TLR7-MyD88-IRF3信号通路对胚胎造血干细胞发育的影响及调控机制研究

Signaling pathways determine embryonic hematopoietic stem cell (HSC) development. Understanding how HSCs form, and in particular the signaling pathways that are involved in embryonic HSC formation is essential for producing HSCs from other cell sources ex vivo, which is a major goal in regenerative medicine. We and others discovered that multiple innate immune and inflammatory signaling pathways regulate HSC formation in the major arteries of mouse and zebrafish embryos. But the mechanisms by which innate immune and inflammatory signaling pathways were activated is unknown and how these pathways guide HSC formation remains unclear. We hypothesized that endogenous retroviruses could activate TLR7-MyD88-IRF3 signaling to enhance inflammatory cytokine expression, and the inflammatory signaling during HSC formation in the embryo might introduce alterations in chromatin accessibility and histone modifications. Here, we proposed to investigate the effect of the TLR7-MyD88-IRF3 signaling on HSC and progenitor formation, uncover the role of the endogenous retroviruses in the activation of the signaling, and explore the potential epigenetic regulation by using the mouse model, CRISPR-Cas9 genome editing, transplantation, flow cytometry and epigenomics. The goal of this proposal is to understand how TLR7-MyD88-IRF3 signaling contributes to the formation of HSCs in the embryo, which will provide useful guidelines for the HSC culture ex vivo.

胚胎造血干细胞发育依赖多种信号通路的精密调控，因此研究信号通路对胚胎造血干细胞发育的调控机制，对体外培养获得造血干细胞具有重要意义。我们和其他团队发现天然免疫和炎症信号通路能调控小鼠和斑马鱼胚胎造血干细胞发育，但是其上游激活和下游作用机制不清。在前期研究基础上，我们提出炎症信号通路在上游被内源性逆转录病毒激活、在下游调节炎症因子表达来改变染色质结构和组蛋白修饰的表观遗传学作用假说。为此，本项目拟利用小鼠模型，研究TLR7-MyD88-IRF3信号通路对胚胎造血干细胞祖细胞发育及炎症因子表达的影响；应用CRISPR-Cas9基因敲除、移植试验、流式细胞术、组学等技术探明TLR7-MyD88-IRF3信号通路被内源性逆转录病毒激活的机制；以及炎症信号通路调节造血干细胞和祖细胞发育的表观遗传学作用机制。本项目的研究有助于我们深入理解胚胎造血干细胞发育的机理，为体外培养获得造血干细胞奠定理论基础。

胚胎造血干细胞发育依赖多种信号通路的精密调控，因此研究信号通路对胚胎造血干细胞发育的调控机制，对体外培养获得造血干细胞具有重要意义。我们和其他团队发现天然免疫和炎症信号通路能调控小鼠和斑马鱼胚胎造血干细胞发育，但是其激活和作用机制不清。在本项目中，我们利用小鼠和斑马鱼模型，研究了TLR7及其信号通路对胚胎造血干细胞发育的作用，我们发现缺失TLR7的胚胎血管内皮细胞发育正常，但是胚胎AGM区域的造血干细胞数量大大减少，直接证明TLR7参与造血干细胞的内皮-造血转换过程。我们还发现TLR7调控造血干细胞发育不依赖街头蛋白MyD88。在缺失TLR7的胚胎中补充IRF7可以让造血干细胞数量回归正常，因此TLR7通过IRF7调控造血干细胞发育。重要的是，我们发现TLR7激动剂R848可以促进造血干细胞生成。这为体外培养造血干细胞提供重要的参考信息，有重要的转化意义。此外，我们还研究了造血转录因子RUNX1在免疫反应中的作用，发现RUNX1可以通过IRF3和STAT1调控干扰素及干扰素激活因子的表达，这些工作增进了人们对造血与免疫相互调控的认识。