Evaluation of Trained Immunity by β-1, 3 (D)-Glucan on Murine Monocytes in Vitro and Duration of Response in Vivo

The β-1, 3 (d)-glucan (β-glucan) present in the cell wall of Candida albicans induces epigenetic changes in human monocytes resulting in primed macrophages exhibiting increased cytokine responsiveness to reinfection. This phenomenon is referred to as trained immunity or innate immune memory. However, whether β-glucan can reprogramme murine monocytes in vitro or induce lasting effects in vivo has yet to be elucidated. Thus, purified murine spleen-derived monocytes were primed with β-glucan in vitro and assessed for markers of differentiation and survival. Important macrophage cell markers during monocyte-to-macrophage differentiation were downregulated and survival enhanced due to partial inhibition of apoptosis. Increased survival and not the β-glucan training effect explained the elevated production of tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) induced by subsequent lipopolysaccharide (LPS) challenge. In vivo, 4 days after systemic administration of β-glucan, mice were more responsive to LPS challenge as shown by the increased serum levels of TNFα, IL-6 and IL-10, an effect shown to be short lived as enhanced cytokine production was lost by day 20. Here, we have characterised murine macrophages derived from β-glucan-primed monocytes based on their surface marker expression and for the first time provide evidence that the training effect of β-glucan in vivo declines within a 3-week period

Targeting and stimulation of the zebrafish (Danio rerio) innate immune system with LPS/dsRNA-loaded nanoliposomes

Herein we report the use of immunostimulant-loaded nanoliposomes (called NLcliposomes) as a strategy to protect fish against bacterial and/or viral infections. This work entailed developing a method for in vivo tracking of the liposomes administered to adult zebrafish that enables evaluation of their in vivo dynamics and characterisation of their tissue distribution. The NLc liposomes, which co-encapsulate poly(I:C) and LPS, accumulate in immune tissues and in immunologically relevant cells such as macrophages, as has been assessed in trout primary cell cultures. They protect zebrafish against otherwise lethal bacterial (Pseudomonas aeruginosa PAO1) and viral (Spring Viraemia of Carp Virus) infections regardless of whether they are administered by injection or by immersion, as demonstrated in a series of in vivo infection experiments with adult zebrafish. Importantly, protection was not achieved in fish that had been treated with empty liposomes or with a mixture of the free immunostimulants. Our findings indicate that stimulation of the innate immune system with co-encapsulated immunostimulants in nano-liposomes is a promising strategy to simultaneously improve the levels of protection against bacterial and viral infections in fish

Behavioural fever is a synergic signal amplifying the innate immune response

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene-environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity-environment interactions are likely to be under strong positive selection

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year

Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

marine drug

Identification of Multipath Genes Differentially Expressed in Pathway-Targeted Microarrays in Zebrafish Infected and Surviving Spring Viremia Carp Virus (SVCV) Suggest Preventive Drug Candidates

<div><p>Spring viremia carp virus (SVCV) is a rhabdovirus seasonally affecting warm-water cyprinid fish farming causing high impacts in worldwide economy. Because of the lack of effective preventive treatments, the identification of multipath genes involved in SVCV infection might be an alternative to explore the possibilities of using drugs for seasonal prevention of this fish disease. Because the zebrafish (<i>Danio rerio</i>) is a cyprinid susceptible to SVCV and their genetics and genome sequence are well advanced, it has been chosen as a model for SVCV infections. We have used newly designed pathway-targeted microarrays 3-4-fold enriched for immune/infection functional-relevant probes by using zebrafish orthologous to human genes from selected pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative analysis of differential expression of genes through 20 pathways in 2-day exposed or 30-day survivors of SVCV infection allowed the identification of 16 multipath genes common to more than 6 pathways. In addition, receptors (Toll-like, B-cell, T-cell, RIG1-like) as well as viral RNA infection pathways were identified as the most important human-like pathways targeted by SVCV infection. Furthermore, by using bioinformatic tools to compare the promoter sequences corresponding to up and downregulated multipath gene groups, we identified putative common transcription factors which might be controlling such responses in a coordinated manner. Possible drug candidates to be tested in fish, can be identified now through search of data bases among those associated with the human orthologous to the zebrafish multipath genes. With the use of pathway-targeted microarrays, we identified some of the most important genes and transcription factors which might be implicated in viral shutoff and/or host survival responses after SVCV infection. These results could contribute to develop novel drug-based prevention methods and consolidate the zebrafish/SVCV as a model for vertebrate viral diseases. </p> </div

VENN diagram between genome-wide and targeted microarrays and comparison of fluorescence intensities obtained by hybridization to targeted microarrays of transcripts from 2- and 30-days after SVCV infection with those obtained from non-infected zebrafish controls.

<p><b>A</b>) A VENN diagram was constructed by comparing unique accession numbers between genome-wide zebrafish vs2 ID019161 microarray of Agilent (43803 probes, 37464 accession numbers) and targeted microarray zfin ID041401 (11586 probes, 8636 unique accession numbers: 2286 and 6350 pathway and keyword sections, respectively). The online software from BioInfoRx (<a href="http://apps.bioinforx.com" target="_blank">http://apps.bioinforx.com</a>) was used. <b>A1</b>, wide-genome zebrafish vs 2 ID019161. <b>A2</b>, pathway section of the targeted microarray zfin ID041401. A3, keyword section of the targeted microarray zfin ID041401. <b>B</b>, <b>C</b>) The zfin ID041401 was used to estimate transcript levels in pooled head kidney and spleen from SVCV-infected and non-infected zebrafish after 2-days (<b>B</b>) or 30-days (<b>C</b>). The Figure shows the range of mean fluorescences obtained from different experiments (6 fish per experiment, n=3 for non-infected zebrafish and for SVCV infected zebrafish after 2-days and n=2 for SVCV infected zebrafish after 30-days). A white straight line shows fold = 1.</p

“Toll-like receptor signaling pathway” of zebrafish genes after SVCV infection.

<p>The gene boxes containing the zebrafish gene short names in <i>italics</i> and their relationships were obtained from the corresponding human KEGG-pathway. All those genes appearing in the Figure were assayed for transcript expression. Capital letters, inputs (black) and outputs (blue) of the pathway. Vertical parallel lines, cellular membranes. Continuous black arrows, activation between gene products. Discontinuous black arrows, inhibition between gene products. Gray form, main pathway. Folds were calculated as described in methods by the formula, fluorescence value 2- or 30-days after infection / fluorescence mean value in non-infected controls. Red <i>italic</i> letters, downregulated transcripts with folds <0.5. Orange <i>italic</i> letters, downregulated transcripts with folds <0.66. Bright green <i>italic</i> letters, upregulated transcripts with folds >2. Dark green <i>italic</i> letters, upregulated transcripts with folds >1.5. Black <i>italic</i> letters, not differentially expressed. Gene short names surrounded by an square, multipath genes. Gray form, downstream pathway beginning with cell membrane toll-like receptors (tlr1, tlr2, tlr4, tlr5b and tlr6). Ovoid brown forms, <i>tlrs</i>. Gene <i>italic</i> letters were colored as follows: red <0.5, orange <0.66, dark green >1.5 and bright green >2 folds. <b>A</b>, 2-days after SVCV infection. <b>B</b>, 30-days after SVCV infection (survivors).</p