Poly-ICLC-induced Blood Brain Barrier protection is independent of Toll-like Receptor 3 Signaling

Introduction: Preconditioning with a TLR3 ligand, polyinosinic polycytidylic acid (poly-ICLC), a synthetic dsRNA, protects against middle cerebral artery occlusion (MCAO). Using an in vitro model of the blood brain barrier (BBB) we found that Poly-ICLC stabilizes the BBB during ischemia through a mechanism involving IFNβ production from glial cells. We assessed the hypothesis that Poly-ICLC stabilization of the BBB and subsequent reduction of ischemic injury requires the presence of TLR3 on both endothelial and glial cells. Methods: BBB was modeled using a transwell co-culture system with primary brain microvascular endothelial cells (BMECs) in the upper chamber and primary mixed glial cells in the lower chamber. Poly-ICLC was administered 24h prior to 5h of oxygen glucose deprivation (OGD), which models stroke in vitro. Transendothelial electric resistance (TEER) and endothelial permeability were evaluated 24h after OGD and IFNβ secretion was assessed by ELISA. For in vivo studies, WT and TLR3 -/- mice were treated with poly-ICLC or vehicle 72h prior to MCAO and infarct volume was determined after 24h of reperfusion. Results: Poly-ICLC administered exclusively to the upper chamber containing BMECs, results in secretion of IFNβ from glial cells in the lower chamber and significant protection against OGD. To determine whether BMEC or glial expression of TLR3 was required for protection we generated BBB models with TLR3 deficient (TLR3-/-) BMECs in co-culture with WT mixed glial cells as well as WT BMECs in co-culture with TLR3 -/- mixed glial cells. Poly-ICLC preconditioning induced protection in the presence or absence of TLR3 suggesting that TLR3 signaling is not required for protection. This finding was supported further by in vivo data showing that poly-ICLC preconditioning protects TLR3 -/- mice against ischemic injury. Conclusions: Our results showed that poly-ICLC interaction with endothelial cells induces IFNβ production in glial cells to confer protection. This process does not require TLR3 expression on endothelial or glial cells suggesting that other dsRNA receptors (MDA5 and RIGI) may be important in poly-ICLC-induced protection.