In previous posts I have commented on the regulatory role that the inflammatory and immune responses may have during regeneration. Whereas in many non-regenerative models wound healing is followed by the formation of a fibrotic scar that blocks further regeneration, scar-free wound healing is a rather conserved feature in regeneration-competent species. It is known for example that, in some contexts, the mammalian embryo is able to heal wounds without forming a fibrotic scar and, consequently, allowing a functional regeneration. During mammalian foetal development this capacity of scarless wound healing is lost as the immune system develops. Some studies have reported that the inhibition of anti-inflammatory factors in mice embryos impairs their capacity to heal wounds without making a fibrotic scar.
A paper from the laboratory of Nadia Rosenthal provides evidences of the requirement of macrophages for successful limb regeneration in salamanders (http://www.ncbi.nlm.nih.gov/pubmed/23690624). Macrophages play important roles as phagocytes in order to remove cell debris after injury as well as acting as a source of pro- and anti-inflammatory factors and growth factors that promote cell migration and proliferation. By checking the expression of a collection of cytokines the authors observed the rapid induction of cytokines, chemokines and inflammatory markers after 1 day of limb regeneration. But they also observed high levels of anti-inflammatory and anti-fibrogenic cytokines at this very early stage. In mammalian wound healing these anti-inflammatory cytokines are induced at later stages of wound healing. The authors then focussed in this early induction of anti-inflammatory factors. First they describe how after just 1 day of regeneration monocytes and macrophages accumulate around the wound region, with their numbers peaking by day 4-6 of regeneration. Next, they used a method that allows the transient in vivo ablation of the macrophages and analysed limb regeneration in that macrophage-free context. Remarkably, when macrophages are eliminated during the early stages of regeneration (before the blastema is formed), limb regeneration is completely blocked. During the first week of regeneration, and before the macrophages return, they observed an increased level of inflammatory cytokines and a reduced level of anti-inflammatory cytokines. In addition, they detected a decrease in the expression of MMP9 and MMP3, two matrix metalloproteinases with an important function for remodelling of the extracellular matrix, several blastema and dedifferentiation markers and the TGF-beta pathway. After macrophage ablation proliferation is also strongly reduced within the stump mesenchyme. Therefore, the authors conclude that depleting the macrophages before amputation disrupts specific gene pathways required for the progression from wound healing to regeneration. In addition, after macrophage depletion those animals displayed fibrotic stumps with extensive collagen I and IV deposition compared to controls, which suggests that macrophages are also regulators of these matrix degrading enzymes. This scar tissue became permanent even after 90 days, despite macrophage repopulation takes place after 2 weeks of treatment. Remarkably, the re-amputation of such limb stumps after macrophage repopulation results in a successful regeneration of a limb.
In summary the authors propose that in salamanders the macrophages are required to orchestrate the early response to injury and activate the limb regeneration program, probably by promoting cell dedifferentiation and the proliferation of progenitor cells needed to rebuild the missing tissues. It remains for future studies whether the early induction of anti-inflammatory factors could be used as therapy for promoting scar-free healing in non-regenerating animals and the subsequent activation of a successful regenerative program.