Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming
Katharina M. Glaser,
Jacob Doon-Ralls,
Nicole Walters,
Xilal Y. Rima,
Angelika S. Rambold,
Eduardo Réategui,
Tim Lämmermann
Affiliations
Katharina M. Glaser
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; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
Jacob Doon-Ralls
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA
Nicole Walters
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA
Xilal Y. Rima
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA
Angelika S. Rambold
Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany
Eduardo Réategui
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; Corresponding author
Tim Lämmermann
Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany; Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, 48149 Münster, Germany; Corresponding author
Summary: 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 in situ have hampered recent progress. We here adapted an in vitro 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.
