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. 2016 Jun;96(6):623-31.
doi: 10.1038/labinvest.2016.40. Epub 2016 Mar 14.

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis

Anthony N Habgood  1 , Amanda L Tatler  1 , Joanne Porte  1 , Sharon M Wahl  2 , Geoffrey J Laurent  3 , Alison E John  1 , Simon R Johnson  1 , Gisli Jenkins  1
Affiliations

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis

Anthony N Habgood et al. Lab Invest. 2016 Jun.

Abstract

Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

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Figures

Figure 1
Figure 1. Slpi−/− mice do not express Slpi mRNA and have enhanced MMP-9 activity in the lung
(a) PCR confirmation of presence of wild-type 490-bp fragment and absence of Neo-cassette fragment 344-bp in wild-type animals (Wt) and absence of wild-type fragment and presence of Neo-cassette in Slpi−/− animals (−/−). Both wild-type and Neo-cassette fragments were present in heterozygous animal (−/+). (b) Slpi gene expression in lung homogenates from wild-type and Slpi−/− animals. Data expressed as mean relative expression (ΔΔCt) ± SEM; n = 3. (c) Neutrophil elastase activity was assessed in lung homogenate 28 days post-bleomycin treatment. Data expressed as mean fluorescence intensity (MFI) ± SEM; n ≥ 2. (d) Ratio of pro and active forms of MMP-2 in BAL supernatant from wild-type and Slpi−/− mice and (e) Ratio of pro and active forms of MMP-9 in BAL supernatant from wild-type and Slpi−/− mice. Mean ± SEM; n = 8. (f) Mmp-9 mRNA levels in lung homogenates 28 days post-bleomycin (BLM). Data expressed as mean relative expression (ΔΔCt) ± SEM; n = 8.
Figure 2
Figure 2. The effect of Slpi deletion on alveolar macrophage TGF-β activation and pulmonary fibrosis
(a) pSmad2 levels in nuclear extracts from BAL cells from Slpi−/− and wild-type mice 28 days post-bleomycin (BLM). Data expressed as mean absorbance at 450 nm ± SEM; n ≥ 5; **P < 0.005 BLM vs. saline. (b) pSmad2 immunohistochemistry. Representative images captured using original magnification x20, scale bars 100 μm. Arrowheads identify positively stained cells within a single, representative, alveolus in saline treated mice only; there is just one arrowhead in the wild-type mouse and three in the Slpi−/− mouse. (c) Masson’s trichrome staining 28 days post-BLM treatment. Representative, whole lung lobe, cross sections. (d) Quantitative assessment of trichrome staining. Data expressed as mean Ashcroft Score ± SEM; n ≥ 3.
Figure 3
Figure 3. The effect of Slpi deletion on weight loss and lung hydroxyproline levels
(a) Body weight during the acute lung injury phase of the model in Slpi−/− compared with wild-type mice. Data expressed as mean percentage of initial (day 0) body mass (%) ± SEM; n ≥ 9; Significant difference determined at day 4 *P < 0.05 Slpi−/− vs. wild-type post-bleomycin (BLM). (b) Lung hydroxyproline levels were assessed at 0, 7, 14 and 28 days following BLM in Slpi−/− vs. wild-type mice. Data expressed as the amount of hydroxyproline per mg of lung tissue (μg/mg) ± SEM; n ≥ 8. *P < 0.05, **P < 0.01 and **** P < 0.0001.
Figure 4
Figure 4. The effect of Slpi deletion on subtype specific collagen synthesis and deposition
(a) Col1α1, Col3α1, Col4α1 and Col6α1 gene expression 0, 7, 14 and 28 days post-bleomycin (BLM). Data expressed as mean relative expression (ΔΔCt) ± SEM; n ≥ 8, *P < 0.05, ** P < 0.01, ***P < 0.001 and **** P < 0.0001. (b) 5 μm thick tissue sections from BLM treated wild-type and Slpi−/− mice were stained with anti-collagen I, anti-collagen III, anti-collagen IV and anti-collagen VI. Low magnification (x10) images were stitched together to visualise whole lung lobes. Images are representative of n ≥ 3 animals. (c) Collagen I and VI deposition was measured in lung tissue sections. Data expressed as mean percentage of total lung tissue comprised of fibrotic lesions greater than 7 500 μm2 containing collagen I and collagen VI (%) ± SEM; n ≥ 3.
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