Project Summary/Abstract Stroke is a prevalent and devastating disease with limited therapeutic options. Inflammation and immune cells are major components in the pathophysiology of ischemic stroke and contribute to acute and delayed tissue injury. However, our incomplete understanding of the factors regulating the immune responses triggered by cerebral ischemia remains a significant obstacle to the development of effective therapeutic interventions based on modulating post-ischemic inflammation. Besides activation of brain resident immune cells, ischemic stroke is characterized by the recruitment of peripheral innate and adaptive immune cells that participate in the inflammatory response and contribute to the damage. Commensal microbiota that populate epithelial surfaces play a defining role in shaping the immune system, the development, maintenance and function of which depends critically on the relative abundance and composition of the different microbial species. In particular, intestinal commensal bacteria, the most abundant symbiotic compartment in the body, have emerged as a potent regulator of the immune system. Remarkably, the gut microbiota influence the development and proliferation of immune cells (γδT cells and Treg), which have been strongly implicated in ischemic brain injury. These observations raise the possibility that the microbiota have the potential of modulating the outcome of cerebral ischemia. The long-term goal of this research program is to elucidate the role of intestinal microbiota in stroke pathobiology and develop the experimental framework for new preventative and therapeutic approaches for ischemic stroke. In the present application, we will test the hypothesis that commensal intestinal microbiota are a critical determinant of stroke outcome by modulating the immune system and inflammatory response to cerebral ischemia. This hypothesis, supported by relevant preliminary results, will be tested by determining: (a) the impact of antibiotic-induced modification of the gut flora (intestinal dysbiosis) on stroke outcome (Aim 1), (b) the changes in the peripheral and brain immune system evoked by such dysbiosis and their role in ischemic brain injury (Aim 2), and (c) the cellular and molecular events leading to this immunomodulation (Aim 3). These goals will be achieved using a mouse model of transient focal cerebral ischemia with assessment of histological and neurological outcome together with a novel model of microbial dysbiosis. This proposal may open the way to new avenues for stroke prevention and therapy based on modulation of the immune system by the gut microbiota, using approaches already in the clinical arena.