The role of interleukin-10 and hyaluronan in murine fetal fibroblast function in vitro: implications for recapitulating fetal regenerative wound healing

Abstract

Background: Mid-gestation fetal cutaneous wounds heal scarlessly and this has been attributed in part to abundant hyaluronan (HA) in the extracellular matrix (ECM) and a unique fibroblast phenotype. We recently reported a novel role for interleukin 10 (IL-10) as a regulator of HA synthesis in the fetal ECM, as well as the ability of the fetal fibroblast to produce an HA-rich pericellular matrix (PCM). We hypothesized that IL-10-mediated HA synthesis was essential to the fetal fibroblast functional phenotype and, moreover, that this phenotype could be recapitulated in adult fibroblasts via supplementation with IL-10 via an HA dependent process.

Methodology/principal findings: To evaluate the differences in functional profile, we compared metabolism (MTS assay), apoptosis (caspase-3 staining), migration (scratch wound assay) and invasion (transwell assay) between C57Bl/6J murine fetal (E14.5) and adult (8 weeks) fibroblasts. We found that fetal fibroblasts have lower rates of metabolism and apoptosis, and an increased ability to migrate and invade compared to adult fibroblasts, and that these effects were dependent on IL-10 and HA synthase activity. Further, addition of IL-10 to adult fibroblasts resulted in increased fibroblast migration and invasion and recapitulated the fetal phenotype in an HA-dependent manner.

Conclusions/significance: Our data demonstrates the functional differences between fetal and adult fibroblasts, and that IL-10 mediated HA synthesis is essential for the fetal fibroblasts' enhanced invasion and migration properties. Moreover, IL-10 via an HA-dependent mechanism can recapitulate this aspect of the fetal phenotype in adult fibroblasts, suggesting a novel mechanism of IL-10 in regenerative wound healing.

Fig 1. Differences in the functional profile of FFB compared to AFB.

A) Rate of metabolism, B) apoptosis, C) migration and D) invasion were compared between FFB and AFB. Bar plots represent average±SD. Asterisks denote statistically significant differences between the groups (* p<0.05, ** p<0.01; Student’s t-test; n = 3 per group at similar passage number; each experiment was conducted in triplicates with cells from independent isolations).

Fig 2. The role of IL-10 and hyaluronan in the functional profile of FFB.

A) Rate of metabolism, B) apoptosis, C) migration and D) invasion were compared between FFB and IL-10 knockout FFB to determine if IL-10 is essential to the functional profile of the FFB, and between FFB and FFB in presence of 4-MU to determine if hyaluronan is essential to the functional profile of the FFB. Bar plots represent average±SD. Asterisks denote statistically significant differences between the groups (* p<0.05, ** p<0.01; Student’s t-test and ANOVA; n = 3 per group at similar passage number; each experiment was conducted in triplicates with cells from independent isolations).

Fig 3. The role of IL-10 in recapitulating the fetal functional profile of AFB.

A) Rate of metabolism, B) apoptosis, C) migration and D) invasion were compared between AFB, AFB+IL-10, and FFB to determine if IL-10 treatment can recapitulate the fetal functional profile in the FFB. IL-10 treatment recapitulated the migration and invasion aspects of the fetal-type fibroblast phenotype in AFB, but metabolism and apoptosis rates were not impacted by IL-10. Bar plots represent average±SD. Asterisks denote statistically significant differences between the groups (* p<0.05, ** p<0.01; Student’s t-test and ANOVA; n = 3 per group at similar passage number; each experiment was conducted in triplicates with cells from independent isolations).

Fig 4. The essential role of HA in IL-10 mediated recapitulation of the fetal-type migration and invasion profile in AFB.

A) migration and B) invasion were compared between AFB, AFB+IL-10, and AFB+IL-10 in the presence of 4-MU to determine if IL-10 mediated recapitulation of fetal-type fibroblast migration and invasion in AFB is HA dependent. The increase in migration and invasion with IL-10 supplementation was abrogated with competitive inhibition of HA synthases in AFB. Bar plots represent average±SD. Asterisks denote statistically significant differences between the groups (* p<0.05, ** p<0.01, *** p<0.001; Student’s t-test and ANOVA; n = 3 per group at similar passage number; each experiment was conducted in triplicates with cells from independent isolations).

Fig 5. FFB have a distinct functional profile from AFB.

A) FFB demonstrates decreased rates of metabolism and apoptosis and increased rates of cellular migration and invasion compared to AFB. IL-10 treatment can recapitulate the fetal functional phenotype of enhanced migration and invasion in AFB. B) Mechanistically, our previous data has shown that fetal fibroblasts synthesize more HA and HA-rich pericellular matrices (PCM) compared to AFB. IL-10 in the fetus has a novel role in the regulation of the HA-rich ECM synthesis. This is the first report that demonstrates that IL-10 mediated HA synthesis regulates the migration and invasion of the FFB, we posit this regulation, in part, facilitates the fetal regenerative response. In comparison to this, AFBs produce less HA and PCM and contribute to scar formation in postnatal wounds. IL-10 treatment increases HA synthesis and HA-rich PCM in AFB and revert them to a more fetal-type dynamic phenotype with increased migration and invasion, and recapitulate fetal phenotype in postnatal wounds.