From the Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University of Mainz (JGU), Germany (S.A., E.W., G.P., H.F., F.B., M.M., F.M., A.G., M.B., M.S., I.B., K.G., K.K., K.J., C.R.).
Institute for Pharmacy & Biochemistry, Johannes Gutenberg University of Mainz, Germany (S.A.).
Arterioscler Thromb Vasc Biol. 2020 Sep;40(9):2279-2292. doi: 10.1161/ATVBAHA.120.314491. Epub 2020 Jul 2.
Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-β) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model.
Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.
中性粒细胞的募集和中性粒细胞胞外诱捕网（NET）的形成导致急性肠系膜梗死的致死性。为了研究肠道微生物群在急性肠系膜梗死中的影响，我们使用无菌小鼠模型研究肠道共生菌是否使中性粒细胞对形成中性粒细胞胞外诱捕网（NETosis）的反应性增强。
我们应用肠系膜缺血再灌注（I/R）损伤模型对无菌（GF）和定植 C57BL/6J 小鼠进行研究。通过活体成像，我们定量了 I/R 损伤肠系膜小静脉中白细胞黏附和 NET 形成。肠道微生物群的定植或单定植增加了白细胞的黏附，这依赖于 TLR4（Toll-like receptor-4）/TRIF（TIR-domain-containing adapter-inducing interferon-β）途径。尽管 I/R 损伤后 GF 小鼠肠系膜小静脉中中性粒细胞的聚集减少，但与常规饲养或定植最小微生物群落改变的沙氏杆菌菌群的小鼠相比，I/R 损伤后 NETosis 显著增强。此外，GF 小鼠和抗生素处理的小鼠的中性粒细胞的 LPS（脂多糖）诱导的 NETosis 增加。GF 中性粒细胞中增强的 TLR4 信号传导归因于 TLR4 表达的增加和 IRF3（干扰素调节因子 3）磷酸化的增加。同样，在缺血前和缺血后，抗生素处理的常规饲养的小鼠的中性粒细胞的 NET 形成增加。在缺乏 TLR 接头 TRIF 的常规饲养的小鼠中，I/R 损伤中的 NETosis 增加被消除。GF 小鼠通过饮用水给予 LPS 可减少体外和肠系膜 I/R 损伤模型中 LPS 诱导的 NETosis，支持肠内 LPS 的脱敏作用。
总之，我们的研究结果表明，肠道微生物群抑制了肠系膜 I/R 损伤中 NETing 中性粒细胞的高反应性，同时通过增强中性粒细胞募集来确保免疫监视。
