SUMMARY Inflammation, dysregulated innate immune responses, and loss of barrier function underlie several skin conditions including psoriasis, atopic dermatitis, hidradenitis suppurativa, and pachyonychia congenita. Compelling evidence now shows that signals regulating inflammation and innate immunity are produced and secreted by stressed keratinocytes making up the skin epidermis. How the relevant signals are generated in keratinocytes, their cellular targets, and the pathways involved in ensuring proper regulation of inflammation and innate immunity in healthy skin tissue, and their dysregulation in disease, remain poorly understood. This multi-PI proposal takes advantage of the complementary expertise of two established investigators and is supported by a strong body of unpublished findings, obtained collaboratively, showing that (i) keratinocyte- derived and keratin-dependent signals regulate the spatial and temporal pattern of neutrophil influx into skin undergoing sterile inflammation; (ii) keratinocytes transiently adapt to prior exposures and are able to amplify responses in the setting of repeated aggressions; (iii) the stress response keratin 17 (K17) acts as a positive regulator of neutrophil infiltration by mediating the sustained activity of protein kinase C a (PKCa) in activated keratinocytes; and (iv) keratin 16 (K16) - another stress response keratin – acts as a negative regulator of neutrophil infiltration and inflammation. We hypothesize that stressed keratinocyte-derived signals engage and regulate cellular effector of innate immunity in skin, and that this is highly relevant to several inflammatory skin disorders. We further hypothesize that K16 and K17 act through unique cis-acting determinants to differentially regulate PKCa activity, neutrophil infiltration and other aspects of innate immunity in stressed skin tissue. The proposal tests these hypotheses through three specific aims. Aim 1 seeks to compare the spatiotemporal pattern of immune cell infiltration and PKCa activity in mouse skin null for either keratin 16 or 17 following exposure to stressors. These animals studies will be corroborated by studies in N-TERT human keratinocytes in culture. Aim 2 seeks to identify, in cell culture models, the cis- acting determinants responsible for the differential impact of K16 and K17 protein on PKCa activity, neutrophil stimulation and other cellular aspects of inflammation and innate immunity. Finally, Aim 3 will define the contribution of other key cellular effectors of innate immunity, specifically macrophages, in mediating the keratinocyte- and keratin-dependent amplification of neutrophil influx in stressed skin in vivo. The mechanistic insight that we stand to gain from completing this project is predicted to lead to a deeper understanding of the pathophysiology of chronic skin inflammatory disorders, yield novel and more reliable biomarkers and, possibly, new strategies to treat skin diseases.