Novel TNF Secretion Assays - PROJECT SUMMARY Tumor necrosis factor (TNF or TNFa) is produced and secreted by a variety of eukaryotic cells [e.g., macrophages, T cells, mast cells (MCs), epithelial cells, etc.] to regulate a broad spectrum of biological processes. Excessive TNF, on the other hand, gives rise to allergic inflammation, septic shock, autoimmune diseases and chronic pain. Synthesized as a type II membrane protein, the full-length, membrane-bound TNF (mTNF) utilizes conventional secretory carriers to reach the cell surface, where the soluble form (sTNF) is released into the extracellular space upon Adam17-mediated proteolytic cleavage. It is widely accepted that the trimeric sTNF mediates local/long distance signaling whereas any mTNF trimers remaining at the cell surface are responsible for contact-dependent signaling. However, evidence from our lab and others’ suggests that mTNF can be delivered to MVBs (multivesicular bodies) and released in association with exosomes (nanovesicles originated from within MVBs) when MVBs fuse with the plasma membrane. This implies that mTNF is also capable of local/long distance signaling. Notably, sTNF and mTNF have distinct preferences for different TNF receptors and can trigger distinct pathophysiological responses. For example, while mTNF seems subservient to sTNF in autoimmune inflammation, it is mTNF, not sTNF, that acts as the major culprit in colitis or lung inflammation in animal models. Therefore, it has become essential to dissect the two modes of TNF release to develop more specific and effective interventions to expand and evolve the current collection of TNF-modulating therapeutics. The objective of this proposal is to build a set of sensitive assays to distinguish sTNF release from mTNF release, and to help researchers delineate the regulatory and/or catalytic mechanisms underlying TNF secretion. By tagging a fluorescent protein at the N-terminus (cytoplasmic side) of TNF in RBL-2H3 MCs, we have generated preliminary data that exosomal mTNF release could be assessed via the fluorescent signal in the supernatant of culture cells. Meanwhile, there is evidence that a dominant negative TNF variant (DN-TNF) can intercalate into sTNF trimers but not mTNF trimers. Based on these observations, we hypothesize that a set of novel assays can be developed/established to differentiate and dissect the two modes of TNF secretion. To test the hypothesis, we will pursue two specific aims. Aim1 will establish a fluorescence-based assay to exclusively measure mTNF release. Aim2 will develop a FRET-based assay to exclusively measure sTNF release. We expect to use these assays to demonstrate that mTNF release is conserved in TNF producing cells and that the two modes of TNF release can be differentially regulated by different types of pathophysiological stress.
