Appearance of claudin-5+ leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles

Abstract

BackgroundThe mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation.MethodsHigh-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels.ResultsA subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE.ConclusionsDuring neuroinflammation, CLN-5+ leukoc...

Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions

“…EMV can deliver and transfer their contents (miRNA, mRNA, proteins) to target cells. Besides, EMV derived from different stimuli have shown different or even adverse effects on the recipient cells depending on their distinct contents (Wang et al, 2013; Pan et al, 2016; Paul et al, 2016). In this study, we generated EMV from HBMEC under TNF-α plus SD stimulation to mimic ischemia and inflammation in ischemic diseases, and examined the functional role of these EMV on HBVSMC.…”

Claudins in the brain: Unconventional functions in neurons

“…Claudins‐1,3,5,11,12,19, occludin, Zona occludens‐1 (ZO‐1) and tricellulin have been identified as key proteins in making these neural barriers . An example of claudins involved in maintaining normal brain function comes from the fact that Claudin‐5 positive leukocytes have been detected in the brain during neuroinflammation . Claudin‐5 also regulates tumor cell motility across the BBB, Indicating the involvement of Claudin‐5 in brain metastasis .…”

“…41 An example of claudins involved in maintaining normal brain function comes from the fact that Claudin-5 positive leukocytes have been detected in the brain during neuroinflammation. 42 Claudin-5 also regulates tumor cell motility across the BBB, Indicating the involvement of Claudin-5 in brain metastasis. 43 Activation of matrix metalloproteinases (MMPs) opens the BBB by degrading tight junction proteins, including Claudins-5 and occludin.…”

Extracellular vesicles and microvascular pathology: Decoding the active dialogue

“…The modulation of TJPs by MV has been further studied in the context of neuroinflammation: the transfer of a major TJP, CLN‐5, from brain endothelium to leukocytes via endothelial MV has been elegantly demonstrated by flow cytometry and confocal imaging. Moreover, endothelial MV transferred CLN‐5 at sites of leukocyte‐endothelial contact along the BBB, a mechanism by which trans‐endothelial migration may be enhanced, through the formation of temporary TJP bridges between these two cell types . Finally, in contrast to these pro‐inflammatory effects at the level of the BBB, endothelial MV can prove to be important protective elements.…”

“…Moreover, endothelial MV transferred CLN-5 at sites of leukocyte-endothelial contact along the BBB, a mechanism by which trans-endothelial migration may be enhanced, through the formation of temporary TJP bridges between these two cell types. 93 appreciate better the duality of EV, that is, can we define what will make a given EV sub-population, in a given setting, deleterious or protective?…”