嗜酸性粒细胞通过调节巨噬细胞极化减轻脂多糖诱导的急性肺损伤

目的：观察体外嗜酸性粒细胞（Eos）对巨噬细胞极化的影响，以及体内注射嗜酸性粒细胞对脂多糖（LPS） 诱导的急性肺损伤（ALI）的作用及其相关机制。方法：原代培养小鼠巨噬细胞，在嗜酸性粒细胞存在或不存 在的情况下，将细胞暴露于脂多糖和干扰素-γ（IFN-γ）以模拟ALI，采用荧光定量PCR 检测各组巨噬细胞中促 炎及抗炎因子的表达，采用免疫印迹法检测巨噬细胞过氧化物酶增殖物激活受体γ（PPARγ）蛋白的表达；将 PPARγ siRNA（si-PPARγ）转染小鼠巨噬细胞，采用免疫印迹法检测巨噬细胞的极化情况；Balb/c 小鼠随机分为 对照组、ALI 组和ALI+Eos 组，ALI 组为通过气管内注射LPS 诱导ALI，ALI+Eos 组给予尾静脉注射嗜酸性粒细 胞，对照组尾静脉注射生理盐水。监测动物7 d 的存活情况。在LPS 注射后3 h 和24 h 处死动物，通过组织学评估 左肺损伤程度，荧光定量PCR 检测右肺促炎和抗炎细胞因子mRNA水平，采用免疫印迹法检测右肺组织PPARγ 蛋白的表达，流式细胞术检测肺泡巨噬细胞诱导型一氧化氮合酶（iNOS）和CD206 的百分比。结果：与嗜酸性 粒细胞共培养后，用LPS+IFN-γ 刺激巨噬细胞，其肿瘤坏死因子-α（TNF-α）和白细胞介素（IL）-6 mRNA水平 呈剂量依赖性下降；而IL-10 mRNA 水平和PPARγ 活性呈剂量依赖性增加。巨噬细胞与嗜酸性粒细胞共培养后， 其iNOS 表达增加和重组人精氨酸酶1（Arg1）表达减少，呈剂量依赖性逆转。转染si-PPARγ 抑制了嗜酸性粒细 胞促进巨噬细胞向M2表型分化的作用。静脉注射嗜酸性粒细胞显著提高了ALI 小鼠的存活率，且显著改善小鼠 的肺组织损伤，降低肺组织IL-6 和TNF-α mRNA水平，增加IL-10 mRNA 水平和PPARγ 活性。此外，嗜酸性粒 细胞干预增加了肺泡巨噬细胞CD206 的百分比，而iNOS 的百分比明显降低。结论：嗜酸性粒细胞通过促进巨噬 细胞M2型极化改善LPS 诱导的ALI。

Eosinophils attenuate lipopolysaccharide-induced acute lung injury by modulating macrophage polarization

Objective To observe the effect of eosinophils（Eos） on the polarization of macrophages in vitro， and eosinophils injection in vivo on lipopolysaccharide（ LPS）-induced acute lung injury（ ALI） and related mechanisms. Methods In primary culture， mouse macrophages were exposed to LPS and interferon-γ（ INF-γ）in the presence or absence of eosinophils to simulate ALI. Fluorescence quantitative PCR was used to detect the expression of pro-inflammatory and anti-inflammatory factors in each group of macrophages， and the expression of peroxidase proliferator-activated receptor-γ（ PPARγ） protein in macrophages was detected by Western blotting， and PPARγ siRNA（ si-PPARγ） was used for transfecting mouse macrophages， and the polarization of macrophages was detected by Western blotting. Balb/c mice were randomly divided into control group， ALI group and ALI+Eos group. In the ALI group， acute lung injury was induced by intratracheal injection of LPS. The ALI+Eos group was given a tail vein injection of eosinophils， and the control group was given a tail vein injection of normal saline. The survival of animals was monitored for 7 days. Animals were sacrificed 3 h and 24 h after LPS injection， left lung injury was evaluated by histology. Fluorescence quantitative PCR was used to detect the right lung pro-inflammatory and antiinflammatory cytokine mRNA levels， and Western blotting was used to detect the expression of PPARγ protein inright lung tissue， and percentage of inducible nitric oxide synthase （iNOS） and CD206 in the alveolar macrophages were detected by flow cytometry. Results — 383 — After co-cultivation with eosinophils， the levels of tumor necrosis factor-α（ TNF-α） and interleukin-6（ IL-6） mRNA in macrophages were decreased with LPS+IFN-γ dose-dependent manner ； while interleukin 10（ IL-10） mRNA level and PPAR-γ activity increased in a dose-dependent manner. In addition， the increase of iNOS expression and the decrease of recombinant arginase 1（ Arg1） expression reversed in a dose-dependent manner after macrophages were co-cultured with eosinophils. Transfection of si-PPARγ inhibited the role of eosinophils in promoting macrophage M2 phenotype differentiation. In vivo， intravenous injection of eosinophils significantly increased the survival rate of ALI mice， and reduced LPS-induced lung injury. Intravenous injection of eosinophils reduced IL-6 and TNF-α mRNA levels in lung tissue， and increased IL-10 mRNA level and PPAR-γ activation. In addition， eosinophil therapy increased the percentage of CD206 in alveolar macrophages， while the percentage of iNOS decreased significantly. Conclusion Eosinophils ameliorate LPS-induced ALI by promoting macrophage M2 polarization.