Bulk RNA-seq analysis of WT and tristetraprolin (TTP)-knockout basophils treated with antigen/IgE stimulation

Basophils play crucial roles in type 2 immune responses, such as chronic allergic inflammation and protective immunity against parasites. However, the molecular mechanisms regulating basophil activation and effector molecule production remain poorly understood. To investigate the role of RNA-binding proteins (RBPs), we first analyzed the gene expression of CCCH zinc finger proteins in antigen/IgE-stimulated basophils. Among these proteins, we identified that Zfp36, encoding tristetraprolin (TTP), was the most significantly upregulated gene. Therefore, we focused on the functions of TTP in basophils. We generated TTP-KO mice using the CRISPR-Cas9 method and conducted bulk RNA-seq analysis of antigen/IgE-stimulated basophils from wild-type (WT) and TTP-knockout (TTP-KO) mice. TTP-KO basophils exhibited elevated mRNA expression of pro-inflammatory mediators, such as Il4, Il13, Areg, Ccl3, and Cxcl2, compared to WT basophils. These data suggest that TTP is a key regulator of basophil activation, controlling the mRNA expression of inflammatory mediators. To examine the effect of TTP deficiency on gene expression profiles in antigen/IgE-stimulated basophils, WT and TTP-KO basophils sensitized with hapten TNP-specific IgE were treated with control OVA for 2 hours or TNP-conjugated OVA for 2 and 4 hours and then subjected to bulk RNA-seq analysis.

嗜碱性粒细胞（Basophils）在2型免疫应答中发挥关键作用，例如参与慢性过敏性炎症以及介导抗寄生虫保护性免疫。然而，调控嗜碱性粒细胞活化及效应分子产生的分子机制仍知之甚少。为探究RNA结合蛋白（RNA-binding proteins, RBPs）的功能，我们首先分析了抗原/IgE刺激的嗜碱性粒细胞中CCCH型锌指蛋白的基因表达情况。在这类蛋白中，我们发现编码三联素蛋白（tristetraprolin, TTP）的Zfp36是上调最为显著的基因。据此，我们将研究重点聚焦于TTP在嗜碱性粒细胞中的功能。我们通过CRISPR-Cas9技术构建了TTP敲除（TTP-KO）小鼠，并对野生型（wild-type, WT）与TTP敲除（TTP-KO）小鼠的抗原/IgE刺激的嗜碱性粒细胞开展了批量RNA测序（bulk RNA-seq）分析。结果显示，相较于野生型嗜碱性粒细胞，TTP-KO嗜碱性粒细胞的促炎介质（如Il4、Il13、Areg、Ccl3及Cxcl2）的mRNA表达水平显著升高。上述数据表明，TTP是嗜碱性粒细胞活化的关键调控因子，可通过调控促炎介质的mRNA表达发挥作用。为进一步考察TTP缺失对抗原/IgE刺激的嗜碱性粒细胞基因表达谱的影响，我们将经半抗原TNP特异性IgE致敏的野生型与TTP-KO嗜碱性粒细胞分别以对照OVA处理2小时，或以TNP偶联OVA分别处理2小时和4小时，随后进行批量RNA测序分析。