Finding Closure: Regulated Irf6 Expression is Required for Proper Wound Healing In Vivo

Abstract

Chronic wounds affect 6.5 million people in the US, yet little is known about the genetic and molecular mechanisms regulating wound healing. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration, all processes Interferon Regulatory Factor 6 (IRF6) has been shown to regulate in keratinocytes. Embryos deficient for Irf6 demonstrated impaired ex‐vivo embryonic wound closure, and Irf6‐deficient keratinocytes displayed decreased cellular migration resulting in delayed closure in an in vitro scratch assay. Furthermore, individuals with IRF6 mutations exhibited increased likelihood of wound healing complications following surgical repair. Therefore, we hypothesize that altered expression of Irf6 will lead to impaired wound healing in adult mice. Since Irf6−/− mice are perinatal lethal, we utilized two Irf6 mutant lines: an Irf6+/− line with decreased Irf6 expression, and a mouse line with ectopic expression of the Irf6 gene under the control of a keratin 14 promoter (K14‐Irf6). K14‐Irf6 leads to overexpression of Irf6 in the basal layer of the epidermis. The adult mice were wounded with a 6 mm biopsy punch, and the wounds were assessed at either 4 or 7 days post‐wounding. Following morphometric analysis, our results indicate that at day 4, Irf6+/− mice displayed an increase in epidermal area, epidermal volume, and wound volume compared to the wild type and K14‐Irf6 mice. By day 7 the K14‐Irf6 displayed a larger wound area and wound volume as well as impaired epidermal closure compared to wild type and Irf6+/− mice, and Irf6+/− displayed a larger percentage of epidermis in the wound compared to wild type and K14‐Irf6. To determine if changes in epidermal volume could be partially attributed to changes in keratinocyte proliferation, immunohistochemistry for PCNA was performed. Our results show an increase in proliferation in the basal keratinocytes of the migratory tongue in Irf6+/− mice compared with the two other groups (P = 0.06). We also performed Masson's Trichrome staining to determine levels of granulation tissue organization. While very little collagen deposition was present 4 days post‐wounding across the three groups, we observed a trend toward decreased collagen staining and granulation tissue organization in Irf6+/− samples compared with wild type and K14‐Irf6 samples. Together, these results demonstrate that the perturbation of the levels of Irf6 (either positively or negatively) leads to defects throughout the entire period of wound healing. The morphometric analysis data suggests that underexpression of Irf6 being more detrimental in early stages of wound healing (day 4) and overexpression being more detrimental in late stages of wound healing (day 7). Ultimately, these data will provide molecular mechanisms for the increased wound healing complications observed in patients with IRF6 mutations.Support or Funding InformationR01 AR067739 (NIH)