Essential Role of the Anti-Inflammatory Cytokine IL-10 in the Fetal Regenerative Phenotype is Mediated Via Stat3 and Hyaluronan Synthase: Implications for Scarless Wound Healing

Abstract

Introduction: Mid-gestation fetal skin heals without scar with high levels of hyaluronan (HA) in the extracellular matrix (ECM) and an attenuated inflammatory response. We have previously demonstrated an essential role of interleukin-10 (IL-10) in fetal wound healing and the ability of fetal fibroblasts (FFb) to produce a large HA-rich pericellular matrix (PCM). These data include 1)elevated IL-10 levels in fetal skin, 2)IL-10-/- fetal wounds heal with scar, and 3)IL-10 induces postnatal wounds to heal scarlessly. Combining these data with accepted IL-10 signaling pathways, we hypothesize that the regenerative effects of IL-10 in the fetus are partially mediated through HA-rich PCM formation dependent on IL-10 receptor 1 (IL-10R1), STAT3 and hyaluronan synthase (HAS1-3) function. We further hypothesize IL-10 can recapitulate this fetal regenerative PCM phenotype in adult fibroblasts (AFb). Methods: to determine the role of IL-10 in FFb PCM production, murine IL-10-/- (E14.5) fibroblasts were cultured for 24 hours and PCM assessed. to determine if FFb produced PCM is HA rich, FFB were treated with hyaluronidase. Next in a sequential series of loss-of-function experiments, murine FFb (E14.5) were treated with inhibitors of IL-10R1 (IL-10R1 neutralizing Ab), STAT3 (STA-21) and HAS 1-3 (4-methyumbelliferone, 4-MU) or PBS and analyzed for PCM production. AFb were cultured with IL-10 (200ng/ml) and PCM assessed. PCM was quantified using erythrocyte particle exclusion assay and computer assisted morphometric analysis which generated a PCM area/cell area ratio. Data expressed as mean±SEM. Results: FFb produce a robust PCM (2.82+1.02). Treatment with hyaluronidase results in a significantly attenuated PCM in FFb (FFb with treatment 1.60±0.11 vs FFb 2.79±0.35;p<0.01). FFB produce a significantly greater PCM than IL10-/- FFb (FFb 2.82±1.02 vs IL10-/- 1.58±0.15;p<0.01). Blockade of IL-10R1 significantly reduces PCM formation compared to FFb (FFb with IL-10R1Ab 1.41±0.05 vs FFb 2.82±1.02;p=0.01). Inhibition of STAT3 by STA-21 results in decreased PCM formation (FFb with STA21 1.24±0.08 vs FFb 2.82±1.02;p<0.001). Attenuation of HAS1-3 function by 4-MU results in decreased PCM formation (FFb with 4-MU 1.63±0.16 vs FFb 2.82±1.02;p<0.01). Treatment with IL-10 significantly increases PCM production in adult Fb (AFb 1.84±0.40 vs AFb+IL-10 2.77±0.72;p<0.01) to levels which recapitulate the fetal regenerative PCM phenotype (AFb+IL-10 2.77±0.72vs FFb 2.82±1.02;p=NS). Conclusions: IL-10 is essential to the fetal fibroblast's ability to produce an HA-rich PCM. FFb production of the PCM is dependent on IL-10R1, STAT3 and HA synthase function. IL-10 can recapitulate PCM component of the fetal regenerative phenotype in adult fibroblasts. These data suggest that IL-10 plays a unique role in the fetal regenerative response, not only as an immunomodulatory agent, but as a regulator of the ECM.