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

BACKGROUND: Cerebral 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. METHODS: Male 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. RESULTS: Blocking 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. CONCLUSIONS: Blocking 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.