Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Macrophages stimulated with LPS and Poly (I:C)

ABSTRACT: 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 Âµm 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 Âµg/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 Âº 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 Âµg/ml of LPS from E. coli (serotype 026:B6, Sigma) or 10 Âµg/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.

ORGANISM(S): Oncorhynchus mykiss

SUBMITTER: Carmen DoÃ±ate

PROVIDER: E-GEOD-13197 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 Î¼l 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

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 ÂºC. 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 Âµg 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).

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).

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).

Project description:Metabolic processes and sexual maturation closely interact during the long-distance reproductive migration of many fish species to their spawning grounds. In the present study, we have for the first time used exercise experimentally to investigate the effects on sexual maturation in rainbow trout. Pubertal autumn-spawning seawater-raised female rainbow trout Oncorhynchus mykiss (n=26; 50-cm, 1.5-kg) were rested or swum at a near optimal speed of 0.75 body-lengths per second in a 6,000 L swim-flume under natural reproductive conditions (16 Â°C fresh-water, starvation, 8h-light:16h-dark photoperiod). Fish were sampled after arrival and subsequently after 10 days (resting or swimming 307 km) and 20 days (resting or swimming 636 km). Ovarian development was significantly reduced in the swimmers. Analysis of the expression of key factors in the reproductive axis included pituitary kiss1-receptor, lh and fsh and ovarian lh-receptor, fsh-receptor, aromatase and vitellogenin-receptor (vtgr). Swimmers had lower pituitary lh and ovarian vtgr expression than resters. Furthermore, the number of late vitellogenic oocytes was lower in swimmers than in resters, probably resulting from the lower vtgr expression, and vitellogenin plasma levels were higher. Therefore, swimming exercise suppresses oocyte development possibly by inhibiting vitellogenin uptake. Transcriptomic changes that occurred in the ovary of exercised fish were investigated using a salmonid cDNA microarray platform. Protein biosynthesis and energy provision were among the sixteen functional categories that were all down-regulated in the ovary. Down-regulation of the transcriptomic response in the ovary illustrates the priority of energy reallocation and will save energy to fuel exercise. A swimming-induced ovarian developmental suppression at the start of vitellogenesis during long-term reproductive migration may be a strategy to avoid precocious muscle atrophy. In order to simulate the natural reproductive conditions of anadromous salmonids, experiments were performed with sea water-raised rainbow trout, Oncorhynchus mykiss. Resters and swimmers were starved during the experiment, seawater was replaced by fresh water and photoperiod was changed. These changes were expected to affect reproductive parameters but as âbackgroundâ effect because both resters as swimmers were experiencing these conditions at the same time in the same set-up. Any additional effects in swimming fish would thus be caused by swimming only. Pubertal autumn spawning rainbow trout (n=26; ~50 cm, ~1.5 kg) were purchased from a Danish exporter (Frederiksvaerk Aleexport) where they had been raised for 2 years in freshwater followed by 4 months in sea water cages at 10 â°. They were transferred by truck within 16 h to Spakenburg (Spakenburg Paling, The Netherlands) and subsequently in the same water in a similar holding tank to the swim-flume at Leiden University (The Netherlands). The next day, 5 randomly chosen fish were sampled as âstartâ-group while others were randomly divided into a ârestâ-group (n=10) and a âswimâ-group (n=11). During the following 4 days, the brackish 10 â° water was stepwise replaced by freshwater at 16 Â°C and photoperiod was changed from 16L:8D to 8L:16D. Fish were then acclimatized for two days to their new conditions. Subsequently, swimmers were first swum at a speed of 0.33 body-lengths per second (BL/s) which was increased the next day to the final, near optimal speed of 0.75 BL/s (0.34 m/s or 29.4 km/day). Fish were sampled after 10 days (5 resters and 5 swimmers) and 20 days (5 resters and 6 swimmers). At sampling, fish were anesthetized using oil of cloves that was commercially purchased from a drugstore (diluted 1:10 in ethanol and used at a dosage of 1.5 ml/l). Fish were euthanized by decapitation and dissected for the ovary which was flash frozen in liquid nitrogen. RNA was isolated from pituitary and ovary samples according to the TRIZOL reagent protocol by Invitrogen (Baro, Spain). Isolated RNA was DNAse treated using RQ1 DNAse Promega (Madison, USA) and reverse transcribed using Superscript IIITM (Baro, Spain) according to the manufacturerâs protocol. Microarray analyses were performed on ovarian tissue for 20-days-swimmers vs. 20-days-resters. A rainbow trout cDNA microarray platform was used that was previously validated and described by Koskinen et al., 2004ab, Krasnov et al., 2005, MacKenzie et al., 2006ab, 2008 and Djordjevic et al., 2009, and deposited in GEO under accession number GPL1212, the original cDNA microarray (SFA2.0 chip), and GPL6154; the updated 1.8 K platform. This platform compromises 1818 genes representing 366 functional categories. Total RNA was extracted from flash frozen samples from experimental fish of the 20-days-swim group (n=5) and 20-days-rest group (n=5) like described before. Total RNA corresponding to pooled samples from the swim or rest groups was labeled with Cye3 and Cye5-dCTP (GE Healthcare, Barcelona, Spain) using SuperScript III reverse transcriptase (Invitrogen, Baro, Spain) and oligo(dT) primer. In our experience, reverse labeling combined with multiple replication of spots provide robust normalization and high statistical power of analyses. cDNA was purified with MicroconYM30 (Millipore). The slides were pretreated with 1% BSA(fraction V), 20 Ã SSC, 10% SDS (30 min at 50Â°C) and washed with 2 Ã SSC (3 min) and 0.2 Ã SSC (3 min) and hybridized overnight in cocktail containing 50 Ã Denhardt's, 20 Ã SSC, 10% SDS, 10Î¼g/Î¼l polyadenylate and 10 Î¼g/Î¼l yeast tRNA. All chemicals were from Sigma-Aldrich. Scanning was performed with ScanArray 5000 and images were processed with QuantArray (GSI Luminomics). The measurements in spots were filtered by criteria I/B â¥ 3 and (I-B)/(SI + SB) â¥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. After subtraction of mean background, LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios (expression ratio) were analyzed with Student's t-test (P <0.05). Validation of the microarray was performed by Q-PCR of 6 differentially expressed genes (DEGs) as described above. The used primers were either published (Alpha-globin I-1: Donate Jimeno, 2008) or designed, again by using the Genamics software.

Project description:The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor Î± (TNFÎ±) and other immune factors, are produced and act on the reproductive system. However, TNFÎ± is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNFÎ± in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). In control and recombinant trout TNFÎ± (rtTNFÎ±)-treated granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNFÎ± on follicle contraction and testosterone production in preovulatory trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNFÎ±-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.Treatment with rtTNFÎ± induces ovarian cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNFÎ± causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNFÎ± induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. In view of these results, we propose that TNFÎ± could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction. Reproductively mature female brown trout (Salmo trutta) from a cultured stock at the Piscifactoria de BagÃ (Generalitat de Catalunya, BagÃ , Spain) were kept under natural conditions of temperature and photoperiod. Fish at the preovulatory stage (according to the position of the germinal vesicle (GV) in oocytes that were cleared using a solution previously described), were anesthesized in 3-aminobenzoic acid ethyl ester (0.1 g/l; Sigma, Alcobendas, Spain) dissolved in fresh water, and the fish were sacrificed by concussion prior to the collection of the ovaries. The dissected ovaries were immediately used for the various in vitro assays. After dissection, brown trout preovulatory ovaries were placed in HankÂ´s balanced salt solution (HBSS) and individual ovarian follicles were manually separated with forceps from each ovary on ice, as previously described. To collect ovarian tissue for RNA extraction, preovulatory follicles from each of a total of three females were incubated (400 follicles/50 ml) in HBSS-BSA in the absence or presence of rtTNFÎ± (100 ng/ ml, dissolved directly in HBSS-BSA), at 15ÂºC for 24 h with gentle shaking (100 rpm). At the end of the incubation follicles (previously de-yolked by gentle pressure) were removed, flash frozen in liquid nitrogen and stored at -80ÂºC until assayed.

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Macrophages stimulated with LPS and Poly (I:C)

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