Polyphenol oxidase cross-linking enhances whey protein-induced systemic food allergy by regulating miRNA in CD4⁺ T cells

Whey protein (WP) contains two major allergenic proteins, α-lactalbumin and β-lactoglobulin, which significantly impact its incorporation and application in food products. Current research primarily focuses on the dynamic changes in allergenicity during the processing of individual protein components. To simulate realistic conditions in food processing, this study aims to investigate the effect of polyphenol oxidase cross-linking on the allergenicity of complex protein matrices. Our findings indicate that mice receiving polyphenol oxidase-crosslinked WP (CL-WP) exhibited more severe systemic food allergic reactions, characterized by decreased body temperature and significantly increased serum levels of specific IgE and mMCPT-1 compared to the WP group. Furthermore, mice in the CL-WP group displayed more pronounced intestinal injury. Flow cytometry results showed a significant decrease in CD103⁺MHC-II⁺CD11c⁺ cells and a significant increase in the proportion of Th2 cells in the CL-WP group. Simultaneously, the marked elevation of Th2 cytokine levels in spleen cell supernatants further indicated a significant Th2 bias in these mice. Lastly, miRNA sequencing of CD4⁺ T cells from the spleen revealed that among the differentially expressed miRNAs, miR-532-5p was significantly upregulated in both WP and CL-WP groups compared to controls, while miR-322-3p was downregulated in both groups. miR-27a-5p remained unchanged in the WP group but was significantly downregulated in the CL-WP group, and miR-92a-3p was upregulated in the WP group but unchanged in the CL-WP group. These results suggest that miRNAs may serve as potential biomarkers for the severity of milk allergy.