Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium

Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium

Correction: Type V Collagen Induced Tolerance Suppresses Collagen Deposition, TGF-β and Associated Transcripts in Pulmonary Fibrosis.

[This corrects the article DOI: 10.1371/journal.pone.0076451.]

Type V Collagen Induced Tolerance Suppresses Collagen Deposition, TGF-β and Associated Transcripts in Pulmonary Fibrosis

<div><p>Rationale</p><p>Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models.</p><p>Methods</p><p>Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses.</p><p>Results</p><p>Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local <i>Il-17a</i> transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (<i>Tgf-β, Il-1β, Pdgfb</i>), matrix (<i>Acta2, Col1a2, Col3a1, Lox</i>, <i>Itgb1/6, Itga2/3</i>) and members of the TGF-β superfamily (<i>Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb</i>).</p><p>Conclusions</p><p>Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.</p></div

Tolerance induction of col(V) suppresses T lymphocytes activation and associated pro-inflammatory/pro-fibrotic cytokine expression.

<p>(<b>A</b>) 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>B</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. (<b>C</b>, <b>D</b>) Plasma samples from (<b>A</b>) 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. (<b>E</b>) At day 14 post bleomycin injury, whole lung homogenates from bleomycin-injured mice+col(V) treatment were quantified for <i>il-17a</i> mRNA expression and normalized for <i>β-actin</i>. Values represent mean ± SEM. n = 5 mice/group; compared to bleomycin group, * p<0.01; one-way ANOVA, post hoc test: Bonferroni.</p

Tolerance induction of Col(V) protects against bleomycin-induced fibrosis.

<p>(<b>A</b>) Col(V) overexpression was detected in 21 day post bleomycin injury by labeling with rhodamine and counterstained with DAPI. (Original magnification, 10×). (<b>B</b>) Schematic illustration of the experimental design. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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×. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076451#pone.0076451.s002" target="_blank">Figure S2A</a>: 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×.</p

Tolerance induction of Col(I) does not confer protection against bleomycin-induced fibrosis.

<p>(<b>A</b>) Schematic illustration of the experimental design. (<b>B</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. (<b>C</b>) 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×. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076451#pone.0076451.s002" target="_blank">Figure S2B</a>: 1×).</p

Schematic representation of pathways affected by intrapulmonary instillation of col(V).

<p>Schematic representation of pathways affected by intrapulmonary instillation of col(V).</p

Col(V) treatment downregulates integrins, TGF-β and other pro-fibrotic cytokines.

<p>At day 28 post bleomycin injury, lung tissues were homogenized and cDNA was analyzed for fibrosis-specific gene expression analyses.</p

Col(V) treatment downregulates TGF-β associated receptors, signaling molecules and transcription factors.

<p>At day 28 post bleomycin injury, lung tissues were homogenized and cDNA was analyzed for fibrosis-specific gene expression analyses.</p

Col(V) treatment protects against established fibrosis.

<p>(<b>A</b>) Schematic illustration of the experimental design. (<b>B</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×. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076451#pone.0076451.s002" target="_blank">Figure S2C</a>: 1×). (<b>C</b>) 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.</p