Mechanisms for generating cargo specificity in inflammasome-mediated exosome secretion

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PROJECT SUMMARY Exosomes mediate intercellular communication and their roles in a growing range of human disease is becoming increasingly evident. In preliminary studies, our lab has discovered that both virus- and LPS-induced exosome production is related to inflammasome activation resulting in cleavage of the Rab7 effector protein, RILP. The loss of RILP from the Rab7 complex reroutes intracellular trafficking toward the cell surface. This results in enhanced exosome secretion, due in part to a redistribution of multivesicular bodies throughout the cellular periphery. Formation of the cleaved form of RILP (cRILP) can also induce unique exosomal cargo loading, leading to the selective enrichment of specific pro-inflammatory miRNAs. Furthermore, inhibiting RILP cleavage can abrogate the proinflammatory actions generated by cRILP while specifically facilitating an anti-inflammatory response. This proposal will examine the hypothesis that enhanced exosome production that results from inflammatory disease is a consequence of inflammasome-triggered casepase-1 activation and subsequent cleavage of the trafficking adapter protein RILP. Cleaved RILP subsequently reprograms secretory events promoting stimuli-specific exosome formation and release. We will investigate this hypothesis with the following specific aims: Aim 1 will identify the mechanism by which cRILP regulates selective miRNA cargo loading. We hypothesize that the various forms of RILP may interact with and/or complex with specific subsets of RNA binding proteins that regulate miRNA cargo loading. Aim 2 will define the relationship between cRILP, RNABPs, and components of the ESCRT pathway. This aim will explore the hypothesis that cRILP hijacks the machinery required for exosome biogenesis leading to altered multivesicular body trafficking and subsequent exosome release. Aim 3 will examine the functional consequences of RILP manipulation on exosome secretion. This aim will identify how RILP manipulation, in both human monocytes and mouse models, can affect the outcome of various disease states to ultimately lead to the artificial modulation of exosome production and thus, the immune response. The ultimate goal of this research is to provide a detailed understanding of how exosomes are produced/secreted in response to pathogenic stimuli but also to describe the mechanisms that inflammatory stimuli use to specifically induce selective exosome cargo secretion. This knowledge can then be used to provide the tools to manipulate exosomes for therapeutic benefits.