Inhibition of JNK Signaling During Mast Cell Differentiation Promotes Altered Cytokine Secretion and Degranulation Profiles

Abstract

Mast cells are large granulated leukocytes active in both innate and acquired immunity. These cells differentiate from hematopoietic stem cells, found in the medullary cavity of long trabecular bones. Increased inflammatory activity and numbers of mast cells contribute to allergic disease, which is of increasing concern worldwide. C‐jun N‐terminal kinase (JNK) is a major signaling node in the map kinase pathway that has been shown to contribute to IL‐3 signaling, known to promote murine mast cell differentiation. The aim of this research is to determine the role of JNK signaling in mast cell differentiation. Primary bone marrow‐derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of a JNK inhibitor (JNK‐IN‐8). Following removal of the inhibitor for 5 days, phenotypic and functional characteristics of BMMCs were probed using flow cytometry, ELISA, and β‐hexominidase release assays. Flow cytometric analysis of surface expression of the mast cell marker c‐kit, and levels of IgE bound to FcɛRI, a measure of mast cell sensitization, were unaffected by impaired JNK signaling compared to cells grown in the absence of inhibitor over mast cell differentiation, suggesting a normal mast cell phenotype with the potential to be activated via the allergen‐bound IgE‐dependent pathway. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL‐6 (p<0.05) and IL‐13 (p<0.05) and a non‐significant impairment in TNF and CCL1 secretion at 24 hrs post‐activation compared to cells grown in the absence of inhibitor. Degranulation was also significantly decreased (by 25%; p<0.05) in BMMCs grown under JNK inhibition. To probe an epigenetic mechanistic role for histone acetylation changes in JNK‐impaired differentiation, expression of HDAC and HAT enzymes were considered by qPCR and western blotting, but were not modulated by JNK. These results suggest that JNK plays an important role in the development of a fully functional mast cell in the context of IgE‐mediated allergic inflammation and may warrant consideration as a strategy to regulate mast cell development in associated pathologies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario and Brock University.