Department of Biochemistry and Molecular Biology, Health Science Center of Shenzhen University, Shenzhen, Guangdong 518060, P.R. China.
Central Laboratory, Shenzhen Nanshan Hospital, Shenzhen, Guangdong 518083, P.R. China.
Int J Mol Med. 2020 Aug;46(2):718-728. doi: 10.3892/ijmm.2020.4613. Epub 2020 May 21.
Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus fumigatus (Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease from Af (Af RNASET2) induces M2‑type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcription‑quantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS‑1 and CAS‑2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2‑related cytokines interleukin (IL)‑4, IL‑10, and IL‑13 were upregulated in mice immunized with rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il‑10, and Il‑13]; however, there was no difference in cells treated with rAf RNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL‑10 in macrophage culture supernatant were also increased following stimulation with rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2‑induced IL‑10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2‑induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.
变应性支气管肺曲霉病（ABPA）是一种对烟曲霉（Af）暴露的过敏免疫反应，通过尚未阐明的机制诱导强烈的辅助性 T 细胞 2（Th2）反应。本研究旨在探讨 Af 核糖核酸酶 2（Af RNASET2）诱导 M2 型巨噬细胞极化产生 Th2 免疫反应的假说。在原核 pET 系统中表达和纯化重组 Af RNASET2（rAf RNASET2），并用 rAf RNASET2 免疫 BALB/c 小鼠进行体内分析。用 rAf RNASET2 体外处理 RAW264.7 巨噬细胞，通过流式细胞术、逆转录-定量 PCR 和 Western blot 分析评估 M2 极化因子的表达水平。结果预测，成熟的 Af RNASET2 蛋白（382 个氨基酸；GenBank 编号 MN593022）包含两个保守的氨基酸序列（CAS）结构域，称为 CAS-1 和 CAS-2，这也是 RNASET2 家族蛋白的特征。用 rAf RNASET2 免疫的小鼠中，Th2 相关细胞因子白细胞介素（IL）-4、IL-10 和 IL-13 的蛋白表达水平上调。用 rAf RNASET2 处理的 RAW264.7 巨噬细胞 M2 因子[精氨酸酶 1、IL-10 和 IL-13]的 mRNA 表达水平增加；然而，在用核糖核酸酶抑制剂（RNasin）失活的 rAf RNASET2 处理的细胞中没有差异。用 rAf RNASET2 刺激后，巨噬细胞培养上清液中 IL-10 的蛋白表达水平也升高。此外，rAf RNASET2 上调 RAW264.7 细胞中磷酸化丝裂原活化蛋白激酶（MAPK）的表达，而 MAPK 抑制剂减弱 rAf RNASET2 诱导的 RAW264.7 细胞中 IL-10 的表达。综上所述，本研究表明 rAf RNASET2 活性高是 rAf RNASET2 诱导巨噬细胞 M2 极化所必需的，并提示 Af RNASET2 在 ABPA 发病机制中具有重要的免疫调节作用。
