Introduction: Stroke triggers a robust inflammatory response, allowing leukocytes to cross the blood brain barrier. T cells infiltrate in an antigen-independent manner early after injury, leading to additional neuronal injury. The pro-inflammatory cytokine, interferon-gamma (IFNγ), upregulates programed death ligand-1 (PD-L1) expression. Engagement of PD-L1 and programed death-1 (PD-1) “calls off” the cytolytic activity of infiltrated T cells. The immunosuppressive activity of PD-L1 is modulated by ubiquitination and glycosylation. Glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces degradation of PD-L1. To date, the role of PD-L1 in endothelial cells (ECs) remains elusive.
Hypothesis: PD-L1 in EC reduces neuroinflammation via protection of microvascular integrity.
Methods: Human brain ECs, HBEC-5i, were subjected to IFNγ treatment in normal or oxygen-glucose deprivation (OGD) conditions. Western blot, Fluorescence-activated cell sorting analysis, PD-1 binding assays and an in vitro T cell killing assay were performed to investigate EC viability and PD-L1 function. Male mice (12 weeks) were subjected to a 60-minute middle cerebral artery occlusion (MCAO) and given either vehicle or GSK3β inhibitor VIII (4 mg/kg). We examined PD-L1 expression, blood vessel integrity and T cell activation in brain tissue using anti-PD-L1, p-GSK3β, CD31, and Granzyme B antibodies for immunohistochemistry (IHC).
Results: IFNγ strongly induced PD-L1 expression in ECs. However, when ECs were exposed to OGD, IFNγ-mediated PD-L1 upregulation was compromised (from 48.9% to 31.4%). PD-L1 binding affinity of EC was also reduced after OGD (from 33.1% to 1.3%). Consistently, human (n=22) and mice (n=6) brain IHC staining found lower levels of PD-L1 and p-GSK3β in ECs and higher expression of Granzyme B in stroke samples compared to controls (p<0.05). MCAO mice treated with GSK3β inhibitor VIII showed higher PD-L1 expression and smaller infarcts.
Conclusion: This study reveals a protective role of PD-L1 in ECs during early brain injury. Activation of GSK3β by stroke destabilized endothelial PD-L1. Thus, stabilizing PD-L1 or preventing downregulation of PD-L1 may maintain microvascular integrity and reduce neuroinflammation after stroke.