Cellular and molecular mechanisms of skin wound healing.

Wound healing is a complex process that involves the coordinated actions of many different tissues and cell lineages. It requires tight orchestration of cell migration, proliferation, matrix deposition and remodelling, alongside inflammation and angiogenesis. Whereas small skin wounds heal in days, larger injuries resulting from trauma, acute illness or major surgery can take several weeks to heal, generally leaving behind a fibrotic scar that can impact tissue function. Development of therapeutics to prevent scarring and successfully repair chronic wounds requires a fuller knowledge of the cellular and molecular mechanisms driving wound healing. In this Review, we discuss the current understanding of the different phases of wound healing, from clot formation through re-epithelialization, angiogenesis and subsequent scar deposition. We highlight the contribution of different cell types to skin repair, with emphasis on how both innate and adaptive immune cells in the wound inflammatory response influence classically studied wound cell lineages, including keratinocytes, fibroblasts and endothelial cells, but also some of the less-studied cell lineages such as adipocytes, melanocytes and cutaneous nerves. Finally, we discuss newer approaches and research directions that have the potential to further our understanding of the mechanisms underpinning tissue repair.

Differential Requirement of Gata2a and Gata2b for Primitive and Definitive Myeloid Development in Zebrafish.

Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model.

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling.

Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.

CXCL12a/CXCR4b acts to retain neutrophils in caudal hematopoietic tissue and to antagonize recruitment to an injury site in the zebrafish larva.

Neutrophils are a major component of the innate immune response and the most abundant circulating cell type in humans and zebrafish. The CXCL12/CXCR4 ligand receptor pair plays a key role in neutrophil homeostasis, controlling definitive hematopoiesis and neutrophil release into circulation. Neutrophils overexpressing CXCR4 respond by migrating towards sources of CXCL12, which is abundant in hematopoietic tissues. However, the physiological role of CXCL12/CXCR4 signaling during inflammatory responses remains unknown. Here, we show that zebrafish mutants lacking functional CXCL12a or CXCR4b show disrupted granulopoiesis in the kidney and increased number of circulating neutrophils. Additionally, CXCL12a and CXCR4b mutants display exacerbated recruitment of neutrophils to wounds and not to infections, and migrating neutrophils to wounds show increased directionality. Our results show that CXCL12a/CXCR4b signaling antagonizes wound-induced inflammatory signals by retaining neutrophils in hematopoietic tissues as a part of a balance between both inflammatory and anti-inflammatory cues, whose dynamic levels control neutrophils complex migratory behavior.

Macrophage Recruitment Contributes to Regeneration of Mechanosensory Hair Cells in the Zebrafish Lateral Line.

In vertebrates, damage to mechanosensory hair cells elicits an inflammatory response, including rapid recruitment of macrophages and neutrophils. While hair cells in amniotes usually become permanently lost, they readily regenerate in lower vertebrates such as fish. Damage to hair cells of the fish lateral line is followed by inflammation and rapid regeneration; however the role of immune cells in this process remains unknown. Here, we show that recruited macrophages are required for normal regeneration of lateral line hair cells after copper damage. We found that genetic ablation or local ablation using clodronate liposomes of macrophages recruited to the site of injury, significantly delays hair cell regeneration. Neutrophils, on the other hand, are not needed for this process. We anticipate our results to be a starting point for a more detailed description of extrinsic signals important for regeneration of mechanosensory cells in vertebrates. J. Cell. Biochem. 117: 1880-1889, 2016. © 2016 Wiley Periodicals, Inc.

Electroablation: a method for neurectomy and localized tissue injury.

BACKGROUND: Tissue injury has been employed to study diverse biological processes such as regeneration and inflammation. In addition to physical or surgical based methods for tissue injury, current protocols for localized tissue damage include laser and two-photon wounding, which allow a high degree of accuracy, but are expensive and difficult to apply. In contrast, electrical injury is a simple and inexpensive technique, which allows reproducible and localized cell or tissue damage in a variety of contexts. RESULTS: We describe a novel technique that combines the advantages of zebrafish for in vivo visualization of cells with those of electrical injury methods in a simple and versatile protocol which allows the study of regeneration and inflammation. The source of the electrical pulse is a microelectrode that can be placed with precision adjacent to specific cells expressing fluorescent proteins. We demonstrate the use of this technique in zebrafish larvae by damaging different cell types and structures. Neurectomy can be carried out in peripheral nerves or in the spinal cord allowing the study of degeneration and regeneration of nerve fibers. We also apply this method for the ablation of single lateral line mechanosensory neuromasts, showing the utility of this approach as a tool for the study of organ regeneration. In addition, we show that electrical injury induces immune cell recruitment to damaged tissues, allowing in vivo studies of leukocyte dynamics during inflammation within a confined and localized injury. Finally, we show that it is possible to apply electroablation as a method of tissue injury and inflammation induction in adult fish. CONCLUSIONS: Electrical injury using a fine microelectrode can be used for axotomy of neurons, as a general tissue ablation tool and as a method to induce a powerful inflammatory response. We demonstrate its utility to studies in both larvae and in adult zebrafish but we expect that this technique can be readily applied to other organisms as well. We have called this method of electrical based tissue ablation, electroablation.

Antiangiogenic, antimigratory and antiinflammatory effects of 2-methoxyestradiol in zebrafish larvae.

2-Methoxyestradiol (2ME), an endogenous metabolite of 17ß-estradiol, has been previously reported to possess antiangiogenic and antitumor properties. Herein, we demonstrate that the effects of this antiangiogenic steroid can be readily assayed in live zebrafish, introducing a convenient and robust new model system as a screening tool for both single cell and collective cell migration assays. Using the in vitro mammalian endothelial cell line EA.hy926, we first show that cell migration and angiogenesis, as estimated by wound assay and tube formation respectively, are antagonized by 2ME. In zebrafish (Danio rerio) larvae, dose-dependent exposure to 2ME diminishes (1) larval angiogenesis, (2) leukocyte recruitment to damaged lateral line neuromasts and (3) retards the lateral line primordium in its migration along the body. Our results indicate that 2ME has an effect on collective cell migration in vivo as well as previously reported anti-tumorigenic activity and suggests that the molecular mechanisms governing cell migration in a variety of contexts are conserved between fish and mammals. Moreover, we exemplify the versatility of the zebrafish larvae for testing diverse physiological processes and screening for antiangiogenic and antimigratory drugs in vivo.

A high-throughput chemically induced inflammation assay in zebrafish.

BACKGROUND: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. RESULTS: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. CONCLUSIONS: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.