Epithelial Deletion of Sulf2 Exacerbates Bleomycin-Induced Lung Injury, Inflammation, and Mortality

Abstract

Epithelial injury has been proposed to be the initiating factor in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have shown previously that heparan sulfate 6-O-endosulfatase (Sulf) 2 is overexpressed in the hyperplastic type II alveolar epithelial cells (AECs) in the IPF lungs. By removing 6-O-sulfates from specific heparan sulfate intrachain sites, Sulf2 modulates the functions of many growth factors and cytokines. In this study, we hypothesized that Sulf2 plays a regulatory role in alveolar epithelial injury and repair, using the murine bleomycin model. Consistent with our findings in human IPF lungs, bleomycin treatment in mice resulted in up-regulation of Sulf2 mRNA in whole-lung extracts and overexpression of Sulf2 protein in type II AECs on lung tissue sections. Sulf2 protein was detectable in bronchoalveolar lavage fluid at baseline, and its level was significantly increased after bleomycin exposure. To study the role of Sulf2 in alveolar injury and repair in vivo, we generated a doxycycline-inducible epithelial-specific Sulf2 conditional knockout (Sulf2 CKO) mouse line. After bleomycin exposure, Sulf2 CKO mice exhibited enhanced neutrophil infiltration in the lung, with elevated levels of total protein, lactate dehydrogenase, and cytokines (granulocyte colony-stimulating factor and interferon-γ-inducible protein 10) in bronchoalveolar lavage fluid compared with wild-type littermates. We further showed that both the p53-p21 DNA damage response and the transforming growth factor-β1 signaling pathway were up-regulated in Sulf2 CKO mice compared with wild-type. Finally, Sulf2 CKO mice suffered increased mortality after bleomycin exposure. In conclusion, Sulf2 expression in type II AECs plays a protective role in epithelial injury, inflammation and mortality.

Keywords: Sulf2; bleomycin; heparan sulfate; p53; type II alveolar epithelial cell.

Figure 1.

Overexpression of heparan sulfate 6-O-endosulfatase 2 (Sulf2) in bleomycin-induced lung injury in mice. (A) Sulf2 mRNA expression in total lung extracts from control (PBS, n = 6) and bleomycin (bleo)-treated mice (bleo 7 d, 0.7 U/kg, n = 8; bleo 14 d, 0.5 U/kg, n = 4) were analyzed by quantitative RT-PCR (qRT-PCR). Data were normalized to the expression of housekeeping gene, Tbp (TATA box binding protein). (B) Sulf2 protein in bronchoalveolar lavage fluid (BALF) from control and bleo-treated mice (0.7 U/kg at 7 d) were analyzed by Western blotting. ***P < 0.001. (C) Immunolocalization of Sulf2 in control and bleo-treated mice. (a and b) Immunostaining for Sulf2 (diaminobenzidine, brown) in control mice; arrows, Sulf2-positive bronchial epithelial cells or type II alveolar epithelial cells (AECs); asterisk, a Sulf2-negative type II AEC. (c–f) Double immunostaining for Sulf2 (DAB, brown) and pro-surfactant protein C (SPC) (purple) in bleo-treated mice mice; arrows, Sulf2 and pro-SPC double-positive cells; arrowheads, Sulf2 single-positive cells. (d and e) Enlarged views of insets in c. (f) Another example of Sulf2 and pro-SPC double-positive cells at the bronchoalveolar duct junction. Scale bars: 10 μm and 100 μm.

Figure 2.

Generation of inducible epithelial-specific Sulf2 conditional knockout (CKO) mice. (A) Model. (B) Expression of Sulf1, Sulf2, and HS6ST1 in primary type II AECs from wild-type (WT; n = 7) and Sulf2 CKO mice (n = 5) after doxycycline (dox) administration. Relative mRNA expression in comparison to housekeeping gene, Tbp, is shown. The purity of type II AEC isolation was 80–90% based on pro-SPC expression (data not shown). (C) Expression of Sulf2 in WT and Sulf2 CKO mice after control (PBS) and bleo exposure. Relative mRNA expression normalized to housekeeping gene, Tbp, is shown. *P < 0.05; **P < 0.01; ***P < 0.001. CMV, cytomegalovirus; Cre, cre-recombinase; LoxP, LoxP sites; rtTA, reverse tetracycline transactivator; tetO, tetracycline operator.

Figure 3.

Sulf2 CKO mice exhibit enhanced injury and inflammation after bleo exposure. (A) Hematoxylin and eosin staining of lung sections from control and bleo (0.7 U/kg at 7 d)-treated WT and Sulf2 CKO mice. (B) BAL differential cell count from control and bleo-treated WT and Sulf2 CKO mice. (C) BAL total protein, lactate dehydrogenase (LDH) and cytokine levels from control and bleo-treated WT and Sulf2 CKO mice. Open bars, control; solid bars, bleo-treated mice (0.7 U/kg, 7 d). *P < 0.05; **P < 0.01; ***P < 0.001. G-CSF, granulocyte colony-stimulating factor; IP-10, interferon-γ–inducible protein 10; KC, key chemokine.

Figure 4.

Sulf2 CKO mice exhibit enhanced p53–p21 signaling after bleo exposure. (A) Expression of total and phosphorylated (at serine 15) p53 was examined by Western blotting. (B) The mRNA expression of p21 and Bcl2-associated X protein (Bax) were examined by qRT-PCR and normalized to housekeeping gene, Tbp. Open bars, control; solid bars, bleo-treated mice (0.7 U/kg, 7 d). **P < 0.01; ***P < 0.001. (C) Immunostaining of p21 on lung tissue sections from control and bleo-treated WT and Sulf2 CKO mice. Arrows, p21 positive cells (DAB, brown). Scale bar: 20 μm. Ctrl, control.

Figure 5.

Sulf2 CKO mice exhibit enhanced transforming growth factor (TGF)-β1 signaling after bleo exposure. (A) BAL total TGF-β1 levels were examined by ELISA. (B) Expression of total and phosphorylated Smad2 was examined by Western blotting. (C) The mRNA expression of collagen I was examined by qRT-PCR and normalized to housekeeping gene, Tbp. Open bars, control; solid bars, bleo-treated mice (0.7 U/kg, 7 d). *P < 0.05; **P < 0.01; ***P < 0.001. (D) Mason’s trichrome staining on lung tissue sections revealed enhanced collagen deposition around vasculatures in Sulf2 CKO mice after bleo exposure.

Figure 6.

Type II AECs from Sulf2 CKO mice express increased levels of inflammatory cytokines and DNA damage response genes after bleo exposure. The mRNA expression of G-CSF (A), IP-10 (B), IL-6 (C), KC (D), p21 (E), and Bax (F) was analyzed in freshly isolated type II AECs from control (WT, n = 4; Sulf2 CKO, n = 4) or bleo-challenged mice (WT, n = 4; Sulf2 CKO, n = 4). Open bars, control; solid bars, bleo-treated mice (0.3 U/kg, 4–5 d). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7.

Sulf2 CKO mice suffer increased mortality after bleo exposure. Survival curves of WT and Sulf2 CKO mice treated with bleo at 0.3 and 0.5 U/kg (combined) were analyzed by log-rank (Mantel–Cox) test.