PROJECT SUMMARY/ABSTRACT Allergies mainly occur in barrier tissues and are driven by the concerted action of the immune and nervous systems. Following allergen sensitization, overt disease develops when allergen-primed immune effectors are mobilized to promote inflammation at barrier tissues in response to allergen re-encounter. Barrier dysfunction is central to the etiology of atopic dermatitis and other allergic diseases. Loss of the barrier integrity not only permits allergen penetration across the epithelial layer but also prompts a homeostatic-to-inflammatory shift in epithelial gene expression. Inflammatory signals derived from epithelial cells of barrier-disrupted skin act on nearby immune cells and nerve endings, dictating their dynamics and function. Skin colonization by Staphylococcus aureus has emerged as an important feature of atopic dermatitis, yet it remains unclear exactly how this bacterium contributes to the development of allergic skin disease. The central hypothesis for the proposed research is that S. aureus colonization drives allergen sensitization and allergic inflammation by causing skin barrier damage and triggering epithelial inflammatory signaling. Our preliminary data point to a role for a protease derived from S. aureus in these pathologic processes. To verify this hypothesis, we will pursue the following specific aims: to determine the role of staphylococcal proteolytic activity in skin barrier disruption and percutaneous allergen sensitization (Aim #1); to establish the epithelial signaling pathways linking staphylococcal protease action to the inflammatory gene expression program (Aim #2); and to elucidate the contribution of staphylococcal protease-activated epithelial signaling to reshaping the skin neuroimmune landscape and driving allergic skin inflammation (Aim #3). This research project will leverage the complementary expertise of the participating investigators and technical support from well-established core facilities. The goal of the proposed research is to decipher the molecular underpinnings of atopic dermatitis and other diseases linked to barrier dysfunction and translate this knowledge into effective clinical strategies for their treatment.