scholarly journals Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster

2020 ◽  
Vol 117 (13) ◽  
pp. 7317-7325 ◽  
Author(s):  
Igor Iatsenko ◽  
Alice Marra ◽  
Jean-Philippe Boquete ◽  
Jasquelin Peña ◽  
Bruno Lemaitre
Keyword(s):  
Drosophila Melanogaster ◽  
Defense Mechanism ◽  
Fat Body ◽  
Immune Defense ◽  
In Vivo Model ◽  
Nutritional Immunity ◽  
Iron Sequestration

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor κB (NF-κB) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

Recent findings on oogenesis of Drosophila melanogaster

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 125-138
Author(s):  
F. Giorgi ◽  
J. Jacob
Keyword(s):  
Drosophila Melanogaster ◽  
Fat Body ◽  
Radioactive Tracer ◽  
Superficial Layer ◽  
Ruthenium Red ◽  
Time Interval ◽  
Intracellular Location ◽  
Short Time

Vitellogenic ovaries from Drosophila melanogaster flies have been exposed, either in in vivo or in vitro conditions, to various extracellular tracers in an attempt to determine the possible route of entry of the yolk precursors. Ruthenium red and lanthanum nitrate have been shown to gain access to the oocyte surface by initially passing through the intercellular spaces of the follicle layer. Both these tracers, however, never attain an intracellular location within any of the cells forming the ovarian chamber. Colloidal Thorotrast when injected into adult females has never been detected within any of the ovarian chambers examined, irrespective of their stage. Vitellogenic oocytes exposed to peroxidase in in vivo conditions exhibit the oolemma and all the structural elements present in the cortical ooplasm well labelled within a very short time after the injection. Moreover, with gradually increasing exposure times to peroxidase, the labelled yolk platelets increase progressively in number. At each time interval after the injection, the label over the yolk platelets remains restricted to the superficial layer and never gets into the associated body. The pattern of tritiated lysine incorporation into vitellogenic oocytes has been studied over a period of 20 h. A few hours after injection of the radioactive tracer, the silver grains located over the ooplasm appear distributed at random. A predominant labelling of the yolk platelets as compared to the rest of the ooplasm, becomes evident only with a 6 h delay since the time of injection. When analysed by electrophoresis and isolectrofocusing, the vitellogenic ovary is seen to exhibit a number of protein bands which are common to those of other tissues as, for instance, haemolymph and fat body. The evidence obtained in the present study is discussed in relation to the hypothesis of an extraovarian origin of the yolk precursors and their sequestration into forming yolk platelets.

scholarly journals The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

1994 ◽  
Vol 14 (7) ◽  
pp. 4465-4474 ◽  
Author(s):  
C Antoniewski ◽  
M Laval ◽  
A Dahan ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Fat Body ◽  
Sequence Similarity ◽  
High Concentration ◽  
Direct Role ◽  
Palindromic Structure ◽  
Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

scholarly journals How Neutrophil Extracellular Traps Become Visible

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Nicole de Buhr ◽  
Maren von Köckritz-Blickwede
Keyword(s):  
Electron Microscopy ◽  
Nuclear Dna ◽  
Defense Mechanism ◽  
Neutrophil Extracellular Traps ◽  
Immune Defense ◽  
Extracellular Traps ◽  
Activated Neutrophils ◽  
Microscopy Techniques

Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one handin vitrolive-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital,in vivo, andin situmicroscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages.

Platelet-Derived Microparticles Induce Angiogenesis and Stimulate Post-Ischemic Revascularization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3916-3916
Author(s):  
Olga Dashevsky ◽  
Alexander Brill ◽  
Julia Rivo ◽  
David Varon
Keyword(s):  
Endothelial Cells ◽  
Aortic Ring ◽  
In Vivo Model ◽  
Preliminary Evaluation ◽  
Vegf Receptor ◽  
In Vivo Models ◽  
Ischemic Region

Abstract Platelet attachment to the subcellular matrix at injured sites of the vasculature is followed by their activation and release of microparticles. Platelet-derived microparticles (PMP) have been shown to be involved in the regulation of hemostasis. However, little is known about the role of PMP in the regulation of angiogenesis and related clinical conditions. We have recently demonstrated that platelets as a cellular system induce angiogenic responses both in vitro and in vivo. In the present study, we investigated the potential role of PMP in angiogenesis. A strong dose-dependent pro-angiogenic effect of PMP in the rat aortic ring model (5.3±2.1 mm2 surface covered with sprouting vessels versus 0.24±0.2 mm2 in the control, p<0.001) was observed. This effect was reversed by selective inhibition of VEGF, bFGF and PDGF (surface covered with vessels 0.7±0.5 mm2, 1.7±1.5 mm2, and 2.4±1.2 mm2, respectively, p<0.02 versus control), but not by inhibition of heparanase (5.1±0.8 mm2, p>0.5 versus control). PMP exert their stimulatory effect via PI3-kinase, Src kinase and ERK, whereas protein kinase C seems not to be involved, as judged by the aortic ring sprouting model. Using confocal and electron microscopy, we also demonstrate that PMP bind to non-activated endothelial cells. In addition, PMP markedly increased invasion of human endothelial cells through a layer of matrigel. This effect was abolished by an inhibitor of VEGF receptor tyrosine phosphorylation or laminaran sulfate (heparanase inhibitor). It was also partially reduced by PDGF blocking mAb, whereas blocking of bFGF had no effect. Furthermore, we have demonstrated that PMP induce angiogenesis in an in vivo model, in which beads (30 μl) of 4% agarose gel containing the substances under study were transplanted subcutaneously into mice. Image analysis of the capillary area revealed the following: control beads − 0.2±0.05 mm2, VEGF + bFGF containing beads − 4.8±1.1 mm2, PMP (100 μg/ml) containing beads − 5.1±1.3 mm2, p<0.001 versus control. The latter finding was further supported by immunohistochemical staining of the skin in the vicinity of the beads for von Willebrand factor, a marker of endothelial cells (control − 4.0±3.2, VEGF+bFGF − 12±4.4, PMP − 17±6.5 capillaries per view field, p<0.05 versus control). Finally, we explored the potential effect of PMP in a rat myocardial infarction model. Ischemia was induced by LAD ligation followed by injection of either PMP or PBS into the ischemic region. Preliminary evaluation of the LAD myocardial territory in sham-operated animals revealed 157±42.0 capillaries per view field. In contrast, number of capillaries observed 3 weeks after induction of ischemia was reduced to 34±21.5. When PMP were injected into the ischemic region, there was an increase in capillary number up to 97±27.3. In conclusion, PMP induce angiogenesis in both in vitro and in vivo models. Local injection of PMP into the ischemic myocardium may improve revascularization.

Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4083-4092 ◽  
Author(s):  
Frédéric Adam ◽  
Alexandre Kauskot ◽  
Paquita Nurden ◽  
Eric Sulpice ◽  
Marc F. Hoylaerts ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Blood Perfusion ◽  
Collagen Matrix ◽  
In Vivo Model ◽  
Wild Type ◽  
Platelet Secretion

Abstract The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.

scholarly journals Staphylococcus aureususes the bacilliredoxin (BrxAB)/ bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

2019 ◽  
Author(s):  
Nico Linzner ◽  
Vu Van Loi ◽  
Verena Nadin Fritsch ◽  
Quach Ngoc Tung ◽  
Saskia Stenzel ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Defense Mechanism ◽  
Redox Homeostasis ◽  
Stress Monitoring ◽  
Macrophage Infection ◽  
Disulfide Reductase ◽  
Mixed Disulfides

ABSTRACTStaphylococcus aureusis a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism ofS. aureusfor detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed asS-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was proposed to function as BSSB reductase. Here, we investigated the role of the bacilliredoxin BrxAB/BSH/YpdA pathway inS. aureusCOL under oxidative stress and macrophage infection conditionsin vivoand in biochemical assaysin vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in theS. aureusCOLypdAdeletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (EBSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reducedEBSHupon recovery from oxidative stress. In addition, theypdAmutant was impaired in H2O2detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2stressin vitroand inside murine J-774A.1 macrophages in infection assaysin vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation ofS-bacilithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis inS. aureusduring recovery from oxidative stress and under infections.

scholarly journals CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage

Stroke ◽  
2021 ◽  
Author(s):  
Yueyang Liu ◽  
Xiaohang Che ◽  
Haotian Zhang ◽  
Xiaoxiao Fu ◽  
Yang Yao ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Kinase Inhibitor ◽  
Neuronal Damage ◽  
Neuronal Cell ◽  
In Vivo Model ◽  
Autophagic Flux ◽  
Autophagosome Formation

Background and Purpose: CAPN1 (calpain1)—an intracellular Ca 2+ -regulated cysteine protease—can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose–deprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusion–operated rats and oxygen-glucose deprivation–exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia–mediated autophagy-lysosomal pathway defects and neuronal damage.

scholarly journals BEL β-Trefoil Reduces the Migration Ability of RUNX2 Expressing Melanoma Cells in Xenotransplanted Zebrafish

Molecules ◽  
2020 ◽  
Vol 25 (6) ◽  
pp. 1270 ◽  
Author(s):  
Maria Teresa Valenti ◽  
Giulia Marchetto ◽  
Massimiliano Perduca ◽  
Natascia Tiso ◽  
Monica Mottes ◽  
...  
Keyword(s):  
Melanoma Cells ◽  
Migration And Invasion ◽  
In Vivo Model ◽  
Wild Mushroom ◽  
Migration Ability ◽  
Melanoma Patients ◽  
Annual Mortality

RUNX2, a master osteogenic transcript ion factor, is overexpressed in several cancer cells; in melanoma it promotes cells migration and invasion as well as neoangiogenesis. The annual mortality rates related to metastatic melanoma are high and novel agents are needed to improve melanoma patients’ survival. It has been shown that lectins specifically target malignant cells since they present the Thomsen–Friedenreich antigen. This disaccharide is hidden in normal cells, while it allows selective lectins binding in transformed cells. Recently, an edible lectin named BEL β-trefoil has been obtained from the wild mushroom Boletus edulis. Our previous study showed BEL β-trefoil effects on transcription factor RUNX2 downregulation as well as on the migration ability in melanoma cells treated in vitro. Therefore, to better understand the role of this lectin, we investigated the BEL β-trefoil effects in a zebrafish in vivo model, transplanted with human melanoma cells expressing RUNX2. Our data showed that BEL β-trefoil is able to spread in the tissues and to reduce the formation of metastases in melanoma xenotransplanted zebrafish. In conclusion, BEL β-trefoil can be considered an effective biomolecule to counteract melanoma disease.

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