Distinct toll-like receptor signaling in the salamander response to tissue damage.

BACKGROUND: Efficient wound healing or pathogen clearance both rely on balanced inflammatory responses. Inflammation is essential for effective innate immune-cell recruitment; however, excessive inflammation will result in local tissue destruction, pathogen egress, and ineffective pathogen clearance. Sterile and nonsterile inflammation operate with competing functional priorities but share common receptors and overlapping signal transduction pathways. In regenerative organisms such as the salamander, whole limbs can be replaced after amputation while exposed to a nonsterile environment. In mammals, exposure to sterile-injury Damage Associated Molecular Patterns (DAMPS) alters innate immune-cell responsiveness to secondary Pathogen Associated Molecular Pattern (PAMP) exposure. RESULTS: Using new phospho-flow cytometry techniques to measure signaling in individual cell subsets we compared mouse to salamander inflammation. These studies demonstrated evolutionarily conserved responses to PAMP ligands through toll-like receptors (TLRs) but identified key differences in response to DAMP ligands. Co-exposure of macrophages to DAMPs/PAMPs suppressed MAPK signaling in mammals, but not salamanders, which activate sustained MAPK stimulation in the presence of endogenous DAMPS. CONCLUSIONS: These results reveal an alternative signal transduction network compatible with regeneration that may ultimately lead to the promotion of enhanced tissue repair in mammals.

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.

The Use of Live Cell Imaging and Automated Image Analysis to Assist With Determining Optimal Parameters for Angiogenic Assay in vitro.

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.

Methods for axolotl blood collection, intravenous injection, and efficient leukocyte isolation from peripheral blood and the regenerating limb.

The vertebrate immune system comprises both adaptive and innate immune cells with distinct functions during the resolution of inflammation and wound healing after tissue injury. Recent evidence implicates a requirement for innate immune cells from the myeloid lineage during the early stages of limb regeneration in the Mexican axolotl. Understanding the functions of innate and adaptive immune cells in the axolotl has been hampered by a lack of approaches to isolate and analyze these cells. Here we describe a protocol to isolate myeloid cells from the regenerating axolotl limb that incorporates intravenous delivery of physiological labels. In addition we provide a protocol to enrich for leukocytes in the peripheral blood. These protocols produce single-cell suspensions that can be analyzed using flow cytometry or sorted into specific subsets using fluorescent-activated cell sorting (FACS). FACS is a routine approach to sort cells based on their physical characteristics as well as their cell surface antigen repertoire. Isolated cell populations can then be analyzed in a wide range of downstream assays to facilitate a greater understanding of leukocyte biology in the axolotl.