PROJECT SUMMARY Chronic itch is debilitating symptom that severely limits quality of life and affects up to 20% of the population. Despite this, there are no FDA-approved drugs specifically indicated for the treatment of chronic itch. Our current understanding of chronic itch pathophysiology largely derives from studying inflammatory disorders such as atopic dermatitis (i.e. eczema). However, how chronic itch arises in conditions that lack overt cutaneous inflammation is poorly understood. Chronic pruritus of unknown origin (CPUO) clinically lacks such overt skin inflammation, accounts for up to 40% of all chronic itch cases and lacks effective treatments. Improper barrier function due to dry, aging skin has been proposed as a key driving factor of itch in CPUO, yet what may mediate this is unknown. Notwithstanding this, our lab has brought recognition of this condition to the forefront in a series of recent publications. IL-33 is an `alarmin' released from keratinocytes upon damage or stress. Our preliminary studies show that IL- 33 is elevated in the sera of patients with CPUO compared to healthy controls. Further, a recent study showed that IL-33 is required for the development of dry skin itch in a murine model that recapitulates several of key aspects of CPUO. However, the mechanisms by which IL-33 promotes itch and how this process is regulated remains poorly understood. IL-33 is a potent inducer of immune cell responses, however, we and others have found that IL-33 can directly activate sensory neurons. Thus, in Aim 1, we will determine if IL-33 is a key mediator of a direct epithelial-neuronal axis in dry skin itch using novel mice we have generated. The activity of IL- 33 is dramatically enhanced upon cleavage by proteases. Our preliminary data suggest that the serine protease KLK7, important for barrier homeostasis, promotes chronic itch through an unknown mechanism. In Aim 2, we will evaluate if KLK7 cleaves IL-33, and enhances IL-33's capacity to induce itch. Our long-term goal is to determine whether IL-33 and KLK7 are therapeutic targets in CPUO. The overall objective of our proposal is to identify the role of IL-33 and KLK7 in promoting dry skin itch. Our central hypothesis is that IL-33 represents a direct epithelial-neuronal mediator of itch that is regulated by KLK7. The rationale for this proposal is that once it is understood how IL-33 and KLK7 promote itch in dry skin, these mechanisms can be harnessed to create effective and novel therapies for CPUO and other dry skin-related conditions.