Intestinal Microbiota Affect Stroke Outcome by Modulating the Dendritic Cell-regulatory T Cell Axis

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PROJECT SUMMARY Stroke is a devastating disease and leading cause of death and disability in the United States. Ischemic stroke results in massive activation of numerous immune cells that can infiltrate the brain following blood-brain-barrier breakdown. The gut microbiota has previously been identified as a significant factor affecting outcome and severity of ischemic stroke in clinical studies and animal models. However, mechanisms underlying the modulatory role of microbiota on immune cells following stroke remain unclear. Dendritic cells (DCs) act as the bridge between innate and adaptive immunity, with their ability to sample material from the intestinal lumen and shape T-cell responses. Antibiotic-induced alteration of microbiota in mice results in stroke neuroprotection in mice following middle cerebral artery occlusion model of ischemic stroke compared to control mice carrying conventional microbiota, which are similarly treated but carry antibiotic-resistant microbiota resulting in microbiota similar to that of naïve mice. This effect is attributed to the greater capacity of intestinal and mesenteric lymph node dendritic cells of mice carrying “altered” microbiota to induce T-regulatory cells (Tregs) in the small intestine which subsequently suppress destructive pro-inflammatory IL-17+ γδ T cells that traffic to the brain following stroke. Using our in vitro model to simulate intestinal DC-T cell interactions, we show that priming naïve DCs with isolated contents from the small intestine (SIC) of mice carrying “altered” or “conventional” microbiota and subsequent co-culture with CD4 cells similarly induces greater proportions of Tregs following SIC from mice carrying “altered” microbiota compared to SIC from mice with “conventional” microbiota. This proposal seeks to elucidate the mechanism by which altering microbiota may result in changes in pattern-recognition receptors or toll-like receptor ligands that are responsible for a DC-tolerizing phenotype and Treg induction observed with microbiota alteration in mice. Using a variety of in vitro and in vivo approaches, I aim to identify the DC receptors and signaling machinery responsible for sensing these luminal contents and producing a tolerogenic phenotype, determine DC-produced signals/cytokines necessary for intestinal Treg induction, and establish the role of pro- inflammatory IL-6 in stroke neuroprotection vs poor stroke outcome in mice carrying “altered” or “conventional” microbiota. In summary, I seek to understand how intestinal DC receptor ligands that are microbiota-dependent can act as regulators of intestinal immunity and stroke outcome, as well as identify potential therapeutic targets.