Controlling Mast Cell Inflammatory Mediators Secretion with Botulinum Neurotoxins

Mast cells are key mediators of allergic and inflammatory responses that exert their actions through the regulated exocytosis of preformed mediators stored in secretory granules and, the de novo synthesis of cytokines which are sorted into vesicles and secreted via constitutive exocytosis. Exocytosis of mast cell mediators requires the function of SNARE complexes. Botulinum neurotoxins, bacterial proteases that inhibit vesicular fusion by cleaving specific SNARE proteins.
The aim of my research was to evaluate the ability of botulinum neurotoxin serotypes B and D to inhibit mediator secretion from mast cells. I hypothesized that through their cleavage of VAMP3, these proteases could indirectly inhibit antigen-evoked degranulation by interfering with IgE receptors trafficking and directly inhibit the secretion of cytokines by inhibiting vesicular fusion. The light chain protease domains of the toxins BoNT.LC/B and D tagged with eGFP, were transfected into bone marrow derived mast cells (BMMCs). The BMMCs were then sensitised with anti-DNP-IgE, before being stimulated with either the antigen DNP or in a receptor-independent manner. Degranulation was assessed by quantifying secreted β-hexosaminidase and by flow cytometry analysis of plasma membrane levels of CD107a. Proteolysis of VAMP3 in transfected BMMCs was confirmed by western blotting. The results from these experiments showed that expression of eGFP-BoNT.LC/B and D did not significantly inhibit degranulation. Furthermore, flow cytometry analysis of cellular levels of the cytokine TNF-α, as well as ELISA quantification of secreted TNF-α, IL-6 and IL-13 from transfected BoNT.LC/B and D BMMCs showed that these cytokines was also not impaired by the loss of VAMP-3. Taken together the results of my research indicate that the VAMP isoforms affected by these serotypes are not essential for mast cell mediator secretion and that novel engineered proteases with altered SNARE specificity will be required if these toxins are to be used to control inflammation and hypersensitivity.