Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis.This work was funded by grants from the National Institutes of Health (T32-AI055396, A.J.P.; A154503 and A136396, L.R.) and the National Health and Medical Research Council (637394, 1044754, and 1069284, G.J.L.), a post-doctoral fellowship from the Taiwan National Science Council (NSC97-2917-I-564-109, C-T.Y.), and an Australian Postgraduate Award and Walter and Eliza Hall Institute of Medical Research Edith Moffatt Scholarship (F.E.). The Australian Regenerative Medicine Institute is supported by funds from the State Government of Victoria and the Australian Federal Government. L.R. is a recipient of the NIH Director’s Pioneer Award and a Wellcome Trust Principal Research Fellowship.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chom.2015.06.00

Immunoresponsive Gene 1 Augments Bactericidal Activity of Macrophage-Lineage Cells by Regulating β-Oxidation-Dependent Mitochondrial ROS Production

SummaryEvidence suggests the bactericidal activity of mitochondria-derived reactive oxygen species (mROS) directly contributes to killing phagocytozed bacteria. Infection-responsive components that regulate this process remain incompletely understood. We describe a role for the mitochondria-localizing enzyme encoded by Immunoresponsive gene 1 (IRG1) during the utilization of fatty acids as a fuel for oxidative phosphorylation (OXPHOS) and associated mROS production. In a zebrafish infection model, infection-responsive expression of zebrafish irg1 is specific to macrophage-lineage cells and is regulated cooperatively by glucocorticoid and JAK/STAT signaling pathways. Irg1-depleted macrophage-lineage cells are impaired in their ability to utilize fatty acids as an energy substrate for OXPHOS-derived mROS production resulting in defective bactericidal activity. Additionally, the requirement for fatty acid β-oxidation during infection-responsive mROS production and bactericidal activity toward intracellular bacteria is conserved in murine macrophages. These results reveal IRG1 as a key component of the immunometabolism axis, connecting infection, cellular metabolism, and macrophage effector function

PGE₂ production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo

Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site—either by apoptosis and efferocytosis or by reverse migration away from the wound—for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve, due to loss of macrophage-dependent handling of eicosanoid prostaglandin E2 (PGE2) that drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous PGE synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors during injury, causing an increase in Alox12 production and switching toward anti-inflammatory eicosanoid signaling. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 (lipoxin A4) in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease

Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro

Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a targeting moiety (TM) that binds to the surface of the microbial target, and an effector moiety (EM) that interacts with chemoattractant receptors on human neutrophils. We report that the bifunctional compounds enhance the interactions between primary human neutrophils and A. fumigatus in vitro, using three microfluidic assay platforms. The bifunctional compounds significantly enhance the recruitment of neutrophils, increase hyphae killing by neutrophils in a uniform concentration of drug, and decrease hyphal tip growth velocity in the presence of neutrophils compared to the antifungal targeting moiety alone. We validated that the bifunctional compounds are also effective in vivo, using a zebrafish infection model with neutrophils expressing the appropriate EM receptor. We measured significantly increased phagocytosis of A. fumigatus conidia by neutrophils expressing the EM receptor in the presence of the compounds compared to receptor-negative cells. Finally, we demonstrate that treatment with our lead compound significantly improved the antifungal activity of neutrophils from immunosuppressed patients ex vivo. This type of bifunctional compounds strategy may be utilized to redirect the immune system to destroy fungal, bacterial, and viral pathogens

Zebrafish models for studying macrophage function and fungal infection

© 2011 Dr. Felix Eiran John EllettCells of the innate immune system provide the first active line of defence against invading pathogens. In response to injury, they actively migrate to the wound site, where they act to phagocytose microbes and excrete antimicrobial compounds. Antigen presenting cells, such as macrophages and dendritic cells, also act as a bridge between the innate and adaptive immune system by processing and presenting antigen to lymphocytes. In zebrafish, macrophages and neutrophils are specified early in development, and are active from 1-2 days post fertilisation, although the adaptive immune system is not functional until 2 weeks post fertilisation. The embryonic zebrafish therefore provides a useful model to study the development and function of the innate immune system in isolation. To identify new regulators of primitive macrophage development, a forward genetic mutagenesis screen was performed. A mutant displaying reduced numbers of macrophages was mapped by others to a mutation in the alk3a gene, which encodes a Type I BMP-receptor. It is preposed, based on previous studies, that Alk3a and Alk8 form a tetramer with Type II receptors to bind BMP2/7 dimer ligands during specification of mesoderm into haematopoietic precursors. To study the function of innate immune cells in response to infection, a novel zebrafish fungal infection model was developed. Penicillium marneffei is a thermally dimorphic opportunistic fungal pathogen that represents a serious and increasingly common AIDS-defining infection in Southeast Asia. The ectothermic biology and in vivo imaging strengths of the embryonic zebrafish model were considered well suited for studies of host-pathogen interactions and the role of fungal dimorphism in virulence. The zebrafish fungal infection model was used to describe both hyphal and yeast morphological forms during infection. Cells of the zebrafish innate immune system were actively engaged in interactions with fungal cells during infection, replicating many aspects of human infection, including intracellular yeast growth at higher temperatures. Infection of the haematopoiesis failure mutant cloche suggested a relationship between the onset of yeast morphology at higher temperatures and the presence of myeloid cells. Infected embryos displayed significantly increased numbers of neutrophils, a phenotype that was abrogated by targeting of the G-CSF receptor (csf3r) transcript by antisense morpholino oligonucleotides (MOs). Myeloperoxidase deficient zebrafish durif mutants exhibited an enhanced neutrophil response to infection, consistent with studies in mice. Knockdown of spi1 with MOs reduced production of early myeloid cells, predominantly macrophages. This led to an unexpected acceleration of spore destruction during the first 24 hour of infection. This was not rescued by MO-mediated knockdown of the complement factor c9, which encodes the pore-forming multimer of the membrane attack complex (MAC). Studies of fungal virulence in a P. marneffei knockout strain simAΔ suggested that this yeast-expressed gene was important for fungal survival in this model. One of the major roadblocks to imaging host-pathogen interactions in zebrafish has been the lack of an available transgenic line with macrophage-specific fluorophore expression with which to study the behaviour of macrophages in vivo. Equivalent neutrophil-specific transgenic lines based on the mpx and lyz promoters have been available for a number of years, and have been invaluable to imaging in zebrafish infection models. In these studies, a macrophage-specific promoter was identified, mpeg1, and used to develop a number of transgenes expressed specifically in zebrafish macrophages. Use of the Gal4/UAS system facilitated combination of the mpeg1-Gal4FF driver with UAS-driven effector lines, such as those used for inducible tissue-specific ablation. These lines represent the first macrophage-specific transgenes in zebrafish and will be highly useful in many fields of zebrafish-based research

SEPSIS-neutrophil-spontaneous motility

"Ellett_data analysis codes.7z" contains several files with code for image analysis, cell tracking, and motility pattern analysis, for microfluidic assay that measures neutrophil spontaneous motility in whole blood. <div>"Ellett_Numerical Values Fig 2-6.xlsx" contains all numerical values used for generating the graphs in figures 2 to 6. </div><div><br></div

Convergent and Divergent Migratory Patterns of Human Neutrophils inside Microfluidic Mazes

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways

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

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

Taking a machine learning approach to optimise prediction of vaccine hesitancy in high income countries

Understanding factors driving vaccine hesitancy is crucial to vaccination success. We surveyed adults (N = 2510) from February to March 2021 across five sites (Australia = 502, Germany = 516, Hong Kong = 445, UK = 512, USA = 535) using a cross-sectional design and stratified quota sampling for age, sex, and education. We assessed willingness to take a vaccine and a comprehensive set of putative predictors. Predictive power was analysed with a machine learning algorithm. Only 57.4% of the participants indicated that they would definitely or probably get vaccinated. A parsimonious machine learning model could identify vaccine hesitancy with high accuracy (i.e. 82% sensitivity and 79–82% specificity) using 12 variables only. The most relevant predictors were vaccination conspiracy beliefs, various paranoid concerns related to the pandemic, a general conspiracy mentality, COVID anxiety, high perceived risk of infection, low perceived social rank, lower age, lower income, and higher population density. Campaigns seeking to increase vaccine uptake need to take mistrust as the main driver of vaccine hesitancy into account