The AhR–TLR4 axis in non-IgE-mediated Cow's milk allergy: a systematic review with integrated multi-omics corroboration

Background & aims: Non-IgE-mediated cow's milk allergy (non-IgE-CMPA) is the most prevalent form of CMPA in infancy; however, its pathogenesis is still poorly understood, which impedes the development of targeted nutritional strategies. Recent evidence suggests a connection between gut dysbiosis and immune dysregulation. Therefore, this systematic review, incorporating multi-omics corroboration, sought to clarify a pathogenic axis driven by dysbiosis and involving the aryl hydrocarbon receptor (AhR) and Toll-like receptor 4 (TLR4). Furthermore, the review aimed to evaluate related predictive biomarkers and potential therapeutic approaches. Methods: We synthesized evidence from 39 studies of infants (0-3 years) with physician-confirmed non-IgE-CMPA, following PRISMA guidelines. To visually corroborate key mechanistic pathways at cellular resolution, we performed an integrative analysis of publicly available single-cell RNA sequencing datasets from pediatric intestinal biopsies. Results: Non-IgE-mediated CMPA exhibits a distinct gut dysbiosis signature, characterized by decreased Bifidobacterium and increased Enterobacteriacea. This dysbiosis is associated with reduced microbial AhR ligands and TLR4 pathway in intestinal epithelial cells. A B/E ratio <0.5 at 3 months of age predicted persistent allergy (HR = 1.9, AUC = 0.82). scRNA-seq data confirmed IEC-specific upregulation of TLR4 co-receptors (CD14, LY96), activation of the NLRP3 inflammasome, and reduced expression of intestinal barrier integrity markers. Synbiotic intervention (LGG + HMOs) was associated with a 67% resolution of symptoms. Conclusion: Early-life gut dysbiosis may disrupt AhR-TLR4 crosstalk, leading to NLRP3-mediated inflammation and barrier dysfunction in non-IgE-mediated CMPA. Bradford Hill criteria suggest a causal relationship for this pathway. Furthermore, the B/E ratio holds promise for early risk stratification, and synbiotics represent a potential mechanism-guided nutritional strategy for restoring microbial-immune homeostasis. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/view/CRD42025104533, PROSPERO CRD420251045333.