Chronic non-healing wounds are a major complication of diabetes, which impacts 1 in 10 people worldwide. Dying cells in the wound perpetuate the inflammation and contribute to dysregulated tissue repair 1-3 . Here, we reveal the membrane transporter Slc7a11 as a molecular 'brake' on efferocytosis, the process by which dying cells are removed, and that inhibiting Slc7a11 can accelerate wound healing. First, transcriptomics of efferocytic dendritic cells identified upregulation of several Slc7 gene family members. In further analyses, pharmacological inhibition, siRNA knockdown, or deletion of Slc7a11 enhanced dendritic cell efferocytosis. Interestingly, Slc7a11 was highly expressed in skin dendritic cells, and scRNAseq of inflamed skin showed Slc7a11 upregulation in innate immune cells. In a mouse model of excisional skin wounding, loss of Slc7a11 expression or inhibition accelerated healing dynamics and reduced apoptotic cell load in the wound. Mechanistic studies revealed a link between Slc7a11, glucose homeostasis, and diabetes. Slc7a11-deficient dendritic cells relied on glycogen store-derived aerobic glycolysis for improved efferocytosis, and transcriptomics of efferocytic Slc7a11-deficient dendritic cells identified genes linked to gluconeogenesis and diabetes. Further, Slc7a11 expression was higher in the wounds of diabetic-prone db/db mice, and targeting Slc7a11 accelerated their wound healing. The faster healing was also linked to the release of TGF- family member GDF15 from efferocytic dendritic cells. Collectively, Slc7a11 is a negative regulator of efferocytosis, and removing this brake improves wound healing, with significant implications for diabetic wound management.

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1β response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

Fibroblasts are a major component of the microenvironment of most solid tumours. Recent research elucidated a large heterogeneity and plasticity of activated fibroblasts, indicating that their role in cancer initiation, growth and metastasis is complex and context-dependent. Here, we performed genome-wide expression analysis comparing fibroblasts in normal, inflammatory and tumour-associated skin. Cancer-associated fibroblasts (CAFs) exhibit a fibrotic gene signature in wound-induced tumours, demonstrating persistent extracellular matrix (ECM) remodelling within these tumours. A top upregulated gene in mouse CAFs encodes for PRSS35, a protease capable of collagen remodelling. In human skin, we observed PRSS35 expression uniquely in the stroma of high-grade squamous cell carcinomas. Ablation of PRSS35 in mouse models of wound-or chemically-induced tumorigenesis resulted in aberrant collagen composition in the ECM and increased tumour incidence. Our results indicate that fibrotic enzymes expressed by CAFs can regulate squamous tumour initiation by remodelling the ECM.

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Due to microRNAs' (miRs) important functions and high potential for disease diagnosis and therapy, increasing efforts have been put to understand their role in wound repair and identify targetable miRs for wound treatment. However, lack of knowledge about miR-mediated gene expression in human wound tissues hinders the recognition of clinically relevant miRs. Here we profiled genome-wide miR and mRNA expression by RNA-sequencing in the same set of samples from human normal acute wounds and chronic non-healing venous ulcers (VU). By integrative analysis of miR and mRNA omics, we unraveled miR-mediated gene regulatory networks specifically associated with different phases of wound repair or VU. We also identified transcriptional factors and miR arm switching events underpinning the dynamically changed miR expression. In this study, we focused on not only a few top changed miRs, but also the miRs with their targetome enriched in the VU gene signature, such as miR-34a-5p, miR-34c-5p, miR-218-5p, miR-7704, miR-424-5p, miR-450-5p, miR-517b-3p, miR-96-5p, and miR-516b-5p. We confirmed these miRs' targetome in human keratinocytes and fibroblasts using microarrays and demonstrated their functional relevance to VU pathology. We presented these datasets at a browsable web portal (https://www.xulandenlab. com/data), providing a rich resource to study the posttranscriptional regulatory underpinnings of human skin wound healing.