Supplementary Material for: Stat3 regulates developmental hematopoiesis and impacts myeloid cell function via canonical and non-canonical modalities

Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis and function of immune cells, with STAT3 disruption associated with human immune-related disorders. These roles have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative non-canonical functional modalities have also been described for STAT3. To further understand the roles of STAT3 gained from lineage-specific mouse knockouts, CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD). Analysis of the KO mutant identified conserved roles for Stat3 within hematopoietic stem cells impacting the development of all lineages throughout primitive and early definitive hematopoiesis, with altered immune cell populations in juveniles. The Stat3 KO mutant was unable to respond to lipopolysaccharide (LPS) or granulocyte colony-stimulating factor (G-CSF), and also exhibited significantly diminished neutrophil migration that correlated with abrogation of the Cxcl8b/Cxcr2 pathway. Many of these phenotypes were not shared by the Stat3 ΔTAD mutant. Indeed, only neutrophil and macrophage development were disrupted in these mutants with neutrophil migration actually increased, while responsiveness to LPS and G-CSF was maintained. This suggests that Stat3 participates in innate immune cell development and function through both canonical and non-canonical modalities, providing additional insights for relevant diseases.

信号转导与转录激活因子3（STAT3）广泛参与免疫细胞的发育、稳态维持与功能调控，其功能缺失与人类免疫相关疾病紧密关联。既往研究认为，STAT3的上述功能源于其作为多种细胞因子下游诱导型转录因子的经典作用模式，但目前也已有研究报道STAT3存在非经典的功能发挥方式。为进一步明晰通过谱系特异性小鼠基因敲除实验所揭示的STAT3功能，本研究采用CRISPR/Cas9技术构建了保守的斑马鱼Stat3蛋白突变体：一类为功能丧失型基因敲除（KO）突变体，另一类为缺失包含转录激活结构域在内的C端序列的突变体（ΔTAD）。对KO突变体的分析显示，Stat3在造血干细胞中具有保守作用，可影响原始造血与早期定型造血过程中所有造血谱系的发育，并导致幼鱼阶段免疫细胞群体比例异常。Stat3 KO突变体无法响应脂多糖（LPS）与粒细胞集落刺激因子（G-CSF），同时中性粒细胞迁移能力显著降低，该现象与Cxcl8b/Cxcr2通路的功能丧失密切相关。而诸多上述表型并未在Stat3 ΔTAD突变体中出现。进一步研究发现，该突变体仅会干扰中性粒细胞与巨噬细胞的发育，且中性粒细胞迁移能力反而有所提升，同时仍可维持对LPS与G-CSF的应答能力。上述结果表明，Stat3可通过经典与非经典两种途径参与先天免疫细胞的发育与功能调控，为相关免疫疾病的研究提供了新的见解。