Sustained activation of NLRP3 inflammasome and release of neutrophil extracellular traps (NETs) impair wound healing of diabetic foot ulcers (DFUs). Our previous study reported that milk fat globule epidermal growth factor VIII (MFG-E8) attenuates tissue damage in systemic lupus erythematosus. However, the functional effect of MFG-E8 on “NLRP3 inflammasome-NETs” inflammatory loop in wound healing of diabetes is not completely elucidated. In this study, neutrophils from DFU patients are susceptible to undergo NETosis, releasing more NETs. The circulating levels of NET components neutrophil elastase and proteinase 3 and inflammatory cytokines IL-1β and IL-18 were significantly elevated in DFU patients compared with healthy controls or diabetic patients, in spite of higher levels of MFG-E8 in DFU patients. In Mfge8^−/− diabetic mice, skin wound displayed exaggerated inflammatory response, including leukocyte infiltration, excessive activation of NLRP3 inflammasome (release of higher IL-1β, IL-18, and TNF-α), largely lodged NETs, resulting in poor angiogenesis and wound closure. When stimulated with high-dose glucose or IL-18, MFG-E8-deficient neutrophils release more NETs than WT neutrophils. After administration of recombinant MFG-E8, IL-18-primed NETosis of WT or Mfge8^−/− neutrophils was significantly inhibited. Furthermore, NET and mCRAMP (component of NETs, the murine equivalent of cathelicidin LL-37 in human)-mediated activation of NLRP3 inflammasome and production of IL-1β/IL-18 were significantly elevated in Mfge8^−/− macrophages compared with WT macrophages, which were also significantly dampened by the administration of rmMFG-E8. Therefore, our study demonstrated that as inhibitor of the “NLRP3 inflammasome-NETs” inflammatory loop, exogenous rMFG-E8 improves angiogenesis and accelerates wound healing, highlighting possible therapeutic potential for DFUs.

NLRP3 炎性体的持续激活和中性粒细胞胞外陷阱 (NET) 的释放会损害糖尿病足溃疡 (DFU) 的伤口愈合。我们之前的研究报告称，乳脂球表皮生长因子 VIII (MFG-E8) 可减轻系统性红斑狼疮的组织损伤。然而，MFG-E8 对糖尿病伤口愈合中“NLRP3 炎性体-NET”炎症循环的功能作用尚未完全阐明。在这项研究中，DFU 患者的中性粒细胞容易发生 NETosis，释放更多的 NET。尽管 DFU 患者的 MFG-E8 水平较高，但与健康对照或糖尿病患者相比，DFU 患者的 NET 成分中性粒细胞弹性蛋白酶和蛋白酶 3 以及炎性细胞因子 IL-1β 和 IL-18 的循环水平显着升高。在Mfge8 ^−/−糖尿病小鼠中，皮肤伤口表现出过度的炎症反应，包括白细胞浸润、NLRP3 炎症小体过度激活（释放较高的 IL-1β、IL-18 和 TNF-α）、大部分 NET 滞留，导致血管生成不良和伤口闭合。当用高剂量葡萄糖或 IL-18 刺激时，MFG-E8 缺陷的中性粒细胞比 WT 中性粒细胞释放更多的 NET。施用重组MFG-E8后，WT或Mfge8 ^−/−中性粒细胞的IL-18引发的NETosis被显着抑制。此外，与 WT 巨噬细胞相比， Mfge8 ^-/−巨噬细胞中 NET 和 mCRAMP（NET 的组成部分，鼠类相当于人类的抗菌肽 LL-37）介导的 NLRP3 炎症小体激活和 IL-1β/IL-18 的产生显着升高，这也被 rmMFG-E8 的施用显着抑制。因此，我们的研究表明，作为“NLRP3炎症小体-NETs”炎症循环的抑制剂，外源性rMFG-E8可以改善血管生成并加速伤口愈合，凸显了DFU的可能治疗潜力。