Identification of the Adult Hematopoietic Liver as the Primary Reservoir for the Recruitment of Pro-regenerative Macrophages Required for Salamander Limb Regeneration.

The lack of scar-free healing and regeneration in many adult human tissues imposes severe limitations on the recovery of function after injury. In stark contrast, salamanders can functionally repair a range of clinically relevant tissues throughout adult life. The impressive ability to regenerate whole limbs after amputation, or regenerate following cardiac injury, is critically dependent on the recruitment of (myeloid) macrophage white blood cells to the site of injury. Amputation in the absence of macrophages results in regeneration failure and scar tissue induction. Identifying the exact hematopoietic source or reservoir of myeloid cells supporting regeneration is a necessary step in characterizing differences in macrophage phenotypes regulating scarring or regeneration across species. Mammalian wounds are dominated by splenic-derived monocytes that originate in the bone marrow and differentiate into macrophages within the wound. Unlike mammals, adult axolotls do not have functional bone marrow but instead utilize liver and spleen tissues as major sites for adult hematopoiesis. To interrogate leukocyte identity, tissue origins, and modes of recruitment, we established several transgenic axolotl hematopoietic tissue transplant models and flow cytometry protocols to study cell migration and identify the source of pro-regenerative macrophages. We identified that although bidirectional trafficking of leukocytes can occur between spleen and liver tissues, the liver is the major source of leukocytes recruited to regenerating limbs. Recruitment of leukocytes and limb regeneration occurs in the absence of the spleen, thus confirming the dependence of liver-derived myeloid cells in regeneration and that splenic maturation is dispensable for the education of pro-regenerative macrophages. This work provides an important foundation for understanding the hematopoietic origins and education of myeloid cells recruited to, and essential for, adult tissue regeneration.

Mechanism of Action of Secreted Newt Anterior Gradient Protein.

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.

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.

Age-related changes in tissue macrophages precede cardiac functional impairment.

Cardiac tissue macrophages (cTMs) are abundant in the murine heart but the extent to which the cTM phenotype changes with age is unknown. This study characterizes aging-dependent phenotypic changes in cTM subsets. Using theCx3cr1(GFP/+) mouse reporter line where GFP marks cTMs, and the tissue macrophage marker Mrc1, we show that two major cardiac tissue macrophage subsets, Mrc1-GFP(hi) and Mrc1+GFP(hi) cTMs, are present in the young (<10 week old) mouse heart, and a third subset, Mrc1+GFP(lo), comprises ~50% of total Mrc1+ cTMs from 30 weeks of age. Immunostaining and functional assays show that Mrc1+ cTMs are the principal myeloid sentinels in the mouse heart and that they retain proliferative capacity throughout life. Gene expression profiles of the two Mrc1+ subsets also reveal that Mrc1+GFP(lo) cTMs have a decreased number of immune response genes (Cx3cr1, Lpar6, CD9, Cxcr4, Itga6 and Tgfßr1), and an increased number of fibrogenic genes (Ltc4s, Retnla, Fgfr1, Mmp9 and Ccl24), consistent with a potential role for cTMs in cardiac fibrosis. These findings identify early age-dependent gene expression changes in cTMs, with significant implications for cardiac tissue injury responses and aging-associated cardiac fibrosis.

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 Cx(3)cr1(GFP/+) 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 Cx(3)cr1(GFP/+) 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.

Characterization of pig intercellular adhesion molecule-2 and its interaction with human LFA-1.

Understanding molecular interactions between human leukocytes and porcine endothelium is important for the future success of pig-to-human xenotransplantation. Here we describe the analysis of pig intercellular adhesion molecule-2 (ICAM-2). A 1020-basepair ICAM-2 cDNA generated from pig lung RNA contained an open reading frame (ORF) encoding a 277-amino-acid protein with six potential N-linked glycosylation sites. The mature protein sequence was 55% identical to human ICAM-2, with conservation of five out of six residues critical for binding of the human protein to its ligand LFA-1. Northern blot analysis identified ICAM-2 transcripts of 4.0 and 1.4 kb in cultured pig endothelial cells and mRNA was detected in pig lung, spleen, kidney, liver and heart by RT-PCR. The gene structure and endothelial expression of pig ICAM-2 were strikingly similar to those of its human and mouse counterparts. However, unlike human ICAM-2, expression of pig ICAM-2 on cultured endothelial cells was not down-regulated by treatment with the inflammatory cytokines TNF-alpha and IL-1beta. Pig ICAM-2 expressed on stable transfectants supported firm adhesion of cells expressing human LFA-1. This conservation of function across the species barrier suggests that pig ICAM-2 plays a role in the cellular interactions associated with xenograft rejection.