Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration

Tissue damage induces early recruitment of neutrophils through redox-regulated Src family kinase (SFK) signaling in neutrophils. Redox-SFK signaling in epithelium is also necessary for wound resolution and tissue regeneration. How neutrophil-mediated inflammation resolves remains unclear. In this paper, we studied the interactions between macrophages and neutrophils in response to tissue damage in zebrafish and found that macrophages contact neutrophils and induce resolution via neutrophil reverse migration. We found that redox-SFK signaling through p22phox and Yes-related kinase is necessary for macrophage wound attraction and the subsequent reverse migration of neutrophils. Importantly, macrophage-specific reconstitution of p22phox revealed that macrophage redox signaling is necessary for neutrophil reverse migration. Thus, redox-SFK signaling in adjacent tissues is essential for coordinated leukocyte wound attraction and repulsion through pathways that involve contact-mediated guidance.     

Treatment of Glucocorticoids Inhibited Early Immune Responses and Impaired Cardiac Repair in Adult Zebrafish

Myocardial injury, such as myocardial infarction (MI), can lead to drastic heart damage. Zebrafish have the extraordinary ability to regenerate their heart after a severe injury. Upon ventricle resection, fibrin clots seal the wound and serve as a matrix for recruiting myeloid-derived phagocytes. Accumulated neutrophils and macrophages not only reduce the risk of infection but also secrete cytokines and growth factors to promote tissue repair. However, the underlying cellular and molecular mechanisms for how immune responses are regulated during the early stages of cardiac repair are still unclear. We investigated the role and programming of early immune responses during zebrafish heart regeneration. We found that zebrafish treated with an anti-inflammatory glucocorticoid had significantly reduced heart regenerative capacities, consistent with findings in other higher vertebrates. Moreover, inhibiting the inflammatory response led to excessive collagen deposition. A microarray approach was used to assess the differential expression profiles between zebrafish hearts with normal or impaired healing. Combining cytokine profiling and immune-staining, our data revealed that impaired heart regeneration could be due to reduced phagocyte recruitment, leading to diminished angiogenesis and cell proliferation post-cardiac injury. Despite their robust regenerative ability, our study revealed that glucocorticoid treatment could effectively hinder cardiac repair in adult zebrafish by interfering with the inflammatory response. Our findings may help to clarify the initiation of cardiac repair, which could be used to develop a therapeutic intervention that may enhance cardiac repair in humans to compensate for the loss of cardiomyocytes after an MI.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Mathematical model of a three-stage innate immune response to a pneumococcal lung infection

Pneumococcal pneumonia is a leading cause of death and a major source of human morbidity. The initial immune response plays a central role in determining the course and outcome of pneumococcal disease. We combine bacterial titer measurements from mice infected with Streptococcus pneumoniae with mathematical modeling to investigate the coordination of immune responses and the effects of initial inoculum on outcome. To evaluate the contributions of individual components, we systematically build a mathematical model from three subsystems that describe the succession of defensive cells in the lung: resident alveolar macrophages, neutrophils and monocyte-derived macrophages. The alveolar macrophage response, which can be modeled by a single differential equation, can by itself rapidly clear small initial numbers of pneumococci. Extending the model to include the neutrophil response required additional equations for recruitment cytokines and host cell status and damage. With these dynamics, two outcomes can be predicted: bacterial clearance or sustained bacterial growth. Finally, a model including monocyte-derived macrophage recruitment by neutrophils suggests that sustained bacterial growth is possible even in their presence. Our model quantifies the contributions of cytotoxicity and immune-mediated damage in pneumococcal pathogenesis.     

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.     

Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.     

Regulatory role of endogenous interleukin-10 in cutaneous inflammatory response of murine wound healing.

This study investigated the role of endogenous interleukin (IL)-10 in cutaneous wound healing. Both IL-10 mRNA and protein were detectable in murine incised wounds for 10 days after injury. The IL-10 protein level peaked 3 h after incision, returned to the normal level by 24 h, but increased again to another peak at 72 h. In situ hybridization studies and immunostaining revealed that epidermal cells and infiltrating mononuclear cells were the major source of IL-10. Neutralizing antibody studies demonstrated that IL-10 inhibited the infiltration of neutrophils and macrophages toward the site of injury. IL-10 also inhibited overexpression of C-C chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha) and proinflammatory cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha) in vivo. These results suggest that IL-10 may play an important regulatory role in the phase-specific infiltration of neutrophils and macrophages as well as the cytokine production in the inflammatory response of cutaneous wound healing.     

The cell biology of regeneration

Regeneration of complex structures after injury requires dramatic changes in cellular behavior. Regenerating tissues initiate a program that includes diverse processes such as wound healing, cell death, dedifferentiation, and stem (or progenitor) cell proliferation; furthermore, newly regenerated tissues must integrate polarity and positional identity cues with preexisting body structures. Gene knockdown approaches and transgenesis-based lineage and functional analyses have been instrumental in deciphering various aspects of regenerative processes in diverse animal models for studying regeneration.     

Tumor necrosis factor-alpha promotes the accumulation of neutrophils and macrophages in skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has been associated with cachexia and is known to regulate multiple inflammatory cell (neutrophil and macrophage) responses. We tested the hypothesis that neutrophils and macrophages accumulate in the extensor digitorum longus (EDL) and soleus muscles of mice after chronic TNF-alpha administration. Murine recombinant TNF-alpha (approximately 100 microg x kg(-1) x day(-1)) in vehicle solution or vehicle solution alone (sham) was administered to C57BL/6 mice for 7 days via osmotic minipumps. In EDL muscles from TNF-alpha-treated mice, neutrophil and macrophage concentrations were elevated seven- and threefold, respectively, compared with sham mice. Neutrophil and macrophage concentrations were also elevated five- and twofold, respectively, in solei of TNF-alpha- relative to sham-treated mice. Treatment with TNF-alpha elevated ubiquitin content by approximately 25% relative to sham values for both the EDL and soleus muscles; however, these elevations were not statistically significant. No differences were observed between TNF-alpha- and sham-treated mice in body weight, food consumption, muscle mass, myofiber cross-sectional area, carbonyl groups, total protein content, or relative abundance of myosin heavy chain protein. Furthermore, no overt signs of muscle injury or regeneration were observed in muscles from TNF-alpha-treated mice in either the EDL or soleus muscles. These observations suggest that 7 days of TNF-alpha administration promote muscle inflammation as indicated by the accumulation of neutrophils and macrophages without overt signs of atrophy, injury, or regeneration.     

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo. Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti‐angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor‐α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.     

Adriamycin promotes macrophage dysfunction in mice

Impaired wound healing contributes to the morbidity and mortality associated with adriamycin chemotherapy. Macrophages are essential for tissue repair and loss of macrophage function leads to impaired wound healing. We recently showed that adriamycin is a potent inducer of thiol oxidation and cell injury in cultured macrophages (FASEB J. 19:1866-1868; 2005). Here we tested the hypothesis that adriamycin also promotes oxidative stress and macrophage dysfunction in vivo. We treated FVB mice twice a week either with low doses of adriamycin (4 mg/kg) or with the same volume of saline by tail vein injection for a total of 8 injections. Wound healing was significantly delayed in adriamycin-treated mice. The number of resident peritoneal macrophages was decreased by 30% and macrophage recruitment in response to thiogycolate stimulation was decreased by 46% in mice treated with adriamycin. LPS-induced TNFalpha and IL-1beta secretion from macrophages of adriamycin-treated mice was decreased by 28.7 and 29.5%, respectively, compared to macrophages isolated from saline-injected mice. Peritoneal macrophages isolated from adriamycin-treated mice also showed increased formation of reactive oxygen species and enhanced protein-S-glutathionylation. In summary, our results show that low cumulative doses of adriamycin are sufficient both to promote sustained thiol oxidative stress and macrophage dysfunction in vivo and to delay tissue repair, suggesting that macrophage dysfunction contributes to impaired wound healing associated with adriamycin chemotherapy.     

Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.     

Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8

Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.     

Aberrant Innate Immune Activation following Tissue Injury Impairs Pancreatic Regeneration

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

The Resolution of Inflammation: A Mathematical Model of Neutrophil and Macrophage Interactions

There is growing interest in inflammation due to its involvement in many diverse medical conditions, including Alzheimer’s disease, cancer, arthritis and asthma. The traditional view that resolution of inflammation is a passive process is now being superceded by an alternative hypothesis whereby its resolution is an active, anti-inflammatory process that can be manipulated therapeutically. This shift in mindset has stimulated a resurgence of interest in the biological mechanisms by which inflammation resolves. The anti-inflammatory processes central to the resolution of inflammation revolve around macrophages and are closely related to pro-inflammatory processes mediated by neutrophils and their ability to damage healthy tissue. We develop a spatially averaged model of inflammation centring on its resolution, accounting for populations of neutrophils and macrophages and incorporating both pro- and anti-inflammatory processes. Our ordinary differential equation model exhibits two outcomes that we relate to healthy and unhealthy states. We use bifurcation analysis to investigate how variation in the system parameters affects its outcome. We find that therapeutic manipulation of the rate of macrophage phagocytosis can aid in resolving inflammation but success is critically dependent on the rate of neutrophil apoptosis. Indeed our model predicts that an effective treatment protocol would take a dual approach, targeting macrophage phagocytosis alongside neutrophil apoptosis.     

Neutrophil signaling during myocardial infarction wound repair

Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.     

Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration

Macrophages and neutrophils are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, which presumably facilitate the regrowth of injured tissues. Taking advantage of the regeneration abilities of zebrafish and the newly generated leukocyte-specific zebrafish lines with labeling of both leukocyte lineages, we assessed the behaviors and functions of neutrophils and macrophages during tail fin regeneration. Live imaging showed that within 6 hours post amputation, the inflammatory stage, neutrophils were the primary cells scavenging apoptotic bodies and small cell debris, although they had limited phagocytic capacity and quickly underwent apoptosis. From 6 hours post amputation on, the resolution and regeneration stage, macrophages became the dominant scavengers, efficiently resolving inflammation and facilitating tissue remodeling and regrowth. Ablation of macrophages but not neutrophils severely impaired the inflammatory resolution and tissue regeneration, resulting in the formation of large vacuoles in the regenerated fins. In contrast, removal of neutrophils slightly accelerates the regrowth of injured fin. Our study documents the differing behaviors and functions of macrophages and neutrophils during tissue regeneration.     

DNase I improves corneal epithelial and nerve regeneration in diabetic mice

DNase I has been reported to improve diabetic wound healing through the clearance of neutrophils extracellular traps (NETs) caused by neutrophil aggregation. However, the function of DNase I on diabetic corneal wound healing remains unclear. Here, we investigated the effect and mechanism of topical DNase I application on diabetic mouse corneal epithelial and nerve regeneration. Corneal epithelial defects, inflammatory response, regeneration‐related signalling pathways, oxidative stress, corneal innervation and sensation were examined and compared between the diabetic and normal mice. The results confirmed firstly the increased NETs production during the delayed corneal epithelial wound healing of diabetic mice, which was significantly improved through either DNase I or Cl‐amidine administration. Mechanistically, DNase I improved inflammation resolution, reactivated epithelial regeneration‐related signalling pathways and attenuated the accumulation of reactive oxygen species (ROS). Moreover, DNase I application also promoted corneal nerve regeneration and restored the impaired corneal sensitivity in diabetic mice. Therefore, these results indicate that topical DNase I application promotes corneal epithelial wound healing and mechanical sensation restoration in diabetic mice, representing the potential therapeutic approach for diabetic keratopathy.