The study of recycling endosomes in innate immune cells

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My research program is focused on the mechanisms of cytokine trafficking in innate immune cells. Trafficking of cytokines to the cell surface involves recycling endosomes (REs), an essential compartment for sorting and transportation of protein cargo to and from the plasma membrane. Recently, REs have been discovered to constitutively traffic the cytokine, tumour necrosis factor-alpha (TNF), from the Golgi to the cell membrane in macrophages. However, the presence and function of REs in trafficking and release of cytokines in granulocytes is poorly understood. Granulocytes are the most abundant circulating innate immune cells in the body, and have the ability to transmigrate into tissues from the blood. Upon tissue infiltration, granulocytes release copious quantities of cytokines, which have potent proinflammatory effects. Thus, granulocytes are powerful secretory cells that function as a double-edged sword in immunity. In addition, granulocytes have robust protein trafficking systems that serve as perfect models for studying the secretion of cytokines.***Objectives*** The long-term objective of this program is to determine pathways of cytokine trafficking in innate immune cells. There is a paucity of literature on cytokine trafficking in innate immune cells, and we have strong evidence for cytokine trafficking via REs in these cells. The specific short-term objectives are to:***1. Characterize the putative REs in granulocytes.***2. Determine the cytokine trafficking pathway via REs and secretory granules for exocytosis.***3. Characterize signaling pathways that regulate release of cytokines from rapidly and slowly recycled compartments of REs.*** We propose to resolve the requirements for the signaling molecules GTPases and SNAREs in the release of protein cargo including cytokines from neutrophils. We will first characterize REs in granulocytes, determine how cytokines are trafficked via GTPases and SNAREs, and identify regulatory molecules required for RE fusion with cell membranes. We will also assess the contribution of recently described rapidly and slowly recycled compartments of REs to cytokine trafficking in innate immune cells. The cytokine of interest is the potent immunomodulatory TNF which is secreted in abundance during stimulation of neutrophils by bacterial lipopolysaccharide (LPS). Using high resolution imaging, we will map the fine distribution of GTPases, SNAREs, granule, and RE-associated proteins during neutrophil secretion of cytokines.*** The findings arising from this proposal will reveal new, undiscovered membrane trafficking compartments in important innate immune cells required to maintain immunity. Understanding the function of REs in innate immune cells will lead to the elucidation of novel trafficking pathways for protein cargo, particularly cytokines, that serve an essential immunomodulatory function.**