R. Ranjan1, S. Balaji1, S. S. Bhattacharya1, C. M. Moles1, N. Hann1, A. F. Shaaban1, P. Bollyky3, T. M. Crombleholme2, S. G. Keswani1 1Cincinnati Children’s Hospital Medical Center,Laboratory Of Regenerative Wound Healing, Division Of Pediatric, General And Thoracic Surgery,Cincinnati, OH, USA 2Children’s Hospital Colorado,Aurora, CO, USA 3Stanford University,Infectious Diseases In The Department Of Medicine,Palo Alto, CA, USA
Introduction: We have previously reported that IL-10 overexpression results in fetal-type regenerative repair in postnatal wounds via a STAT3 dependent mechanism. It is unclear which cellular compartment of the wound is responsible for IL-10’s effects. The fibroblast is the main cellular mediator of scarring and has a significant role in the fetal regenerative phenotype. Taken together, we hypothesize that IL-10’s regenerative effects are dependent on fibroblast-specific STAT3 signaling.
Methods: We developed inducible STAT3 knockdown models which were Fibroblast specific (Col1a2-Cre), Keratinocyte specific (Krt14-Cre) and Skin specific (UBC-Cre). IL-10 was overexpressed (lentiviral-CMV-IL-10;106 T.U.) and 4 mm wounds were created and evaluated at 28 days in all models. In a gain of function experiment, we used a fibroblast transplant model in which the fibroblast is the only cell in the wound capable of responding to IL-10. Syngeneic fibroblasts expressing IL-10 or GFP were transplanted into the wounds of skin specific STAT3 knockout mice and analyzed at 28 days. A quantifiable six-parameter histologic scar scoring scale was developed to evaluate scar formation, with uninjured skin benchmarked to zero. The mean and standard deviation are calculated based on which a 95% confidence interval is reported, p values by ANOVA/ t-test.
Results: In normal controls, IL-10 overexpression resulted in a significant improvement in scar formation compared to the PBS treatment (p<0.0001). Skin-specific STAT3 knockdown resulted in loss of IL-10 effect on scar attenuation to a level that is similar in PBS scar (p=ns), suggesting that IL-10 effects are mediated via STAT3 signaling. Fibroblast-specific STAT3 knockout similarly abrogates IL-10 effects (p=ns), suggesting that fibroblasts are a primary mediator of IL-10 effects. Keratinocyte-specific STAT3 knockout results in a partial attenuation of IL-10 effects (p<.01), suggesting IL-10 effects are in part mediated by keratinocytes. Syngeneic transplant of fibroblasts expressing GFP resulted in normal scarring (p=ns), but transplant of fibroblasts overexpressing IL-10 resulted in a significant improvement in scar reduction (p<0.01), albeit less than the effect of lenti-il-10 treatment.
Conclusion: IL-10 effects on attenuating scar formation are primarily mediated via STAT3 signaling in dermal fibroblasts. However, the partial loss of effect on dermal appendages suggests potential epidermal–dermal interactions may be important. Understanding these fundamental mechanisms will permit the development of novel clinically translatable regenerative therapies.
