Institute of Parasitology, Justus -Liebig University Giessen, Giessen, Germany.
CIBAV Research Group, Facultad de Ciencias Agrarias, Universidad de Antioquia (UdeA), Medellín, Colombia.
Front Immunol. 2022 Aug 11;13:842482. doi: 10.3389/fimmu.2022.842482. eCollection 2022.
The protozoan parasite is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by stages. So far, the metabolic requirements of -triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in sporozoite-induced NETosis.
原生动物寄生虫是牛球虫病的病原体，牛球虫病是一种具有全球重要意义的肠道疾病，它严重影响着牛的生产力。以前的研究表明，牛中性粒细胞胞外诱捕网（NET）形成——多形核中性粒细胞（PMN）的一种重要的早期宿主固有效应机制——是由 阶段诱发的。到目前为止，尚未明确 触发 NET 形成的代谢需求。我们在这里研究了 PMN 的早期糖酵解和线粒体反应，以及 pH 值、不同代谢途径、P2 受体介导的嘌呤能信号转导以及单羧酸转运蛋白 1 和 2（MCT1、MCT2）在 裂殖子诱导的 NET 形成中的作用。基于 Seahorse 的实验揭示了 PMN 的耗氧量（OCR）和早期糖酵解反应的快速诱导，这反映了 PMN 在与裂殖子接触时的即时激活和代谢变化。通过使用 2-氟-2-脱氧-D-葡萄糖（FDG）、6-二氮-5-氧-L-正亮氨酸（DON）、二氯乙酸钠（DCA）、噻唑胺（OT）、草氨酸钠（OXA）和寡霉素 A（OmA）来靶向糖酵解和能量生成的化学抑制实验研究了这些代谢变化对 NET 形成的影响，分别阻断糖酵解、谷氨酰胺分解、丙酮酸脱氢酶激酶、丙酮酸脱氢酶、乳酸脱氢酶和线粒体 ATP 合酶。总的来说，裂殖子诱导的 NET 形成在 PMN 用 OmA 和 OXA 预处理后显著减少，这表明 ATP 和乳酸介导的代谢途径起着关键作用。因此，我们还研究了细胞外 pH 值、MCT1、MCT2 和嘌呤能受体抑制剂（AR-C141900、AR-C155858、可可碱和 NF449，分别）的影响。用后者抑制剂预处理可阻断暴露的牛 PMN 中裂殖子触发的 DNA 释放。本报告首次提供了证据，证明碳水化合物相关代谢途径和嘌呤能受体在 裂殖子诱导的 NETosis 中起着关键作用。
