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Staphylococcus aureus is a major pathogen associated with important diseases in humans and animals. Macrophages are a key component of the innate immune response to S. aureus infection and play a major role in disease outcomes. To investigate the adaptive evolution of S. aureus in response to macrophages, we developed an experimental infection assay. S. aureus strains representing major human epidemic clones were passaged many times in a macrophage cell line, accumulating mutations in an array of genomic loci. Phenotypic analysis revealed the emergence of a lineage exhibiting increased survival in macrophages and human blood, and resistance to vancomycin. The evolved lineage exhibited a previously undescribed small colony variant (SCV) phenotype characterized by hyper-pigmentation, which resulted from a missense mutation in rsbW . Notably, the novel SCV was a conditional adaptive trait that was unstable in nutrient-replete conditions in vitro , rapidly converting from hyper-pigmented SCV to a non-pigmented large colony variant via spontaneous sigB deletion events. Importantly, we identified similar deletions in the genome sequences of a limited number of clinical S. aureus isolates from public databases, indicating that related events may occur during clinical infection. Experimental infection of zebrafish did not reveal a difference in virulence between parent and novel SCV but demonstrated an in vivo fitness cost for the compensatory sigB deletion events. Taken together, we report an experimental evolutionary approach for investigating bacterial innate immune cell interactions, revealing a conditional adaptation that promotes S. aureus survival in macrophages and resistance to vancomycin., Importance: Staphylococcus aureus is an important human bacterial pathogen. The host response to S. aureus involves the production of innate immune cells such as macrophages which are important for fighting infection. Here we report a new model of experimental evolution for studying how S. aureus can evade killing by macrophages. We identified a novel adaptive phenotype that promotes survival in macrophages and blood and resistance to antibiotics. The phenotype is lost rapidly upon growth in nutrient-rich conditions via disruption of the alternative sigma factor sigB, revealing a conditional niche-specific fitness advantage. Genomic analysis of clinical isolates suggests similar adaptations may occur during human infections. Our model may be used broadly to identify adaptations of S. aureus to the innate immune response., Competing Interests: The authors declare no conflict of interest.

RNASET2-deficient leukodystrophy is a rare infantile white matter disorder mimicking a viral infection and resulting in severe psychomotor impairments. Despite its severity, there is little understanding of cellular mechanisms of pathogenesis and no treatments. Recent research using the rnaset2 mutant zebrafish model has suggested that microglia may be the drivers of the neuropathology, due to their failure to digest apoptotic debris during neurodevelopment. Therefore, we developed a strategy for microglial replacement through transplantation of adult whole kidney marrow-derived macrophages into embryonic hosts. Using live imaging, we revealed that transplant-derived macrophages can engraft within host brains and express microglia-specific markers, suggesting the adoption of a microglial phenotype. Tissue-clearing strategies revealed the persistence of transplanted cells in host brains beyond embryonic stages. We demonstrated that transplanted cells clear apoptotic cells within the brain, as well as rescue overactivation of the antiviral response otherwise seen in mutant larvae. RNA sequencing at the point of peak transplant-derived cell engraftment confirms that transplantation can reduce the brain-wide immune response and particularly, the antiviral response, in rnaset2-deficient brains. Crucially, this reduction in neuroinflammation resulted in behavioral rescue-restoring rnaset2 mutant motor activity to wild-type (WT) levels in embryonic and juvenile stages. Together, these findings demonstrate the role of microglia as the cellular drivers of neuropathology in rnaset2 mutants and that macrophage transplantation is a viable strategy for microglial replacement in the zebrafish. Therefore, microglia-targeted interventions may have therapeutic benefits in RNASET2-deficient leukodystrophy., Competing Interests: Competing interests statement:H.A.R. works as a consult for Vigil Neuroscience Inc. Vigil had no role in study design, data collection and interpretation, or the decision to submit this work for publication.

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for infections in people living with chronic respiratory conditions, such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Traditionally, in people with chronic respiratory disorders, P. aeruginosa infection has been managed with a combination of inhaled and intravenous antibiotic therapies. However, due in part to the prolonged use of antibiotics in these people, the emergence of multi-drug resistant P. aeruginosa strains is a growing concern. The development of anti-virulence therapeutics may provide a new means of treating P. aeruginosa lung infections whilst also combatting the AMR crisis, as these agents are presumed to exert reduced pressure for the emergence of drug resistance as compared to antibiotics. However, the pipeline for developing anti-virulence therapeutics is poorly defined, and it is currently unclear as to whether in vivo and in vitro models effectively replicate the complex pulmonary environment sufficiently to enable development and testing of such therapies for future clinical use. Here, we discuss potential targets for P. aeruginosa anti-virulence therapeutics and the effectiveness of the current models used to study them. Focus is given to the difficulty of replicating the virulence gene expression patterns of P. aeruginosa in the CF and NCFB lung under laboratory conditions and to the challenges this poses for anti-virulence therapeutic development.

Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we have undertaken multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a (ukp1a), uroplakin 2 (upk2) and uroplakin 3b (upk3b) genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated ukp1a expression in the zebrafish pronephros and cloaca from 96 h post-fertilization. Haematoxylin and Eosin staining of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder that leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a, Uroplakin 2 and GATA3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage and intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans, and offer the former as a model system to study disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Senescence drives the onset and severity of multiple ageing-associated diseases and frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP + cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP + cells display other markers of senescence, including senescence-associated β-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP + cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems., (© 2023 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

People with activated PI3 kinase delta syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models. Zebrafish embryos provide an ideal system to study neutrophils and macrophages. We used CRISPR-Cas9 and CRISPR-Cpf1, with oligonucleotide-directed homologous repair, to engineer zebrafish equivalents of the two most prevalent human APDS1 disease mutations. These zebrafish pik3cd alleles dominantly caused excessive neutrophilic inflammation in a tail-fin injury model. They also resulted in total body neutrophilia in the absence of any inflammatory stimulus but normal numbers of macrophages. Exposure of zebrafish to the PI3Kδ inhibitor CAL-101 reversed the total body neutrophilia. There was no apparent defect in neutrophil maturation or migration, and tail-fin regeneration was unimpaired. Overall, the finding is of enhanced granulopoeisis, in the absence of notable phenotypic change in neutrophils and macrophages., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Staphylococcus aureus is a human commensal and also an opportunist pathogen causing life threatening infections. During S. aureus disease, the abscesses that characterise infection can be clonal, whereby a large bacterial population is founded by a single or few organisms. Our previous work has shown that macrophages are responsible for restricting bacterial growth such that a population bottleneck occurs and clonality can emerge. A subset of phagocytes fail to control S. aureus resulting in bacterial division, escape and founding of microabscesses that can seed other host niches. Here we investigate the basis for clonal microabscess formation, using in vitro and in silico models of S. aureus macrophage infection. Macrophages that fail to control S. aureus are characterised by formation of intracellular bacterial masses, followed by cell lysis. High-resolution microscopy reveals that most macrophages had internalised only a single S. aureus, providing a conceptual framework for clonal microabscess generation, which was supported by a stochastic individual-based, mathematical model. Once a threshold of masses was reached, increasing the number of infecting bacteria did not result in greater mass numbers, despite enhanced phagocytosis. This suggests a finite number of permissive, phagocyte niches determined by macrophage associated factors. Increased understanding of the parameters of infection dynamics provides avenues for development of rational control measures., (© 2023. The Author(s).)

Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo . Here, we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration, and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Herman, Wright, Marriott, McCaughran, Bowden, Collins, Renshaw and Prince.)

Background: Telomerase, the enzyme capable of elongating telomeres, is usually restricted in human somatic cells, which contributes to progressive telomere shortening with cell-division and ageing. T and B-cells cells are somatic cells that can break this rule and can modulate telomerase expression in a homeostatic manner. Whereas it seems intuitive that an immune cell type that depends on regular proliferation outbursts for function may have evolved to modulate telomerase expression it is less obvious why others may also do so, as has been suggested for macrophages and neutrophils in some chronic inflammation disease settings. The gut has been highlighted as a key modulator of systemic ageing and is a key tissue where inflammation must be carefully controlled to prevent dysfunction. How telomerase may play a role in innate immune subtypes in the context of natural ageing in the gut, however, remains to be determined., Results: Using the zebrafish model, we show that subsets of gut immune cells have telomerase-dependent"hyper-long" telomeres, which we identified as being predominantly macrophages and dendritics (mpeg1.1 + and cd45 + mhcII + ). Notably, mpeg1.1 + macrophages have much longer telomeres in the gut than in their haematopoietic tissue of origin, suggesting that there is modulation of telomerase in these cells, in the gut. Moreover, we show that a subset of gut mpeg1.1 + cells express telomerase (tert) in young WT zebrafish, but that the relative proportion of these cells decreases with ageing. Importantly, this is accompanied by telomere shortening and DNA damage responses with ageing and a telomerase-dependent decrease in expression of autophagy and immune activation markers. Finally, these telomerase-dependent molecular alterations are accompanied by impaired phagocytosis of E. coli and increased gut permeability in vivo., Conclusions: Our data show that limiting levels of telomerase lead to alterations in gut immunity, impacting on the ability to clear pathogens in vivo. These are accompanied by increased gut permeability, which, together, are likely contributors to local and systemic tissue degeneration and increased susceptibility to infection with ageing., (© 2022. The Author(s).)

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis., Competing Interests: The authors have declared that no competing interests exist.

Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly silenced by the host genome, they have been found to be activated in multiple disease states, such as auto-inflammatory disorders and neurological diseases. However, the numerous copies in mammalian genomes and the lack of tools to study them make defining their role in health and diseases challenging. In this study, we identified eight copies of the zebrafish endogenous retrovirus zferv. We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single-cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and, eventually, death in individuals with cystic fibrosis (CF), a genetic disease caused by biallelic mutations in the CF transmembrane conductance regulator (CFTR) gene. Therefore, we examined whether roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defense, might provide a beneficial effect in the context of CF. Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immunopathophysiology, combined with murine and human approaches, we sought to determine the effects of roscovitine on innate immune responses to tissue injury and pathogens in the CF condition. We show that roscovitine exerts antiinflammatory and proresolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial reactive oxygen species (ROS)-mediated neutrophil trafficking by reducing DUOX2/NADPH-oxidase activity and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. It is important to note that, although roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo , we found that treatment with roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/NADPH oxidase-dependent ROS production, roscovitine reduces the number of neutrophils at infection sites and, consequently, compromises granuloma formation and maintenance, favoring extracellular multiplication of M. abscessus and more severe infection. Our findings bring important new understanding of the immune-targeted action of roscovitine and have significant therapeutic implications for safely targeting inflammation in CF.

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus , the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection., Competing Interests: The authors have declared that no competing interests exist.

Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury., Competing Interests: HI, CL, SA, FK, KH, AK, JH, CM, MK, VK, NH, GL, SR, PE No competing interests declared, (© 2021, Isles et al.)

Macroautophagy/autophagy functions to degrade cellular components and intracellular pathogens. Autophagy receptors, including SQSTM1/p62, target intracellular pathogens. Staphylococcus aureus is a significant pathogen of humans, especially in immunocompromise. S. aureus may use neutrophils as a proliferative niche, but their intracellular fate following phagocytosis has not been analyzed in vivo. In vitro , SQSTM1 can colocalize with intracellular Staphylococcus aureus , but whether SQSTM1 is beneficial or detrimental in host defense against S. aureus in vivo is unknown. Here we determine the fate and location of S. aureus within neutrophils throughout zebrafish infection. We show Lc3 and Sqstm1 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil and that Lc3 marking of bacterial phagosomes within neutrophils may precede bacterial degradation. Finally, we show Sqstm1 is important for controlling cytosolic bacteria, demonstrating for the first time a key role of Sqstm1 in autophagic control of S. aureus in neutrophils. Abbreviations: AR: autophagy receptor; CFU: colony-forming unit; CHT: caudal hematopoietic tissue; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LWT: london wild-type: lyz: lysozyme; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification; UBD: ubiquitin binding domain.

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus , multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus -infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus -infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci . Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus infects ∼30% of the human population and causes a spectrum of pathologies ranging from mild skin infections to life-threatening invasive diseases. The strict host specificity of its virulence factors has severely limited the accuracy of in vivo models for the development of vaccines and therapeutics. To resolve this, we generated a humanised zebrafish model and determined that neutrophil-specific expression of the human C5a receptor conferred susceptibility to the S. aureus toxins PVL and HlgCB, leading to reduced neutrophil numbers at the site of infection and increased infection-associated mortality. These results show that humanised zebrafish provide a valuable platform to study the contribution of human-specific S. aureus virulence factors to infection in vivo that could facilitate the development of novel therapeutic approaches and essential vaccines., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Staphylococcus aureus is a member of the human commensal microflora that exists, apparently benignly, at multiple sites on the host. However, as an opportunist pathogen it can also cause a range of serious diseases. This requires an ability to circumvent the innate immune system to establish an infection. Professional phagocytes, primarily macrophages and neutrophils, are key innate immune cells which interact with S. aureus , acting as gatekeepers to contain and resolve infection. Recent studies have highlighted the important roles of macrophages during  S. aureus  infections, using a wide array of killing mechanisms. In defense,  S. aureus  has evolved multiple strategies to survive within, manipulate and escape from macrophages, allowing them to not only subvert but also exploit this key element of our immune system. Macrophage- S. aureus  interactions are multifaceted and have direct roles in infection outcome. In depth understanding of these host-pathogen interactions may be useful for future therapeutic developments. This review examines macrophage interactions with  S. aureus throughout all stages of infection, with special emphasis on mechanisms that determine infection outcome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pidwill, Gibson, Cole, Renshaw and Foster.)

During inflammation, dysregulated neutrophil behaviour can play a major role in a range of chronic inflammatory diseases, for many of which current treatments are generally ineffective. Recently, specific naturally occurring tanshinones have shown promising anti-inflammatory effects by targeting neutrophils in vivo, yet such tanshinones, and moreover, their isomeric isotanshinone counterparts, are still a largely underexplored class of compounds, both in terms of synthesis and biological effects. To explore the anti-inflammatory effects of isotanshinones, and the tanshinones more generally, a series of substituted tanshinone and isotanshinone analogues was synthesised, alongside other structurally similar molecules. Evaluation of these using a transgenic zebrafish model of neutrophilic inflammation revealed differential anti-inflammatory profiles in vivo, with a number of compounds exhibiting promising effects. Several compounds reduce initial neutrophil recruitment and/or promote resolution of neutrophilic inflammation, of which two also result in increased apoptosis of human neutrophils. In particular, the methoxy-substituted tanshinone 39 specifically accelerates resolution of inflammation without affecting the recruitment of neutrophils to inflammatory sites, making this a particularly attractive candidate for potential pro-resolution therapeutics, as well as a possible lead for future development of functionalised tanshinones as molecular tools and/or chemical probes. The structurally related β-lapachones promote neutrophil recruitment but do not affect resolution. We also observed notable differences in toxicity profiles between compound classes. Overall, we provide new insights into the in vivo anti-inflammatory activities of several novel tanshinones, isotanshinones, and structurally related compounds., Competing Interests: The authors have declared that no competing interests exist.

Inflammation-related progressive lung destruction is the leading causes of premature death in cystic fibrosis (CF), a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR). However, therapeutic targeting of inflammation has been hampered by a lack of understanding of the links between a dysfunctional CFTR and the deleterious innate immune response in CF. Herein, we used a CFTR-depleted zebrafish larva, as an innovative in vivo vertebrate model, to understand how CFTR dysfunction leads to abnormal inflammatory status in CF. We show that impaired CFTR-mediated inflammation correlates with an exuberant neutrophilic response after injury: CF zebrafish exhibit enhanced and sustained accumulation of neutrophils at wounds. Excessive epithelial oxidative responses drive enhanced neutrophil recruitment towards wounds. Persistence of neutrophils at inflamed sites is associated with impaired reverse migration of neutrophils and reduction in neutrophil apoptosis. As a consequence, the increased number of neutrophils at wound sites causes tissue damage and abnormal tissue repair. Importantly, the molecule Tanshinone IIA successfully accelerates inflammation resolution and improves tissue repair in CF animal. Our findings bring important new understanding of the mechanisms underlying the inflammatory pathology in CF, which could be addressed therapeutically to prevent inflammatory lung damage in CF patients with potential improvements in disease outcomes., (Copyright © 2020 Bernut, Loynes, Floto and Renshaw.)

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo , using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis , Mycobacterium tuberculosis , and another established intracellular pathogen, Staphylococcus aureus . Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis , the etiological agent of human TB.

The contribution of microglia in neurological disorders is emerging as a leading disease driver rather than a consequence of pathology. RNAseT2-deficient leukoencephalopathy is a severe childhood white matter disorder affecting patients in their first year of life and mimicking a cytomegalovirus brain infection. The early onset and resemblance of the symptoms to a viral infection suggest an inflammatory and embryonic origin of the pathology. There are no treatments available for this disease as our understanding of the cellular drivers of the pathology are still unknown. In this study, using a zebrafish mutant for the orthologous rnaset2 gene, we have identified an inflammatory signature in early development and an antiviral immune response in mature adult brains. Using the optical transparency and the ex utero development of the zebrafish larvae we studied immune cell behavior during brain development and identified abnormal microglia as an early marker of pathology. Live imaging and electron microscopy identified that mutant microglia displayed an engorged morphology and were filled with undigested apoptotic cells and undigested substrate. Using microglia-specific depletion and rescue experiments, we identified microglia as drivers of this embryonic phenotype and potential key cellular player in the pathology of RNAseT2-deficient leukoencephalopathy. Our zebrafish model also presented with reduced survival and locomotor defects, therefore recapitulating many aspects of the human disease. Our study therefore placed our rnaset2 mutant at the forefront of leukodystrophy preclinical models and highlighted tissue-specific approaches as future therapeutic avenues., (© 2020 The Authors. Glia published by Wiley Periodicals, Inc.)

Antimicrobial therapy has provided the main component of chemotherapy against bacterial pathogens. The effectiveness of this strategy has, however, been increasingly challenged by the emergence of antimicrobial resistance which now threatens the sustained utility of this approach. Humans and animals are constantly exposed to bacteria and have developed effective strategies to control pathogens involving innate and adaptive immune responses. Impaired pathogen handling by the innate immune system is a key determinant of susceptibility to bacterial infection. However, the essential components of this response, specifically those which are amenable to re-calibration to improve host defense, remain elusive despite extensive research. We provide a mini-review focusing on therapeutic targeting of microbicidal responses in macrophages and neutrophils to de-stress reliance on antimicrobial therapy. We highlight pre-clinical and clinical data pointing toward potential targets and therapies. We suggest that developing focused host-directed therapeutic strategies to enhance "pauci-inflammatory" microbial killing in myeloid phagocytes that maximizes pathogen clearance while minimizing the harmful consequences of the inflammatory response merits particular attention. We also suggest the importance of One Health approaches in developing host-based approaches through model development and comparative medicine in informing our understanding of how to deliver this strategy., (Copyright © 2020 Watson, Russell, Baillie, Dhaliwal, Fitzgerald, Mitchell, Simpson, Renshaw and Dockrell.)

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Introduction: Pirfenidone has been shown to reduce the decline in forced vital capacity (FVC) compared to placebo in patients with idiopathic pulmonary fibrosis (IPF). Previous studies have suggested that patients with a more rapid decline in FVC during the period before starting pirfenidone experience the greatest benefit from treatment. The purpose of this retrospective observational study was to investigate the response to pirfenidone in IPF patients, comparing two groups stratified by the annual rate of decline in FVC % predicted prior to treatment., Methods: Using the rate of decline in FVC % predicted in the 12 months prior to pirfenidone, patients were stratified into slow (<5%) or rapid (≥5%) decliner groups. Comparisons in the lung function response to pirfenidone in these two groups were performed., Results: Pirfenidone resulted in no statistically significant reduction in the median annual rate of decline in FVC or FVC % predicted. In the rapid decliners, pirfenidone significantly reduced the median (IQR) annual rate of decline in FVC % predicted (-8.7 (-14.2 - -7.0) %/yr vs 2.0 (-7.1 - 6.0) %/yr; n=17; p<0.01). In the slow decliners, pirfenidone did not reduce the median (IQR) annual rate of decline in FVC % predicted (-1.3 (-3.2 - 1.3) %/yr vs -5.0 (-8.3 - -0.35) %/yr; n=17; p=0.028)., Conclusions: We demonstrate the greater net effect of pirfenidone in IPF patients declining rapidly. We suggest that using an annual rate of decline in FVC of <5% and ≥5% may be useful in counselling patients with regard to pirfenidone treatment. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (2): 218-224) ., (Copyright: © 2020 SARCOIDOSIS VASCULITIS AND DIFFUSE LUNG DISEASES.)

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate ('nanobiotics') deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease., Competing Interests: AR, KH, KH, AT, HI, CT, DS, JR, NK, AR, KH, CT, IS, WZ, AM, MW, DD, SR, LP No competing interests declared, CS is an employee of GlaxoSmithKline Research and Development Ltd. The author declares no other competing interests exist., (© 2019, Rahman et al.)

The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.

Prognosticating idiopathic pulmonary fibrosis (IPF) is challenging, in part due to a lack of sensitive biomarkers. A recent article in Thorax described how hyperpolarised xenon magnetic resonance spectroscopy may quantify regional gas exchange in IPF lungs. In a population of patients with IPF, we find that the xenon signal from red blood cells diminishes relative to the tissue/plasma signal over a 12-month time period, even when the diffusion factor for carbon monoxide is static over the same time period. We conclude that hyperpolarised 129 Xe MR spectroscopy may be sensitive to short-term changes in interstitial gas diffusion in IPF., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae-which express a non-functional zebrafish myeloperoxidase-the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus has colonized humans for at least 10 000 years, and today inhabits roughly a third of the population. In addition, S. aureus is a major pathogen that is responsible for a significant disease burden, ranging in severity from mild skin and soft-tissue infections to life-threatening endocarditis and necrotizing pneumonia, with treatment often hampered by resistance to commonly available antibiotics. Underpinning its versatility as a pathogen is its ability to evade the innate immune system. S. aureus specifically targets innate immunity to establish and sustain infection, utilizing a large repertoire of virulence factors to do so. Using these factors, S. aureus can resist phagosomal killing, impair complement activity, disrupt cytokine signalling and target phagocytes directly using proteolytic enzymes and cytolytic toxins. Although most of these virulence factors are well characterized, their importance during infection is less clear, as many display species-specific activity against humans or against animal hosts, including cows, horses and chickens. Several staphylococcal virulence factors display species specificity for components of the human innate immune system, with as few as two amino acid changes reducing binding affinity by as much as 100-fold. This represents a major issue for studying their roles during infection, which cannot be examined without the use of humanized infection models. This review summarizes the major factors S. aureus uses to impair the innate immune system, and provides an in-depth look into the host specificity of S. aureus and how this problem is being approached.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis., Competing Interests: The authors have declared that no competing interests exist.