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Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

ABSTRACT: 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.

SUBMITTER: Glaser KM

PROVIDER: S-EPMC10777075 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

Glaser Katharina M KM Doon-Ralls Jacob J Walters Nicole N Rima Xilal Y XY Rambold Angelika S AS Réategui Eduardo E Lämmermann Tim T

iScience 20231216 1

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 <i>in situ</i> have hampered recent progress. We here adapted an <i>in vitro</i> swarming-on-a-chip platform for the use with ...[more]

PMID: 38205244

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Project description:BackgroundThe aberrant formation of neutrophil extracellular traps (NETs) has been implicated in ulcerative colitis (UC), a chronic recurrent intestinal inflammation. Cyclosporine A (CsA) is now applied as rescue therapy for acute severe UC. In addition, it has been certained that CsA inhibits the formation of NETs in vitro and the mechanism of which was still vague. The study aimed to explore the mechanism CsA inhibits the NETs formation of colitis in vivo and in vitro.MethodsNETs enrichment in clinical samples was analyzed using databases from Gene Expression Omnibus and verified in our center. Dextran sulfate sodium (DSS)-induced acute colitis mice model was used to investigate the effect of CsA on NETs of colonic tissue expression. To clarify the mechanism, intracellular energy metabolites were examined by Liquid Chromatograph Mass Spectrometer, and reactive oxygen species (ROS) levels were examined by fluorescence intensity in neutrophils treated with CsA after LPS stimulation. The transcriptional level and activity of G6PD of neutrophils were also assessed using qRT-PCR and WST-8. RNA Sequencing was used to detect differentially expressed genes of neutrophils stimulated by LPS with or without CsA. The expression levels of related proteins were detected by western blot.ResultsNETs enrichment was especially elevated in moderate-to-severe UC patients compared to HC. NETs expression in the colon from DSS colitis was decreased after CsA treatment. Compared with neutrophils stimulated by LPS, NETs formation and cellular ROS levels were decreased in LPS + CsA group. Cellular ribulose 5-phosphate and NADPH/NADP + related to the pentose phosphate pathway (PPP) were reduced in LPS + CsA group. In addition, CsA could decrease G6PD activity in neutrophils stimulated with LPS, and the results were further verified by inhibiting G6PD activity. At last, P53 protein was highly expressed in LPS + CsA group compared with the LPS group. Intracellular G6PD activity, ROS level and NETs formation, which were downregulated by CsA, could be reversed by a P53 inhibitor.ConclusionOur results indicated CsA could alleviate the severity of colitis by decreasing the formation of NETs in vivo. In vitro, CsA reduced ROS-dependent NETs release via downregulating PPP and cellular ROS levels by decreasing G6PD activity directly by activating the P53 protein.

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Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

Publications

Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

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