Immunomodulatory Effect of Kappa Opioid Receptor Agonists on Mast Cells and Food Allergy Models

<p><strong>Mast cells are tissue-resident immune cells widely present in various tissues, such as the gastrointestinal lining, skin, and respiratory epithelium. Their ability to store and rapidly release potent mediators, including inflammatory cytokines and proteases, within minutes of activation makes mast cells a crucial first line of defence in protecting the host against threats and regulating pathological conditions such as food allergies.</strong></p><p>During food allergy, mast cells release a variety of inflammatory mediators responsible for the rapid onset of gastrointestinal symptoms, including diarrhoea, abdominal pain, and vomiting. Currently, there is no cure for food allergies, and existing treatments focus primarily on symptom management, often with side effects like drowsiness, nausea, and headaches. Consequently, allergen avoidance remains the most effective strategy, but it can lead to nutritional deficiencies, particularly in children allergic to milk and eggs. Thus, alternative and more effective treatments are urgently needed.</p><p>Kappa opioid receptors (KORs) are widely expressed throughout the human body, including in the brain, spinal cord, gastrointestinal tract, and on various immune cells. Previous studies have shown that KOR modulation using KOR-stimulating compounds significantly improved allergic diarrhoea in a mouse model, and this beneficial effect was prevented when KOR was blocked. These findings highlight the potential role of KOR in food allergy. Although the underlying mechanisms of KOR modulation in food allergy remain unclear, it is hypothesised that KOR activation may alleviate allergic reactions by regulating immune cells, particularly mast cells, and by directly affecting gastrointestinal processes such as motility and fluid absorption.</p><p>In this thesis, I investigated the effects of KOR modulation using high-affinity KOR agonists, such as U50,488H and nalfurafine, in both in vitro and in vivo models. I optimised a high-purity and functional protocol for generating bone marrow-derived mast cells (BMMCs) to explore the direct effects of KOR agonists on mast cell activation in vitro. All tested KOR agonists, including nalfurafine and U50,488H, successfully inhibited mast cell activation to levels comparable to the positive control, clemastine fumarate. Notably, nalfurafine emerged as the most potent agonist, effectively inhibiting mast cell activation at a concentration as low as 0.5 nM, while U50,488H required a much higher concentration of 1000 nM to achieve similar effects. Interestingly, both agonists exhibited an inverted bell-shaped dose-response curve, with high efficacy at lower concentrations but diminished effects at higher concentrations. Moreover, the inhibitory effects of KOR agonists on mast cell activation were abolished in KOR-deficient BMMCs, confirming that the effects were mediated through the KOR pathway and validating the specificity of the agonists.</p><p>In addition to the in vitro experiments, I developed a food allergy model to evaluate the in vivo effects of nalfurafine and U50,488H. Both agonists significantly improved clinical symptoms during disease onset, demonstrating their potential as therapeutic agents for managing food allergy symptoms. However, when nalfurafine was administered prophylactically before immunisation, there was no significant effect on the manifestation of food allergies. This suggests that while nalfurafine is effective at alleviating symptoms once the disease is established, it does not prevent the initial sensitisation process that drives food allergies.</p><p>This thesis also highlights the impact of genetic differences between inbred mouse strains C57BL/6 and BALB/cByJ in both BMMC cultures and the food allergy model. BMMCs from the BALB/cByJ strain were less efficient in culture generation compared to those from the C57BL/6 strain. Conversely, in the food allergy model, C57BL/6 mice exhibited milder food allergy symptoms compared to BALB/cByJ mice. These findings underscore the importance of considering genetic background when designing and interpreting immunological experiments.</p><p>Overall, the findings presented in this thesis provide valuable insights into the role of KOR in modulating immune responses, particularly in the context of mast cell activation and food allergy. Furthermore, the study emphasises the influence of genetic background on experimental outcomes, with notable differences between C57BL/6 and BALB/cByJ mice in both BMMC culture efficiency and the severity of food allergy symptoms. These findings advance our understanding of KOR modulation in immune responses and suggest potential avenues for developing targeted therapies to manage food allergy and other mast cell-related conditions.</p>