Activated alveolar epithelial cells initiate fibrosis through secretion of mesenchymal proteins

Abstract

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-β-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Figure 1

Generation and validation of mice with lung epithelial-specific deletion of Col1a1. A: Schematic of floxed Col1a1 (Col1a1^f/f) mice generated by inserting loxP sites flanking exons 2 to 5 in the Col1a1 gene. Cre-mediated recombination of the floxed Col1a1 leads to removal of exons 2 to 5, shift in the translational reading frame in subsequent exons, and permanent inactivation of collagen I expression. B: MEFs isolated from Col1a1^f/f mice have deletion of collagen I expression after AdCre, whereas cells treated with AdGFP or WT Col1a1 MEFs treated with AdGFP or AdCre have normal expression of collagen I by immunoblot analysis. C: Lung epithelial cell-specific and permanent deletion of Col1a1 is achieved with the use of transgenic mice that carry the SPC-rtTA and tetO-Cre. Triple transgenic SPC-rtTA/tetO-Cre/Col1a1^f/f mice are abbreviated SCcol. The SPC promoter yields rtTA expression specifically within lung epithelial cells, in the presence of doxycycline, rtTA activates the tetO-CMV promoter, leading to expression of cre recombinase and permanent deletion of DNA flanked by loxP sites. Deletion within lung epithelial cells is confirmed by PCR with the use of primers that encompass the floxed region. Intact floxed Col1a1 yields a 2.2-kb PCR product, and removal of the floxed Col1a1 yields a 0.5-kb PCR product. D and E: Uninjured lungs from SCcol (E) mice have normal histology compared with littermate genotype control mice (D), by H&E staining. F: AECs from SCcol mice have diminished EMT-induced expression of collagen I by immunoblot analysis compared with AECs from littermate control mice lacking one of the three transgenes. Primary lung fibroblasts from SCcol mice have similar collagen I expression compared with control lung fibroblasts, verifying robust lung epithelial-specific deletion of Col1a1. Original magnification: ×200 (D and E). AdCre, adenoviral-mediated expression of Cre; AdGFP, adenovirus expressing green fluorescent protein; AEC, Alveolar epithelial cell; Col1a1, collagen, type I, alpha 1; Ctrl, control; EMT, epithelial-mesenchymal transition; Fib, fibroblast; MEF, murine embryonic fibroblast; SPC-rtTA, surfactant proteins-C promoter-reverse tetracycline transactivator; tetO-Cre, tetO-cytomegalovirus promoter-Cre recombinase; WT, wild-type.

Figure 2

Lung epithelial cell-derived type I collagen contributes to fibrogenesis. A: Lung sections from littermate control and SCcol mice 3 weeks after bleomycin injection, stained with H&E and trichrome. Original magnification, ×100. Genotype control mice have robust fibrosis compared with SCcol mice. B: Whole lung lysate from mice 3 weeks after treatment with saline or bleomycin was analyzed for collagen I by immunoblot analysis. Control mice have robust induction of collagen I after bleomycin compared with SCcol mice. C: Hydroxyproline assay from entire lungs 3 weeks after saline or bleomycin in SCcol (black bars) or littermate control (white bars) mice. SCcol mice have less fibrosis after bleomycin (n = 6 to 15 per group). ^∗P < 0.05 versus control bleomycin. Bleo, bleomycin; Sal, saline; SCcol, triple transgenic surfactant proteins-C promoter-reverse tetracycline transactivator/tetO-cytomegalovirus promoter-Cre recombinase/homozygous floxed collagen, type I, alpha 1.

Figure 3

Lung epithelial cell-derived collagen I inhibits sustained lung inflammation. A: Survival of littermate control (white circles; n = 25) and SCcol (black circles; n = 18) mice after bleomycin. SCcol mice have a trend toward less survival. B and C: BAL from control (white bars) and SCcol (black bars) mice at 0, 7, and 14 days after bleomycin injury was analyzed for protein (B), and cell count (C) shows that SCcol and control mice have similar increases in proteins and cell count 1 week after bleomycin, whereas SCcol mice have greater persistence of protein, n = 5 to 7 per group. ^∗P < 0.05 versus control bleomycin. BAL, bronchoalveolar lavage; SCcol, triple transgenic surfactant proteins-C promoter-reverse tetracycline transactivator/tetO-cytomegalovirus promoter-Cre recombinase/homozygous floxed collagen, type I, alpha 1.

Figure 4

Lung epithelial cell-deletion of collagen I attenuates collagen I accumulation acutely after bleomycin treatment. A: Whole lung mRNA was analyzed for Col1a1 mRNA 1 week after bleomycin or saline, n = 4 to 9. ^∗P < 0.05 versus control saline. Difference between control (white bars) and SCcol (black bars) bleo is not statistically significant. P = 0.3. B: mRNA from AECs isolated 1 week after bleomycin or saline, n = 4. ^†P < 0.01 versus control saline; ^‡P < 0.01 versus control bleomycin. C: Whole lung mRNA was analyzed with primers specific for a truncated Col1a1 mRNA missing exons 2 to 5 (Col1a1 Δex2-5), forward primer bridged the adjacent exons 1 and 6, and reverse primer was within exon 8. Col1a1 Δex2-5 was detected in SCcol mice 1 week after bleomycin, indicating native promoter activation and transcription of the recombined Col1a1 gene within lung epithelial cells after bleomycin. D: Immunoblot of 50 μL of a 1-mL BAL from littermate control and SCcol mice 1 week after saline or bleomycin. Bleomycin induces collagen I accumulation in BAL of control mice. BAL of SCcol mice have less collagen I accumulation after bleomycin. E: One week after bleomycin, SCcol mice have attenuated activation of the collagen receptor DDR2 observed by immunopreciptiation of whole lung lysate for DDR2 followed by immunoblot analysis for phosphotyrosine. Immunoblot of 1% of the whole lung lysate indicates increase in DDR2 expression after bleomycin in SCcol and control mice. AEC, alveolar epithelial cell; BAL, bronchoalveolar lavage; Bleo, bleomycin; Ctrl, control; DDR2, discoidin domain receptor 2; Sal, saline; SCcol, triple transgenic surfactant proteins-C promoter-reverse tetracycline transactivator/tetO-cytomegalovirus promoter-Cre recombinase/homozygous floxed collagen, type I, alpha 1.

Figure 5

Alveolar epithelial cell-derived factors promote fibroblast activation. A: DDR2 phosphorylation of collagen I-deleted fibroblasts by BAL from control and SCcol mice collected 1 week after saline or bleomycin injury. Cells treated with 10 μg/mL exogenous type I collagen and cells treated with BAL from bleomycin-injured control mice have induction of DDR2 phosphorylation. B: Reverse transcription-PCR of primary lung fibroblasts from Col1a1^f/f mice treated with AdGFP, AdCre, or AdCre plus exogenously added type I collagen (collagen I-coated plus 10 μg/mL added to media). Loss of collagen I expression led to reduction in expression of several fibroblast activation markers; the expression of these genes was restored by exogenous collagen. C: Immunoblot of lung fibroblasts stimulated with plain media, CM from AECs cultured on Matrigel (AEC CM), CM from AECs cultured on FN (EMT CM), and media supplemented with 4 ng/mL TGF-β. TGF-β and EMT CM activate fibroblast expression of α-SMA and collagen I. TGF-β promotes smad2 phosphorylation but EMT CM does not. D: Immunoblot of lung fibroblasts stimulated with plain media, CM from WT AECs cultured on FN (WT CM), smad3-null AECs (Sm3-CM) cultured on FN, and WT AECs cultured on FN with addition of TGFβ receptor inhibitor SB431542 (SB), 10 μmol/L (WT + SB CM). E: Mouse fibrosis reverse transcription-PCR array of AECs cultured on Matrigel, FN, or FN with addition of SB. Expression of genes on FN versus Matrigel (black) and FN with SB versus Matrigel (red) are shown. Blue lines indicate fivefold differential expression. Several genes of interest are indicated. AdCre, adenovirus expressing Cre; AdGFP adenovirus expressing green fluorescent protein; AEC, alveolar epithelial cell; BAL, bronchoalveolar lavage; Bleo, bleomycin; CM, conditioned media; Col I, type I collagen; Col1a1^f/f, homozygous floxed Col1a1; Col1a2, collagen, type I, alpha 2; Col3a1, collagen, type III, alpha 1; ctgf, connective tissue growth factor; Ctrl, control; DDR2, discoidin domain receptor-2; EMT, epithelial-mesenchymal transition; FN, fibronectin; GAPDH, glyceraldehyde phosphate dehydrogenase; lox, lysyl oxidase; Sal, saline; SB, transforming growth factor β receptor inhibitor SB431542; SCcol, triple transgenic surfactant proteins-C promoter-reverse tetracycline transactivator/tetO-cytomegalovirus promoter-Cre recombinase/homozygous floxed collagen, type I, alpha 1; SMA, smooth muscle actin; sma, smooth muscle actin; TGF-β, transforming growth factor β; tsp, thrombospondin; WT, wild-type.