肺间质巨噬细胞M2型极化及其对哮喘小鼠Th2反应的影响

Depending on the microenvironment, macrophages can be polarized to various distinct subsets and their heterogeneity of circulating monocytes may predefine their polarization fate once they arrive at tissues. Polarized macrophages have been broadly classified as M1 or classically activated macrophages and M2 or alternatively activated mcrophages. However, in the presence of immune complexes, macrophages become regulatory cells producing high levels of interleukin-10. Macrophages are among the most abundant cells in respiratory tract and can be divided into alveolar macrophages and interstitial macrophages. Alveolar macrophages have been extensively studied, but the phenotype and in vivo function of interstitial macrophages have been incompletely characterized. Recently it was shown that pulmonary interstitial macrophages producing high levels of interleukin-10, considered as a regulatory macrophages, can prevent airway allergy under normal conditions. In our previous study, pulmonary interstitial macrophages from a mouse model of asthma expressed the phenotypic markers of M2 or alternatively activated macrophages, including CD206 and arginase 1 and showed reduced expression of IL-10, a major feature of regulatory macrophages, compared with the PBS-treated mice. On the other hand, iNOS expression of these macrophages, a marker of M1 or clasically activated macrophages, and IL-12 produced by these macrophages, released by M1 or classically activated macrophages, were similar in a mouse model of asthma compared with the PBS-treated mice. Meanwhile, it was found the significantly high expression of transglutaminase 2 (TG2) mRNA in pulmonary interstitial macrophages from a mouse model of asthma. Recent study has shown that an inhibitor of TG2, R2 peptide, reduced airway inflammation and airway hyperresponsiveness in a mouse model of asthma. As a result, it was hypothesized that pulmonary interstitial macrophages undergo a phenotypic swich from a regulatory macrophages under normal conditions to M2 or alternative activation state and M2-polarized pulmonary interstitial macrophages induce Th2 response through TG2 in a mouse model of asthma. For the purpose, wildtype-, IL-4Rα-/-- and TG2-/--BALB/c mice were used to establish a mouse model: (1) To investigate polarization state of pulmonary interstitial macrophages in a mouse model of asthma; (2) To investigate its role of M2-polarized pulmonary interstitial macrophages in Th2 response in a mouse model of asthma; (3) To investigate M2-polarized pulmonary interstitial macriphages induce Th2 response through TG2 in a mouse model of asthma. The aim of this study is to investigate that M2-polarized pulmonary interstitial macrophages induce Th2 response through TG2 in asthma, and interventions which therapeutic approaches can be targeted toward changing the functional phenotype of lung interstitial macrophages could be beneficial for treatment of asthma.

巨噬细胞依赖微环境刺激表现为不同的表型和功能状态。正常小鼠肺间质巨噬细胞为调节性肺间质巨噬细胞，通过产生IL-10防止发生Th2反应。课题组初步发现哮喘小鼠肺间质巨噬细胞表现为M2型肺间质巨噬细胞的表型特征，即精氨酸酶1和CD206表达水平明显增高，IL-10表达水平明显降低；同时肺间质巨噬细胞转谷氨酰胺酶2（TG2）表达水平明显增高。由于抑制TG2 活性可以降低哮喘小鼠气道炎症和气道高反应性，因此假设哮喘肺间质巨噬细胞发生M2型极化，表现为M2型肺间质巨噬细胞的表型和功能特征，通过TG2参与哮喘Th2反应。为此，建立野生型和不同基因敲除小鼠哮喘模型，观察：①哮喘小鼠肺间质巨噬细胞的极化状态；②肺间质巨噬细胞M2型极化对哮喘小鼠Th2反应的影响；③肺间质巨噬细胞M2型极化参与哮喘小鼠Th2反应是否依赖TG2。探讨哮喘肺间质巨噬细胞是否发生M2型极化，并通过TG2参与哮喘Th2反应。

本项目探讨卵清蛋白（OVA）诱导的哮喘肺间质巨噬细胞是否发生M2型极化，并通过转谷氨酰胺酶2（TG2）参与哮喘Th2反应。为此，建立野生型小鼠哮喘模型：（1）观察OVA诱导的哮喘小鼠肺间质巨噬细胞的极化状态；（2）观察肺间质巨噬细胞M2型极化对哮喘小鼠Th2反应的影响；（3）初步观察肺间质巨噬细胞M2型极化通过TG2参与哮喘小鼠Th2反应。结果发现：（1）OVA诱导的哮喘小鼠肺CD206+F4/80+CD11c- 巨噬细胞数量增加；OVA诱导的哮喘小鼠肺CD206+F4/80+CD11c- 巨噬细胞替代活化标志表达水平增高，表现为肺CD206+F4/80+CD11c- 巨噬细胞精氨酸酶1（Arg 1）mRNA和Arg 1蛋白表达水平增高；OVA诱导的哮喘小鼠肺间质巨噬细胞培养上清液趋化因子CCL17、CCL22和CCL24表达水平增高；哮喘小鼠肺间质巨噬细胞IL-10、IL-10 mRNA表达水平降低，培养上清液IL-10表达水平降低；OVA诱导的哮喘小鼠肺间质巨噬细胞精氨酸酶1活性增强；（2）抗F4/80单克隆抗体可以选择性去除OVA诱导的哮喘小鼠肺间质巨噬细胞；抗F4/80单克隆抗体减少哮喘小鼠肺间质巨噬细胞Arg 1蛋白表达水平；抗F4/80单克隆抗体减少哮喘小鼠Th2细胞百分比；抗F4/80单克隆抗体减轻哮喘小鼠气道炎症反应；抗F4/80单克隆抗体减少哮喘小鼠肺泡灌洗液炎性细胞浸润和炎性细胞因子分泌水平；抗F4/80单克隆抗体降低哮喘小鼠气道高反应性；抗F4/80单克隆抗体减少哮喘小鼠肺组织转录因子T-bet 和GATA-3表达水平；过继转移野生型BALB/c哮喘小鼠肺间质巨噬细胞可以促进正常小鼠Th2反应；（3）OVA诱导的野生型BALB/c哮喘小鼠肺间质巨噬细胞TG2 mRNA和TG2蛋白表达水平增高；过继转移野生型BALB/c哮喘小鼠肺间质巨噬细胞可以促进正常小鼠TG2 mRNA和TG2蛋白表达。.实验按照项目资助计划书进行实施，目前已发表论文4篇，其中SCI源1篇，国内核心3篇；国内学术会议交流2次；毕业硕士研究生3名。