Determining the Roles of Q-SNAREs in Mast Cell Tumor Necrosis Factor Exocytosis

As evidence accumulates on the contributions of mast cell function to the development of allergic disorders, cardiovascular and neurological pathologies, and cancers, there is a growing recognition of the importance of mast cell-derived TNF to mast cell-mediated pathophysiology. However, our understanding of the mechanisms that regulate TNF exocytosis in mast cells remains woefully inadequate, and expanding this knowledge would undoubtedly aid in the development of therapeutics to specifically target mast cell TNF secretion. To explore the roles to SNAREs in TNF release in mast cells, I used Stx2 KO BMMCs as well as Stx3 and Stx11 KD RBL-2H3 cells in secretion assays to determine the consequences of depleting these isoforms on TNF secretion. The resulting data indicates that while Stx2 KO does not impact either TNF secretory pathway, Stx3 KD partially inhibited pre-formed TNF secretion, and Stx11 KD substantially increased pre-formed TNF secretion and TNF production in activated mast cells. I also designed and utilized a strategy to engineer Stx2, -4, -6, and –11 KO RBL-2H3 cells using Cytosine Base Editing, and successfully generated heterozygous KO cell lines of all four genes. The results of these investigations have revealed that Stx2 may be dispensable for mast cell exocytosis in general potentially significant roles for Stx3 and Stx11 in mast cell TNF exocytosis which must be investigated further.