Food-derived peptides drive non-IgE-mediated type 2 immune responses - PROJECT SUMMARY Food allergies affect an estimated 33 million people in the United States, including IgE-mediated, non-IgE- mediated, and mixed forms, with prevalence steadily increasing. Despite this burden, diagnostics remain blunt: IgE-mediated disease is defined by allergen-specific IgE tests, while non-IgE disease still defaults to elimination diets and oral food challenges. Currently, strict avoidance of the allergenic food remains the standard of care. There is an urgent need to identify the food-derived molecular triggers of type 2 inflammation in allergies to enable mechanism-based therapies. A core group of fewer than ten foods (e.g., cow's milk, eggs, peanuts, soy) accounts for most allergic reactions, yet the molecular basis for why only certain dietary proteins become allergenic remain unclear. Using a machine learning-guided screen based on physicochemical profiles of type 2 immunity modulators (e.g., neuropeptide substance P), we identified a cow's milk αs2-casein peptide (AS2P), absent in breast milk, with predicted agonistic activity to MRGPRX2/MrgprB2, a potent activator on mast cells. Intravenous administration of cow's AS2P induced rapid anaphylactoid reactions in naïve mice, whereas the alternative milk camel's homolog elicited no detectable inflammatory response. Furthermore, topical administration of cow's milk AS2P in a model of ear atopic dermatitis (AD) elicited exacerbated dermal inflammation as measured by ear thickness. Notably, local skin inflammation modulated mast cells in the colon, but not in the small intestine, leading to upregulation of CD200R3, a key activating receptor on a subset of mast cells and basophils, thereby highlighting a previously unrecognized skin-gut pathway. Based on our new preliminary data, we hypothesize that cow's milk-derived peptides such as AS2P activate mast cells through MRGPRX2/B2 to trigger non–IgE mediated food allergy. Using interdisciplinary approaches, we propose to generate a detailed understanding of how cow's milk, and alternative animal milk, peptides mediate systemic and local allergic responses independently of IgE. In Aim 1, we will characterize mast cells responses to MRGPRX2/B2-mediated activation by cow's milk AS2P, and other livestock milk homologs. Further we will assess the systemic immunological effects triggered by the milk-derived MRGPRX2/B2 peptide agonists after naïve and secondary exposure. In Aim 2, we will determine how activation of skin MrgprB2+ mast cells alters mast cell composition and function in the colon. We will further assess whether these peripheral changes are protective or promote inflammation in the setting of intestinal injury. Together, these studies will uncover a previously unrecognized, IgE-independent mast cell activation pathway driven by milk-derived peptides, contributing to the establishment of the molecular mechanisms needed to transform diagnosis and treatment of food allergy.
