Multiplexed Detection of Secreted Cytokines at near-Molecular Resolution Elucidates Macrophage Polarization Heterogeneity.
Multiplexed Detection of Secreted Cytokines at near-Molecular Resolution Elucidates Macrophage Polarization Heterogeneity.
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DOI：
10.1021/acs.analchem.1c02222
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发表时间：
2021-12
影响因子：
7.4
通讯作者：
Vanessa Herrera;S. J. Hsu;Veena Y Naveen;Wendy F Liu;Jered B. Haun
中科院分区：
文献类型：
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作者：
Vanessa Herrera;S. J. Hsu;Veena Y Naveen;Wendy F Liu;Jered B. Haun
监测单细胞的蛋白质分泌可以为细胞如何响应其微环境提供重要的见解。这对于免疫细胞尤其如此，免疫细胞可以表现出很大程度的反应异质性。微制造的孔阵列提供了一种强大且通用的方法来评估单个细胞的细胞因子、趋化因子和生长因子的分泌，但是检测灵敏度被限制在每个细胞10，000的量级的高水平。最近，我们报道了一种基于量子点的免疫测定法，将细胞因子TNF-α的检测限降低到接近单细胞水平的浓度。在这里，我们将这种检测方法适用于三个额外的目标，同时保持高检测灵敏度。具体来说，我们使用具有不同发射光谱的量子点检测MCP-1、TGF-β、IL-10和TNF-α，每种量子点显示出每孔1-10 fM或1-2个分子范围内的检测阈值。然后，我们定量了来自单个巨噬细胞的所有四种蛋白质的分泌，所述单个巨噬细胞用脂多糖（LPS）刺激朝向促炎状态或用LPS和白细胞介素4（IL-4）两者刺激朝向促愈合状态。我们发现MCP-1和TGF-β主要仅以高水平分泌（> 10，000分子/细胞），而大量细胞以较低水平分泌IL-10和TNF-α，只能使用我们的方法检测到。随后的主成分和聚类分析显示，分泌谱可被分类为完全促炎性，包括MCP-1和/或TNF-α，或显示促愈合和促炎特征的更微妙的反应。我们的研究结果突出了细胞系体外刺激后巨噬细胞表型的异质性和非离散性。未来的工作将集中在通过扩展发射光谱带宽和/或空间条形码捕获抗体来扩展多路复用能力，以及评估体外和体内正常和患病免疫细胞群体的增强检测灵敏度能力。
Monitoring the secretion of proteins from single cells can provide important insights into how cells respond to their microenvironment. This is particularly true for immune cells, which can exhibit a large degree of response heterogeneity. Microfabricated well arrays provide a powerful and versatile method to assess the secretion of cytokines, chemokines, and growth factors from single cells, but detection sensitivity has been limited to high levels on the order of 10,000 per cell. Recently, we reported a quantum dot-based immunoassay that lowered the detection limit for the cytokine TNF-α to concentrations to nearly the single-cell level. Here, we adapted this detection method to three additional targets while maintaining high detection sensitivity. Specifically, we detected MCP-1, TGF-β, IL-10, and TNF-α using quantum dots with different emission spectra, each of which displayed a detection threshold in the range of 1-10 fM or ∼1-2 molecules per well. We then quantified secretion of all four proteins from single macrophage cells that were stimulated toward a pro-inflammatory state with lipopolysaccharide (LPS) or toward a pro-healing state with both LPS and interleukin 4 (IL-4). We found that MCP-1 and TGF-β were predominantly secreted at high levels only (>10,000 molecules/cell), while a substantial number of cells secreted IL-10 and TNF-α at lower levels that could only be detected using our method. Subsequent principal component and cluster analysis revealed that secretion profiles could be classified as either exclusively pro-inflammatory, including MCP-1 and/or TNF-α, or more subtle responses displaying both pro-healing and pro-inflammatory characters. Our results highlight the heterogeneous and nondiscrete nature of macrophage phenotypes following in vitro stimulation of a cell line. Future work will focus on expanding the multiplexing capacity by extending emission spectra bandwidth and/or spatially barcoding capture antibodies, as well as evaluating the enhanced detection sensitivity capabilities with normal and diseased immune cell populations in vitro and in vivo.