Motile Collectors: Platelets Promote Innate Immunity.

Platelets migrate in vitro but the significance of platelet migration in vivo remains unclear. In a recent issue of Cell, Gaertner et al. (2017) demonstrate that active platelet migration in vivo promotes mechano-scavenging of bacterial pathogens and neutrophil activation.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

The cell's dilemma: resolving directional decisions.

How neutrophils make decisions about polarity and migration path in complex tissue environments in situ remains unclear. Hadjitheodorou et al. describe how an internal mechanical regulator might help cells resolve the dilemma of two competing cell fronts.

Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Nonmammalian jawed vertebrates lack lymph nodes but maintain diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing nonhematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen-presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ. This lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system.

Real-time imaging of inflammation and its resolution: It's apparent because it's transparent.

The ability to directly observe leukocyte behavior in vivo has dramatically expanded our understanding of the immune system. Zebrafish are particularly amenable to the high-resolution imaging of leukocytes during both homeostasis and inflammation. Due to its natural transparency, intravital imaging in zebrafish does not require any surgical manipulation. As a result, zebrafish are particularly well-suited for the long-term imaging required to observe the temporal and spatial events during the onset and resolution of inflammation. Here, we review major insights about neutrophil and macrophage function gained from real-time imaging of zebrafish. We discuss neutrophil reverse migration, the process whereby neutrophils leave sites of tissue damage and resolve local inflammation. Further, we discuss the current tools available for investigating immune function in zebrafish and how future studies that simultaneously image multiple leukocyte subsets can be used to further dissect mechanisms that regulate both the onset and resolution of inflammation.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Neutrophil and Macrophage NADPH Oxidase 2 Differentially Control Responses to Inflammation and to Aspergillus fumigatus in Mice.

Aspergillus fumigatus is an important opportunistic fungal pathogen and causes invasive pulmonary aspergillosis in conditions with compromised innate antifungal immunity, including chronic granulomatous disease, which results from inherited deficiency of the superoxide-generating leukocyte NADPH oxidase 2 (NOX2). Derivative oxidants have both antimicrobial and immunoregulatory activity and, in the context of A. fumigatus, contribute to both fungal killing and dampening inflammation induced by fungal cell walls. As the relative roles of macrophage versus neutrophil NOX2 in the host response to A. fumigatus are incompletely understood, we studied mice with conditional deletion of NOX2. When NOX2 was absent in alveolar macrophages as a result of LysM-Cre-mediated deletion, germination of inhaled A. fumigatus conidia was increased. Reducing NOX2 activity specifically in neutrophils via S100a8 (MRP8)-Cre also increased fungal burden, which was inversely proportional to the level of neutrophil NOX2 activity. Moreover, diminished NOX2 in neutrophils synergized with corticosteroid immunosuppression to impair lung clearance of A. fumigatus. Neutrophil-specific reduction in NOX2 activity also enhanced acute inflammation induced by inhaled sterile fungal cell walls. These results advance understanding into cell-specific roles of NOX2 in the host response to A. fumigatus. We show that alveolar macrophage NOX2 is a nonredundant effector that limits germination of inhaled A. fumigatus conidia. In contrast, reducing NOX2 activity only in neutrophils is sufficient to enhance inflammation to fungal cell walls as well as to promote invasive A. fumigatus. This may be relevant in clinical settings with acquired defects in NOX2 activity due to underlying conditions, which overlap risk factors for invasive aspergillosis.

Anomalous diffusion and asymmetric tempering memory in neutrophil chemotaxis.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca2+ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric 'memory' of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration.

Neutrophil migration is essential for inflammatory responses to kill pathogens; however, excessive neutrophilic inflammation also leads to tissue injury and adverse effects. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified 8 miRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (Cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Our results therefore reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide directions in alleviating systemic inflammation.

Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish.

Neutrophils are primary phagocytes of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections, including those by the generally non-pathogenic fungus Aspergillus nidulans The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response to Aspergillus nidulans In a wild-type host, A. nidulans germinates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasive A. nidulans infection despite robust neutrophil infiltration. Expression of subunit p22phox (officially known as CYBA), specifically in neutrophils, does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasive A. nidulans infection.

Neutrophil plasticity in the tumor microenvironment.

Neutrophils act as the body's first line of defense against infection and respond to diverse inflammatory cues, including cancer. Neutrophils display plasticity, with the ability to adapt their function in different inflammatory contexts. In the tumor microenvironment, neutrophils have varied functions and have been classified using different terms, including N1/N2 neutrophils, tumor-associated neutrophils, and polymorphonuclear neutrophil myeloid-derived suppressor cells (PMN-MDSCs). These populations of neutrophils are primarily defined by their functional phenotype, because few specific cell surface markers have been identified. In this review, we will discuss neutrophil polarization and plasticity and the function of proinflammatory/anti-inflammatory and protumor/antitumor neutrophils in the tumor microenvironment. We will also discuss how neutrophils with the ability to suppress T-cell activation, referred to by some as PMN-MDSCs, fit into this paradigm.

Filopodia and focal adhesions: An integrated system driving branching morphogenesis in neuronal pathfinding and angiogenesis.

Single cell branching during development in vertebrates is typified by neuronal branching to form neurites and vascular branches formed by sprouting angiogenesis. Neurons and endothelial tip cells possess subcellular protrusions that share many common features from the morphological to the molecular level. Both systems utilize filopodia as their cellular protrusion organelles and depend on specific integrin-mediated adhesions to the local extracellular matrix for guidance in their pathfinding. We discuss the similar molecular machineries involved in these two types of cell branch formation and use their analogy to propose a new mechanism for angiogenic filopodia function, namely as adhesion assembly sites. In support of this model we provide primary data of angiogenesis in zebrafish in vivo showing that the actin assembly factor VASP participates in both filopodia formation and adhesion assembly at the base of the filopodia, enabling forward progress of the tip cell. The use of filopodia and their associated adhesions provide a common mechanism for neuronal and endothelial pathfinding during development in response to extracellular matrix cues.

Efficacy of Voriconazole against Aspergillus fumigatus Infection Depends on Host Immune Function.

Antifungal therapy can fail in a remarkable number of patients with invasive fungal disease, resulting in significant morbidity worldwide. A major contributor to this failure is that while these drugs have high potency in vitro, we do not fully understand how they work inside infected hosts. Here, we used a transparent larval zebrafish model of Aspergillus fumigatus infection amenable to real-time imaging of invasive disease as an in vivo intermediate vertebrate model to investigate the efficacy and mechanism of the antifungal drug voriconazole. We found that the ability of voriconazole to protect against A. fumigatus infection depends on host innate immune cells and, specifically, on the presence of macrophages. While voriconazole inhibits fungal spore germination and growth in vitro, it does not do so in larval zebrafish. Instead, live imaging of whole, intact larvae over a multiday course of infection revealed that macrophages slow down initial fungal growth, allowing voriconazole time to target and kill A. fumigatus hyphae postgermination. These findings shed light on how antifungal drugs such as voriconazole may synergize with the immune response in living hosts.

Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing.

In immunocompromised individuals, Aspergillus fumigatus causes invasive fungal disease that is often difficult to treat. Exactly how immune mechanisms control A. fumigatus in immunocompetent individuals remains unclear. Here, we use transparent zebrafish larvae to visualize and quantify neutrophil and macrophage behaviors in response to different A. fumigatus strains. We find that macrophages form dense clusters around spores, establishing a protective niche for fungal survival. Macrophages exert these protective effects by inhibiting fungal germination, thereby inhibiting subsequent neutrophil recruitment and neutrophil-mediated killing. Germination directly drives fungal clearance as faster-growing CEA10-derived strains are killed better in vivo than slower-growing Af293-derived strains. Additionally, a CEA10 pyrG-deficient strain with impaired germination is cleared less effectively by neutrophils. Host inflammatory activation through Myd88 is required for killing of a CEA10-derived strain but not sufficient for killing of an Af293-derived strain, further demonstrating the role of fungal-intrinsic differences in the ability of a host to clear an infection. Altogether, we describe a new role for macrophages in the persistence of A. fumigatus and highlight the ability of different A. fumigatus strains to adopt diverse modes of virulence.

Interaction with an endothelial lumen increases neutrophil lifetime and motility in response to P aeruginosa.

Neutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.

Metformin modulates innate immune-mediated inflammation and early progression of NAFLD-associated hepatocellular carcinoma in zebrafish.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) is an increasing clinical problem associated with progression to hepatocellular carcinoma (HCC). The effect of a high-fat diet on the early immune response in HCC is poorly understood, while the role of metformin in treating NAFLD and HCC remains controversial. Herein, we visualized the early immune responses in the liver and the effect of metformin on progression of HCC using optically transparent zebrafish. METHODS: We used live imaging to visualize liver inflammation and disease progression in a NAFLD/NASH-HCC zebrafish model. We combined a high-fat diet with a transgenic zebrafish HCC model induced by hepatocyte-specific activated beta-catenin and assessed liver size, angiogenesis, micronuclei formation and inflammation in the liver. In addition, we probed the effects of metformin on immune cell composition and early HCC progression. RESULTS: We found that a high-fat diet induced an increase in liver size, enhanced angiogenesis, micronuclei formation and neutrophil infiltration in the liver. Although macrophage number was not affected by diet, a high-fat diet induced changes in macrophage morphology and polarization with an increase in liver associated TNFα-positive macrophages. Treatment with metformin altered macrophage polarization, reduced liver size and reduced micronuclei formation in NAFLD/NASH-associated HCC larvae. Moreover, a high-fat diet reduced T cell density in the liver, which was reversed by treatment with metformin. CONCLUSIONS: These findings suggest that diet alters macrophage polarization and exacerbates the liver inflammatory microenvironment and cancer progression in a zebrafish model of NAFLD/NASH-associated HCC. Metformin specifically affects the progression induced by diet and modulates the immune response by affecting macrophage polarization and T cell infiltration, suggesting possible effects of metformin on tumor surveillance. LAY SUMMARY: This paper reports a new zebrafish model that can be used to study the effects of diet on liver cancer. We found that a high-fat diet promotes non-resolving inflammation in the liver and enhances cancer progression. In addition, we found that metformin, a drug used to treat diabetes, inhibits high-fat diet-induced cancer progression in this model, by reducing diet-induced non-resolving inflammation and potentially restoring tumor surveillance.

Live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues.

Cell motility is required for diverse processes during immunity and inflammation. Classically, leukocyte motility is defined as an amoeboid type of migration, however some leukocytes, like macrophages, also employ a more mesenchymal mode of migration. Here, we sought to characterize the mechanisms that regulate neutrophil and macrophage migration in vivo by using real-time imaging of leukocyte motility within interstitial tissues in zebrafish larvae. Neutrophils displayed a rounded morphology and rapid protease-independent motility, lacked defined paxillin puncta, and had persistent rearward polarization of stable F-actin and the microtubule network. By contrast, macrophages displayed an elongated morphology with reduced speed and increased directional persistence and formed paxillin-containing puncta but had a less-defined polarization of the microtubule and actin networks. We also observed differential effects of protease inhibition, microtubule disruption and ROCK inhibition on the efficiency of neutrophil and macrophage motility. Taken together, our findings suggest that larval zebrafish neutrophils and macrophage display distinct modes of migration within interstitial tissues in vivo.

Neutrophils in the Tumor Microenvironment.

Neutrophils are the first responders to sites of acute tissue damage and infection. Recent studies suggest that in addition to neutrophil apoptosis, resolution of neutrophil inflammation at wounds can be mediated by reverse migration from tissues and transmigration back into the vasculature. In settings of chronic inflammation, neutrophils persist in tissues, and this persistence has been associated with cancer progression. However, the role of neutrophils in the tumor microenvironment remains controversial, with evidence for both pro- and anti-tumor roles. Here we review the mechanisms that regulate neutrophil recruitment and resolution at sites of tissue damage, with a specific focus on the tumor microenvironment. We discuss the current understanding as to how neutrophils alter the tumor microenvironment to support or hinder cancer progression, and in this context outline gaps in understanding and important areas of inquiry.

A physician-scientist preceptorship in clinical and translational research enhances training and mentorship.

BACKGROUND: Dual degree program MD/PhD candidates typically train extensively in basic science research and in clinical medicine, but often receive little formal experience or mentorship in clinical and translational research. METHODS: To address this educational and curricular gap, the University of Wisconsin Medical Scientist Training Program partnered with the University of Wisconsin Institute for Clinical and Translational Research to create a new physician-scientist preceptorship in clinical and translational research. This six-week apprentice-style learning experience-guided by a physician-scientist faculty mentor-integrates both clinical work and a translational research project, providing early exposure and hands-on experience with clinically oriented research and the integrated career of a physician-scientist. Five years following implementation, we retrospectively surveyed students and faculty members to determine the outcomes of this preceptorship. RESULTS: Over five years, 38 students and 36 faculty members participated in the physician-scientist preceptorship. Based on student self-assessments (n = 29, response rate 76%), the course enhanced competency in conducting translational research and understanding regulation of clinical research among other skills. Mentor assessments (n = 17, response rate 47%) supported the value of the preceptorship in these same areas. Based on work during the preceptorship, half of the students produced a peer-reviewed publication or a meeting abstract. At least eleven peer-reviewed manuscripts were generated. The preceptorship also provided a structure for physician-scientist mentorship in the students' clinical specialty of choice. CONCLUSION: The physician-scientist preceptorship provides a new curricular model to address the gap of clinical research training and provides for mentorship of physician-scientists during medical school. Future work will assess the long-term impact of this course on physician-scientist career trajectories.