Bacterial extracellular vesicles in microbiota-brain communication and hypertension

Project Summary Disruption of the normal gut microbiota composition, termed gut dysbiosis, is an underlying cause of hypertension (HT). The pathway where dysbiosis leads to HT involves gut inflammation, neuroinflammation in the cardiovascular control centers in brain, and ultimately HT. This pathway from gut microbiota to HT requires communications via the microbiota-gut-brain axis (GBA), a multiple component system for the communication between the gut and the brain. Although a number of mechanisms have been described as messengers for GBA communications, our understanding of the GBA in the context of HT is lacking. Extracellular vesicles (EVs) released from bacteria represent a route for inter-kingdom communication through which the gut microbiota influences host. In this proposal, we will develop the idea that extracellular vesicles (EVs) derived from gut microbiota constitute an important component of the GBA communications involved with the development of HT. We now propose the overall hypothesis that the dysbiotic microbiota releases pro-inflammatory bacterial EVs that induce gut inflammation, neuroinflammation, and HT. In strong support of this hypothesis, we demonstrate that the cargo of bacterial EVs isolated from normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive stroke prone rats (SHRSP) gut is significantly different. Specifically, the pro- inflammatory bacterial components flagellin and lipopolysaccharide (LPS) are significantly elevated in SHRSP EVs. We show that bacterial EVs gain access to blood and brain. Finally, transplantation of bacterial EVs from SHRSP to WKY by oral gavage leads to increased blood pressure (BP). This proposal will interrogate the role of bacterial EVs as mediators of the GBA by examining the effects of EVs at the epithelial interface and in brain. In Aim 1 we will identify the bacterial origin of bacterial EVs involved in the development of HT and examine the mechanistic role of bacterial EV flagellin and LPS. Using a novel dilution scheme to simplify bacterial communities, we will determine the bacteria producing EVs that contribute to elevated BP. In Aim 2 we will elucidate the role of bacterial EVs on gut epithelial inflammation. Using gut enteroids derived from WKY and SHRSP we will determine the direct effects of bacterial EVs on epithelial inflammation and barrier function. In Aim 3 we will define the role of bacterial EVs on the development of neuroinflammation. Using single cell RNA sequencing we will determine the effects of bacterial EVs on microglia and neurons in the paraventricular nucleus. We will also deplete microglia to investigate the role of neuroinflammation in bacterial EV-induced HT. The focus of this proposal represents a novel area of investigation examining the role of bacterial EVs in the GBA and hypertension. These studies represent a translatable foundation for development of novel approaches for the prevention and treatment of HT.