Project Summary The skin, the largest and most external organ in the body, is routinely subject to inflammatory insults. As such the skin, specifically the epidermis, is tasked with maintaining a physical barrier in the face of pathogenic and toxic insults. Importantly, cutaneous epithelia are replenished by a small pool of epidermal stem cells (EpdSCs) capable of self-renewing and differentiating into the various lineages of the tissue in healthy and disease states. Yet, to date, the lasting impact of inflammation on these precious tissue stem cells has not been examined. My preliminary data indicate that EpdSCs can sense and respond to inflammatory signals, and sustain epithelial hyperproliferation. Intriguingly, I find that inflammation-experienced epidermis is faster at full thickness wound closure than age-matched control skin indicating that inflammation causes long-term changes to tissue fitness and function. Based on these early findings, I hypothesize that EpdSCs are likely altered as a result of inflammation. Using a well-defined murine model of T cell mediated skin inflammation, I aim to systematically dissect specifically how EpdSCs are altered by inflammation and elucidate the consequences of such alterations on tissue fitness. To this end, I will 1) characterize the long-lasting transcriptional and epigenetic changes within EpdSCs and changes to the skin milieu, post-inflammation, 2) assess differences in tissue fitness and function within the post-inflamed epidermis, and lastly, 3) test the functional significance of identified genes and pathways for their contribution to the sustained differences within the inflammation-experienced epidermis. I propose using cell type/stage-specific CreER+; Rosa26YFPfl/fl mice to lineage trace and FACS purify the progenitor and differentiating populations of the epidermis as well as inflammation-experienced epidermal cells. To dissect intrinsic changes within the EpdSCs post-inflammation, I will perform ATAC-Seq in conjunction with transcriptome analysis (RNA-seq). Genes of interest will be those with sustained differential expression after inflammation and which are candidates for direct targets of the transcription factors activated in EpdSCs after inflammation. To determine their functional significance, I will knock them down in vivo using shRNA and an established in utero lentiviral injection system and test their consequences to the rapid immune response. Because EpdSCs are long-lived cells, they represent ideal targets for efficacious long-term interventions. Additionally, because these adult epidermal stem cells are central to the development of cancers, chronic wounds and epidermal aging, as well as disorders associated with accumulating inflammatory stresses, understanding how an epidermal stem cell is impacted by inflammation will provide novel mechanistic insights into the etiology and pathology of these epithelial diseases.