Characterizing the role of tafazzin in allergically activated mast cells
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Introduction & Aim: Allergic inflammatory diseases are a constantly growing health concern in westernized societies. Mast cells, the driving force behind many allergic diseases, modulate various metabolic pathways to carry out their various functions following IgE-FceRI-mediated activation. Tafazzin is a cardiolipin transacylase that works to remodel cardiolipin into its mature form of tetralinoleoyl-cardiolipin. Mature cardiolipin is important for efficient energy production through oxidative phosphorylation. The aim of this project is to study the effects of a reduction in tafazzin protein content on IgE-mediated mast cell activation. Methods: Fetal liver-derived mast cells (FLMCs) were treated with 1 μg/mL doxycycline for 5 days to initiate the tafazzin knockdown. Western blotting was used to confirm a reduction in tafazzin protein content. Flow cytometry was used to ensure that the FLMCs expressed both c-kit and FceRI receptors, and that receptor expression was not impacted by the doxycycline treatment. Oxygen consumption rate was measured using a Clark-type electrode. b-hexosaminidase release assays were utilized to assess degranulation during the early phase of the allergic reaction. ELISA assays were used to examine secretion of de novo synthesized inflammatory mediators, while qPCR assays were used to examine gene transcription of these mediators. Results: The doxycycline inducible tafazzin knockdown resulted in a 99.5% reduction in tafazzin protein content. This reduction was accompanied by a 25% reduction in oxygen consumption and a 31.4% reduction in degranulation. Secretion of CCL1, CCL2, and TNF was significantly reduced, CCL9 secretion levels showed a decrease that approached significance. Secretion of CCL3 and IL-6 was not impacted. Gene expression was not impacted for any of the inflammatory mediators measured. Conclusion & Significance: These results suggest that tafazzin may play a role in mast cell oxidative phosphorylation. Functionally, the results suggest that tafazzin plays a role in degranulation and mediator secretion. However, since gene expression was not impacted it is thought that the tafazzin reduction causes these decreases due to an impairment of the exocytosis mechanism. This work adds to the growing field of immunometabolism and improves our understanding of how mast cells modulate metabolic pathways during an allergic inflammatory event.