Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC 27834, United States.
Biochem Biophys Res Commun. 2011 Nov 18;415(2):288-93. doi: 10.1016/j.bbrc.2011.10.043. Epub 2011 Oct 18.
We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPARγ) and the PPARγ-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPARγ. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPARγ plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p=0.0005) increase in ABCG1 expression with no change of PPARγ or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by analysis of bronchoalveolar lavage fluid. Lung compliance was diminished in untreated GMCSF KO mice but improved significantly after lenti-ABCG1 treatment. Data demonstrate that in vivo instillation of lenti-ABCG1 in GM-CSF KO mice is sufficient to restore pulmonary homeostasis by: (1) upregulating ABCG1; (2) reducing intra and extracellular lipids; and (3) improving lung function. Results suggest that the ABCG1 lipid transporter is the key downstream target of GM-CSF-induced PPARγ necessary for surfactant catabolism.
我们已经在患有肺泡蛋白沉积症（PAP）的患者的肺泡巨噬细胞中发现核转录因子过氧化物酶体增殖物激活受体-γ（PPARγ）和 PPARγ 调节的三磷酸腺苷结合盒转运蛋白 G1（ABCG1）的表达降低。PAP 患者也表现出对粒细胞-巨噬细胞集落刺激因子（GM-CSF）的中和抗体，GM-CSF 是 PPARγ 的上调因子。与功能性 GM-CSF 缺乏相关，PAP 肺的特征是表面活性剂充满的肺泡空间和富含脂质的肺泡巨噬细胞。GM-CSF 敲除（KO）小鼠也具有相似的病理学特征。我们之前曾报道，将慢病毒（lenti）-PPARγ 质粒经气管内滴注到 GM-CSF KO 动物中可升高 ABCG1 并减少肺泡巨噬细胞脂质堆积。在这里，我们假设，慢病毒-ABCG1 的滴注可能足以减少 GM-CSF KO 小鼠中的脂质堆积并改善肺功能。动物接受气管内滴注慢病毒-ABCG1 或对照慢病毒增强型绿色荧光蛋白（eGFP）质粒，并在 10 天后收获肺泡巨噬细胞。通过 lenti-eGFP 荧光显示，肺泡巨噬细胞的转导效率为 79％。定量 PCR 分析表明，慢病毒-ABCG1 处理的小鼠肺泡巨噬细胞中 ABCG1 的表达增加了三倍（p=0.0005），而 PPARγ 或 ABCA1 没有变化。对照 lenti-eGFP 和 PBS 处理组的 ABCG1 不变。油红 O 染色检测到慢病毒-ABCG1 处理组的肺泡巨噬细胞中细胞内中性脂质减少。通过分析支气管肺泡灌洗液还发现细胞外胆固醇和磷脂减少。未治疗的 GMCSF KO 小鼠的肺顺应性降低，但经 lenti-ABCG1 治疗后明显改善。数据表明，在 GM-CSF KO 小鼠体内滴注慢病毒-ABCG1 足以通过以下方式恢复肺内稳态：（1）上调 ABCG1；（2）减少细胞内外脂质；和（3）改善肺功能。结果表明，ABCG1 脂质转运蛋白是 GM-CSF 诱导的 PPARγ 的关键下游靶标，是表面活性剂分解代谢所必需的。
