Transgenically-expressed secretoglobin 3A2 accelerates resolution of bleomycin-induced pulmonary fibrosis in mice

Abstract

Background: Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice.

Methods: A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis.

Results: Adult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs.

Conclusions: These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.

Fig. 1

Generation of the SCGB3A2 transgenic mouse line. a Illustration of the construct showing the human SP-C gene promoter cloned into the pUC18 vector with SV40 small T intron and poly A. b Northern Blot analysis. The high level of Scgb3a2 is observed only in lung tissue. The bands indicated by an arrow represent exogenously expressed Scgb3a2 mRNAs. The highest mobility band shown by an arrowhead is derived from endogenous Scgb3a2. c Representative Western blot result showing increased level of SCGB3A2 protein in Scgb3a2-transgenic mouse lungs as compared with wild-type mice. GAPDH was used as a loading control. Lower panel shows the result of quantitation, normalized to GAPDH. N = 3. d qRT-PCR analysis of relative expression levels of Scgb3a2 in lungs of various gestational days and ages of wild-type (WT) and transgenic (TG) mice. e SCGB3A2 levels in BALF of different ages of WT and TG mice. For D, E: N = 5 in each group. The results are shown as the mean ± SD. **P < 0.01, ***P < 0.001 by student t-test in comparison between WT and TG. f Immunohistochemistry for SP-C and SCGB3A2 in 4-month-old WT and TG mouse lungs. Arrows indicate representative positive signals in brown. TG lungs express SCGB3A2 in alveolar type II cells (middle panel) in addition to airway epithelial cells (right panel). Scales are as indicated. g Co-immunofluorescence analysis of TG mouse lungs for SP-C and SCGB3A2. Airway cells express only SCGB3A2 (red, shown by an asterisk) while alveolar type II cells express both SP-C and SCGB3A2 (yellow, shown by arrows). Scales are as indicated

Fig. 2

BLM-induced pulmonary fibrosis in wild-type (WT) and Scgb3a2-transgenic (TG) mice. a Body weight curves of mice intubated and treated with BLM on day 0, followed by necropsy on day 63. Body weights are shown as the percentage of Day 0 weight set as 100 %. *P < 0.05, WT-BLM group vs. TG-BLM, **P < 0.01, BLM-treated groups vs. PBS-treated groups. b Whole lung H&E images of WT and TG lungs at 3 weeks (BLM 3 W), 6 weeks (BLM 6 W), and 9 weeks (BLM 9 W) post-BLM treatment, and their corresponding PBS-treated lungs collected at 3 W (PBS) as control. Magnification 40X images were stitched. c Masson Trichrome staining of lung sections of WT and TG mice at 3, 6, and 9 weeks post-BLM treatment. Magnification, 100X. d Ashcroft scores of BLM-induced damaged areas at 3, 6, and 9 weeks post-BLM treatment. PBS control levels are those from 3 W. e Hydroxyproline content at 3, 6, and 9 weeks post-BLM treatment. PBS control levels are those from 3 W. N > 6 in each group. The results are shown as the mean ± SD. *P < 0.05, NS, not significant. Statistical analysis was carried out by using student’s t-test

Fig. 3

Characterization of BLM-induced pulmonary fibrosis-harboring lungs of wild-type (WT) and Scgb3a2-transgenic (TG) mice. a Number of inflammatory cells in BAL fluids at 3, 6, and 9 weeks post-BLM treatment and PBS control groups collected at 3 weeks. (right panel) Number of monocytes/lymphocytes, neutrophils, and macrophages from 3 weeks post-BLM treatment mice were separately counted. *P < 0.05 by student’s t-test. b qRT-PCR analysis for various mRNA levels. The results are shown as the mean ± SD from N = 7-10. *P < 0.05, **P < 0.01, NS, not significant by student’s t-test

Fig. 4

SCGB3A2 and proinflammatory cytokine levels in lungs of wild-type (WT) and Scgb3a2-transgenic (TG) mice. a Levels of lung Scgb3a2 mRNAs (left panel) and SCGB3A2 proteins in BALF (right panel) in WT and TG mice at 3, 6, and 9 weeks post-BLM treatment and PBS collected at 3 weeks as control. b Representative immunohistochemistry of SCGB3A2 in WT and TG mice at 3 and 6 weeks post-BLM treatment and PBS collected at 3 weeks as control. Arrow indicates representative SCGB3A2 positive signals found in the airway epithelial cells. Scale bar: 50 μm. c qRT-PCR analysis of lung mRNAs for proinflammatory cytokines, IL-1β, IL-6, and TNFα at 3, 6, and 9 weeks post-BLM treatment and PBS as control. N > 6 in each group. *P < 0.05, **P < 0.01 by student’s t-test

Fig. 5

Microarray analysis. a Heat map diagram of differentially expressed genes in normal Scgb3a2-transgenic (TG) lungs vs. wild-type (WT) lungs. All genes >2 fold with P < 0.05 differences were used for the analysis. The average of 4 samples in each group is shown. Green and red indicate down- and up-regulation of gene expression, respectively. The scale is shown at the bottom. Genes found in top three pathways as shown in C are indicated with the fold differences in parenthesis. The number (1, 2, or 3) means the pathway to which indicated genes belong in C. b qRT-PCR analysis of highly up- or down-regulated genes indicated in A using RNAs isolated from untreated WT and TG mouse lungs. The results are shown as the mean ± SD. N > 6, *P < 0.05 by student’s t-test. c Top three pathways, Carbohydrate metabolism (pathway 1), Developmental disorder (pathway 2), and Cellular compromise pathways (pathway 3) determined by Ingenuity Pathway Analysis using genes identified by microarray analysis that have >1.5 fold with P < 0.05 differences