Receptor-Interacting Protein Kinase 2 (RIPK2) Profoundly Contributes to Post-Stroke Neuroinflammation and Behavioral Deficits with Microglia as Unique Perpetrators
Description
Background: Receptor-interacting protein kinase 2 (RIPK2) is a serine/threonine kinase whose activity propagates inflammatory signaling through its association with pattern recognition receptors (PRRs) and subsequent TAK-1, NF-κB, and MAPK pathway activation. The hypoxic and low energy conditions elicited by an ischemic event in the brain lead to rapid cell death of neurons whose blood supply is interrupted. Dead and dying cells release a host of damage-associated molecular patterns (DAMPs) that activate PRRs and initiate an inflammatory immune response. We hypothesize that RIPK2 plays a damaging role in the progression of stroke injury by enhancing the neuroinflammatory response to stroke and that global genetic deletion or microglia-specific conditional deletion of Ripk2 will be protective following ischemic stroke. Methods: Adult (3-6 months) male mice were subjected to 45min of transient middle cerebral artery occlusion (tMCAO) followed by 24h, 48h, or 28 days of reperfusion. Aged male and female mice (18-24mo) were subjected to permanent middle cerebral artery occlusion and sacrificed 48h later. Infarct volumes were calculated using TTC staining (24-48h) or Cresyl violet staining (28d). Sensorimotor tests (weight grip, vertical grid, and open field) were performed at indicated timepoints. Blood-brain barrier (BBB) damage, tight junction proteins, matrix metalloproteinase-9 (MMP-9), and neuroinflammatory markers were assessed via Western blotting, ELISA, immunohistochemistry, and RT-qPCR. Differential gene expression profiles were generated through bulk RNA sequencing and nanoString®. Results: Global genetic deletion of Ripk2 resulted in decreased infarct sizes and reduced markers of neuroinflammation 24h after stroke compared to wild-type controls. Ripk2 global deletion also improved both acute and long-term behavioral outcomes with powerful effects on reducing infarct volume and mortality at 28d post-stroke. Conditional deletion of microglial Ripk2 (KO) partially recapitulated our results in global Ripk2 deficient mice, showing reductive effects on infarct volume and improved behavioral outcomes within 48h of injury. Finally, we identified a differential gene expression profile in microglia derived from global Ripk2 knockout mice, as well as a differential genetic profile in the ipsilateral cortex of μKO animals during acute stroke injury. Conclusion: These results reveal a hitherto unknown role for RIPK2 in the pathogenesis of ischemic stroke injury, with microglia playing a distinct role. This study identifies RIPK2 as a potent propagator of neuroinflammatory signaling, highlighting its potential as a therapeutic target for post-stroke intervention.