Stomatin promotes neutrophil degranulation and vascular leakage in the early stage after severe burn via enhancement of the intracellular binding of neutrophil primary granules to F-actin

Abstract

Background: The pathophysiology of severe burn injuries in the early stages involves complex emergency responses, inflammatory reactions, immune system activation, and a significant increase in vascular permeability. Neutrophils, crucial innate immune cells, undergo rapid mobilization and intricate pathophysiological changes during this period. However, the dynamic alterations and detailed mechanisms governing their biological behavior remain unclear. Stomatin protein, an essential component of the cell membrane, stabilizes and regulates the membrane and participates in cell signal transduction. Additionally, it exhibits elevated expression in various inflammatory diseases. While Stomatin expression has been observed in the cell and granule membranes of neutrophils, its potential involvement in post-activation functional regulation requires further investigation.

Methods: Neutrophils were isolated from human peripheral blood, mouse peripheral blood, and mouse bone marrow using the magnetic bead separation method. Flow cytometry was used to assess neutrophil membrane surface markers, ROS levels, and phagocytic activity. The expression of the Stomatin gene and protein was examined using quantitative real-time polymerase chain reaction and western blotting methods, respectively. Furthermore, the enzyme-linked immunosorbent assay was used to evaluate the expression of neutrophil-derived inflammatory mediators (myeloperoxidase (MPO), neutrophil elastase (NE), and matrix metalloproteinase 9 (MMP9)) in the plasma. Images and videos of vascular leakage in mice were captured using in vivo laser confocal imaging technology, whereas in vitro confocal microscopy was used to study the localization and levels of the cytoskeleton, CD63, and Stomatin protein in neutrophils.

Results: This study made the following key findings: (1) Early after severe burn, neutrophil dysfunction is present in the peripheral blood characterized by significant bone marrow mobilization, excessive degranulation, and impaired release and chemotaxis of inflammatory mediators (MPO, NE, and MMP9). (2) After burn injury, expression of both the stomatin gene and protein in neutrophils was upregulated. (3) Knockout (KO) of the stomatin gene in mice partially inhibited neutrophil excessive degranulation, potentially achieved via reduced production of primary granules and weakened binding of primary granules to the cell skeleton protein F-actin. (4) In severely burned mice, injury led to notable early-stage vascular leakage and lung damage, whereas Stomatin gene KO significantly ameliorated lung injury and vascular leakage.

Conclusions: Stomatin promotes neutrophil degranulation in the early stage of severe burn injury via increasing the production of primary granules and enhancing their binding to the cell skeleton protein F-actin in neutrophils. Consequently, this excessive degranulation results in aggravated vascular leakage and lung injury.

Keywords: Burn injury; Degranulation; Stomatin; Vascular leakage.