Unconventional TNF release from mast cells

The National Institute of General Medical Sciences awarded The University of Southern Mississippi $562,443 on August 3, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate the origin, secretion, and function of membrane-bound tumor necrosis factor (mTNF) bearing extracellular vesicles released from mast cells.

The research dissects unconventional TNF release pathways in mast cells, focusing on how mTNF localizes to multivesicular body-like granules and associates with extracellular vesicles during secretion. The work challenges the conventional understanding that TNF is released only as a soluble proteolytic product, with significant implications for understanding inflammatory and immune disorders where mast cell-derived TNF plays a pathogenic role. The investigator's long-term goal is to identify mast cell exocytosis signaling pathways that could serve as therapeutic targets for mast cell diseases.

Work is performed at Hattiesburg, Mississippi, with a period of performance from August 3, 2026, through July 31, 2029. The award is classified as a Project Grant, an assistance type typical of NIGMS's portfolio of investigator-initiated basic research supporting early-stage and at-risk investigators.

Abstract

Mast cell (MC)-derived TNF (tumor necrosis factor) plays vital roles in innate and adaptive immunity, but when in excess, causes various inflammatory or immune disorders. Unlike other TNF producing cells, MCS pre-store TNF in cytoplasmic granules, which can undergo rapid exocytosis upon MC activation. MC activation also promotes TNF synthesis, and newly synthesized TNF is secreted constitutively through either recycling endosomes or golgi-derived vesicles. Both regulated and constitutive TNF secretion/exocytosis require specific interactions between snares (rooted either in vesicles/granules or the plasma membrane) and their cognate partners such as MUNC13S and MUNC18S. However, due in part to the redundant pathways, the precise molecular mechanism underlying TNF release in MC is still elusive. Very recently, using an original secretion assay to study the acute discharge of prestored TNF, we revealed that a full-length, membrane-bound form of TNF (mtnf), localized to MVB (multivesicular body, a late endosome)-like granules, is secreted in association with extracellular vesicles (EVS), challenging the common belief that TNF is only releasable as a soluble form (stnf; result of tace-mediated proteolytic processing). This has significant pathophysiological implications because mtnf and stnf have been shown to differ in biochemical and signaling properties. Our long-term goal is to delineate the signaling pathways specific to MC exocytosis that could be targets for treatment of MC diseases. The overall objective of this proposal is to dissect the origin, secretion and function of the newly discovered mtnf- bearing EVS. Based on our localization and functional studies of MUNC13 in RBL-2H3 cells (a tumor analog of mucosal MCS) and preliminary finding that (MC-derived) mtnf-bearing EVS trigger pro-inflammatory responses in macrophages, we hypothesize that unconventional secretion of mtnf from MC is mediated by MUNC13D to promote local and long-distance signaling. To test this hypothesis, we (in collaboration with DR. Soman abraham at duke university) will exploit a multifaceted strategy to pursue the following three specific aims. Aim 1 will uncover the origin of mtnf-bearing EVS. Aim 2 will differentiate the roles MUNC13 homologs in TNF exocytosis. Aim 3 will assess the signaling properties of mtnf. Our characterization of the unconventional mtnf secretion from MCS will reveal the molecular regulation that distinguishes mtnf release from stnf release and shed light on new strategies to combat TNF-linked diseases that are still difficult to treat.