Tissue-resident macrophages contribute to ear hole regeneration of early postnatal mice

Healing of ear holes made in early postnatal mice represents an accessible model for the study of the regeneration of multiple tissues in mammals. This regenerative process involves three phases: wound healing, blastema formation, and re-differentiation. Classical models for regeneration studies have implicated macrophages as key cells for regeneration progression. In this study macrophage distribution and macrophage depletion experiments were conducted to evaluate whether these cells have a role during ear hole regeneration in early postnatal mice. Using the pan-macrophage markers CD68 and F4/80, we discriminate infiltrating from tissue-resident macrophages. In addition to expressing F4/80, tissue-resident macrophages also showed the presence of iNOS, a marker of pro-inflammatory macrophages. Unexpectedly, depletion of macrophages by clodronate liposomes administration during the wound healing stage of ear hole regeneration of early postnatal-age mice resulted in an increased number of infiltrating CD68+ cells and a deletion of F4/80+ macrophages with the subsequent delay in re-epithelialization and blastema formation. Under this experimental condition, IL10 was not affected during the wound healing phase, but its levels decreased when the re-population of F4/80+ tissue-resident macrophages was evident in the regenerating tissues. In addition, the administration of clodronate liposomes during the re-differentiation stage accelerated the maturation of regenerating elastic cartilage. These results indicate that F4/80+ tissue-resident macrophages control the infiltration of determined immune cells and are the main macrophage subpopulation that contributes to the regeneration of ear holes made in early postnatal mice.