Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil's ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.

Human erythrocyte fragmentation during ex-vivo pig organ perfusion.

Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex-vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet-sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in-vivo organ xenotransplant.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus.

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching.

Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.

Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provide evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death.

Autosomal recessively inherited glucocerebrosidase 1 (GBA1) mutations cause the lysosomal storage disorder Gaucher's disease (GD). Heterozygous GBA1 mutations (GBA1(+/-)) are the most common risk factor for Parkinson's disease (PD). Previous studies typically focused on the interaction between the reduction of glucocerebrosidase (enzymatic) activity in GBA1(+/-) carriers and alpha-synuclein-mediated neurotoxicity. However, it is unclear whether other mechanisms also contribute to the increased risk of PD in GBA1(+/-) carriers. The zebrafish genome does not contain alpha-synuclein (SNCA), thus providing a unique opportunity to study pathogenic mechanisms unrelated to alpha-synuclein toxicity. Here we describe a mutant zebrafish line created by TALEN genome editing carrying a 23 bp deletion in gba1 (gba1(c.1276_1298del)), the zebrafish orthologue of human GBA1. Marked sphingolipid accumulation was already detected at 5 days post-fertilization with accompanying microglial activation and early, sustained up-regulation of miR-155, a master regulator of inflammation. gba1(c.1276_1298del) mutant zebrafish developed a rapidly worsening phenotype from 8 weeks onwards with striking reduction in motor activity by 12 weeks. Histopathologically, we observed marked Gaucher cell invasion of the brain and other organs. Dopaminergic neuronal cell count was normal through development but reduced by >30% at 12 weeks in the presence of ubiquitin-positive, intra-neuronal inclusions. This gba1(c.1276_1298del) zebrafish line is the first viable vertebrate model sharing key pathological features of GD in both neuronal and non-neuronal tissue. Our study also provides evidence for early microglial activation prior to alpha-synuclein-independent neuronal cell death in GBA1 deficiency and suggests upregulation of miR-155 as a common denominator across different neurodegenerative disorders.

Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.

The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.

Real-time whole-body visualization of Chikungunya Virus infection and host interferon response in zebrafish.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.

Blockage of lysophosphatidic acid signaling improves spinal cord injury outcomes.

Evidence suggests a proinflammatory role of lysophosphatidic acid (LPA) in various pathologic abnormalities, including in the central nervous system. Herein, we describe LPA as an important mediator of inflammation after spinal cord injury (SCI) in zebrafish and mice. Furthermore, we describe a novel monoclonal blocking antibody raised against LPA that potently inhibits LPA's effect in vitro and in vivo. This antibody, B3, specifically binds LPA, prevents it from interacting with its complement of receptors, and blocks LPA's effects on the neuronal differentiation of human neural stem/progenitor cells, demonstrating its specificity toward LPA signaling. When administered systemically to mice subjected to SCI, B3 substantially reduced glial inflammation and neuronal death. B3-treated animals demonstrated significantly more neuronal survival upstream of the lesion site, with some functional improvement. This study describes the use of anti-LPA monoclonal antibody as a novel therapeutic approach for the treatment of SCI.

mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.

Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.

Passive redirection filters minimize red blood cell contamination during neutrophil chemotaxis assays using whole blood.

Neutrophils are the most numerous white blood cells and are the first to arrive at sites of inflammation and infection. Thus, neutrophil behavior provides a comprehensive biomarker for antimicrobial defenses. Several microfluidic tools have been developed to test neutrophil chemotaxis, phagocytosis, extrusion of extracellular traps, etc. Traditional tools rely on purified neutrophil samples, which require lengthy and expensive isolation procedures from large volumes of blood. In the absence of such isolation, visualizing neutrophils in blood is complicated by the overwhelming number of red blood cells (RBCs), which outnumber neutrophils by 1000 : 1. Recently, several microfluidic technologies have been designed to analyze neutrophils directly in blood, by separating neutrophils on selectin coated surfaces before the migration assay or blocking the advance of RBCs with the moving neutrophils. However, RBC contamination remains an issue, albeit with a reduced ratio, down to 1 : 1. Here, we present an RBC-debulking strategy for neutrophil assays based on microscale passive redirection filters (PRFs) that reduce RBC contamination down to as few as a 1 : 17 RBC to neutrophil ratio. We compare the performance of different PRF designs and measure changes in neutrophil chemotaxis velocity and directionality following immune stimulation of whole blood.

Leading edge competition promotes context-dependent responses to receptor inputs to resolve directional dilemmas in neutrophil migration.

Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.

Fusobacterium nucleatum dissemination by neutrophils.

Recent studies uncovered that Fusobacterium nucleatum (Fn), a common, opportunistic bacterium in the oral cavity, is associated with a growing number of systemic diseases, ranging from colon cancer to Alzheimer's disease. However, the pathological mechanisms responsible for this association are still poorly understood. Here, we leverage recent technological advances to study the interactions between Fn and neutrophils. We show that Fn survives within human neutrophils after phagocytosis. Using in vitro microfluidic devices, we determine that human neutrophils can protect and transport Fn over large distances. Moreover, we validate these observations in vivo by showing that neutrophils disseminate Fn using a zebrafish model. Our data support the emerging hypothesis that bacterial dissemination by neutrophils is a mechanistic link between oral and systemic diseases. Furthermore, our results may ultimately lead to therapeutic approaches that target specific host-bacteria interactions, including the dissemination process.

Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

Multifactorial assessment of neutrophil chemotaxis efficiency from a drop of blood.

Following injury and infection, neutrophils are guided to the affected site by chemoattractants released from injured tissues and invading microbes. During this process (chemotaxis), neutrophils must integrate multiple chemical signals, while also responding to physical constraints and prioritizing their directional decisions to generate an efficient immune response. In some clinical conditions, human neutrophils appear to lose the ability to chemotax efficiently, which may contribute both directly and indirectly to disease pathology. Here, a range of microfluidic designs is utilized to test the sensitivity of chemotaxing neutrophils to various perturbations, including binary decision-making in the context of channels with different chemoattractant gradients, hydraulic resistance, and angle of approach. Neutrophil migration in long narrow channels and planar environments is measured. Conditions in which neutrophils are significantly more likely to choose paths with the steepest chemoattractant gradient and the most direct approach angle, and find that migration efficiency across planar chambers is inversely correlated with chamber diameter. By sequential measurement of neutrophil binary decision-making to different chemoattractant gradients, or chemotactic index in sequential planar environments, data supporting a model of biased random walk for neutrophil chemotaxis are presented.

Microfluidic capture of chromatin fibres measures neutrophil extracellular traps (NETs) released in a drop of human blood.

Neutrophils are the largest population of white blood cells in the circulation, and their primary function is to protect the body from microbes. They can release the chromatin in their nucleus, forming characteristic web structures and trap microbes, contributing to antimicrobial defenses. The chromatin webs are known as neutrophil extracellular traps (NETs). Importantly, neutrophils can also release NETs in pathological conditions related to rheumatic diseases, atherosclerosis, cancer, and sepsis. Thus, determining the concentration of NETs in the blood is increasingly important for monitoring patients, evaluating treatment efficacy, and understanding the pathology of various diseases. However, traditional methods for measuring NETs require separating cells and plasma from blood, are prone to sample preparation artifacts, and cannot distinguish between intact and degraded NETs. Here, we design a microfluidic analytical tool that captures NETs mechanically from a drop of blood and measures the amount of intact NETs unbiased by the presence of degraded NETs in the sample.

Neutrophil profiles of pediatric COVID-19 and multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory illness characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigenemia, cytokine storm, and immune dysregulation. In severe COVID-19, neutrophil activation is central to hyperinflammatory complications, yet the role of neutrophils in MIS-C is undefined. Here, we collect blood from 152 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 78 pediatric controls. We find that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism that is distinct from the neutrophil interferon-stimulated gene (ISG) response we observe in pediatric COVID-19. Moreover, we observe extensive spontaneous neutrophil extracellular trap (NET) formation in MIS-C, and we identify neutrophil activation and degranulation signatures. Mechanistically, we determine that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Our findings suggest that hyperinflammatory presentation during MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia, driven by uncontrolled neutrophil activation and NET release in the vasculature.

Damage-Induced Calcium Signaling and Reactive Oxygen Species Mediate Macrophage Activation in Zebrafish.

Immediately after a wound, macrophages are activated and change their phenotypes in reaction to danger signals released from the damaged tissues. The cues that contribute to macrophage activation after wounding in vivo are still poorly understood. Calcium signaling and Reactive Oxygen Species (ROS), mainly hydrogen peroxide, are conserved early wound signals that emanate from the wound and guide neutrophils within tissues up to the wound. However, the role of these signals in the recruitment and the activation of macrophages is elusive. Here we used the transparent zebrafish larva as a tractable vertebrate system to decipher the signaling cascade necessary for macrophage recruitment and activation after the injury of the caudal fin fold. By using transgenic reporter lines to track pro-inflammatory activated macrophages combined with high-resolutive microscopy, we tested the role of Ca²âº and ROS signaling in macrophage activation. By inhibiting intracellular Ca²âº released from the ER stores, we showed that macrophage recruitment and activation towards pro-inflammatory phenotypes are impaired. By contrast, ROS are only necessary for macrophage activation independently on calcium. Using genetic depletion of neutrophils, we showed that neutrophils are not essential for macrophage recruitment and activation. Finally, we identified Src family kinases, Lyn and Yrk and NF-κB as key regulators of macrophage activation in vivo, with Lyn and ROS presumably acting in the same signaling pathway. This study describes a molecular mechanism by which early wound signals drive macrophage polarization and suggests unique therapeutic targets to control macrophage activity during diseases.