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Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Site-specific differences in skin response to pathogens and in the course of cutaneous inflammatory diseases are well appreciated. The composition and localization of cutaneous leukocytes has been studied extensively using histology and flow cytometry. However, the precise three-dimensional (3D) distribution of distinct immune cell subsets within skin at different body sites requires visualization of intact living skin. We used intravital multiphoton microscopy in transgenic reporter mice in combination with quantitative flow cytometry to generate a 3D immune cell atlas of mouse skin. The 3D location of innate and adaptive immune cells and site-specific differences in the densities of macrophages, T cells, and mast cells at four defined sites (ear, back, footpad, and tail) is presented. The combinatorial approach further demonstrates an as yet unreported age-dependent expansion of dermal gamma-delta T cells. Localization of dermal immune cells relative to anatomical structures was also determined. Although dendritic cells were dispersed homogeneously within the dermis, mast cells preferentially localized to the perivascular space. Finally, we show the functional relevance of site-specific mast cell disparities using the passive cutaneous anaphylaxis model. These approaches are applicable to assessing immune cell variations and potential functional consequences in the setting of infection, as well as the pathogenesis of inflammatory skin conditions.