James W. Godwin

Macrophages are required for adult salamander limb regeneration

The failure to replace damaged body parts in adult mammals results from a muted growth response and fibrotic scarring. Although infiltrating immune cells play a major role in determining the variable outcome of mammalian wound repair, little is known about the modulation of immune cell signaling in efficiently regenerating species such as the salamander, which can regrow complete body structures as adults. Here we present a comprehensive analysis of immune signaling during limb regeneration in axolotl, an aquatic salamander, and reveal a temporally defined requirement for macrophage infiltration in the regenerative process. Although many features of mammalian cytokine/chemokine signaling are retained in the axolotl, they are more dynamically deployed, with simultaneous induction of inflammatory and anti-inflammatory markers within the first 24 h after limb amputation. Systemic macrophage depletion during this period resulted in wound closure but permanent failure of limb regeneration, associated with extensive fibrosis and disregulation of extracellular matrix component gene expression. Full limb regenerative capacity of failed stumps was restored by reamputation once endogenous macrophage populations had been replenished. Promotion of a regeneration-permissive environment by identification of macrophage-derived therapeutic molecules may therefore aid in the regeneration of damaged body parts in adult mammals.

An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP+ population within the adult Cx3cr1GFP/+ knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45+CD11b+GFP+) are distinct from mononuclear CD45+CD11b+GFP+ cells sorted from the spleen and brain of adult Cx3cr1GFP/+ mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

Regeneration, tissue injury and the immune response

The involvement of the immune system in the response to tissue injury has raised the possibility that it might influence tissue, organ or appendage regeneration following injury. One hypothesis that has been discussed is that inflammatory aspects may preclude the occurrence of regeneration, but there is also evidence for more positive roles of immune components. The vertebrate eye is an immunoprivileged site where inflammatory aspects are inhibited by several immunomodulatory mechanisms. In various newt species the ocular tissues such as the lens are regenerative and it has recently been shown that the response to local injury of the lens involves activation of antigen‐presenting cells which traffic to the spleen and return to displace and engulf the lens, thereby inducing regeneration from the dorsal iris. The activation of thrombin from prothrombin in the dorsal iris is one aspect of the injury response that is important in the initiation of regeneration. The possible relationships between the immune response and the regenerative response are considered with respect to phylogenetic variation of regeneration in general, and lens regeneration in particular.

Scar-free wound healing and regeneration in amphibians: immunological influences on regenerative success.

Salamanders and frogs are distinct orders of Amphibians with very different immune systems during adult life, exhibiting varying potential for scar free repair and regeneration. While salamanders can regenerate a range of body parts throughout all stages of life, regeneration is restricted to early stages of frog development. Comparison of these two closely related amphibian orders provides insights into the immunological influences on wound repair, and the different strategies that have evolved either to limit infection or to facilitate efficient regeneration. After injury, cells of the immune system are responsible for the removal of damaged cells and providing a cohort of important growth factors and signaling molecules. Immune cells not only regulate new vessel growth important for supplying essential nutrients to damaged tissue but, modulate the extracellular matrix environment by regulating fibroblasts and the scarring response. The profile of immune cell infiltration and their interaction with local tissue immune cells directly influences many aspects of the wound healing outcomes and can facilitate or prevent regeneration. Evidence is emerging that the transition from wound healing to regeneration is reliant on immune cell engagement and that the success of regeneration in amphibians may depend on complex interactions between stem cell progenitors and immune cell subsets. The potential immunological barriers to mammalian regeneration are discussed with implications for the successful delivery of stem cell therapeutic strategies in patients.

Macrophages in cardiac homeostasis, injury responses and progenitor cell mobilisation.

Macrophages are an immune cell type found in every organ of the body. Classically, macrophages are recognised as housekeeping cells involved in the detection of foreign antigens and danger signatures, and the clearance of tissue debris. However, macrophages are increasingly recognised as a highly versatile cell type with a diverse range of functions that are important for tissue homeostasis and injury responses. Recent research findings suggest that macrophages contribute to tissue regeneration and may play a role in the activation and mobilisation of stem cells. This review describes recent advances in our understanding of the role played by macrophages in cardiac tissue maintenance and repair following injury. We examine the involvement of exogenous and resident tissue macrophages in cardiac inflammatory responses and their potential activity in regulating cardiac regeneration.

Extracellular matrix considerations for scar-free repair and regeneration: insights from regenerative diversity among vertebrates.

The extracellular matrix (ECM) is an essential feature of development, tissue homeostasis and recovery from injury. How the ECM responds dynamically to cellular and soluble components to support the faithful repair of damaged tissues in some animals but leads to the formation of acellular fibrotic scar tissue in others has important clinical implications. Studies in highly regenerative organisms such as the zebrafish and the salamander have revealed a specialist formulation of ECM components that support repair and regeneration, while avoiding scar tissue formation. By comparing a range of different contexts that feature scar-less healing and full regeneration vs. scarring through fibrotic repair, regenerative therapies that incorporate ECM components could be significantly enhanced to improve both regenerative potential and functional outcomes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.

Chasing the recipe for a pro-regenerative immune system.

Identification of the key ingredients and essential processes required to achieve perfect tissue regeneration in humans has so far remained elusive. Injury in vertebrates induces an obligatory wound response that will precede or overlap any regeneration specific program or scarring outcome. This process shapes the cellular and molecular landscape of the tissue, influencing the success of endogenous repair pathways or for potential clinical intervention. The involvement of immune cells is also required for aspects of development extending beyond the initial inflammatory phase of wounding. It has now become clear from amphibian, fish and mammalian models of tissue injury that the type of immune response and the profile of immune cells attending the site of injury can act as the gatekeepers that determine wound repair quality. The heterogeneity among innate and adaptive immune cell populations, along with the developmental origin of these cells, form key ingredients affecting the potential for downstream repair and the suppression of fibrosis. Cell-to-cell interactions between immune cells, such as macrophages and T cells, with stem cells and mesenchymal cells are critically important for shaping this process and these exchanges, are in turn influenced by the type of injury, tissue location and developmental stage of the organism. Developmentally, mouse cardiac regeneration is restricted to early stages of postnatal life where the balance of innate to adaptive immune cells may be poised towards regeneration. In the injured adult mouse liver, specific macrophage subsets improve repair while other bone marrow derived cells can exacerbate injury. Other studies using genetically diverse mice have shown enhanced regeneration in certain strains, restricted to specific tissues. This enhanced repair is linked with expression of genes such as Insulin-like Growth Factor- 1 (IGF-1) and activin (Act 1), that both play important roles in shaping the immune system. Immune cells are now appreciated to have powerful influences on critical cell types required for regeneration success. The winning recipe for tissue regeneration is likely to be found ultimately by identifying the genetic elements and specific cell populations that limit or allow intrinsic potential. This will be essential for developing therapeutic strategies for tissue regeneration in humans.

The promise of perfect adult tissue repair and regeneration in mammals: Learning from regenerative amphibians and fish

Regenerative medicine promises to greatly impact on human health by improving repair outcomes in a range of tissues and injury contexts. Successful therapies will rely on identifying both intrinsic and extrinsic biological circuits that control wound healing, proliferation, cell survival, and developmental cell fate. Animals such as the zebrafish and the salamander display powerful examples of near‐perfect regeneration and scar‐free healing in a range of injury contexts not attained in mammals. By studying regeneration in a range of highly regenerative species that maintain regenerative potential throughout life, many instructive and permissive factors have been identified that could assist in the development of regenerative therapies. This review highlights some of the recent observations in immune regulation, epigenetic regulation, stem cell mobilization, and regenerative signatures that have improved our understanding of the regenerative process. Potential opportunities in harnessing this knowledge for future translation into the clinic are discussed.

Isolation and analysis of single cells from the mouse heart

The adult mouse heart is comprised of a highly heterogeneous cell population. Isolation and effective cellular and molecular analysis of various cell types are critical for understanding cardiac development, homeostasis and disease. Moreover, strategies to isolate and analyse the complex inflammatory and tissue remodelling cell types that follow cardiac injury are particularly important for development of strategies to improve cardiac repair. Here we describe in detail how non-cardiomyocytes can be successfully isolated from the mouse heart. In addition, we describe how these isolation methods can be effectively coupled with flow cytometry, fluorescence activated cell sorting and/or magnetic-labelling to analyse and enrich cells for subsequent cellular or molecular analyses.

Tissue factor expression in newt iris coincides with thrombin activation and lens regeneration

Lens regeneration in adult salamanders occurs at the pupillary margin of the mid-dorsal iris where pigmented epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. It is not understood how the injury caused by removal of the lens (lentectomy) in one location is linked to initiating the response in a different spatial location (dorsal iris) and to this particular sector. We propose that the blood provides a link between the localised coagulation and signal transduction pathways that lead to regeneration. A transmembrane protein (tissue factor) is expressed in a striking patch-like domain in the dorsal iris of the newt that localises coagulation specifically to this location, but is not expressed in the axolotl, a related species that does not show thrombin activation after lentectomy and cannot regenerate its lens. Our hypothesis is that tissue factor expression localises the initiation of regeneration through the activation of thrombin and the recruitment of blood cells, leading to local growth factor release. This is the first example of gene expression in a patch of cells that prefigures the location of a regenerative response, and links the immune system with the initiation of a regenerative program.