A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS
Lisa Johann, … , Markus Schwaninger, Ari Waisman
Lisa Johann, … , Markus Schwaninger, Ari Waisman
Published October 19, 2023
Citation Information: J Clin Invest. 2023;133(24):e168314. https://doi.org/10.1172/JCI168314.
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Research Article Autoimmunity Neuroscience Article has an altmetric score of 3

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS

Abstract

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Authors

Lisa Johann, Sasha Soldati, Kristin Müller, Josephine Lampe, Federico Marini, Matthias Klein, Eva Schramm, Nathalie Ries, Carsten Schelmbauer, Ilaria Palagi, Khalad Karram, Julian C. Assmann, Mahtab A. Khan, Jan Wenzel, Mirko H.H. Schmidt, Jakob Körbelin, Dirk Schlüter, Geert van Loo, Tobias Bopp, Britta Engelhardt, Markus Schwaninger, Ari Waisman

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Figure 7

ICOSL regulates T cell adhesion to pMBMECs.

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ICOSL regulates T cell adhesion to pMBMECs. (A–C) Static adhesion assay ...
(A–C) Static adhesion assay of T cells on pMBMECs. pMBMECs were stimulated with IL-1β for 72 hours and incubated for 30 minutes with α-ICOSL or control antibody. T cells were labelled with CellTracker Green CMFDA dye and added for 30 minutes to the pMBMECs on a shaking platform. After washing, images were taken and adhering cells were counted. Data is representative of 2 individual experiments with n = 6 per group. (A) Representative microscopic images of 2D2 Th1 cells (green) adhering to the pMBMEC monolayer (nuclear stain blue, DAPI). (B) Quantification of number of adhered 2D2 Th1 cells per field of view (FOV). (C) Quantification of number of adhered 2D2 Th17 cells per FOV. (D–G) Analysis of T cell postarrest behavior on pMBMECs. Data is pooled from 4 individual experiments. pMBMECs were stimulated with IL-1β for 72 hours and incubated for 30 minutes with α-ICOSL or control antibody. 2D2 Th1 T cell postarrest behavior was assessed by live cell imaging under physiological flow. (D) Number of arrested Th1 cells per FOV. (E) Quantification of T cell postarrest behavior. Each category is shown as fraction of the sum of the categorized cells. (F) Crawling distance of T cells that successfully completed diapedesis on IL-1β-stimulated isotype or α-ICOSL treated pMBMECs under flow conditions during 20 minutes observation time shown in μm. (G) Crawling speed of T cells that successfully completed diapedesis on IL-1β-stimulated isotype or α-ICOSL treated pMBMECs under flow conditions shown in μm/min. Data in F and G is pooled from 4 individual experiments with n = 86–102 tracks analyzed. Data was analyzed using 2-tailed unpaired Student’s t test (B–D, F, and G) or 2-way ANOVA with Šidák’s multiple comparison test (E). *P<0.05, **P<0.01.