Development of innate immune memory by non-immune cells during Staphylococcus aureus infection depends on reactive oxygen species.

Introduction: The mechanisms underlying innate immune memory (trained immunity) comprise epigenetic reprogramming of transcriptional pathways associated with alterations of intracellular metabolism. While the mechanisms of innate immune memory carried out by immune cells are well characterized, such processes in non-immune cells, are poorly understood. The opportunistic pathogen, Staphylococcus aureus, is responsible for a multitude of human diseases, including pneumonia, endocarditis and osteomyelitis, as well as animal infections, including chronic cattle mastitis that are extremely difficult to treat. An induction of innate immune memory may be considered as a therapeutic alternative to fight S. aureus infection. Methods: In the current work, we demonstrated the development of innate immune memory in non-immune cells during S. aureus infection employing a combination of techniques including Enzyme-linked immunosorbent assay (ELISA), microscopic analysis, and cytometry. Results: We observed that training of human osteoblast-like MG-63 cells and lung epithelial A549 cells with ß-glucan increased IL-6 and IL-8 production upon a stimulation with S. aureus, concomitant with histones modifications. IL-6 and IL-8 production was positively correlated with an acetylation of histone 3 at lysine 27 (H3K27), thus suggesting epigenetic reprogramming in these cells. An addition of the ROS scavenger N-Acetylcysteine, NAC, prior to ß-glucan pretreatment followed by an exposure to S. aureus, resulted in decreased IL-6 and IL-8 production, thereby supporting the involvement of ROS in the induction of innate immune memory. Exposure of cells to Lactococcus lactis resulted in increased IL-6 and IL-8 production by MG-63 and A549 cells upon a stimulation with S. aureus that was correlated with H3K27 acetylation, suggesting the ability of this beneficial bacterium to induce innate immune memory. Discussion: This work improves our understanding of innate immune memory in non-immune cells in the context of S. aureus infection. In addition to known inducers, probiotics may represent good candidates for the induction of innate immune memory. Our findings may help the development of alternative therapeutic approaches for the prevention of S. aureus infection.

Comparison of interferometric light microscopy with nanoparticle tracking analysis for the study of extracellular vesicles and bacteriophages.

Research on extracellular vesicles (EVs) and bacteriophages (phages) has been steadily expanding over the past decades as many of their roles in medicine, biology, and ecosystems have been unveiled. Such interest has brought about the need for new tools to quantify and determine the sizes of these biological nanoparticles. A new device based on interferometric light microscopy (ILM), the Videodrop, was recently developed for this purpose. Here, we compared this new device to two nanoparticle tracking analysis (NTA) devices, the NanoSight and the ZetaView, for the analysis of EVs and phages. We used EVs isolated from bacteria, fecal samples, bovine milk and human cells, and phages of various sizes and shape, ranging from 30 to 120 nm of diameter. While NTA instruments correctly enumerated most phages, the Videodrop detected only the largest one, indicating a lower sensitivity threshold compared to the NTA devices. Nevertheless, the performance of the Videodrop compared favourably to that of the NTA devices for the determination of the concentration of eukaryotic EV samples. The NanoSight instrument provided the most precise size distributions but the Videodrop was by far the most time-saving device, making it worthy of consideration for studies conducted on a large number of samples composed of nanoparticles larger than 90 nm.

Krypton in the Chassigny meteorite shows Mars accreted chondritic volatiles before nebular gases.

Volatile elements are thought to have been delivered to Solar System terrestrial planets late in their formation through accretion of chondritic meteorites. Mars can provide information on inner Solar System volatile delivery during the earliest planet formation stages. We measured krypton isotopes in the martian meteorite Chassigny, representative of the planet's interior. We found chondritic krypton isotope ratios, which imply early incorporation of chondritic volatiles. The atmosphere of Mars has different (solar-type) krypton isotope ratios, indicating that it is not a product of magma ocean outgassing or fractionation of interior volatiles. Atmospheric krypton instead originates from accretion of solar nebula gas after formation of the mantle but before nebular dissipation. Our observations contradict the common hypothesis that during planet formation, chondritic volatile delivery occurred after solar gas acquisition.

Deep-mantle krypton reveals Earth's early accretion of carbonaceous matter.

Establishing when, and from where, carbon, nitrogen and water were delivered to Earth is a fundamental objective in understanding the origin of habitable planets such as Earth. Yet, volatile delivery to Earth remains controversial1-5. Krypton isotopes provide insights on volatile delivery owing to their substantial isotopic variations among sources6-10, although pervasive atmospheric contamination has hampered analytical efforts. Here we present the full suite of krypton isotopes from the deep mantle of the Galápagos and Iceland plumes, which have the most primitive helium, neon and tungsten isotopic compositions11-16. Except for 86Kr, the krypton isotopic compositions are similar to a mixture of chondritic and atmospheric krypton. These results suggest early accretion of carbonaceous material by proto-Earth and rule out any combination of hydrodynamic loss with outgassing of the deep or shallow mantle to explain atmospheric noble gases. Unexpectedly, the deep-mantle sources have a deficit in the neutron-rich 86Kr relative to the average composition of carbonaceous meteorites, which suggests a nucleosynthetic anomaly. Although the relative depletion of neutron-rich isotopes on Earth compared with carbonaceous meteorites has been documented for a range of refractory elements1,17,18, our observations suggest such a depletion for a volatile element. This finding indicates that accretion of volatile and refractory elements occurred simultaneously, with krypton recording concomitant accretion of non-solar volatiles from more than one type of material, possibly including outer Solar System planetesimals.

Reconstruction of the birth of a male sex chromosome present in Atlantic herring.

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an ∼500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination.

Chaperone-Mediated Autophagy in the Light of Evolution: Insight from Fish.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol.

Transcriptomic Analysis of Trout Gill Ionocytes in Fresh Water and Sea Water Using Laser Capture Microdissection Combined with Microarray Analysis.

Fish gills represent a complex organ composed of several cell types that perform multiple physiological functions. Among these cells, ionocytes are implicated in the maintenance of ion homeostasis. However, because the ionocyte represents only a small percent of whole gill tissue, its specific transcriptome can be overlooked among the numerous cell types included in the gill. The objective of this study is to better understand ionocyte functions by comparing the RNA expression of this cell type in freshwater and seawater acclimated rainbow trout. To realize this objective, ionocytes were captured from gill cryosections using laser capture microdissection after immunohistochemistry. Then, transcriptome analyses were performed on an Agilent trout oligonucleotide microarray. Gene expression analysis identified 108 unique annotated genes differentially expressed between freshwater and seawater ionocytes, with a fold change higher than 3. Most of these genes were up-regulated in freshwater cells. Interestingly, several genes implicated in ion transport, extracellular matrix and structural cellular proteins appeared up-regulated in freshwater ionocytes. Among them, several ion transporters, such as CIC2, SLC26A6, and NBC, were validated by qPCR and/or in situ hybridization. The latter technique allowed us to localize the transcripts of these ion transporters in only ionocytes and more particularly in the freshwater cells. Genes involved in metabolism and also several genes implicated in transcriptional regulation, cell signaling and the cell cycle were also enhanced in freshwater ionocytes. In conclusion, laser capture microdissection combined with microarray analysis allowed for the determination of the transcriptional signature of scarce cells in fish gills, such as ionocytes, and aided characterization of the transcriptome of these cells in freshwater and seawater acclimated trout.

Effects of iron on rainbow trout gill cells in primary culture.

This study investigated the effects of iron in the form of iron sulphate (FeSO(4)·7H(2)O), over the range 0.01-1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO(4) did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO(4) reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.

Obtaiment of Pudu (Pudu pudu) deer embryos by the somatic nuclear transfer technique

RESUMEN: Los objetivos de este trabajo fueron los de producir embriones de pudú, obtenidos por la transferencia de núcleos de fibroblastos de la oreja de pudú en ovocitos de un rumiante domésticos que es el bovino. Para posteriormente en un trabajo futuro proceder a la transferencia de embriones de pudú, al útero de hembras receptoras sincronizadas de otra especie. Se obtuvieron biopsias de 1 mm aproximadamente del borde externo de la orejas de dos ciervos pudu machos del jardín zoológico Buin-Zoo, Santiago de Chile. Las líneas celulares han sido establecidas y conservadas según los protocolos utilizados para las bovinos. Los ovocitos son obtenidos por punción del complejo cúmulos-ovocito (COC).desde ovarios de vacas recuperados del matadero. Cada ovocito es enucleado y fusionado con un fibroblasto aislado insertado bajo la zona pelúcida. La fusión de membranas celulares es obtenida por choques eléctricos. En cuanto a la cronología, observamos que al segundo día se forma una etapa de dos blastómeras, al tercer día mórulas de 8 a 16 células, y desde el cuarto día se ha diferenciado como blastocisto, el cuál al séptimo día termina por eclosionar de la zona pelúcida.La obtención de blastocistos embrionarios indica que es posible obtener embriones de pudú mediante clonaje heteroespecífico, aunque, el porcentaje de éxito obtenido es relativamente bajo. Queda aun por verificar la viabilidad de los embriones así obtenidos después de la transferencia in útero.