Abstract
The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.
Highlights
The blood brain barrier (BBB) plays an integral role in maintaining central homeostasis by acting as a physical barrier between the central nervous system (CNS) and the periphery
neutrophil-derived microvesicles (NMV) isolated from the blood of healthy volunteers were analyzed using ZetaView nanoparticle tracking analysis (NTA) revealed they were heterogenous with a mean size of 226 nm (Figure 1A), confirming previous characterization studies carried out in our laboratory [29]
We demonstrate that NMV are internalized by human brain endothelial cells in vitro, significantly altering the transcriptomic profile and leading to increased monolayer permeability, suggesting a mechanism whereby the peripheral response to infection can induce brain microvascular endothelial cells (BMEC) dysfunction
Summary
The blood brain barrier (BBB) plays an integral role in maintaining central homeostasis by acting as a physical barrier between the central nervous system (CNS) and the periphery. Animal models have demonstrated that peripheral activation of immune cells by inflammatory stimuli such as lipopolysaccharide (LPS) can impact BBB permeability, increasing microglial activation [12,13,14,15,16] Together, these studies support a role for systemic inflammation in modulating BBB function and increasing neuroinflammation within the CNS. Patients with dementia have significantly higher levels of circulating neutrophils than non-neurological controls [18], and their activation status correlates with AD progression [19] While these studies indicate a role for neutrophils in AD, neutrophil extravasation is not routinely associated with AD pathology, and the effect of neutrophils on the BBB is currently unknown. The study aimed to determine whether NMV interact with BMEC, their mechanism of internalization and NMV-induced transcriptomic changes in brain endothelial cells
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