Stroke is the fifth leading cause of mortality in the United States and the leading cause of permanent disability in older adults. Thrombolytic therapy that causes reperfusion is the only FDA approved therapy for patients that have suffered an occlusive stroke. However, this treatment is only used in 5% of reported cases. Myeloid cell modification leading to modulation of the inflammatory response is a potential treatment in stroke, but to date has not successfully been translated into a viable treatment. Previous research has shown that myeloid cell modification to an anti-inflammatory phenotype is protective in cerebral ischemia/reperfusion (I/R). Immune cell metabolism has recently been shown to be a major factor involved in immune cell modulation. Itaconate, the metabolic intermediate that is produced by the enzyme aconitate decarboxylase (ACOD1) from the TCA cycle intermediate aconitate, has been shown to be protective in a cardiac model of ischemia/reperfusion by using dimethyl itaconate (DMI) a cell-permeable form. DMI has shown to exert anti- inflammatory effects on macrophages, and to decrease reactive oxygen species (ROS) formation in vitro. However, the role of the endogenous itaconate production by ACOD1 has not been addressed. Our preliminary data shows that germline ACOD1 deletion leads to increased ischemia/reperfusion induced infarct size following transient MCA occlusion (tMCAo). We confirm that DMI and 4-octyl itaconate (4-OI) decrease inflammatory genes in vitro. We propose to test the role of ACOD1 in stroke and determine what cell types are important by using ACOD1 floxed animals and myeloid, microglial and astrocyte specific knockout. We will also test whether DMI or 4-OI can be protective in stroke. We will quantify infarct size in wild type or ACOD1 cell- specific knockout mice with and without treatment of DMI or 4-OI in tMCAo to determine effects in this model. The production of itaconate has been shown to contribute to alteration of metabolism with decreases in oxidative phosphorylation, the buildup of intracellular succinate and increase in glycolysis. We propose to use metabolomics analysis to measure intracellular metabolite intermediates such as succinate, metabolic flux and reactive oxygen species. Upon completion of this fellowship project, these studies will allow for better understanding of the mechanism behind itaconates protective effects on ischemia/reperfusion injury and give better insight into possible treatments for patients that suffer from ischemic stroke.
Our long-term goal is to understand the role that macrophages and other inflammatory cells have in the disease process after stroke. Understanding the mechanisms of therapeutic benefit by itaconate and the effects that it has on specific cell types will allow us to identify the important cell types involved in the disease process. It will also allow us to identify new ways to modulate the inflammatory response to develop new therapeutic strategies for the treatment of stroke.
