Neutrophil extracellular traps aggravate neutrophil reverse transendothelial migration during traumatic brain injury

It is appreciated that neutrophils infiltrating infected tissues can undergo reverse trans-endothelial migration (rTEM) to re-enter the bloodstream, contributing to the dissemination of inflammatory responses. However, the specific regulatory mechanisms of rTEM under mechanical injury condition are still poorly understood. Here, in a murine mechanical injury model of traumatic brain injury (TBI), we observed rTEM phenomenon in neutrophil during the acute phase of TBI using in vivo LY6G-Biotin/Streptavidin (STREPT) crosslinking labeling experiment. We also found that neutrophils undergoing rTEM primarily migrated to tibial bone marrow after TBI. Further exploration indicated that neutrophils undergoing rTEM highly express intercellular cell adhesion molecule-1 (ICAM-1), and the levels of ICAM-1 on peripheral blood neutrophils are significantly positively correlated with the levels of neutrophil extracellular traps (NETs) in damaged brain tissue. Subsequently, we performed endothelial/neutrophil Double-Transwell assay to simulate neutrophil rTEM in vitro, in which we confirmed that the formation of NETs significantly reduces the expression of junctional adhesion molecule C (JAM-C) between endothelial cells and promotes the expression of ICAM-1 on neutrophils. These effects can be significantly antagonized by the NETs inhibitor Cl-Amidine. Cl-Amidine treatment in TBI mice significantly reduced the proportion of neutrophils undergoing rTEM in peripheral blood and their distribution in peripheral tibia, subsequently facilitating long-term neurological recovery and inflammatory resolution after TBI. These results collectively showed that NETs promote rTEM of neutrophils within the injured brain tissue by degrading endothelial JAM-C and upregulating the expression of neutrophil ICAM-1, leading to reverse migration to peripheral organs.