Trout macrophages: Control vs. Escherichia coli PGNs from strains O111:B4 and K12
Ontology highlight
ABSTRACT:
ORGANISM(S): Oncorhynchus mykiss
SUBMITTER: Sebastian Boltana
PROVIDER: E-GEOD-22330 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. Transcript profiling was assessed using function-targeted cDNA microarrays hibridation (n = 36) to differential responses to both PGNs that are both time and treatment dependen over trout macrophages. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 shows a high similarity with a general LPS response where multiple functional classes are related to ribosome biogenesis or cellular metabolism
2010-06-16 | GSE22330 | GEO
Project description:This study describes the development and validation of the Aquagenomic Sparus aurata oligonucleotide-microarray (SAQ) based on the Agilent Technology system (eArray) to provide a platform for studies on the gene expression of gilthead seabream. The platform developed used all available public ESTs stored and annotated in the Aquagenomic Consortium seabream library (10K). In fish, lipopolysaccharide (LPS) gives a robust cytokine response that is stimulated by crude LPS preparations, some component of the LPS complex are responsible for this stimulation. Peptidoglycan (PGN) is a component of G-negative bacteria (found as a contaminant of crude LPS preparations) able to be recognized by macrophages inducing depth transcriptional modulations and a strong inflammatory response. For microarray analysis, head kidney macrophage cultures were used (N = 36 fish). Each cell culture was stimulated with equal concentrations of PGN and LPS from E. coli O111:B4 strain (10 ug/mL): non-stimulated cell cultures (control n = 9 fish), stimulated during 6 h with LPS (n = 9), and stimulated during 1 h (n = 9), and 6 h (n = 9) with PGN. A loop microarray design approach was used for the study. All experimental RNA samples were labelled with a single colour dye (Cy3) and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Our microarray analyses identified differential transcriptional modulations in macrophages stimulated with both LPS and PGN at the level of differentially activated RNA transcripts related with the regulation of transcriptional program, prostaglandin synthesis or highlighting the expression of responsive gene-cassettes tightly related to LPS-PGN host recognition. A total of 43,398 oligonucleotide probes were used to construct a high-density seabream microarray based on the Agilent 4 × 44 K design format. Microarray hybridization validation was made by analyzing the gene expression profiles in primary cultures of seabream macrophages (MC). 7,285 transcripts with annotated sequences were spotted in triplicate onto the slide (total probes 21,855), as well as 8,377 ESTs without annotation, 183 enriched sequences (gene bank) with 15 replicated probes (total probes 2,745), and finally 1,417 internal control probes of Agilent (N = 43,398).
2011-04-22 | E-GEOD-28610 | biostudies-arrayexpress
Project description:The aim of the current study was to investigate the response of in vitro differentiated trout macrophages, a primary cell culture system widely used in fish immunology research, to lipopolysaccharide (LPS) and to the analog viral ds(RNA) Poly I:C. To that end we have used a salmonid-specific microarray platform enriched with immune-related genes. The results clearly suggest that the molecular mechanisms involved in the response of macrophages to LPS and Poly (I:C) are specific in some signaling pathways related to cell communication, signal transduction and kinase cascades. Nevertheless, macrophages stimulated with bacterial or viral PAMPs also activate common transcription factors. Trout macrophages were isolated using the protocol previously described Head kidneys were dissected, placed in sterile 100 µm nylon mesh cell-strainers and homogenized in the presence of DMEM (PAA Laboratories) containing high glucose, 10% heat inactivated FCS (PAA Laboratories) and Primocin antibiotic (100 µg/ml, Invivogen). The homogenates were plated on 60 mm poly-D-lysine (Sigma) treated cell culture Petri plates, 3 ml per plate. The cultures were kept in an incubator at 16 º C and 5% CO2. Non-adhering cells were removed after 24 hours and new medium was added, then adherent cells were incubated for another 4 days. After 5 total days of culture, medium was removed and test cells were stimulated adding fresh medium containing 10 µg/ml of LPS from E. coli (serotype 026:B6, Sigma) or 10 µg/ml of Poly (I:C) (Invivogen), whereas medium free of stimulant was added in control plates. All culture plates were incubated for 12h. Six independent experiments (six different fish) were performed for each condition. Total RNA was extracted from the cultures using 1 ml of Tri Reagent (Sigma), following the manufacturerâ??s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.
2010-06-25 | E-GEOD-13197 | biostudies-arrayexpress
Project description:We have employed whole genome microarray expression profiling to identify genes differentially expressed in cord blood purified neutrophils after a short-term exposure to peptidoglycan (PGN). PGN induced gene expressions of neonatal neutrophils were measured at 4 hours. Three biological replicates were performed for each treatment group.
2013-09-04 | E-GEOD-40636 | biostudies-arrayexpress
Project description:The effect of dietary immunostimulation in the immune organs, head kidney and spleen, of rainbow trout (Oncorhynchus mykiss), was investigated using a salmonid-specific microarray platform enriched with immune-related genes. Immunostimulant-diet feeding significantly changed transcriptomic expression profiles: larger reduction rather than induction was observed, with significant regulation in genes and functional GO categories related to remodeling processes and immune and hematopoietic activities. The results revealed that Immunostimulant-diets hava effect in the transcriptome of cultured fish. Keywords: spleen, head kidney, immunostimulats, transcriptomic response, trout Adult rainbow trout (Oncorhynchus mykiss) were randomly distributed in four fibreglass tanks (25 fish per tank). During the feeding trial, trouts were hand-fed once a day with either the control diet or the immunodiet, in duplicate tanks, following manufacturer’s indication of 1 g food/fish for 4 weeks. After the 4 weeks, 4 fish of each tank (8 for each diet) were sampled for microarray analysis. Tissues removed for RNA extraction were frozen in liquid nitrogen and stored at -80 ºC. Total RNA was extracted from the organs using 1 ml of Tri Reagent (Sigma) per 100 mg of tissue, following the manufacturer’s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes. RNA was pooled within treatments (n=8), and 15 µg of control and test were labeled with Cy3- and Cy5-dCTP (Amersham Pharmacia) using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer (Promega), and cDNA was purified with Microcon YM30 (Millipore). Dye swap method was used, therefore each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. Scanning was performed with Axon scanner 4000B and images were processed with GenePix Pro 6.0. After subtraction of mean background, and LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Student’s t-test (p<0.01) and genes were ranked by log(p-level). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes. The functional categories of Gene Ontology were compared with regulated genes (p<0.01) by the sums of ranks (Student’s t-test, p<0.05) and the statistical significance of over represented functional categories was assessed using the Yates correction to Chi square test (corrected p<0.05).
2008-08-27 | E-GEOD-11949 | biostudies-arrayexpress
Project description:The focus of this study was to investigate the response of cultured rainbow trout erythrocytes to lipopolysaccharide (LPS) and to the analog viral dsRNA Poly(I:C) using a salmonid-specific microarray platform enriched with immune-related genes. Although the transcriptomic response measured as the total number of differentially expressed genes was higher in LPS, the intensity response, in terms of genes with a FC>2, was greater in Poly(I:C) treated cells. These two treatments shared the regulation of some genes, but not alls followed the same pattern. Over representation of Gene Ontology functional categories showed us that Poly(I:C) treatment produced a immune response whereas LPS stimulation affect biological processes specially. Trout erythrocytes were isolated from blood by twice Ficoll 1.007 density gradient centrifugations. For primary cell culture, erythrocytes were resuspended in DMEM (PAA Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories) and the antibiotic Primocin (100 μg/ml, Invivogen) at a density of 7.5x106 cells/ml in six well cell culture plates (NUNC) and cultured at 18 ºC and 5% CO2. Erythrocytes were stimulated with LPS from E. coli (serotype 026:B6, Sigma) and Poly(I:C) (Invivogen) at 50 μg/ml, each treatment had their own control plate. All culture plates were incubated for 24h. Total RNA was extracted from the cultures using 1 ml of Tri Reagent (Sigma) following the manufacturer’s instructions with minor modifications. Quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.
2010-06-16 | E-GEOD-22379 | biostudies-arrayexpress
Project description:The response of the trout, O.mykiss, head kidney to bacterial lipopolysaccharide (LPS) or active and attenuated infectious hematopoietic necrosis virus (IHNV and attINHV respectively) intraperoniteal challenge, 24 and 72 hours post-injection, was investigated using a salmonid-specific cDNA microarray. The head kidney response to i.p. LPS-induced inflammation in the first instance displays an initial stress reaction involving suppression of major cellular processes, including immune function, followed by a proliferative hematopoietic-type/biogenesis response 3 days after administration. The viral response at the early stage of infection highlights a suppression of hematopoietic and protein biosynthetic function and a stimulation of immune response. In fish infected with IHNV a loss of cellular function including signal transduction, cell cycle and transcriptional activity 72 hours after infection reflects the tissue-specific pathology of IHNV infection. attIHNV treatment on the other hand shows a similar pattern to native IHNV infection at 24 hours however at 72hours a divergence from the viral response is seen and replace with a recovery response more similar to that observed for LPS is observed. Rainbow trout, Oncorhynchus mykiss were intraperitoneally injected with saline (control) and LPS (6mg/Kg), or with 100 μl of a 106 pfu/ml dilution of IHNV or attIHNV or culture medium (negative control). At defined time points, 24 and 72 hours post i.p. injection, animals (total n=6), control and experimental, were selected from each tank (n=2) and sacrificed. Head kidneys (HK) were immediately dissected out, pooled and processed for total RNA purification using Tri Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturerâs protocol.The design of the microarray includes 1380 genes printed in six replicates each. Random clones from common and subtracted cDNA libraries (976) were compared with the known vertebrate proteins using blastx and 686 genes were identified; the functional annotations were transferred from the putative homologs. These clones were supplemented with 297 genes selected by the categories of Gene Ontology. Overall, each microarray was enriched in a number of functional classes, such as stress and defense response (145 and 105 genes, respectively), cell cycle (62 genes), signal transduction (114 genes), chaperone activity (41 genes), and apoptosis (79 genes). Total RNA obtained from head kidney tissue was verified for quantity and integrity by denaturing electrophoresis and labeling with Cy3- and Cy5-dCTP (Amersham Pharmacia) was completed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer, and cDNA was purified with Microcon YM30 (Millipore). We used a dye swap experimental design and each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignment was reversed. All head kidney samples were analyses using the dye swap protocol. Scanning was performed with ScanArray 5000 and images were processed with QuantArray (GSI Luminomics. After subtraction of mean background, locally weighted non-linear regression (Lowess) normalization was performed separately for each slide. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Studentâs t-test (p<0.01). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes
2010-06-25 | E-GEOD-10272 | biostudies-arrayexpress
Project description:The effect of dietary immunostimulation in the portals of entry, intestine and gills, of rainbow trout (Oncorhynchus mykiss), was investigated using a salmonid-specific microarray platform enriched with immune-related genes. IS-diet feeding significantly changed transcriptomic expression profiles: larger reduction rather than induction was observed, with significant changes in genes and functional GO categories related to remodeling processes and antigen presentation. The results revealed that one of the main effects of IS-diets in trout is the increase of genes involved in antigen recognition in epithelial cells of gills. Adult rainbow trout (Oncorhynchus mykiss) were randomly distributed in four fibreglass tanks (25 fish per tank). During the feeding trial, trouts were hand-fed once a day with either the control diet or the immunodiet, in duplicate tanks, following manufacturer’s indication of 1 g food/fish for 4 weeks. After the 4 weeks, 4 fish of each tank (8 for each diet) were sampled for microarray analysis. Tissues removed for RNA extraction were frozen in liquid nitrogen and stored at -80 ºC. Total RNA was extracted from the organs using 1 ml of Tri Reagent (Sigma) per 100 mg of tissue, following the manufacturer’s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes. RNA was pooled within treatments (n=8), and 15 µg of control and test were labeled with Cy3- and Cy5-dCTP (Amersham Pharmacia) using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer (Promega), and cDNA was purified with Microcon YM30 (Millipore). Dye swap method was used, therefore each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. Scanning was performed with Axon scanner 4000B and images were processed with GenePix Pro 6.0. After subtraction of mean background, and LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Student’s t-test (p<0.01) and genes were ranked by log(p-level). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes. The functional categories of Gene Ontology were compared with regulated genes (p<0.01) by the sums of ranks (Student’s t-test, p<0.05) and the statistical significance of over represented functional categories was assessed using the Yates correction to Chi square test (corrected p<0.05).
2008-03-31 | E-GEOD-10981 | biostudies-arrayexpress
Project description:Rainbow trout (Oncorhynchus mykiss) were fed during 4 weeks with either a control diet or an immunostimulant diet and then injected with LPS to investigate the effect of dietary immunostimulation in the portals of entry, intestine and gills, using a salmonid-specific microarray platform enriched with immune-related genes. IS-diet feeding significantly changed transcriptomic expression profiles in response to LPS: significant changes in genes and functional GO categories related to remodeling processes and antigen presentation were different for both diets. The results revealed that one of the main effects of IS-diets in trout is the increase of genes involved in antigen recognition and in adaptive immunity. Keywords: gills, intestine, immunostimulats, transcriptomic response, trout Adult rainbow trout (Oncorhynchus mykiss) were randomly distributed in four fibreglass tanks (25 fish per tank). During the feeding trial, trouts were hand-fed once a day with either the control diet or the immunodiet, in duplicate tanks, following manufacturerâ??s indication of 1 g food/fish for 4 weeks. After the 4 weeks, 5 fish were injected with LPS and 5 fish were injected with PBS. After 24h, samples were taken for microarray analysis. Tissues were removed for RNA extraction and frozen in liquid nitrogen, then stored at -80 ºC. Total RNA was extracted from the organs using 1 ml of Tri Reagent (Sigma) per 100 mg of tissue, following the manufacturerâ??s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes. RNA was pooled within treatments (n=5), and 15 µg of control and test were labeled with Cy3- and Cy5-dCTP (Amersham Pharmacia) using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer (Promega), and cDNA was purified with Microcon YM30 (Millipore). Dye swap method was used, therefore each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. Scanning was performed with Axon scanner 4000B and images were processed with GenePix Pro 6.0. After subtraction of mean background, and LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Studentâ??s t-test (p<0.01) and genes were ranked by log(p-level). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes. The functional categories of Gene Ontology were compared with regulated genes (p<0.01) by the sums of ranks (Studentâ??s t-test, p<0.05) and the statistical significance of over represented functional categories was assessed using the Yates correction to Chi square test (corrected p<0.05).
2010-06-25 | E-GEOD-12961 | biostudies-arrayexpress
Project description:Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGN) are major components of bacterial cell walls and possess immunity-stimulating activities in metazoans and plants. Here, we show that PGN sensing in Arabidopsis thaliana requires three LysM domain proteins, LYM1, LYM3 and CERK1. A 24 DNA microarray study using toral RNA from three Arabidopsis mutants (lym1-1, lym3-1, cerk1-2) as well as wild type treated with water or peptidoglycan.
2011-11-10 | E-GEOD-28004 | biostudies-arrayexpress