Podoplanin neutralization reduces thrombo-inflammation in experimental ischemic stroke by inhibiting interferon/caspase-1/GSDMD in microglia.

Abstract

BackgroundCerebral ischemia/reperfusion (I/R) injury involves the interaction between thrombosis and inflammatory pathways. The aim of this study was to explore the therapeutic effect of podoplanin neutralizing antibody (α-PDPN, clone 8.1.1) on I/R-induced thrombo-inflammation in a mouse model of ischemic stroke.MethodsMale C57BL/6 mice (weight: 22–25 g, aged 6–8 weeks, n=114) were subjected to transient middle cerebral artery occlusion (MCAO) and administered intracerebroventricular injection of α-PDPN (29 µg). Stroke outcomes and microvascular thromboses were examined by immunohistochemistry (IHC) and western blot analysis. In vitro, microglia BV2 cells were pre-treated with α-PDPN and then subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) insult. The microglia culture medium (MCM) was co-cultured with vascular endothelial b.End3 cells. The MCM-induced bEnd.3 cells dysfunction were examined by western blot assays and IHC.ResultsBlocking PDPN decreased the infarct size and ameliorated neurological deficit after MCAO without enhancing the risk of intracerebral hemorrhage. In addition, α-PDPN treatment significantly alleviated thrombus formation in the cerebral microvasculature. Furthermore, treatment with α-PDPN attenuated I/R-induced caspase-1 and gasdermin D expression in vivo and in vitro. The MCM containing α-PDPN reduced the expressions of von Willebrand factor and intercellular cell adhesion molecule-1 in bEnd.3 cells. Moreover, RNA sequencing analysis showed that α-PDPN decreased interferon signaling pathways in BV2 cells.ConclusionsBlocking PDPN can alleviate thrombo-inflammation in acute ischemic stroke by inhibiting caspase-1 expression in microglia, and indirectly reduce endothelial cell dysfunction. These data indicated the beneficial effects of blocking podoplanin during stroke in mice.