Glaser Katharina M, Doon-Ralls Jacob, Walters Nicole, Rima Xilal Y, Rambold Angelika S, Réategui Eduardo, Lämmermann Tim
Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany.
International Max Planck Research School for Immunobiology, Epigenetics and Metabolism (IMPRS-IEM), 79108 Freiburg, Germany.
iScience. 2023 Dec 16;27(1):108656. doi: 10.1016/j.isci.2023.108656. eCollection 2024 Jan 19.
Neutrophil swarming is an essential process of the neutrophil response to many pathological conditions. Resultant neutrophil accumulations are hallmarks of acute tissue inflammation and infection, but little is known about their dynamic regulation. Technical limitations to spatiotemporally resolve individual cells in dense neutrophil clusters and manipulate these clusters have hampered recent progress. We here adapted an swarming-on-a-chip platform for the use with confocal laser-scanning microscopy to unravel the complexity of single-cell responses during neutrophil crowding. Confocal sectioning allowed the live visualization of subcellular components, including mitochondria, cell membranes, cortical actin, and phagocytic cups, inside neutrophil clusters. Based on this experimental setup, we identify that chemical inhibition of the Arp2/3 complex causes cell death in crowding neutrophils. By visualizing spatiotemporal patterns of reactive oxygen species (ROS) production in developing neutrophil swarms, we further demonstrate a regulatory role of the metabolic pentose phosphate pathway for ROS production and neutrophil cluster growth.
中性粒细胞聚集是中性粒细胞对许多病理状况作出反应的一个重要过程。由此产生的中性粒细胞聚集是急性组织炎症和感染的标志，但对其动态调节却知之甚少。在密集的中性粒细胞簇中对单个细胞进行时空分辨并操纵这些簇的技术限制阻碍了近期的研究进展。我们在此采用了一种用于共聚焦激光扫描显微镜的芯片上聚集平台，以揭示中性粒细胞拥挤过程中单细胞反应的复杂性。共聚焦切片能够实时观察中性粒细胞簇内的亚细胞成分，包括线粒体、细胞膜、皮质肌动蛋白和吞噬杯。基于此实验设置，我们发现对Arp2/3复合物的化学抑制会导致拥挤的中性粒细胞死亡。通过观察发育中的中性粒细胞群中活性氧（ROS）产生的时空模式，我们进一步证明了代谢性磷酸戊糖途径对ROS产生和中性粒细胞簇生长的调节作用。
