Abstract 72: The Innate Immunity Receptor CD36 Contributes to Ischemic Brain Injury by Regulating Post-ischemic Caspase-1 Activity and Interleukin-1b Production in Microglia

Abstract

CD36, an innate immunity receptor enriched in microglia, and IL-1β, a cytokine produced by the inflammasome, play a key role in ischemic brain injury by mediating the inflammatory component of the damage (J Neurosci, 35:4674, 2015; Nat Rev Immunol, 5:629, 2005). However, a link between post-ischemic CD36 activation and IL-1β has not been established. Microglial CD36 could contribute to ischemic brain injury by increasing the post-ischemic production of IL-1β. To test this hypothesis, male wild type (WT) and CD36 –/– mice were subjected to transient middle cerebral artery occlusion (tMCAO), and infarct volume was assessed in cresyl violet-stained brain sections 72 hrs later. At 18 hrs after tMCAO, the expression of IL-1β mRNA in microglia, isolated by cell sorting, increased equally in WT (3.4±0.9) and CD36 –/– mice (3.6±0.8, fold increase; p>0.05; N=3 pools, 3 mice/pool). However, the levels of mature IL-1β protein, measured by cytometric bead arrays in the ischemic hemisphere, were higher in WT mice (104±30 pg/g) than in CD36 –/– mice (59±14 pg/g; p<0.05 from WT; n=5/group). Since caspase-1 regulates active IL-1β levels by cleaving the mature form from its pro-peptide, we next analyzed microglial caspase-1 activity using a flow cytometry-based assay. The percentage of active caspase-1 was greater in WT (4.0±0.2%) than in CD36 –/– microglia (2±0.2 %; p= 0.001; n=5/group). Therefore, the reduction in infarct volume in CD36 –/– mice could be due to reduced caspase-1 and attendant IL1β signaling. Consistent with this hypothesis, pretreatment with recombinant IL-1β receptor antagonist (2 μg into the cerebral ventricles) decreased infarct volume in WT (47±5 vs. 32±5 mm 3 ; p<0.5; n=8-5), but not in CD36 –/– mice (23±6 vs. 21±5 mm 3 ; p> 0.5; n=5). We conclude that CD36 localized to microglia is a key determinant of post-ischemic IL-1β production by regulating caspase-1 activity. Although the CD36 ligand(s) and the molecular mechanisms linking CD36 to caspase-1 activation remain to be established, this study identifies microglial CD36 signaling as a previously unrecognized pathway for inflammasome activation and IL-1β production after cerebral ischemia, and a viable target to mitigate the damaging effects of post-ischemic inflammation.