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\"How do the billions of connections between neurons in our brain change as we learn and remember? This is the story of the discovery and the discoverer of synaptic pruning, the process of synapse elimination central to making us who we are. Taking the reader from Professor Peter Huttenlocher's childhood in wartime and post-war Germany to his emigration to the US to reunite with his mother and the launch and progress of a career in medicine and research, we uncover the motivations and process of scientific discovery that led to an unexpected leap in our understanding of the human brain. Decades after the discovery, the importance of synaptic pruning to early learning, autism, schizophrenia, Alzheimer's disease and other conditions are now in the process of being uncovered. Accessible examples illustrate how, decades later, researchers are discovering the importance of synaptic pruning in early learning, autism, schizophrenia and Alzheimer's disease.\" -- Back cover.

Neutrophils are critical for host defense against infection. Central to neutrophil function is the infiltration into damaged tissues mediated by a multistep process that requires the coordination between both immune and nonimmune cells. Among factors that facilitate neutrophil recruitment is the chemokine CXCL8, one of the most well-studied chemokines. Here, Hou and Huttenlocher discuss the role of models and tools in advancing chemokine research and recent advances in understanding new functions for CXCL8 and its receptors.

Key Points Complementary models have been developed to study neutrophil migration, including microfluidics and live imaging using mice and zebrafish. Neutrophil migration in response to injury or infection occurs in phases: early recruitment, amplification and resolution. Early-recruited neutrophils modulate the amplification phase both directly and indirectly through the activation of tissue and tissue-resident cells, producing sustained signals such as the CXC-chemokine ligand 8 family chemokines. Activated neutrophils at a site of inflammation do not necessarily undergo apoptosis but in some circumstances might undergo reverse migration away from the site of damage (reverse neutrophil migration) and/or re-enter the circulation (reverse transendothelial migration (rTEM)). Neutrophil forward and reverse migration may be attractive targets for anti-inflammatory therapies. Neutrophils follow a multitude of signals to reach sites of injury or infection. Understanding how this occurs and what the fate of these neutrophils is provides insight into how immune responses are controlled and chronic inflammation is avoided. In this Review, the authors describe the movement of neutrophils during inflammation. Neutrophil migration and its role during inflammation has been the focus of increased interest in the past decade. Advances in live imaging and the use of new model systems have helped to uncover the behaviour of neutrophils in injured and infected tissues. Although neutrophils were considered to be short-lived effector cells that undergo apoptosis in damaged tissues, recent evidence suggests that neutrophil behaviour is more complex and, in some settings, neutrophils might leave sites of tissue injury and migrate back into the vasculature. The role of reverse migration and its contribution to resolution of inflammation remains unclear. In this Review, we discuss the different cues within tissues that mediate neutrophil forward and reverse migration in response to injury or infection and the implications of these mechanisms to human disease.

Candida auris has recently emerged as the first fungal pathogen to cause a global public health threat. The reason this species is causing hospital-associated outbreaks of invasive candidiasis with high mortality is unknown. In this study, we examine the interaction of C. auris with neutrophils, leukocytes critical for control of invasive fungal infections. We show that human neutrophils do not effectively kill C. auris . Compared to Candida albicans , neutrophils poorly recruited to C. auris and failed to form neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. In mixed cultures, neutrophils preferentially engaged and killed C. albicans over C. auris . Imaging of neutrophils in a zebrafish larval model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris compared to C. albicans . Upon encounter with C. albicans in the zebrafish hindbrain, neutrophils produced clouds of histones, suggesting the formation of NETs. These structures were not observed in C. auris infection. Evasion of neutrophil attack and innate immunity offers an explanation for the virulence of this pathogen. IMPORTANCE The emerging fungal pathogen Candida auris has produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to kill C. auris compared to the most commonly encountered Candida species, C. albicans . While neutrophils produce neutrophil extracellular traps (NETs) upon encounter with C. albicans , these antimicrobial structures are not formed in response to C. auris . Using human neutrophils and a zebrafish model of invasive candidiasis, we show that C. auris poorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response to C. auris sheds light on the dismal outcomes for patients with invasive disease. The emerging fungal pathogen Candida auris has produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to kill C. auris compared to the most commonly encountered Candida species, C. albicans . While neutrophils produce neutrophil extracellular traps (NETs) upon encounter with C. albicans , these antimicrobial structures are not formed in response to C. auris . Using human neutrophils and a zebrafish model of invasive candidiasis, we show that C. auris poorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response to C. auris sheds light on the dismal outcomes for patients with invasive disease.

Neutrophils are cells at the frontline of innate immunity that can quickly activate effector functions to eliminate pathogens upon stimulation. However, little is known about the metabolic adaptations that power these functions. Here we show rapid metabolic alterations in neutrophils upon activation, particularly drastic reconfiguration around the pentose phosphate pathway, which is specifically and quantitatively coupled to an oxidative burst. During this oxidative burst, neutrophils switch from glycolysis-dominant metabolism to a unique metabolic mode termed ‘pentose cycle’, where all glucose-6-phosphate is diverted into oxidative pentose phosphate pathway and net flux through upper glycolysis is reversed to allow substantial recycling of pentose phosphates. This reconfiguration maximizes NADPH yield to fuel superoxide production via NADPH oxidase. Disruptions of pentose cycle greatly suppress oxidative burst, the release of neutrophil extracellular traps and pathogen killing by neutrophils. Together, these results demonstrate the remarkable metabolic flexibility of neutrophils, which is essential for their functions as the first responders in innate immunity. Upon activation, neutrophils undergo rapid metabolic reconfigurations towards pentose cycling, which powers their effector functions.

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.

Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas(V12). Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas(V12) using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas(V12) transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas(V12) in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

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.

The veterinary sedative and alpha-2 adrenergic receptor (α2AR) agonist xylazine, found in the illicit opioid supply, is associated with cutaneous wounds in humans. Here, we developed a larval zebrafish model of xylazine-induced tissue damage to investigate the mechanisms by which xylazine affects the skin. Xylazine treatment caused keratinocyte extrusion, tissue-wide skin contraction and disruption of basal keratinocyte cell–cell interactions in zebrafish larvae. Notably, other α2AR agonists did not recapitulate most of these effects. Xylazine was recently described as a kappa opioid receptor (κOR) agonist, and we found that both xylazine and a separate κOR agonist acted directly on epithelial cells to drive cellular contraction and disrupt tissue homeostasis. Our model suggests that xylazine disrupts skin homeostasis through a direct mechanism involving epithelial cells and κOR, which may be of importance for the treatment of these wounds.

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.