The study of recycling endosomes in innate immune cells

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My research program is focused on studying the role of recycling endosomes (REs) in immune cells. Recycling endosomes (REs) are specialized secretory compartments that recycle cell surface receptors through the endocytic and exocytotic pathways. These secretory organelles have been well characterized in numerous cell types. Recently, REs were found to have multiple roles in protein trafficking beyond their originally characterized function in trafficking cell surface receptors. REs have been discovered to constitutively traffic the cytokine, tumour necrosis factor-alpha (TNF-alpha), from the Golgi to the cell membrane in macrophages, a type of innate immune cell. However, the presence of REs and their role in trafficking and release of cytokines in immune cells in general is poorly studied. Many immune cells, including neutrophils and eosinophils (granulocytes), are complex and highly granulated secretory cells capable of releasing a wide array of cytokines. Granulocytes are the most abundant circulating innate immune cell in the body, and are powerful secretory cells that function as a double-edged sword in immunity. These specialized secretory cells have the ability to transmigrate into tissues and release copious quantities of cytokines. Thus, immune cells have robust protein trafficking systems and serve as perfect models for studying RE function and exocytosis of cytokines. Objectives The long-term objective of this study is to determine the presence and function of REs, a novel secretory compartment, in cytokine trafficking in innate immune cells. There is a paucity of literature on RE function in innate immune cells, and yet there is strong evidence for RE existence and function in these cells. The specific short-term objectives are to: 1. Characterize TNF-alpha trafficking in REs in innate immune cells using tools and reagents for their detection. 2. Determine additional cytokines trafficked via REs for secretion in innate immune cells. 3. Characterize signaling pathways that regulate release of cytokines from REs in innate immune cells. Specifically, we will assess the contribution of the recently described rapidly and slowly recycled compartments of REs to cytokine trafficking in neutrophils, macrophages, and eosinophils. Rapid and slow recycling REs can be detected by distinct patterns of expression of specific Rab guanosine triphosphatases (GTPases). The cytokines of interest are numerous and include the potent immunomodulatory TNF-alpha, the neutrophil chemokine interleukin-8, and the recently characterized interleukin-33, all of which are secreted by innate immune cells. We will also determine whether R- and Q-SNAREs are required, and identify the specific SNAREs required for RE fusion with the cell membrane. We propose to resolve the requirements for various Rabs and SNAREs in the release of protein cargo including cytokines from neutrophils. Using high resolution imaging and ultrastructural studies, we will map the fine distribution of Rabs, SNAREs, granule and RE-associated proteins during neutrophil RE exocytosis. The findings arising from this proposal will reveal a new, undiscovered membrane trafficking compartment 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.