Mast cell mediators: their subcellular localization, bacterial influence on their release and expression and their potential value as markers for predicting the severity of allergic reactions

Abstract

Allergic reactions involve the explosive release of inflammatory mediators from mast cells including the proteases tryptase, carboxypeptidase A3 (CPA3) and chymase; and there have been suggestions that dipeptidyl peptidase I (DPPI) could be secreted as well. Our aim has been to investigate the levels of mast cell proteases and a panel of proinflammatory cytokines in serum during allergic reactions provoked by drugs or food to identify and replicate distinct clinico-immunological endotypes of allergic reactions using topological data analysis. In addition, we have investigated factors that can modulate the release and expression of mast cell mediators and their subcellular localization. Patients who had suffered drug- or food-induced allergic reactions were recruited from two separate centers (Southampton, UK and Doha, Qatar). Healthy individuals who had no history of allergic diseases were recruited to serve as controls. Detailed clinical assessment and measurements of mast cell proteases and pro-inflammatory cytokines were performed. The clinical and biochemical parameters were analyzed using topological data analysis. In addition, cells of the LAD2 mast cell line were employed to investigate the effect of bacterial infection on the release and expression of mast cell mediators. This cell line was also employed to investigate the subcellular localization of mast cell proteases through the application of immunocytochemical analysis. There were higher levels of CPA3 in baseline serum samples from patients with more severe historical allergic reactions than in those from healthy individuals (p< 0.0001). CPA3 levels of ≥ 6.5 ng/ml were associated with severe allergic reactions in patients with drug allergies (AUC: 0.61, 95% CI: 0.51-0.71, p= 0.048), and levels of ≥ 3 ng/ml in those with food allergies (AUC: 0.74, 95% CI: 0.62-0.86, p= 0.002). Higher serum levels of CPA3 were seen also in patients with one or more concomitant atopic conditions (including atopic dermatitis, hay fever and asthma) compared to those in healthy subjects (p< 0.0001). A strong association was found between the severity of allergic reactions and concomitant atopic illnesses (p< 0.0001). Topological data analysis allowed identification of four novel clinico-immunological clusters those with: (I) high CPA3 and IL-13 levels, females, drug reactions, more severe historical reactions; (II) high CPA3, low IL-13, males, food reactions, more severe historical reactions; (III) low CPA3 and IL-13 levels, young, females, food reactions, mild historical reactions; and (IV) low CPA3, high IL-13, females, drug reactions, mild historical reactions. These clusters were replicated in both geographical cohorts. In separate studies with cultured LAD2 cells, it was found that S. aureus infection could inhibit IgE- and non-IgE-dependent mast cell degranulation and down-regulate gene expression for major cytokines involved in anti-bacterial defense mechanisms (including that for TNFα, IL-8, and IL-1β). Bacterial exposure also altered the phosphorylation of protein kinases downstream of FcRI engagement. At the subcellular level, DPPI was observed inside the granules of mast cells and colocalized with tryptase, CPA3 and chymase. Our finding that DPPI may be present in association with other mast cell proteases within the granules suggests that DPPI is secreted upon mast cell stimulation and may have extracellular roles in immune modulation. DPPI could thus represent a novel marker for mast cell activation that can enhance the diagnosis of allergic reactions when measured in combination with tryptase and CPA3. The potential for bacterial infection to interfere with mast cell responses could reduce the susceptibility to allergic reactions and as the mechanisms involved deserve consideration as a novel therapeutic approach to prevent development of severe reactions. Serum levels of CPA3 have the potential to predict the severity of allergic reactions to drugs or food. Identification of four multidimensional endotypes underlines the connection between CPA3 and IL-13 levels and their association with clinical features of patients who have drug or food allergies. Their use as biomarkers can help to identify those at particular risk of allergic reactions and allow optimal interventions to be undertaken.

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ORCID for Andrew Walls: orcid.org/0000-0003-4803-4595

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