Regulation of Blood-Brain Barrier Endothelial Cell Hyperpermeability by NLRP3 Inflammasome Inhibition

The blood-brain barrier (BBB) is a semi-permeable and protective barrier of the brain, primarily composed of endothelial cells interconnected by tight junction proteins, that regulates the movement of ions and molecules between the blood and the neural matter. In pathological conditions such as traumatic brain injury (TBI), disruption of the BBB contributes to the leakage of solutes and fluids into the brain parenchyma resulting in the onset of cerebral edema and elevation of intracranial pressure. Recent studies from our lab have demonstrated that the release of the proinflammatory cytokine interleukin 1-β (IL-1β) followed by the activation of matrixmetalloproteinase-9 (MMP-9) is critical to BBB dysfunction following TBI. NLRP3 inflammasome is a multiprotein complex that is critical to the formation of IL-1β. The role of NLRP3 inflammasome in modulating BBB integrity has been recently investigated in our lab. Our results suggest that NLRP3 inflammasome activation is critical to BBB dysfunction and hyperpermeability following TBI. Once activated, the NLRP3 inflammasome is a critical component in the regulation of BBB hyperpermeability, recruiting enzymes such as caspase-1 and subsequent activation of MMP-9, which disrupt tight junction proteins necessary for BBB integrity. Thus, the NLRP3 inflammasome pathway remains a potential target for TBI therapeutics. The objective of this study was to determine the upstream regulators of NLRP3 signaling and BBB hyperpermeability, particularly to determine if extracellular ATP via P2X7R, a purinergic receptor promotes NLRP3 inflammasome activation. Extracellular ATP is a major contributor of secondary injuries following TBI. NLRP3 inflammasome pathway and its impact on BBB endothelial monolayer permeability, tight junction protein expression, and caspase-1 and MMP-9 activation following extracellular ATP-mediated barrier dysfunction was investigated in in human brain microvascular endothelial cells. The effect of benzyl ATP (BzATP)-treatment on tight junction integrity was investigated via zonula occludens-1 (ZO-1) immunofluorescence localization, ZO-1 gene expression by RT-PCR, and caspase-1 and MMP-9 activities by fluorometric assays. Pharmacological inhibition of NLRP3 (using MCC950) resulted in a decrease in BzATP-induced monolayer hyperpermeability, caspase-1 and MMP-9 activities. These effects were independent of changes in cell viability. Our results suggest that extracellular ATP promotes NLRP3 inflammasome activation and subsequent caspase-1 and MMP-9-mediated tight junction disorganization are major pathways that lead to BBB dysfunction and hyperpermeability following conditions such as traumatic brain injury. This project would not have been possible without funding from the National Institute of Health and guidance from Dr. Travis Hein and other collaborators at Texas A&M. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.