Figure 1.
Relative expression of col(I) and col(V) in patients with UIP/IPF and pathologically normal specimens.
(A) Lung tissue sections from UIP/IPF patients and pathologically normal specimens were immunostained with col(I) and col(V) antibodies and their IgG, followed by incubation with rhodamine-anti-rabbit. Nuclei were counterstained with DAPI. (Original magnification, 10×, representative of 4 patients). Corresponding H&E staining is also shown. (B) Pepsin digested lung homogenates (15 µg) and corresponding standards run in a 5% gel and immunoblotted with antibodies against col(V) and col(I). Image shows representative 3 normal and 5 IPF tissues, (C) Densitometry of protein expressions of individual alpha chains of col(I) and col(V) obtained from IPF lung biopsies and pathologically normal specimens. Values represent mean ± SEM of5 normals and 20 IPF specimens (p<0.01; one-way ANOVA, post hoc test: Bonferroni), (D) mRNA expression were determined by qPCR of lung tissue sections of UIP/IPF and pathologically normal specimens. Values represent mean ± SEM; 3 normals and 4 UIP/IPF specimens; (p<0.01; one-way ANOVA, post hoc test: Bonferroni).
Figure 2.
Auto-antibody responses of col(I) and col(V) in plasma of patients with UIP/IPF and normal volunteers.
Col(I) and col(V) antibodies were detected in the plasma by flow cytometry in plasma of patients with UIP/IPF with varying degrees of disease severity (n = 40) and healthy normal volunteers (n = 7). Values are represented as mean ± SEM; *, p<0.05, **, p<0.001; one-way ANOVA, post hoc test: Bonferroni.
Figure 3.
Tolerance induction of Col(V) protects against bleomycin-induced fibrosis.
(A) Col(V) overexpression was detected in 21 day post bleomycin injury by labeling with rhodamine and counterstained with DAPI. (Original magnification, 10×). (B) Schematic illustration of the experimental design. (C) Circulating antibodies specific to col(V) were detected in 21 day post bleomycin injury mice. Values represent mean ± SEM; number of animals: PBS = 5, BLEO = 6 and BLEO+col(V) Neb = 4. Compared to bleomycin group, * p<0.001; ** p<0.01; one-way ANOVA, post hoc test: Bonferroni. (D) Collagen deposition was measured quantitatively by assaying for hydroxyproline concentrations from the whole left lung day 21 post bleomycin injury+col(V) treatment. Values represent mean ± SEM; n = 7 mice/group. Compared to bleomycin group, ** p<0.01; * p<0.05 one-way ANOVA, post hoc test: Newman-Keuhl's. (E) At day 21 post bleomycin injury, lung tissue sections were stained for H&E and Masson's blue trichrome staining. Extensive collagen deposition was observed with bleomycin injury while col(V) nebulized lungs, similar to normal lungs, had collagen deposition around airways and vasculature. Original magnifications: 10×. (Figure S2A: 1×). Lung tissue sections were immunostained against alpha-smooth muscle actin (α-SMA) or IgG. Streptavidin-conjugated horseradish peroxidase was used with 3,3′-diaminobenzidene as substrate (brown) and nuclei were counterstained with hematoxylin (blue). Original magnifications: 20×.
Figure 4.
Tolerance induction of Col(I) does not confer protection against bleomycin-induced fibrosis.
(A) Schematic illustration of the experimental design. (B) Circulating antibodies specific to col(I) were unchanged in 21 day post bleomycin injury mice. Values represent mean ± SEM; number of animals: PBS = 4, BLEO = 4 and BLEO+col(I) Neb = 4. (C) At day 21 post bleomycin injury, lung tissue sections were stained for H&E and Masson's blue trichrome staining. Extensive collagen deposition was observed with bleomycin injury and col(I) nebulized lungs. Original magnifications: 10×. (Figure S2B: 1×).
Figure 5.
Tolerance induction of col(V) suppresses T lymphocytes activation and associated pro-inflammatory/pro-fibrotic cytokine expression.
(A) At day 21 post bleomycin injury, mediastinal lymphocytes were isolated and cultured alone or in the presence of autologous antigen presenting cells (APCs from naïve C57-BL/6 mice) for 48 h. The cells were then radiolabeled with tritiated thymidine for 16 h and assessed for proliferation rates. Values represent mean ± SEM. n = 5 mice/group; Compared to bleomycin group, * p<0.01; one-way ANOVA, post hoc test: Bonferroni. (B) Conditioned media from A was analyzed for Th1/Th2/Th17 cytokines. Values represent mean ± SEM. n = 5 mice/group; Compared to bleomycin group, * p<0.01; one-way ANOVA, post hoc test: Bonferroni. (C, D) Plasma samples from (A) were analyzed for Th1/Th2/Th17 cytokines. Values represent mean ± SEM. n = 5 mice/group; Compared to bleomycin group, * p<0.01; one-way ANOVA, post hoc test: Bonferroni. (E) At day 14 post bleomycin injury, whole lung homogenates from bleomycin-injured mice+col(V) treatment were quantified for il-17a mRNA expression and normalized for β-actin. Values represent mean ± SEM. n = 5 mice/group; compared to bleomycin group, * p<0.01; one-way ANOVA, post hoc test: Bonferroni.
Figure 6.
Col(V) treatment protects against established fibrosis.
(A) Schematic illustration of the experimental design. (B) At day 28 post bleomycin injury, lung tissue sections were stained for H&E and Masson's blue trichrome staining. Extensive collagen deposition was observed with bleomycin injury while col(V) nebulized lungs, similar to normal lungs, had collagen deposition around airways and vasculature. Original magnifications: 10×. (Figure S2C: 1×). (C) Collagen deposition was measured quantitatively by assaying for hydroxyproline concentrations from the whole left lung day 28 and day 14 post bleomycin injury+col(V) treatment. Values represent mean ± SEM; n = 12 mice/group; * p<0.001 for day 28 bleomycin vs. day 28 PBS and day 28 col(V) (Neb); day 14 bleomycin vs. day 14 PBS; day 28 bleomycin vs. day 14 bleomycin; one-way ANOVA, post hoc test: Bonferroni.
Figure 7.
Col(V) treatment downregulates integrins, TGF-β and other pro-fibrotic cytokines.
At day 28 post bleomycin injury, lung tissues were homogenized and cDNA was analyzed for fibrosis-specific gene expression analyses.
Figure 8.
Col(V) treatment downregulates matrix molecules.
At day 28 post bleomycin injury, lung tissues were homogenized and cDNA was analyzed for fibrosis-specific gene expression analyses.
Figure 9.
Col(V) treatment downregulates TGF-β associated receptors, signaling molecules and transcription factors.
At day 28 post bleomycin injury, lung tissues were homogenized and cDNA was analyzed for fibrosis-specific gene expression analyses.
Table 1.
List of bleomycin-induced genes modulated by col(V) treatment.
Figure 10.
Schematic representation of pathways affected by intrapulmonary instillation of col(V).