Neutrophil extracellular traps and associated pathogenesis in TBI: a novel peptide therapeutic strategy. - ABSTRACT Traumatic brain injury (TBI) causes the blood-brain barrier (BBB) dysfunction and transmigration of inflammatory immune cells into the brain, an important mechanism underlying neurovascular damage and neuroinflammation. Adhesion of leukocytes to endothelial cells is a critical step in the migration of leukocytes into injured tissues. Previously, it has been demonstrated that activation of leukocytes, especially neutrophils cause the release of nuclear and granular contents to form an extensive web-like structure of DNA called neutrophil extracellular traps (NET). However, in TBI, the mechanism of injury-induced formation of NET and its mechanistic regulatory role in thrombosis remains elusive. Moreover, it is not clear whether blocking of formation of NET provides better outcomes after TBI. Therefore, an approach to suppress the formation of NET would be a valuable therapeutic strategy and to analyze the efficacy of the therapy in the functional recovery level after TBI. Here, we hypothesize that inhibition of peptidyl arginine deiminase type 4 (PAD4), an enzyme required for NET formation, using PAD4 antagonistic peptide (PAP) will attenuate the formation of NET, NET-induced thrombosis, and promote neovascularization and functional recovery after TBI. In the first aim, we test whether PAP reduces PAD4 expression, inhibits NET formation, and promotes neovascularization. In the second specific aim, we will uncover the molecular mechanisms of the formation of NET-induced thrombosis in TBI and we will dissect the therapeutic role of PAP in depleting NET-dependent thrombosis. We will validate the role of PAD4 in the formation of NET and its role in thrombosis by CRISPR/Cas9 mediated PAD4 gene deletion in human brain microvascular endothelial cells (hBMVECs) and human neutrophil co-culture in vitro and PAD4 knockout (KO) mice (PAD4−/−) in vivo. In addition, we will study the role of different brain cells in NET formation by creating conditional knockout mice by breeding PAD4flox strain with specific brain cell Cre strains. In the third aim, we will use a cohort of behavioral tests that include sensorimotor functions, memory, and psychological stress analyses to validate the role of PAP in promoting functional recovery following TBI. Therefore, in this project, to validate the central hypothesis, these three aims target a subset of events towards unraveling a larger picture of neurovascular remodeling and functional recovery after TBI by attenuating PAD4 activity using a novel small peptide developed in the PI’s lab.
