Brain endothelium and innate immune responses after stroke

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ABSTRACT Endothelial cells reside at the interface of the blood-brain barrier, making them ideally situated to act as a gatekeeper of the systemic immune response to acute brain injury. Recent work in our laboratory has identified a circulating immune regulator, soluble ST2 (sST2), which we propose as a key signal that bridges brain injury and the inflammatory response. We have shown that sST2 is expressed by brain endothelial cells and elevated in both brain tissue and systemic circulation after experimental stroke in rats. Our pilot rat studies demonstrate that sST2 expression peaks at 3 days after stroke during the acute pro-inflammatory phase, and rapidly subsides by day 7 during the transition to the reparative phase of the immune response. In human patients, sST2 plasma level in the first 3 days after stroke is associated with cerebral edema, inflammatory peripheral monocytes, and long-term neurologic outcome after acute neurovascular injury. We hypothesize that the sST2 pathway exemplifies the principle that following acute injury, the brain endothelium secretes signals both locally and into circulation that orchestrate a biphasic innate immune response. We predict that excessive activation of local microglia and blood-derived macrophages due to acutely increased sST2 level augment acute inflammation, increase damage to the blood-brain barrier, and enhance edema formation. We further predict that the rapid decline in sST2 level by day 7 facilitates the transition to reparative inflammation. However, our understanding of the signaling that regulates communication between the ischemic brain and acute myeloid inflammatory cells is incomplete. In Aim 1, we will validate the brain sST2 source by using fluorescence activated cell sorting of brain cells and in situ hybridization. We will functionally characterize the activation profile of tissue resident microglia/macrophages and peripheral myeloid cells through flow cytometry, qPCR array, and cytokine analysis. We will directly perturb the activation state by ex vivo exposure to sST2 or its neuroprotective ligand, IL-33. We will also examine how these findings vary in the setting of aged animals, which has an important impact on the inflammatory response. In Aim 2, we will directly test our hypothesis that sST2 plays a causal role by studying an sST2 knockout rat that we generated via CRISPR-Cas9. We will complement these studies by knocking down sST2 level in vivo using AAV9-shRNA delivered to the brain. In each model system, we will assess the effect of reduced or absent sST2 on brain-resident and circulating innate immune cells, anticipating an attenuation of the acute pro-inflammatory response and an augmentation of the reparative response. We will further examine how reducing sST2 level alters brain edema and brain water content. Taken together, this study provides a critical opportunity to answer fundamental questions about the signaling role of endothelium in the pathophysiology of brain edema and determine the role of sST2.