M. M. Rae1, T. Lu1, C. M. Moles1, X. Wang1, M. Fahrenholtz1, H. Li1, P. Duann1, P. Bollylky2, S. Balaji1, S. G. Keswani1 1Texas Children’s Hospital And Baylor College Of Medicine,Division Of Pediatric Surgery,Houston, TX, USA 2Stanford University School Of Medicine,Infectious Diseases And Microbiology And Immunology,Stanford, CA, USA
Introduction:
Our lab has shown a significant role for the anti-inflammatory cytokine IL-10 in regulating inflammation and ECM production, thereby attenuating fibrosis in skin wounds. Previous reports have shown, paradoxically, that wounds in IL-10-/- mice heal faster, with increased rates of re-epithelialization as compared to wildtype mice. However, these wounds were not controlled for contraction and wound environment. Therefore, we sought to determine the role of contraction on IL-10’s wound healing and anti-fibrotic effects in a controlled moist wound environment.
Methods:
Full thickness excisional 6mm wounds were made in IL-10+/+ and IL-10-/- mice that were controlled for contraction using a silicone stent. A consistent and moist wound environment was provided by semi-occlusive dressing Tegaderm. Wounds were serially photographed at 3, 5 and 7d and harvested at 7d and 14d post wounding, then examined for epithelial gap, granulation tissue (H&E), myofibroblasts (a-SMA staining) and leucocyte infiltration (CD45). Data is presented as mean+/-SD, n=5 wounds/group/time point; p-value by ANOVA.
Results:
Macroscopic appearance of unstented wounds with no dressing showed accelerated wound closure in IL-10-/- mice by day 7 compared to controls(IL-10+/+). This effect was lost when a semi-occlusive wound dressing was applied to unstented wounds to create a consistent moist wound environment, with no significant differences observed in re-epithelialization(IL-10-/- 2380±508.4 vs IL-10+/+ 2480.4.7±824.6, p=ns), epithelial gap (IL-10-/- 1433.7±558.1 vs IL-10+/+ 1436.2±527.7, p=ns), granulation tissue (IL-10-/- 1.65±0.5 vs IL-10+/+ 1.21±0.4, p=ns), or CD45 positive cells (IL-10-/- 10.1%±6.2 vs IL-10+/+ 8.9%±4.2, p=ns). In unstented wounds, a-SMA was abundantly expressed at the wound margins, but in IL-10-/- wounds, a-SMA was present throughout the granulation tissue and extended into deep dermal layers, whereas a-SMA expression was less pronounced in the IL-10+/+ wound bed. Stenting of wounds, which controlled for the contractility of mouse skin, significantly delayed wound healing. However, there was no statistical difference in either epithelial gap (IL-10-/- 4883.5±610.8 vs. IL-10+/+ 4152.2.7±480.6, p=ns) or granulation tissue (IL-10-/- 0.33±0.1 vs. IL-10+/+ 0.55±0.2, p=ns) at day 7, although stented IL-10-/- wounds exhibited increased a-SMA density. Interestingly, the CD45+ cellular infiltrate significantly increased in stented IL-10-/- mice as compared to unstented (IL-10-/- 10.1%±6.2 vs. IL-10-/- with stent 32.8%±15.6, p<0.01), but did not change in IL-10+/+ mice. Finally, wounds in IL-10-/- mice also developed significantly more scar tissue and much thicker epidermis on day 14 compared to IL-10+/+ mice.
Conclusion:
IL-10 expression does not delay normal wound healing of skin wounds when wounds are controlled for contraction and moist environment. However, the loss of IL-10 leads to increased fibrosis. This data signifies a previously unrecognized role for endogenously expressed IL-10 contributing to the tissue repair response.