Oral Neutrophils Characterized: Chemotactic, Phagocytic, and Neutrophil Extracellular Trap (NET) Formation Properties
Oral Neutrophils Characterized: Chemotactic, Phagocytic, and Neutrophil Extracellular Trap (NET) Formation Properties
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DOI：
10.3389/fimmu.2019.00635
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发表时间：
2019-03-29
影响因子：
7.3
通讯作者：
Nicu, Elena A.
中科院分区：
文献类型：
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作者：
Moonen, Carolyn G. J.;Hirschfeld, Josefine;Nicu, Elena A.
维持口腔健康在一定程度上是由中性粒细胞(PMN)的免疫监视和抗菌功能管理的，PMN作为口腔PMN(OPMN)从循环系统通过口腔粘膜组织迁移。在任何微生物丰富的生态系统中，如口腔，PMN迁移到各种外源趋化物质，吞噬细菌，并产生中性粒细胞外陷阱(Net)，以固定和消除病原体。通过静脉穿刺法从循环中获得的PMN(以下称为cPMN)已被广泛使用各种功能分析方法进行研究。我们的目的是研究OPMN在维持口腔健康方面的潜力，并因此将其趋化和抗菌功能与cPMN进行比较。为了建立趋化、吞噬和网络形成能力，从没有明显口腔炎症的健康受试者中分离出OPMN和cPMN。使用Insall小室和视频显微镜分析对趋化物质fMLP的定向趋化作用。用流式细胞仪检测FMLP的表达。用流式细胞仪分析PMN与热灭活的FITC标记微生物孵育后的吞噬功能。此外，采用琼脂平板法对大肠杆菌(EC)进行杀灭试验。在PMA或RPMI刺激(非刺激对照)后，用SYTOX(TM)Green荧光定量测定oPMN和cPMN形成的净值。与cPMN相反，oPMN对fMLP的趋化反应与对照组无差异(cPMN的平均速度+/-扫描电子显微镜：0.79+/-0.24；oPMN的平均速度：0.10+/-0.07微米/分钟)。通过显著降低fMLP受体的表达，可以解释oPMN对fMLP定向运动能力的减弱。观察到OPMN增加了对各种微生物的黏附和内化。无论是在刺激条件下还是在刺激条件下，OPMN形成的Net数都是受刺激cPMN的13倍。与cPMN相比，oPMN对EC的细胞内杀伤能力有限。总之，OPMN对fMLP的高效趋化能力已经耗尽，这可能是通过口腔组织进入口腔的结果，是一个高度敌意的生态系统。总体而言，OPMN的行为与多动症和沮丧的杀戮是一致的。然而，OPMN最有可能有助于维持一个平衡的口腔生态系统，因为它们在进入口腔后仍有能力将微生物内化，同时保持其丰富的净产量。
Maintenance of oral health is in part managed by the immune-surveillance and antimicrobial functions of polymorphonuclear leukocytes (PMNs), which migrate from the circulatory system through the oral mucosal tissues as oral PMNs (oPMNs). In any microorganism-rich ecosystem, such as the oral cavity, PMNs migrate toward various exogenous chemoattractants, phagocytose bacteria, and produce neutrophil extracellular traps (NETs) to immobilize and eliminate pathogens. PMNs obtained from the circulation through venipuncture (hereafter called cPMNs) have been widely studied using various functional assays. We aimed to study the potential of oPMNs in maintaining oral health and therefore compared their chemotactic and antimicrobial functions with cPMNs. To establish chemotactic, phagocytic, and NET forming capacities, oPMNs and cPMNs were isolated from healthy subjects without obvious oral inflammation. Directional chemotaxis toward the chemoattractant fMLP was analyzed using an Insall chamber and video microscopy. fMLP expression was assessed by flow cytometry. Phagocytosis was analyzed by flow cytometry, following PMN incubation with heat-inactivated FITC-labeled micro-organisms. Furthermore, agar plate-based killing assays were performed with Escherichia coli (Ec). NET formation by oPMNs and cPMNs was quantified fluorimetrically using SYTOX (TM) Green, following stimulation with either PMA or RPMI medium (unstimulated control). In contrast to cPMNs, the chemotactic responses of oPMNs to fMLP did not differ from controls (mean velocity +/- SEM of cPMNs: 0.79 +/- 0.24; of oPMNs; 0.10 +/- 0.07 micrometer/min). The impaired directional movement toward fMLP by oPMNs was explained by significantly lower fMLP receptor expression. Increased adhesion and internalization of various micro-organisms by oPMNs was observed. oPMNs formed 13 times more NETs than stimulated cPMNs, in both unstimulated and stimulated conditions. Compared to cPMNs, oPMNs showed a limited ability for intracellular killing of Ec. In conclusion, oPMNs showed exhausted capacity for efficient chemotaxis toward fMLP which may be the result of migration through the oral tissues into the oral cavity, being a highly "hostile" ecosystem. Overall, oPMNs' behavior is consistent with hyperactivity and frustrated killing. Nevertheless, oPMNs most likely contribute to maintaining a balanced oral ecosystem, as their ability to internalize microbes in conjunction with their abundant NET production remains after entering the oral cavity.