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

Overview of attention for article published in Journal of Neuroinflammation, November 2016
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Title
Appearance of claudin-5+ leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles
Published in
Journal of Neuroinflammation, November 2016
DOI 10.1186/s12974-016-0755-8
Pubmed ID
Authors

Debayon Paul, Valentina Baena, Shujun Ge, Xi Jiang, Evan R. Jellison, Timothy Kiprono, Dritan Agalliu, Joel S. Pachter

Abstract

The 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. High-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. A 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. During neuroinflammation, CLN-5(+) leukocytes appear in the CNS, and both CLN-5(+) leukocytes and CLN-5(+) EVs are detected in the blood. As endothelial cells transfer CLN-5(+) to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.

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Mendeley readers

The data shown below were compiled from readership statistics for 76 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United Kingdom 1 1%
Italy 1 1%
Unknown 74 97%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 18 24%
Researcher 16 21%
Student > Bachelor 9 12%
Student > Master 6 8%
Student > Doctoral Student 5 7%
Other 10 13%
Unknown 12 16%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 18 24%
Neuroscience 10 13%
Medicine and Dentistry 10 13%
Agricultural and Biological Sciences 7 9%
Immunology and Microbiology 5 7%
Other 12 16%
Unknown 14 18%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 06 October 2017.
All research outputs
#9,487,254
of 11,877,834 outputs
Outputs from Journal of Neuroinflammation
#972
of 1,357 outputs
Outputs of similar age
#199,570
of 272,984 outputs
Outputs of similar age from Journal of Neuroinflammation
#7
of 15 outputs
Altmetric has tracked 11,877,834 research outputs across all sources so far. This one is in the 11th percentile – i.e., 11% of other outputs scored the same or lower than it.
So far Altmetric has tracked 1,357 research outputs from this source. They receive a mean Attention Score of 4.9. This one is in the 15th percentile – i.e., 15% of its peers scored the same or lower than it.
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We're also able to compare this research output to 15 others from the same source and published within six weeks on either side of this one. This one is in the 26th percentile – i.e., 26% of its contemporaries scored the same or lower than it.