Evidence 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.
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•GC-driven C/EBPβ and JAK/STAT pathways regulate macrophage irg1 expression•Irg1-depleted macrophages have reduced mROS-dependent bactericidal activity•Macrophages utilize fatty acid β-oxidation to “fuel” Irg1-dependent mROS production•Murine macrophages require β-oxidation for elevated mROS and bactericidal activity
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Once thought to be a feature exclusive to lymphocyte-driven adaptive immunity, immune memory has also been shown to operate as part of the innate immune system following infection to provide an ...elevated host response to subsequent pathogenic challenge. This evolutionarily conserved process, termed ‘trained immunity’, enables cells of the innate immune system to ‘remember’ previous pathogen encounters and mount stronger responses to the same, or different, pathogens after returning to a non-activated state. Here we show that challenging larval zebrafish, that exclusively rely on innate immunity, with live or heat-killed Salmonella typhimurium provides protection to subsequent infection with either Salmonella typhimurium or Streptococcus iniae, that lasts for at least 12 days. We also show that larvae injected with β-glucan, the well-known trigger of trained immunity, demonstrate enhanced survival to similar live bacterial infections, a phenotype supported by increased cxcl8 expression and neutrophil recruitment to the infection site. These results support the conservation of a trained immunity-like phenotype in larval zebrafish and provide a foundation to exploit the experimental attributes of larval zebrafish to further understand this form of immunological memory.
•Bacterial infection protects larval zebrafish to subsequent infections.•β-glucan stimulation protects larval zebrafish to subsequent infections.•Evidence of trained immunity in larval zebrafish.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish ...lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.
Metastatic cancer cells typically fail to halt migration on contact with non-cancer cells. This invasiveness is in contrast to normal mesenchymal cells that retract on contact with another cell. Why ...cancer cells are defective in contact inhibition of locomotion is not understood. Here, we analyse the dynamics of prostate cancer cell lines co-cultured with fibroblasts, and demonstrate that a combinatorial code of Eph receptor activation dictates whether cell migration will be contact inhibited. The unimpeded migration of metastatic PC-3 cells towards fibroblasts is dependent on activation of EphB3 and EphB4 by ephrin-B2, which we show activates Cdc42 and cell migration. Knockdown of EphB3 and EphB4 restores contact inhibition of locomotion to PC-3 cells. Conversely, homotypic collisions between two cancer cells results in contact inhibition of locomotion, mediated by EphA-Rho-Rho kinase (ROCK) signalling. Thus, the migration of cancer cells can switch from restrained to invasive, depending on the Eph-receptor profile of the cancer cell and the reciprocal ephrin ligands expressed by neighbouring cells.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Lymphatic vessels are known to be derived from veins; however, recent lineage‐tracing experiments propose that specific lymphatic networks may originate from both venous and non‐venous sources. ...Despite this, direct evidence of a non‐venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay‐like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA‐L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non‐venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging‐based evidence of a non‐venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context‐dependent.
Synopsis
The zebrafish facial lymphatics have three progenitor populations; one of these is non‐venous in origin and arises from a late‐forming angioblast population. This is the first time a non‐venous lymphatic progenitor has been described using live cell imaging.
The zebrafish facial lymphatics form through the sequential addition of three progenitor populations in a relay mechanism.
One of these progenitor populations, called the ventral aorta lymphangioblast (VA‐L), does not arise from a blood vessel, instead it arises directly from an angioblast population near the ventral aorta (VA‐A).
The dorsal segment of the VA‐A expresses Prox1 and contributes towards facial lymphatic development while the ventral segment contributes to the hypobranchial artery.
The zebrafish facial lymphatics have three progenitor populations; one of these is non‐venous in origin and arises from a late‐forming angioblast population. This is the first time a non‐venous lymphatic progenitor has been described using live cell imaging.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. ...Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.
Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a ...proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1β and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.
Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to ...form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.
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•Identification of an unexpected role for the ADPKD gene Pkd1 in lymphatic development•Pkd1 regulates endothelial cell sprouting and cell-cell junctions in vitro•Pkd1 regulates cell polarity and cell-cell junctions in developing vessels in mice
The earliest steps in lymphatic vascular development are increasingly well defined, but the processes and molecules that control ongoing network morphogenesis remain to be understood. In this study, Coxam et al. show that the autosomal dominant polycystic kidney disease (ADPKD) gene Pkd1 regulates lymphatic endothelial cell polarity, shape, and adhesion during migration, vessel morphogenesis, and lymphatic vascular network elaboration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed ...to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish
, they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation.
The innate immune system can
previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed "trained immunity." Trained innate immune ...cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where β-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of β-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to β-glucan stimulation.
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