Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor (G-CSF) through its receptor G-CSFR are essential for antibacterial innate defense. However, the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear. N⁶-methyladenosine (m⁶A) RNA modification and the related demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, but their roles in neutrophil production and mobilization are still unknown. We used cecal ligation and puncture (CLP)-induced polymicrobial sepsis to model systemic bacterial infection, and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization. ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice. In addition, Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation, which led to fewer neutrophils at the infection site than in their wild-type littermates. During bacterial infection, ALKBH5 imprinted production- and mobilization-promoting transcriptome signatures in both mouse and human neutrophils. Mechanistically, ALKBH5 erased m⁶A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR, consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils. The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA, and the binding strength declined upon bacterial infection, accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils. Considering these results collectively, we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m⁶A demethylation-dependent posttranscriptional regulation, indicating that m⁶A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.

粒细胞集落刺激因子 (G-CSF) 通过其受体 G-CSFR 触发紧急粒细胞生成和中性粒细胞动员，这对于抗菌先天防御至关重要。然而，对于内在调节 G-CSFR 表达和中性粒细胞抗菌反应至关重要的表观遗传修饰剂仍不清楚。 N ⁶ -甲基腺苷 (m ⁶ A) RNA 修饰和相关的去甲基化酶 alkB 同源物 5 (ALKBH5) 是免疫和炎症的关键表观遗传调节因子，但它们在中性粒细胞产生和动员中的作用仍不清楚。我们使用盲肠结扎穿刺 (CLP) 诱导的多种微生物败血症来模拟全身细菌感染，我们报告说 ALKBH5 是紧急粒细胞生成和中性粒细胞动员所必需的。 ALKBH5 耗竭显着损害了脓毒症小鼠骨髓中未成熟中性粒细胞的产生。此外， Alkbh5缺陷型脓毒症小鼠在骨髓中表现出较高的成熟中性粒细胞保留率以及向循环中释放的中性粒细胞有缺陷，这导致感染部位的中性粒细胞比野生型同窝小鼠少。在细菌感染期间，ALKBH5 在小鼠和人类中性粒细胞中留下了促进生产和动员的转录组特征。从机制上讲，ALKBH5 消除了CSF3R mRNA 上的 m ⁶ A 甲基化，以增加 G-CSFR 的 mRNA 稳定性和蛋白表达，从而上调细胞表面 G-CSFR 表达和中性粒细胞中的下游 STAT3 信号传导。 RIP-qPCR 结果证实 ALKBH5 与CSF3R mRNA 直接结合，并且结合强度在细菌感染时下降，这是细菌感染的中性粒细胞上 G-CSFR 表达下降的原因。综合考虑这些结果，我们定义了 ALKBH5 通过 m ⁶ A 去甲基化依赖性转录后调节从本质上驱动中性粒细胞产生和动员的新作用，表明中性粒细胞中的 m ⁶ A RNA 修饰是治疗细菌感染和中性粒细胞减少症的潜在靶标。