A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS
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
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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Research Article Autoimmunity Neuroscience

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 1

CNS EC-A20 plays a protective role in CNS autoimmunity.

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CNS EC-A20 plays a protective role in CNS autoimmunity. (A) Breeding str...
(A) Breeding strategy for generation of A20ΔCNS-EC mice. Conditional deletion of Tnfaip3 in A20ΔCNS-EC mice is achieved through TAM injections. (B) Validation of Tnfaip3 deletion in pMBMECs from A20ΔCNS-EC mice by RT-PCR. Tnfaip3 mRNA levels are presented relative to control (n = 3–4 mice per group). (C) Western blot analysis of A20 protein in cultured primary brain endothelial cells from A20ΔCNS-EC and littermate control mice. Protein expression of A20 is normalized to actin levels and presented relative to the expression in controls (n = 5 mice per group). (D–H) Adoptive transfer-EAE (AT-EAE) disease in A20ΔCNS-EC mice and A20fl/fl littermates. Clinical signs of EAE were monitored daily. Data is pooled from 2 independent experiments with n = 15–17 mice per group. (D) Probability of symptom-free survival is shown as Kaplan Meier curve. (E) Clinical signs of EAE are shown as mean clinical disease scores ± SEM. (F) AUC, (G) maximum clinical scores, and (H) day of onset analyses of clinical course shown in (E). Every circle represents a single mouse. (I–L) AT-EAE disease was induced in A20ΔEC lacking A20 in all endothelial cells driven by the Cdh5-CreERT2 and A20fl/fl littermate controls as described before. Clinical signs of EAE were monitored daily. Data is pooled from 2 independent experiments with n = 11–12 mice per group. (I) Clinical signs of EAE are shown as mean clinical disease scores ± SEM. (J) AUC, (K) maximum clinical scores, and (L) day of onset analyses of clinical course shown in (I). Every circle represents a single mouse. Statistical significance was determined by 2-tailed unpaired Student’s t test (B, C, F–H, and J–L) or Log-rank (Mantel-Cox) test (D). *P<0.05, **P<0.01, ****P<0.0001.