Surfactant protein D inhibits lipid-laden foamy macrophages and lung inflammation in chronic obstructive pulmonary disease

Abstract

Increased levels of surfactant protein D (SP-D) and lipid-laden foamy macrophages (FMs) are frequently found under oxidative stress conditions and/or in patients with chronic obstructive pulmonary disease (COPD) who are also chronically exposed to cigarette smoke (CS). However, the roles and molecular mechanisms of SP-D and FMs in COPD have not yet been determined. In this study, increased levels of SP-D were found in the bronchoalveolar lavage fluid (BALF) and sera of ozone- and CS-exposed mice. Furthermore, SP-D-knockout mice showed increased lipid-laden FMs and airway inflammation caused by ozone and CS exposure, similar to that exhibited by our study cohort of chronic smokers and COPD patients. We also showed that an exogenous recombinant fragment of human SP-D (rfhSP-D) prevented the formation of oxidized low-density lipoprotein (oxLDL)-induced FMs in vitro and reversed the airway inflammation and emphysematous changes caused by oxidative stress and CS exposure in vivo. SP-D upregulated bone marrow-derived macrophage (BMDM) expression of genes involved in countering the oxidative stress and lipid metabolism perturbations induced by CS and oxLDL. Our study demonstrates the crucial roles of SP-D in the lipid homeostasis of dysfunctional alveolar macrophages caused by ozone and CS exposure in experimental mouse emphysema, which may provide a novel opportunity for the clinical application of SP-D in patients with COPD.

Keywords: Alveolar macrophages; Chronic obstructive pulmonary diseases; Cigarettes; Lipid metabolism; Ozone; Surfactant protein D.

Fig. 1

Ozone induced lung inflammation and increased the level of SP-D in serum and BALF. A BALB/c female mice were exposed to 1 and 3 ppm ozone for 3 h 3 times per week. After exposure, lung function was determined using Snapshot150 perturbation on a FlexiVent (SCIREQ). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA vs. the air group. B H&E staining of lung sections. Scale bar = 100 µm. C Alveolar well length was measured by the MLI. D BALF cell counts included total cells, macrophages (macro), neutrophils (neu), basophils (baso), and lymphocytes (lymph). The data represent the mean ± SEM. **p ≤ 0.01; ***p ≤ 0.001, Student’s unpaired t test. The levels of E TNF-α, IL-6 and CXCL1 in the BALF and F SP-D in serum and BALF were measured by ELISA. The data represent the mean ± SEM. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s unpaired t test

Fig. 2

Depletion of SP-D exacerbated lung inflammation in ozone-exposed mice. In ozone-exposed C57BL/6J female mice, A the levels of SP-D in serum and BALF were measured by ELISA. B Lung function was determined using Snapshot150 perturbation on FlexiVent (SCIREQ). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA vs. the air group. C Alveolar well length was measured by the MLI. D H&E staining of lung sections. E BALF cell counts included total cells, macrophages (macro), neutrophils (neu), basophils (baso), and lymphocytes (lymph). The data represent the mean ± SEM. **p ≤ 0.01; ***p ≤ 0.001, Student’s unpaired t test. F Cells in BALF were stained with Liu and observed by light microscopy. Scale bar = 100 µm. G The levels of TNF-α, IL-6, and CXCL1 were measured by ELISA. The data represent the mean ± SEM. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s unpaired t test

Fig. 3

Exogenous SP-D alleviated lung function and inflammation and reduced cell infiltration. A Ozone-exposed BALB/c female mice were treated with SP-D or PBS once per week, and lung function was determined using Snapshot150 perturbation on a FlexiVent (SCIREQ). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA vs. the air group. B H&E staining of lung sections. Scale bar = 100 µm. C Alveolar well length was measured by the MLI. D BALF cell counts included total cells, macrophages (macro), neutrophils (neu), basophils (baso), and lymphocytes (lymph). The data represent the mean ± SEM. **p ≤ 0.01; ***p ≤ 0.001, Student’s unpaired t test. E The levels of SP-D in serum and BALF. F TNF-α, IL-6, and CXCL1 were measured by ELISA. The data represent the mean ± SEM. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s unpaired t test

Fig. 4

SP-D pretreatment decreased ROS production and early-stage apoptotic cells and induced the expression of Nrf2. A Bioluminescence imaging of ROS production in the lungs of air-, ozone-, and cigarette smoke-exposed COPD C57BL/6J female mice was performed with an L-012 luminescent probe (IVIS Lumina LT Series III, PerkinElmer, USA). B A549, BES-2B cells, and BMDMs were pretreated with SP-D followed by H₂O₂. ROS production was examined by DCFDA. C Early-stage apoptosis (annexin V⁺ and PI⁻) in A549 and BES-2B cells was measured by flow cytometry. D Percentage of cells in early apoptosis (A549 and BEAS-2B). E A549 cells were pretreated with 1, 2, 5, or 10 μg/ml SP-D and then treated with H₂O₂. Nrf2 protein expression was examined by western blotting. SP-D pretreatment yielded a dose-dependent increase in Nrf2 expression. Nrf2 expression in the lung tissues of F wild-type and G SP-D^−/− mice was examined by western blotting. H After SP-D administration, Nrf2 expression in the lung tissue was examined by IHC. Scale bar = 100 µm. BMDMs were treated with medium (m), recombinant full length-SP-D (SP-D), and cigarette extract (CS) or were pretreated with SP-D and then treated with cigarette extract (SP-D + CS). I In a two-fold-change analysis, SP-D + CS regulated the expression of 21 genes compared with CS. J Gene heatmap of log₂-transformed ratios of oxidative stress gene expression. The left gene heatmap shows the log₂ ratios of CS, SP-D + CS, and SP-D treatment normalized to the medium control (CS/m, SP-D + CS/m, and SP-D/m). The right gene heatmap shows the log₂ ratios of SP-D + CS normalized to CS (SP-D + CS/CS)

Fig. 5

Foamy cells were significantly increased in smokers, and COPD and ozone-exposed mice with oxLDL-treated BMDMs exhibited increased inflammation in the lung. A Foamy cells in BALF were stained with oil red O. The percentage of foamy cells was calculated in B never-smokers and non-COPD controls (n = 9). Smokers included current smokers (n = 4), ex-smokers (n = 9), and C smokers with COPD (n = 4). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, Student’s unpaired t test vs. controls. Scale bar = 50 µm. D Ozone-induced foamy macrophages in C57BL/6J female wild-type and SP-D^−/− female mice. Foamy macrophages were examined by oil red O staining. E BMDMs were pretreated with or without SP-D and then treated with LPS, LDL, and oxLDL. Foamy cells were examined by staining with oil red O. Scale bar = 50 µm. F C57BL/6J female mice received PBS only (PBS), medium, or LDL- or oxLDL-treated BMDMs (BM, BML, and BMO) in the air- (A-PBS, A-BM, A-BML, and A-BMO) or ozone- (O-PBS, O-BM, O-BML, and O-BMO) exposed mouse model. G Lung function was determined using Snapshot150 perturbation on a FlexiVent (SCIREQ). The data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA vs. the air group. H H&E staining of lung sections. Scale bar = 50 µm. I Number of total cells in BALF

Fig. 6

SP-D combined with oxLDL alleviated airway inflammation and improved lung function in ozone-exposed mice. A Protocol of adoptive transfer of BMDMs in an ozone-induced C57BL/6J female mouse model of COPD. Bone marrow-derived macrophages (BMDMs) were pretreated with or without SP-D (10 μg/ml) for 6 h and then treated with 50 μg/ml LDL or 50 μg/ml oxLDL for 1 day. The mice received 2 × 10⁵ live cells by intratracheal administration. B Lung function was determined using Snapshot150 perturbation on FlexiVent (SCIREQ). Two-way ANOVA was performed to compare Rrs (1.46 vs. 1.91), Ers (40.00 vs. 51.27), G (10.92 vs. 6.14), and H (40.07 vs. 33.68) in the groups of mice that received oxLDL-induced foamy macrophages pretreated with SP-D (O-SP-D-BMO) vs. the group of mice that received oxLDL-induced foamy macrophages without SP-D treatment (O-BMO) during 200 mg/ml methacholine challenge. The data represent the mean ± SEM. *p < 0.05. C Total cell numbers in BALF. D H&E staining of lung sections. Scale bar = 50 µm. E Protocol of BMDM treatment during LPS plus ozone exposure. F Lung function was determined as described above. G Total cell numbers in BALF. H Foamy macrophages were stained with oil red O and then I quantified. J The levels of CXCL1, IL-1β, TNF-α, and IL-6 were measured by ELISA. The data represent the mean ± SEM. K H&E staining of lung sections. Scale bar = 100 (upper) and 200 (lower) µm

Fig. 7

SP-D reduced oxLDL-induced ROS production and CXCL1 expression, as well as the metabolism of several lipids, oxidative stress, and the regulation of inflammatory gene expression. A BMDMs were pretreated with 5, 10, or 20 μg/ml SP-D, followed by treatment with 50 μg/ml LDL or oxLDL. ROS production was examined by DCFDA. B BMDMs were pretreated with SP-D for 1 day and then washed 3 times with PBS. Then, the cells were treated with LDL or oxLDL for 1 day. The supernatant was collected, and the production of CXCL1, CCL2, and IL-6 was measured by ELISA. RNA expression was analyzed by RNA-seq. The left gene heatmap shows the log2 ratios of LDL, oxLDL, SP-D + oxLDL and SP-D groups normalized to the medium control (LDL/m, oxLDL/m, SP-D + oxLDL/m, and SP-D/m). The right gene heatmap shows the log₂ ratios of SP-D + oxLDL treatment normalized to oxLDL treatment (SP-D + oxLDL/oxLDL). The genes independently regulate C oxidative stress, D cytokine activity, and E lipid metabolic processes. F The RNA expression levels of ABCA1, ABCG1, CD36, and SR-A were assessed by real-time quantitative PCR (qPCR) and normalized to GAPDH. The results are expressed as the mean fold change compared to the medium

Fig. 8

Surfactant protein D inhibits lipid-laden foamy macrophages and lung inflammation in chronic obstructive pulmonary disease. SP-D deficiency exacerbates airway inflammation, lipid-laden macrophage accumulation, and emphysematous alveolar destruction in ozone- and CS-exposed lungs. Local instillation of a recombinant fragment of human surfactant protein D (rfhSP-D) alleviates oxidative stress and CS-induced airway inflammation, decreases FM formation, and causes emphysematous changes in recipient mice. The biological benefits of SP-D in ozone- and CS-induced COPD may be caused by the inhibitory effect of SP-D on ROS production in dysfunctional AMs and the restoration of lipid metabolism and cellular machinery of reverse lipid transport in lipid-laden FMs