). Scale bars = 100 μm.
- Figure S7. BMS induces mild chronic fibrosis in D1CC, D1BC, and DBA/1J mice.
Weak IP progression in D1CC, D1BC alone, and their genetic background DBA/1J mice treated with BMS. (A) Quantitative analysis of Ashcroft score at week 14 for DBA/1J, D1CC, D1BC, and D1CC×D1BC mice (averaged from scores by two individuals, Y Miura and S Kanazawa, performed in a blinded manner). The results represent means ± SE from five mice (DBA/1J and D1CC) or three mice (D1BC and D1CC×D1BC) per group. Asterisk shows P < 0.05, compared with D1CC×D1BC mice. (B) Histochemical staining with hematoxylin and eosin. Representative histopathological micro-graphs of Masson’s trichrome stained sections from BMS-induced D1CC×D1BC mice. In acute phase at week 2, many infiltrated cells were observed. In the chronic phase at week 14, honeycombing was indicated by arrows. Scale bar = 500 μm.
- Figure S8. Elevated αSMA expression after BMS administration.
(A) Immunohistochemical staining for αSMA (DAB staining) at weeks 0, 2, and 14. Scale bar = 100 μm. (B) Expression of αSMA at weeks 0, 2, 8, and 14 was determined by WB and ImageJ, Fiji. Data are presented means ± SE of three mice.
- Figure S9. Invasive bronchiolar epithelial cells with less epithelial mesenchymal transition in chronic phase of BMS model.
Immunostaining for epithelial cells using E-cadherin (green) and S100A4 (red) as an epithelial mesenchymal transition marker (red). Scale bar = 50 μm.
- Figure 4. Extension of fibroblasts rather than proliferation in fibrotic foci in chronic phase.
(A, B, C, D, E) Expression of (A) vimentin, (B) PDGFRα, (C) PDGFRβ, (D) PDGFα, and (E) PDGFβ at weeks 0, 2, 8, and 14 determined by WB and ImageJ, Fiji. Data are presented means ± SE of three mice for each week. Asterisks shows *P < 0.05, **P < 0.01, and ***P < 0.001, compared with 0 wk. (F, G, H) Representative immunostaining at week 14 showing (F) fibrotic area (Col1, yellow), bronchiolar epithelial cells (E-cadherin (E-cad), green), and AEC2s and bronchiolar epithelial cells (SP-C, red), (G) fibrotic area (Col1, white), PDGFRα (red) and PDGFRβ (green), and (H) Ki67 (red), PDGFRα (green), and PDGFRβ (white). Scale bars = 50 μm. All specimens for immunostaining were obtained at week 14.
- Figure 5. Invasive bronchiolar epithelial cells in chronic phase.
(A, B) Immunostaining for invasive bronchiolar epithelial cells using (A) E-cadherin (E-cad, green) and MMP7 (red) or (B) E-cadherin (E-cad, green) and N-terminal proteolytic fragments of Laminin, γ2pf (red). (C, D, E) In situ hybridization for invasive bronchiolar epithelial cells at weeks 0 and 14. (C, D, E) Scgb1a1 (green) and Krt5 (red), (D) Scgb1a1 (green) and Sftpc (red), and (E) Scgb1a1 (green), Tgfβ1 (white), and Col1a1 (red). Each arrow indicates Krt5 (red), Scgb1a1 (green), and Krt5 and Scgb1a1 (yellow)-positive cells in C a and b. Scale bars = 50 μm (white) and 25 μm (yellow). (F, G) E-cadherin and (G) uteroglobin expression determined by WB and ImageJ, Fiji. Data are presented means ± SE of three mice. Asterisks show *P < 0.05, compared with week 0.
- Figure S10. Representative Western blotting images of indicated proteins.
Protein expression in whole lung extracts determined by Western blot and visualized by ECL chemiluminescence system. The relative signal intensity for each protein was determined using tubulin-α as a loading control (shown for one blot only, bottom). Details about antibodies were mentioned in the Materials and Methods section. Grouped data from three mice at weeks 0, 2, 8, and 14 is shown in Fig 1E–G (SP-A, SP-C, SP-D), Fig S9 (αSMA), Fig 3G (Type I collagen), Fig 3J (TGF-β), Fig 4 (Vimentin, PDGFRα, PDGFRβ, PDGFα, and PDGFβ), Fig 5 (E-cadherin and uteroglobulin), and Fig 6 (CD31, VEGFA, and PEDF). Arrows indicate each protein. Tubulin-a (55 kD) was used as an internal control for each.
- Figure 6. Poor reconstitution of blood vessels in chronic phase.
(A, B) Immunohistochemical staining for VEGF (green), CD31 (red), and PDGFRβ (white) or (B) VEGF (green) and E-cadherin (Ecad, red) at weeks 0, 2, 8, and 14. (C, D, E) CD31, (D) VEGFA, and (E) PEDF expression was determined by WB and ImageJ, Fiji. Data are presented means ± SE of three mice for each week, 0, 2, 8 and 14. Asterisk shows *P < 0.05, compared with week 0. (F) Representative hematoxylin eosin staining (upper) and immunohistochemical staining for VEGF (green), CD31 (red), and PDGFRβ (white) (lower). “a” indicates hyperplastic area with bronchiolar epithelial cells, whereas “b” indicates hyperplastic interstitial area at week 14. Arrows indicate CD31-positive cells. (G) Representative subpleural images at weeks 0 and 14 showing blood vessels, visualized using Microfil and micro-CT. (H) The total volume was calculated for each range of microvessel diameter in the whole lung after ex vivo Microfil application. Data are presented as means ± SE of three mice for each week, 0 and 14. (A, B, F, G) Scale bars = 50 μm (white), 1 mm (yellow).
- Figure 7. Chronological features in iUIP mouse model.
(1) Serum SP-D levels immediately increased in the acute phase, whereas total lung SP-D increased towards the chronic phase rather than the acute phase. (2) Cell death and double-strand breaks in alveolar epithelial cells were induced by BMS administration. (3) Bimodal fibrosis was found in the acute and the chronic phases. Honeycombing was observed from the intermediate to chronic phases. (4) Fibroblast expansion rather than growth was evident in the acute phase. (5) Invasive epithelial cells, which expressed MMP7 and γ2pf, were observed in the intermediate and the chronic phases. (6) Some invasive epithelial cells expressed both E-cadherin and S100A4, suggesting that epithelial mesenchymal transition occurring in the intermediate and chronic phases. (7) Poor vascularization was evidenced by reduction of CD31 and VEGF expression throughout the entire period after BMS administration. PEDF expression was up-regulated only in the acute phase.
Supplementary Materials
