Pioneer neutrophils release chromatin within in vivo swarms.

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.

Evaluation of the anti-inflammatory effects of synthesised tanshinone I and isotanshinone I analogues in zebrafish.

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 ß-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.

Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation.

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.

Expression and regulation of drug transporters in vertebrate neutrophils.

There remains a need to identify novel pro-resolution drugs for treatment of inflammatory disease. To date, there are no neutrophil-specific anti-inflammatory treatments in clinical use, perhaps due to our lack of understanding of how drugs access this complex cell type. Here we present the first comprehensive description and expression of both major classes of drug transporters, SLC and ABC, in resting human blood neutrophils. Moreover, we have studied the expression of these carriers in the tractable model system, the zebrafish (Danio rerio), additionally examining the evolutionary relationship between drug transporters in zebrafish and humans. We anticipate that this will be a valuable resource to the field of inflammation biology and will be an important asset in future anti-inflammatory drug design.

Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen.

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available 'cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection.

PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org.

Zebrafish as a model for the study of neutrophil biology.

To understand inflammation and immunity, we need to understand the biology of the neutrophil. Whereas these cells can readily be extracted from peripheral blood, their short lifespan makes genetic manipulations impractical. Murine knockout models have been highly informative, and new imaging techniques are allowing neutrophils to be seen during inflammation in vivo for the first time. However, there is a place for a new model of neutrophil biology, which readily permits imaging of individual neutrophils during inflammation in vivo, combined with the ease of genetic and chemical manipulation. The zebrafish has long been the model of choice for the developmental biology community, and the availability of genomic resources and tools for gene manipulation makes this an attractive model. Zebrafish innate immunity shares many features with mammalian systems, including neutrophils with morphological, biochemical, and functional features, also shared with mammalian neutrophils. Transgenic zebrafish with neutrophils specifically labeled with fluorescent proteins have been generated, and this advance has led to the adoption of zebrafish, alongside existing models, by a number of groups around the world. The use of these models has underpinned a number of key advances in the field, including the identification of a tissue gradient of hydrogen peroxide for neutrophil recruitment following tissue injury and direct evidence for reverse migration as a regulatable mechanism of inflammation resolution. In this review, we discuss the importance of zebrafish models in neutrophil biology and describe how the understanding of neutrophil biology has been advanced by the use of these models.

Epidemiologic analysis of nosocomial Salmonella infections in hospitalized horses.

OBJECTIVE: To examine the relationship between abdominal surgery and nosocomial Salmonella infections and the relationship between high caseload in combination with abdominal surgery and nosocomial Salmonella infections in hospitalized horses with signs of gastrointestinal tract disease. ANIMALS: 140 horses. DESIGN: Case-control study. PROCEDURES: To accomplish the first objective, 1 to 4 control horses were matched with each nosocomial case horse on the basis of admission date of a primary case horse. The frequency of abdominal surgery and other investigated exposure factors were compared between nosocomial case horses and control horses. For the second objective, 4 control horses were matched with each nosocomial case horse on the basis of year of admission. The frequency of high caseload (>or=26 inpatients), abdominal surgery, and other factors was compared between nosocomial case horses and control horses. RESULTS: The odds of nosocomial Salmonella infection were 8 times as high (odds ratio=8.2; 95% confidence interval=1.11, 60.24) in horses that underwent abdominal surgery, compared with the odds for horses that did not undergo surgery. High caseload alone or in combination with abdominal surgery was not associated with increased risk of nosocomial Salmonella infection. CONCLUSIONS AND CLINICAL RELEVANCE: Abdominal surgery was identified as a risk factor for nosocomial Salmonella infections in horses. Horses that undergo abdominal surgery require enhanced infection control and preventative care. Risk of nosocomial Salmonella infections may be reduced by implementation of biosecurity measures (such as the use of plastic boots, gloves, and footbaths) immediately after surgery.

Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.

Rickettsia felis in Xenopsylla cheopis, Java, Indonesia.

Rickettsia typhi and R. felis, etiologic agents of murine typhus and fleaborne spotted fever, respectively, were detected in Oriental rat fleas (Xenopsylla cheopis) collected from rodents and shrews in Java, Indonesia. We describe the first evidence of R. felis in Indonesia and naturally occurring R. felis in Oriental rat fleas.