Delayed neutrophil apoptosis during sepsis may impact neutrophil organ accumulation and tissue immune homeostasis. Elucidating the mechanisms underlying neutrophil apoptosis may help identify potential therapeutic targets. Glycolysis is critical to neutrophil activities during sepsis. However, the precise mechanisms through which glycolysis regulates neutrophil physiology remain under-explored, especially those involving the non-metabolic functions of glycolytic enzymes. In the present study, the impact of programmed death ligand-1 (PD-L1) on neutrophil apoptosis was explored. The regulatory effect of the glycolytic enzyme, pyruvate kinase M2 (PKM2), whose role in septic neutrophils remains unaddressed, on neutrophil PD-L1 expression was also explored. Peripheral blood neutrophils were isolated from patients with sepsis and healthy controls. PD-L1 and PKM2 levels were determined by flow cytometry and Western blotting, respectively. Dimethyl sulfoxide (DMSO)-differentiated HL-60 cells were stimulated with lipopolysaccharide (LPS) as an in vitro simulation of septic neutrophils. Cell apoptosis was assessed by annexin V/propidium iodide (annexin V/PI) staining, as well as determination of protein levels of cleaved caspase-3 and myeloid cell leukemia-1 (Mcl-1) by Western blotting. An in vivo model of sepsis was constructed by intraperitoneal injection of LPS (5 mg/kg) for 16 h. Pulmonary and hepatic neutrophil infiltration was assessed by flow cytometry or immunohistochemistry. PD-L1 level was elevated on neutrophils under septic conditions. Administration of neutralizing antibodies against PD-L1 partially reversed the inhibitory effect of LPS on neutrophil apoptosis. Neutrophil infiltration into the lung and liver was also reduced in PD-L1−/− mice 16 h after sepsis induction. PKM2 was upregulated in septic neutrophils and promoted neutrophil PD-L1 expression both in vitro and in vivo. In addition, PKM2 nuclear translocation was increased after LPS stimulation, which promoted PD-L1 expression by directly interacting with and activating signal transducer and activator of transcription 1 (STAT1). Inhibition of PKM2 activity or STAT1 activation also led to increased neutrophil apoptosis. In this study, a PKM2/STAT1-mediated upregulation of PD-L1 on neutrophils and the anti-apoptotic effect of upregulated PD-L1 on neutrophils during sepsis were identified, which may result in increased pulmonary and hepatic neutrophil accumulation. These findings suggest that PKM2 and PD-L1 could serve as potential therapeutic targets.

中文翻译：

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脓毒症期间 PKM2/STAT1 介导的 PD-L1 对中性粒细胞的上调通过抗凋亡作用促进中性粒细胞器官积累

脓毒症期间中性粒细胞凋亡延迟可能影响中性粒细胞器官积累和组织免疫稳态。阐明中性粒细胞凋亡的潜在机制可能有助于确定潜在的治疗靶点。糖酵解对败血症期间的中性粒细胞活动至关重要。然而，糖酵解调节中性粒细胞生理学的确切机制仍未得到充分探索，尤其是那些涉及糖酵解酶的非代谢功能的机制。在本研究中，探讨了程序性死亡配体 1 (PD-L1) 对中性粒细胞凋亡的影响。还探讨了糖酵解酶丙酮酸激酶 M2 (PKM2) 对中性粒细胞 PD-L1 表达的调节作用，其在败血症中性粒细胞中的作用仍未得到解决。从脓毒症患者和健康对照者中分离外周血中性粒细胞。PD-L1 和 PKM2 水平分别通过流式细胞术和蛋白质印迹法测定。用脂多糖 (LPS) 刺激二甲基亚砜 (DMSO) 分化的 HL-60 细胞作为败血症中性粒细胞的体外模拟。通过膜联蛋白 V/碘化丙啶 (annexin V/PI) 染色评估细胞凋亡，并通过蛋白质印迹法测定裂解的 caspase-3 和骨髓细胞白血病-1 (Mcl-1) 的蛋白质水平。通过腹腔注射 LPS (5 mg/kg) 16 小时构建脓毒症体内模型。通过流式细胞术或免疫组织化学评估肺和肝中性粒细胞浸润。在败血症条件下，中性粒细胞的 PD-L1 水平升高。施用针对 PD-L1 的中和抗体可部分逆转 LPS 对中性粒细胞凋亡的抑制作用。在脓毒症诱导后 16 小时，PD-L1-/- 小鼠的肺和肝中性粒细胞浸润也减少了。PKM2 在脓毒症中性粒细胞中上调，并在体外和体内促进中性粒细胞 PD-L1 表达。此外，LPS 刺激后 PKM2 核转位增加，其通过直接相互作用并激活信号转导和转录激活因子 1 (STAT1) 促进 PD-L1 表达。PKM2 活性或 STAT1 激活的抑制也导致中性粒细胞凋亡增加。在这项研究中，确定了 PKM2/STAT1 介导的中性粒细胞 PD-L1 上调以及败血症期间上调 PD-L1 对中性粒细胞的抗凋亡作用，这可能导致肺和肝中性粒细胞积聚增加。