(WHS-I.04) PERINATAL UPREGULATION OF CCL11 IN SKIN FIBROBLASTS IS ESSENTIAL FOR SUBCUTANEOUS ADIPOGENESIS AND WOUND HEALING
Wednesday, May 15, 2024
1:45 PM – 4:00 PM East Coast USA Time
Background: Fibroblasts are the most abundant mesenchymal cells in the dermis. Though traditionally known for its role in extracellular matrix synthesis, recent advances have provided insight into unexpected immunomodulatory properties of skin fibroblasts during homeostasis and in diseased conditions. Here, we hypothesized that immuno-regulatory secretome by cutaneous fibroblasts is age-dependent and tested if CCL11, a chemokine expressed largely by fibroblasts, is necessary for skin tissue homeostasis that may affect the wound healing process. Materials and methods: Publicly available scRNA-seq datasets (GSE189210, GSE183031, GSE172226) were pooled, and fibroblast subsets were categorized as neonatal (P0) or adolescent (P22 and P28) for downstream analysis using Seurat package. Among fibroblast transcriptome, CCL11 upregulation was most notable, which was validated using FACS followed by RT-qPCR and RNAScope on WT C57BL/6N mice. To determine biological significance, CCL11null mice were examined, and skin specimens were compared to P15 and P28 of age. In a series of animal studies, wound healing was examined in WT and CCL11null mice by histologic analysis and flow cytometry. Autogenous fat grafting in CCL11null mice and CCL11 blocking by neutralizing antibody were examined for healing parameters. Animal studies were carried out with N=4-6 mice each, and statistical significance was determined at p<0.05 by one-way ANOVA and T-test.
Results: Bioinformatics analysis demonstrated that CCL11 transcripts were highly upregulated in the fibroblasts of adolescent mice compared to that of perinatal mice. Further validation showed that CCL11 mRNA was minimally expressed in PDGFRa+ fibroblasts from P3 skin, compared to P15 skin. RNAscope analysis confirmed the age-dependent expression of CCL11, which was largely localized to the dermal layer. CCL11 knockout mice showed a significant reduction in subcutaneous adipose layer width compared to wildtype littermates at P28, suggesting a role for adipogenesis in developing skin. Full-thickness wounds in CCL11null mice had fewer aSMA+ myofibroblasts and delayed re-epithelization compared to WT mice. Fat autografting prior to wound induction in CCL11null mice rescued the healing parameters compared to sham control groups, whereas CCL11 neutralization in WT mice did not have an impact on the wound healing process, indicating that wound healing deficit is dependent on intact adipose tissues and not CCL11.
Conclusion: Our study demonstrates that early-life production of CCL11 by dermal fibroblasts is essential for normal adipogenesis, and in turn, cutaneous injury response. The study highlights an important role of fibroblast-derived cytokine production that may unveil pathologic mechanisms behind dysregulated adipogenesis and impaired wound healing.