Targeting Lipid Metabolism in Myeloid Cells to Improve Ischemic Stroke Recovery

Abstract

Globally, more than 67 million people are living with the effects of ischemic stroke. Importantly, many stroke survivors develop a chronic inflammatory response that may contribute to cognitive impairment, a common and debilitating sequela of stroke that is insufficiently studied and currently untreatable. Therefore, the goal of my research has been to develop a novel therapeutic to attenuate chronic inflammation and prevent post-stroke cognitive decline in a preclinical murine model of experimental ischemic stroke. We hypothesize that, following ischemic stroke, lipids derived from myelin debris and other apoptotic or necrotic cell membranes overwhelm the processing capability of infiltrating monocytes and resident microglia in the brain, leading to the formation of lipid-laden foamy macrophages, generation of cholesterol crystals, secretion of pro-inflammatory cytokines, and production of degradative enzymes. Further, we hypothesize that the resultant chronic inflammatory response, coupled with concurrent cell death, causes secondary neurodegeneration and impairs cognitive and locomotor function. To address these hypotheses, the aims of the studies presented herein were three-fold: (1) to characterize the lipidome of chronic stroke infarcts and determine whether the pathogenic lipids are derived from myelin; (2) to determine whether the ablation of lipid metabolic pathways (CD36, NLRP3, C3) impacts stroke recovery; and (3) to determine whether lipid complexation and macrophage reprogramming within infarcts, via the repeated administration of 2-hydroxypropyl-β-cyclodextrin (HPβCD), attenuates chronic inflammation and improves recovery after experimental stroke. We first demonstrate that lipid metabolism is disrupted in chronic stroke infarcts, which causes the accumulation of uncleared lipid debris and correlates with chronic inflammation. Specifically, we illustrate that, coincident to the infiltration of peripheral immune cells, stroke infarcts amass lipids derived from myelin membranes, including sulfatides, sphingomyelins, fatty acids, and cholesterol esters. To our knowledge, these substantial alterations in lipid homeostasis have not been previously recognized or investigated in the context of ischemic stroke. We next investigate the roles of lipid metabolic pathways in ischemic stroke recovery. Specifically, we demonstrate that genetic ablation of Cd36 reduces the accumulation of B lymphocytes and improves locomotor function in the weeks after stroke. We also illustrate that the genetic ablation of Nlrp3 or C3 has negligible effects on chronic inflammation at 7 weeks after stroke; although, mice deficient in Nlrp3 demonstrate marginal improvements in tests of spontaneous locomotor function. Together, these results indicate that Cd36 and, to a lesser extent, Nlrp3 have integral roles in chronic inflammation and locomotor function after ischemic stroke. We then provide a proof of principle that solubilizing and entrapping lipophilic substances using 2-hydroxypropyl-β-cyclodextrin (HPβCD) could be an effective strategy for treating chronic inflammation after ischemic stroke or other central nervous system (CNS) injuries. HPβCD is a U.S. Food and Drug Administration (FDA)-approved cyclic oligosaccharide that promotes liver X receptor (LXR)-mediated transcriptional reprogramming in macrophages and incites anti-inflammatory mechanisms. We illustrate that the repeated administration of HPβCD curtails the chronic inflammatory response to ischemic stroke by reducing lipid accumulation within stroke infarcts. In these preclinical trials, we subcutaneously injected young adult and aged male mice with vehicle or HPβCD three times per week, with treatment beginning one week after ischemic stroke. We demonstrate that chronic stroke infarct and peri-infarct regions in HPβCD-treated mice were characterized by an upregulation of genes involved in lipid metabolism and a downregulation of genes involved in innate and adaptive immunity, reactive astrogliosis, and chemotaxis. Correspondingly, HPβCD reduced the accumulation of lipid droplets, T lymphocytes, B lymphocytes, and plasma cells in stroke infarcts. Repeated administration of HPβCD also preserved NeuN immunoreactivity in the striatum and thalamus and c-Fos immunoreactivity in hippocampal regions. Additionally, HPβCD improved recovery through the protection of hippocampal-dependent spatial working memory and reduction of impulsivity. These results indicate that systemic HPβCD treatment attenuates chronic inflammation and secondary neurodegeneration and prevents post-stroke cognitive decline in a murine model of ischemic stroke. We propose that repurposing HPβCD for the prevention of post-stroke dementia could improve recovery and increase long-term quality of life in stroke sufferers.