Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis
Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis
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DOI：
10.1371/journal.pone.0006209
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发表时间：
2009-07-10
期刊：
影响因子：
3.7
通讯作者：
Kobzik, Lester
中科院分区：
文献类型：
--
作者：
DeLoid, Glen M.;Sulahian, Timothy H.;Kobzik, Lester
肺泡巨噬细胞（AMs）可以通过先天免疫受体（如清道夫受体（SRs））吞噬未被吞噬的病原体，如金黄色葡萄球菌。细胞骨架事件和信号通路参与吞噬的非个性化的细菌可能支配的命运摄入的病原体，但很少表征。我们开发了一种基于高通量扫描细胞术的检测方法，用于在96孔微孔板上定量人血源性am样巨噬细胞对金黄色葡萄球菌的吞噬作用。内化与结合细菌或微球的差异荧光标记允许对扫描细胞术获得的折叠共聚焦堆栈图像进行自动图像分析，并定量总结合颗粒和内化颗粒的百分比。我们比较了经典的SR阻滞剂多肌苷酸、细胞骨架抑制剂细胞松缩素D和诺可唑、信号抑制剂staurosporine、Go 6976、JNK抑制剂I和kn93对一组活的非个体金黄色葡萄球菌菌株（Wood, Seattle 1945 （ATCC 25923）和RN6390）的吞噬作用，以及商业杀死的Wood菌株、热杀死的Wood菌株和乳胶珠的作用。我们的研究结果显示，SR抑制剂多肌苷酸无法阻断任何活的金黄色葡萄球菌菌株的结合，这表明SR介导的对商业杀死的荧光细菌颗粒的摄取并不能准确地模拟与活菌的相互作用。我们还观察到细胞骨架和信号抑制剂对不同金黄色葡萄球菌菌株内化的影响存在异质性。这些数据表明，人巨噬细胞摄取未个性化的活金黄色葡萄球菌不是由SRs介导的，并且介导金黄色葡萄球菌吞噬的细胞力学和信号传导过程各不相同。研究结果还证明了高通量扫描细胞术技术在更详细地研究金黄色葡萄球菌和其他生物吞噬作用方面的潜在效用。
Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Go 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.