Comparison of Different Mast Cell Types and Their Responses to IL-33 via High Resolution Mass Spectrometry

Abstract

Mast cells (MCs) are tissue resident granulocytes which are vital members of the innate immune system, required for defence against invading pathogens, immunoregulation, and homeostasis (1). To allow this MCs express a number of innate immune receptors including the IL-33 receptor. Further to their roles in innate immunity, MCs can contribute to humoral responses, a function enabled by the MC’s expression of the high affinity IgE receptor (2–4). MCs express the heterodimeric IL-33 receptor, ST2-IL1-RAcP, in high abundance on their surface (5). IL-33 is able to induce MC synthesis and secretion of pro-inflammatory cytokines and chemokines including IL-6, IL-13, TNF, GM-CSF, CCL3, and CCL4 via activation of MyD88-dependent MAPK signalling (5–7). Dysregulation of IL-33-ST2 MC activation is heavily implicated in a number of conditions including allergic conditions, IBDs, COPD, and more (8–13). Thus the study of MCs and their functional responses to IL-33 is highly important in both progressing understanding of these conditions and the development of therapeutics (14–17). The yield of primary ‘ex vivo’ MCs from tissue is poor, making them unsuitable for wide scale investigation (18). As a result, MC models and culture systems are an invaluable tool in MC research. However the biological and functional variation between models and the impact of such differences on stimuli-induced functional responses is not fully understood.

This study aimed to better characterise three widely used murine MC culture systems, bone marrow-derived, foetal liver-derived, and peritoneal-derived MCs (BMMCs, FLMCs, and PMCs) and their responses to IL-33. High resolution mass spectrometry was used to investigate the basal and IL-33 stimulated FLMC, BMMC, and PMC proteomes, additionally this method was employed to elucidate the roles of major MAPKs, p38 and ERK1/2, downstream of IL-33 stimulation in BMMCs by using p38 inhibitor, VX-745, and MEK1/2 inhibitor, which serves to block ERK1/2 activation, PD184352 (19,20).

Key MC granule proteins, including MC specific proteases Tpsb1, Cma1, Cpa3, and Mcpt4, and protein translation machinery were found to be differentially expressed in the different MC models, with PMCs expressing the highest levels of granule proteins but the lowest levels of anabolic pathway machinery. IL-33 did not upregulate granule protein expression with the exception of serglycin (Srgn) which is critical to granule development, integrity and storage of effector molecules including histamine and serotonin which are vital to MC function.

IL-33-mediated extensive remodelling of the FLMC, BMMC and PMC proteomes including shared and model-specific responses. IL-33 caused an overall trend toward protein upregulation which aligned with observed IL-33-dependent increase in FLMC, BMMC, and XV PMC mass and increase in gene ontology (GO) terms related to protein translation and synthesis. Expression of genes identified in these GO terms was consistently highest in FLMCs. Notably, a high proportion of the IL-33-induced changes to the BMMC proteome were found to be p38 dependent, in addition to the regulation of a select group of proteins being ERK1/2 dependent.

Histamine promotes inflammation as a potent agent of bronchoconstriction and vasodilation, and a chemotactic factor for neutrophils and eosinophils which is highly implicated in the pathogenesis of allergic diseases and MC activation disorders (MCADs), whilst peripheral serotonin plays key roles in regulation of heart rate, GI function and immunomodulation (14,21-23). IL-33-mediated FLMC, BMMC, and PMC increase in expression of the rate limiting enzymes Hdc and Tph1 which are required for the synthesis of histamine and peripheral serotonin respectively. In BMMCs, IL-33 induced Hdc in a p38-dependent manner, while ERK1/2 played a smaller role, whereas IL-33-mediated Tph1 expression was p38-dependent, with a partial role for MK2/3 downstream seen, however this pathway was seen to be ERK1/2-independent. IL-1β is crucial to the inflammatory response via induction of pro-inflammatory cytokine and chemokine synthesis, and induction of Th17 differentiation (24,25). Here, IL-33 mediated IL-1β upregulation across all three cell types. In the BMMCs IL-33 induced IL-1β synthesis and secretion in a p38-MK2/3 dependent pathway.

Additionally, early work which sought to investigate the role of MSK1/2 downstream of IL-33 stimulation of BMMCs established IL-33 induced granzyme C expression in an MSK1/2-CREB dependent manner downstream of p38 and ERK1/2 activation via flow cytometry, immunoblotting, and qPCR. Further investigation using proteomics however found little difference between IL-33 wild type and MSK1/2 KO PMC proteomes.

The outcomes of this research demonstrate important differences between the proteomes of the different MC models used here, in addition to demonstrating that IL-33 induces global changes to the FLMC, BMMC, and PMC proteomes which includes shared and cell-specific responses, a high proportion of which were seen to be p38-dependent in the BMMCs.

Date of Award	2025
Original language	English
Awarding Institution	University of Dundee
Supervisor	Simon Arthur (Supervisor) & Ian Ganley (Supervisor)