哮喘小鼠脾脏来源CD4^+ T细胞促进体外培养巨噬细胞的M2极化

Spleen-derived CD4^+ T cells of asthmatic mice promote M2 polarization of macrophages in vitro

目的探讨哮喘小鼠脾脏来源CD4^+T细胞体外对小鼠骨髓来源巨噬细胞(BMDM)极化的影响及其机制。方法建立粉尘螨变应原诱导的过敏性哮喘小鼠动物模型。于末次激发24h后,取小鼠肺中叶、HE染色观察炎症改变,运用实时荧光定量PCR检测肺、脾脏组织中miR-155-5p水平;免疫磁珠分选哮喘小鼠脾脏CD4^+T细胞,实时荧光定量PCR检测哮喘小鼠脾脏CD4^+T细胞miR-155-5p水平,同时用流式细胞术检测哮喘小鼠脾脏中CD4^+IFN-γ^+Th1细胞和CD4^+IL-4^+Th2细胞比例的变化;实时荧光定量PCR检测正常组和哮喘组小鼠肺组织中M2相关标志基因精氨酸酶1(Arg1)、类几丁质酶3样分子3(YM1/Chi3l3)、类抵抗素α(retnlα/FIZZ1)的mRNA水平;哮喘小鼠脾脏来源CD4^+T细胞与BMDM进行共培养,实时荧光定量PCR检测BMDM的M2相关标志基因Arg1、YM1、FIZZ1的mRNA水平;通过瞬时转染的方法,将miR-155-5p抑制剂(miR-155-5p-inhibitor)和阴性对照分别转染入哮喘小鼠脾脏CD4^+T细胞后,再与BMDM进行共培养48h后,实时定量PCR测定BMDM中的Arg1、YM1、FIZZ1的mRNA水平。结果与对照组相比,哮喘组小鼠肺、脾脏组织中miR-155-5p水平增加,脾脏CD4^+T细胞miR-155-5p水平升高,且Th2细胞比例上升,肺组织中Arg1、YM1、FIZZ1表达上调;哮喘组小鼠脾脏CD4^+T细胞与BMDM体外共培养后,BMDM的Arg1、YM1、FIZZ1的mRNA水平上调,而转染miR-155-5p-inhibitor后的脾脏CD4^+T细胞并未明显影响体外共培养的BMDM的M2标志基因表达。结论哮喘小鼠脾脏来源的CD4^+T细胞能够促进体外共培养的巨噬细胞向M2极化。

Objective To investigate the effect of asthmatic mouse spleen-derived CD4^+ T cells on the polarization of bone marrow-derived macrophages(BMDMs) in vitro and its mechanism. Methods An animal model of allergic asthma induced by Dermatophagoides farinae allergen was established in mice. After the last challenge lasting 24 hours, the middle lobe of mouse lung was taken and HE staining was used to observe its inflammatory changes. The levels of miR-155-5p in the lung and spleen as well as spleen CD4^+ T cells were detected by real-time quantitative PCR(qRT-PCR). The proportions of CD4^+IFN-γ^+ Th1 cells and CD4^+IL-4^+ Th2 cells in the spleen of asthmatic mice were detected by flow cytometry. The mRNA expression levels of M2 macrophage marker genes arginase 1(Arg1), chitinase-like molecule 3(YM1/Chi3l3) and resistance-like α(Retnlα/FIZZ1) in the lung were examined by qRT-PCR. Spleen-derived CD4^+ T cells from the asthmatic mice were co-cultured in vitro with BMDMs for 48 hours, and then the mRNA expression levels of Arg1, YM1, and FIZZ1 in the BMDMs were detected by qRT-PCR. The spleen CD4^+ T cells of the asthmatic mice were transfected with miR-155-5p inhibitor or the negative control, and then co-cultured with BMDM for 48 hours. The qRT-PCR was used to further determine the expression levels of Arg1, YM1, FIZZ1 in BMDMs. Results Compared with the control group, the levels of miR-155-5p in the lung, spleen and spleen CD4^+ T cells of asthmatic mice increased, and the proportion of Th2 cells in asthmatic mouse spleen also increased. The expression levels of the M2 macrophage marker genes Arg1, YM1 and FIZZ1 were up-regulated in the lung of asthmatic mice compared to the control group. After co-culture of spleen CD4^+ T cells from asthmatic mice with BMDMs in vitro, the mRNA expression levels of M2 marker genes Arg1, YM1 and FIZZ1 of BMDMs were up-regulated. While transfected with miR-155-5p-inhibitor, the spleen CD4^+ T cells of asthmatic mice did not significantly affect the M2 marker gene expression of the BMDMs. Conclusion The spleen-derived CD4^+ T cells of asthmatic mice can promote the polarization of co-cultured macrophages towards M2 phenotype in vitro.