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Salmon Macrophage early response to Aeromonas salmonicida

ABSTRACT: Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Enriched macrophages isolated from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0 h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0 h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response. Keywords: time course

ORGANISM(S): Salmo salar

PROVIDER: GSE8526 | GEO | 2008/02/27

SECONDARY ACCESSION(S): PRJNA101653

REPOSITORIES: GEO

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Project description:Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Enriched macrophages isolated from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0 h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0 h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response. Keywords: time course Enriched macrophages from 24 responder fish that showed positive respiratory burst in response to phorbol myristate acetate were plated in individual wells of 96-well flat-bottom polystyrene tissue culture plates. A. salmonicida were added to the macrophages, and incubated for 0.5, 1.0 or 2.0 h. Control wells received 10 ul of HBSS. Three replicate infections were performed for each type of bacteria. Hybridizations were carried out in duplicate, reversing the fluors for each sample on the second chip.

Project description:Aliivibrio salmonicida causes âcold-water vibriosisâ (or âHitra diseaseâ) in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and an âomics approach to study how A. salmonicida responds to hydrogen peroxide (H2O2). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H2O2 response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis, microarray analysis and 2D gel electrophoresis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down-regulated genes are distributed among all functional gene categories, but a striking number of â¥2 fold up-regulated genes encode proteins involved in detoxification or DNA protection and repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADH/NADPH. Our predictions and âomics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation. Two-condition experiment, cells grown in LB medium (control samples) vs. cells grown under oxidative stress (H2O2) (stimulated samples). Samples collected from three different timepoints (15, 30 and 60 min). Technical replicates for each timepoint: 3 control, 3 stimulated, independently grown and harvested. One replicate per array.

Project description:The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt+ B subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of infection with Aeromonas was highly variable across cell types, with the largest changes observed in resident macrophages and infiltrating monocytes, followed by resting mature B cells. Our differential expression analysis offers evidence for a strong inflammatory response to bacteria in the spleen driven by macrophages and monocytes, and the recruitment of splenic igt+ B cells to the gut. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

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Salmon Macrophage early response to Aeromonas salmonicida

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