Macrophages stimulated with LPS and Poly (I:C)
Ontology highlight
ABSTRACT:
ORGANISM(S): Oncorhynchus mykiss
PROVIDER: GSE13197 | GEO | 2009/12/09
SECONDARY ACCESSION(S): PRJNA109493
REPOSITORIES: GEO
Similar Datasets
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: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.
2010-06-17 | GSE22379 | GEO
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:Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. Keywords: time course For each treatment (Sigma LPS, LIST LPS, and media only), three biological replicates (separate macrophage cultures and RNA isolations) were profiled. Each sample was labeled with Cy3 and Cy5 and co-hybridized with Stratagene Universal Mouse Reference (dye flip techical replicates). Expression at 2 timepoints (6 and 24 hours post-treatment) was assessed.
2010-10-01 | E-GEOD-21548 | biostudies-arrayexpress
Project description:Molecular Pathways and Transcriptional Networks Involved in the Macrophage Response to LPS, poly(I:C) and CpG DNA Stimulation Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. For each treatment (LPS, polyIC, CpG DNA, media only), three biological replicates (separate macrophage cultures and RNA isolations) were profiled. Each sample was labeled with Cy3 and Cy5 and co-hybridized with Stratagene Universal Mouse Reference (dye flip techical replicates). Expression at 2 timepoints (6 and 24 hours post-treatment) was assessed.
2009-06-30 | E-GEOD-15066 | biostudies-arrayexpress
Project description:Molecular Pathways and Transcriptional Networks Involved in the Macrophage Response to LPS, poly(I:C) and CpG DNA Stimulation Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations.
2009-07-01 | GSE15066 | GEO
Project description:Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. Keywords: time course
2010-10-01 | GSE21548 | GEO
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. Keywords: Viral and LPS transcriptomic response, IHNV, Trout
2008-02-07 | GSE10272 | GEO
Project description:Recent studies reported the inhibitory properties of LAIR-1 in different inflammatory diseases, including allergic and viral airway inflammation. However, the implication of LAIR-1 in ALI/ARDS remains unknown. Poly(I:C), a synthetic analog of (ds)RNA was administrated intranasally to mimic acute inflammation in viral infections and therefore induce experimental ALI/ARDS. RNA sequencing approach was used to explore and compare lung macrophage transcriptomic profiles in wild-type and LAIR-1 knock-out mice.
2023-09-01 | GSE216259 | GEO
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
2008-10-02 | GSE12961 | GEO