The Role of ASK1 in Allergen-mediated Mast Cell Signaling and Activation-dependent Inflammatory Responses

Abstract

The prevalence of allergies has been increasing at alarming rates and identifying key targets in allergen-induced mast cell-mediated inflammation is crucial for therapeutic development. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK) that is involved in various cellular responses, including oxidative stress, high calcium concentrations and receptor-mediated inflammation. ASK1 has been known to be a key player in various inflammatory-based pathologies, such as liver, kidney, and cardiovascular disease, and has been investigated in various immune cells such as neutrophils, macrophages, and dendritic cells. However, its role in IgE-FcERI-activated mast cells remains elusive. The purpose of this project is to expand on current knowledge of MAPK signaling and the role of ASK1 in mast cell-mediated allergic inflammation. Bone marrow-derived mast cell and fetal liver-derived mast cells were sensitized with TNP-BSA-specific IgE antibodies and stimulated following treatment with various GS-444217 treatment concentrations. GS-444217 (ASK1-IN) is a potent and ATP-selective ASK1 inhibitor. Following incubation of various inhibitor concentrations, IgE-mediated mast cell degranulation responses were significantly reduced in both BMMC and FLMC models. Other mast cell signaling responses in BMMCs, such as protein/mRNA expression, cytokine/chemokine secretion, receptor expression, and cell viability were also investigated. Through western blotting, our results show that GS-444217 does not alter JNK, p38, ERK, or ASK1 protein levels. Phosphorylation of ASK1 could not be detected, however the presence of ASK1 in mast cells has been identified. qPCR data also shows that there were no alterations in TNF-a-, IL-6-, CCL2-, and CCL3 mRNA expression following ASK1-IN treatment. Interestingly, contrary to mRNA expression levels, 5µM ASK1-IN treatment caused a significant reduction in CCL1, IL-6, and IL-13 secretion. Lastly, two key receptors associated with IgE-mediated mast cell activation – c-kit and FcERI – showed no changes in expression following inhibitor treatment. Cell metabolic activity was also investigated to ensure cell viability and no significant changes occurred compared to controls. Our results suggest that ASK1 may play a role as an upstream regulator in secretory mechanisms in mast cell-directed allergic inflammation and may warrant future consideration as a therapeutic target candidate.