Summary/Abstract: Ticks are ancient hematophagous arthropods that transmit pathogens of public health and veterinary importance. During feeding, they disrupt host homeostasis through salivary molecules that perturb inflammation, blood coagulation and nociception. For more than 100 years, tick saliva has fascinated researchers with its anti-hemostatic, analgesic, and anti-inflammatory properties. Over the previous funding period, we used orthogonal approaches and a cell-centric perspective to better understand the intricate biology occurring between ticks and their hosts. We uncovered that tick extracellular vesicles (EVs) carry bioactive molecules and mediate interspecies communication with mammals. We discovered that tick EVs affect dendritic epidermal T cells (DETCs) and promote distinct outcomes of microbial transmission to mammals. DETCs are a subset of murine γδ T cells located in the skin epidermis that have a critical role in wound healing. DETCs interact with highly specialized epithelial cells (keratinocytes), which comprise approximately 95% of the epidermal layer in the skin. Under homeostasis, epidermal cells differentiate and migrate to the skin surface where they are shed while being replenished at the basal layer or stratum basale. Upon an injury, wound healing occurs in four stages of tissue repair and replacement: hemostasis, inflammation, proliferation, and remodeling. Unfortunately, the epidermis has been mostly neglected when studying the relationship between ticks and their mammalian hosts. For the next iteration of this project (R01 competing renewal), we will investigate the effect of tick EVs on the DETC-keratinocyte dialogue. We will also examine how tick EVs affect the wound healing function of keratinocytes during feeding. Single-cell RNA sequencing, flow cytometry, cell sorting, and murine genetics will be used to uncover how DETCs and keratinocytes communicate during feeding of an ectoparasite. Lineage tracing combined with drug perturbations and allied with sophisticated intravital imaging techniques will be used to track the proliferation, differentiation, and migration of epidermal cells. Our central hypothesis states that tick EVs disrupt the dialogue occurring between DETCs and keratinocytes during feeding altering epidermal wound healing. In Aim #1 of this proposal, we will investigate the effect of tick EVs on the crosstalk between DETCs and keratinocytes. In Aim #2 of this grant application, we will ascertain how tick EVs affect epidermal wound healing during a tick bite and microbial transmission. Collectively, we will provide a distinct biological insight and fill a current gap in knowledge for ectoparasitology and microbial diseases by describing how ticks affect the epidermal environment during feeding.