LPS-induced TIR domain complex acetylation activated TLR4/MAL/MyD88 signal pathway in sepsis [scRNA-Seq]

Among the diseases caused by Toll-like receptor 4 (TLR4) abnormal activation by bacterial endotoxin, sepsis is the most dangerous one. The reprogramming of macrophages plays a crucial role in orchestrating the pathogenesis of sepsis. However, the precise mechanism underlying TLR4 activation in macrophages remained incompletely understood. Our studies revealed that upon lipopolysaccharide (LPS) stimulation, CREB-binding protein (CBP) was recruited to the TLR4 signalosome complex and resulted in pronounced acetylation in the TIR domains of TLR4, Myeloid differentiation factor 88 (MyD88) and MyD88 adapter-like (MAL), which significantly enhanced the activation of the NF-κB signaling pathway and polarization of M1 macrophages. In sepsis patients, significantly elevated TLR4-TIR acetylation was detected in CD16+ monocytes combined with elevated expression of M1 macrophage markers and production of pro-inflammatory cytokines. In contrast, histone deacetylase 1 (HDAC1) served as a key deacetylase in the deacetylation of the TIR domain complex. The inhibition of HDAC1 accelerated sepsis-associated syndromes, while the inhibition of CBP alleviated this process. Overall, our findings highlighted the crucial role of TIR domain complex acetylation in the regulation of inflammatory immune response and suggested that the reversible acetylation of the complex emerged as a promising therapeutic target for M1 macrophages during the progression of sepsis. The purified monocytes from sepsis were labeled with TLR4-acK and FITC-conjugated secondary antibody, then positively stained cells were enriched through the BD FACSAria™ III Cell Sorter.

在由细菌内毒素引发Toll样受体4（Toll-like receptor 4, TLR4）异常活化所导致的疾病中，脓毒症是最为凶险的一种。巨噬细胞重编程在调控脓毒症的发病机制中发挥关键作用，但目前巨噬细胞中TLR4活化的精确分子机制仍未完全阐明。本研究发现，在脂多糖（lipopolysaccharide, LPS）刺激下，CREB结合蛋白（CREB-binding protein, CBP）被招募至TLR4信号小体复合物，进而引发TLR4、髓系分化因子88（Myeloid differentiation factor 88, MyD88）以及MyD88接头样蛋白（MyD88 adapter-like, MAL）的TIR结构域发生显著乙酰化修饰，这一过程显著增强了NF-κB信号通路的活化以及M1型巨噬细胞的极化。在脓毒症患者体内，CD16+单核细胞中可检测到TLR4-TIR乙酰化水平显著升高，同时伴随M1型巨噬细胞标志物表达上调与促炎细胞因子生成增加。与之相反，组蛋白去乙酰化酶1（histone deacetylase 1, HDAC1）作为关键去乙酰化酶参与TIR结构域复合物的去乙酰化修饰。抑制HDAC1会加剧脓毒症相关综合征，而抑制CBP则可缓解该病理进程。综上，本研究结果凸显了TIR结构域复合物乙酰化在炎症免疫应答调控中的关键作用，并提示该复合物的可逆乙酰化修饰有望成为脓毒症进程中M1型巨噬细胞的潜在治疗靶点。从脓毒症患者体内分离纯化的单核细胞经TLR4-acK一抗与异硫氰酸荧光素（fluorescein isothiocyanate, FITC）标记的二抗孵育标记后，通过BD FACSAria™ III细胞分选仪富集阳性染色细胞。