Abstract The skin of AD patients is often colonized by S. aureus strains that produce superantigens (SAg), primarily staphylococcal enterotoxin B (SEB). There is a positive association between S. aureus skin colonization and food allergy in AD. The mechanism of this association is unknown. We have made the observation that epicutaneous (EC) application of ovalbumin (OVA) and SAg producer S. aureus, or OVA and SEB, results in the selective exaggeration of anaphylaxis to oral challenge with OVA compared to EC application of OVA alone. Moreover, it results in exaggerated systemic anaphylaxis to oral challenge with BSA-TNP in mice passively sensitized with IgE anti-TNP, indicating that the enhancement of food anaphylaxis was non-antigen-specific and determined by factors beyond differences in IgE Ab levels or affinity. We propose to dissect the mechanisms of SEB enhancement of IgE mediated oral anaphylaxis. Preliminary data show that enhanced susceptibility to oral anaphylaxis in mice EC exposed to OVA+SEB is associated with elevated levels of serum IL-4, dependent on IL-4 and IL-4R expression by intestinal epithelial cells (IECs),and accompanied by increased intestinal permeability (IP). Enhanced susceptibility is inhibited by Divertin, a small molecule that suppresses intestinal absorption of antigen via the paracellular pathway by blocking the recruitment of myosin light chain kinase (MLCK) to the peri-junctional actinomyosin ring, where it disrupts epithelial tight junctions. This suggests a critical role for MLCK in food allergy. In addition, the data show that EC application of SEB causes a massive influx of basophils in skin-draining lymph nodes (dLNs) that was dependent on CD40 keratinocyte (KC)-derived IL-33, and T cells. The recruited basophils enhanced the ability of dendritic cells (DCs) from skin dLNs to drive Th2 polarization. Pretreatment of DCs in vitro with IL-4 also promoted their capacity to drive Th2 polarization. We propose to test the hypothesis that SEB from S. aureus that colonizes AD skin binds to CD40 on KCs and triggers caspase 8 mediated cleavage and release of bioactive IL-33 which induces IL-3 release by T cells leading to recruitment of basophils in dLNs. There, basophil-derived IL-4 promotes the Th2 polarizing ability of DCs that have captured antigen encountered in the skin. These events drive a rise in systemic levels of Th2 derived IL-4. Increased IL-4 signaling in IECs synergizes with mediators released by MCs to promote MLCK dependent barrier loss by causing redistribution of tight junction proteins. The resulting increased antigen absorption triggers a forward amplification cycle of MC activation that exaggerates allergy to foods against which the patient has been sensitized. The studies proposed will define the mechanisms by which S. aureus skin colonization aggravates food allergy and will uncover a central role of MLCK in this disease. They may lead to novel therapies for food allergy that would target S....