PROJECT SUMMARY Wound care is essential for patients with burn injury. Wound healing is commonly linked with itch and pain. After burn injury, monocyte-derived Langerhans cells (LCs) are recruited from the bone marrow to the epidermis and may play a role in wound healing. Additionally, itch and pain are mediated by the free nerve endings of nociceptive sensory neurons, which are often located close to resident LCs in the epidermis. However, our understanding of the role of LCs in modulating wound healing, itch, and pain is particularly limited because approaches to selectively manipulate the activity of LCs have not been established. Here, we hypothesize that distinct subsets of LCs mediate wound healing, itch, and pain. To test this hypothesis, we have successfully applied optogenetics to directly control the activity of LCs by establishing a mouse line that selectively expresses light-sensitive cation channels (ChR2) in LCs. We have also successfully applied chemogenetics to directly control the activity of LCs by establishing a mouse line that selectively expresses Gq- or Gi-biased Designer Receptors Exclusively Activated by Designer Drugs (Gq- or Gi-DREADD) in LCs. The long-term goal of this application is to advance our understanding of the molecular mechanisms behind wound healing, itch, and pain regulated by LC- sensory neuron interaction. Aim 1 will determine the role of LC-sensory neuron interaction in wound healing using hematopoietic chimeras in which monocyte-derived LCs selectively express Gq- or Gi- DREADD and transgenic mice in which sensory neurons selectively express Gq- or Gi-DREADD. Aim 2 will determine the contribution of LCs to postburn itch and pain using hematopoietic chimeras in which resident LCs selectively express ChR2 or Gi-DREADD, single-cell RNAseq, and in vitro calcium imaging from LCs. This proposal will help us to understand completely new roles for LCs and how they may regulate wound healing and wound-related itch and pain. The successful completion of this proposal would provide a unique approach to treating wound healing, wound-related itch, and pain by targeting LCs.