Muñoz-Caro Tamara, Gibson Amanda J, Conejeros Iván, Werling Dirk, Taubert Anja, Hermosilla Carlos
Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, Germany.
Escuela de Medicina Veterinaria, Facultad de Recursos Naturales y Medicina Veterinaria, Universidad Santo Tomás, Talca 3460000, Chile.
Pathogens. 2021 Jan 24;10(2):118. doi: 10.3390/pathogens10020118.
Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to . The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation.
TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites.
we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation.
This is the first report on -induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.
牛多形核中性粒细胞（PMN）组成性表达Toll样受体（TLR）TLR2和TLR4，并且已证明在暴露于……后会产生中性粒细胞胞外陷阱（NETs）。本研究调查了TLR2和TLR4在子孢子识别与摄取、白细胞介素-8产生及中性粒细胞胞外陷阱（NET）形成中的作用。
通过流式细胞术分析暴露于活的羧基荧光素琥珀酰亚胺酯（CFSE）染色子孢子的PMN上的TLR表达。在不存在或存在TLR抗体的情况下，评估暴露于不同子孢子制剂和抗原的PMN的上清液中的白细胞介素-8分泌。将细胞暴露于子孢子制剂，并使用荧光素酶报告基因测定法评估转录因子NF-κB的激活情况。进行免疫荧光分析以研究暴露于活子孢子的牛PMN上的TLR2和TLR4表面表达及NET形成。
我们观察到TLR2和TLR4表达显著增加，TLR2的表达平均增加幅度大于TLR4。这种上调既未抑制也未促进牛PMN对子孢子的吞噬作用。活子孢子与抗TLR2单克隆抗体一起导致白细胞介素-8产生显著增强。与暴露于热灭活子孢子和抗原的TLR4/MD2-HEK细胞相比，TLR2-HEK细胞中NF-κB的激活更强。免疫荧光分析显示PMN表面有TLR阳性信号并伴有NET形成。
这是关于牛PMN来源的NETosis过程中诱导TLR2和TLR4伴随表达的首次报道。
