Project description:RNAs were analysed from bone marrow derived dendritic cells from 6 week old birds (control and LPS stimulated), bone marrow derived macrophages from 6 week old birds (control and LPS stimulated) and heterophils isolated from blood of day-old chicks (control and LPS stimulated).
Project description:Here, the m6A modification patterns in chicken liver at the acute stage of LPS-stimulated inflammation and at the normal state were explored via m6A and RNA sequencing and bioinformatics analysis. A total of 7815 m6A peaks distributed in 5066 genes were identified in the normal chicken liver, and were mostly located in the CDS, 3’UTR region and around the stop codon. At 2 h after the LPS intraperitoneal injection, the m6A modification pattern changed, and showed 1200 different m6A peaks. The hyper- and hypo-m6A peaks were differentially located, with the former mostly located in the CDS region and the later in the 3’UTR and in the region near the stop codon. The hyper- or hypo methylated genes were enriched in different GO ontrology and pathways. Co-analysis revealed a significantly positive relationship between the fold change of m6A methylation level and the relative fold change of mRNA expression. Moreover, protein and protein interaction analysis showed that genes with altered m6A methylation and mRNA expression levels were clustered in processes involved in lipid metabolism, immune response, DNA replication and protein ubiquitination. CD18 and SREBP-1 were the two hub genes clustered in the immune process and lipid metabolism, respectively. Hub gene AGPAT2 was suggested to link the immune response and lipid metabolism clusters in the PPI network. This study presented the first m6A map of broiler chicken liver at the acute stage of LPS induced inflammation. The findings may shed lights on the possible mechanisms of m6A-mediated lipid metabolism disorder in inflammation.
Project description:Erythrocytes represent the most abundant cell type of the bloodstream in all vertebrates. The principal feature that differenciates erythrocytes of non-mammals is that unlike mammals they mantain the nucleus in their terminal differentiation. While the main function associated to erythrocytes is the oxygen and carbon dioxide transport, some other functions, no less important, have been attributed to these cells. The focus of this study was to investigate the response of cultured chicken erythrocytes to bacterial lipopolysaccharide (LPS), bacterial peptidoglycan (PGN) and to the analog viral dsRNA Poly(I:C) using a comercial microarray platform from Agilent. Chicken 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 37ºC and 5% CO2. Erythrocytes were stimulated with LPS from E. coli (serotype 0111:B4, Sigma) and Poly(I:C) (Invivogen) at 50 μg/ml, and with peptidoglycan from E.coli (0111:B4, Invivogen) at 5 μg/ml. All culture plates were incubated for 12h. 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 Nanodrop1000 (ThermoScientific) and Bioanalyzer 2100 (Agilent Technologies) respectively. Three biological replicates were used.
Project description:RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA.
Project description:RNA from all normal tissues were pooled in equal amounts and used to create the normal tissue library. RNA from all immune stimulated tissues (Salmonella infection, CT+LPS, and PBC, with and without cycloheximide) were pooled in equal amounts and used to create the immune stimulated tissue library. The subtracted library was created by combining the normal and immune stimulated libraries and by subtracting porcine fibroblast RNA. Keywords: other
Project description:Erythrocytes represent the most abundant cell type of the bloodstream in all vertebrates. The principal feature that differenciates erythrocytes of non-mammals is that unlike mammals they mantain the nucleus in their terminal differentiation. While the main function associated to erythrocytes is the oxygen and carbon dioxide transport, some other functions, no less important, have been attributed to these cells. The focus of this study was to investigate the response of cultured chicken erythrocytes to bacterial lipopolysaccharide (LPS), bacterial peptidoglycan (PGN) and to the analog viral dsRNA Poly(I:C) using a comercial microarray platform from Agilent.
Project description:Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS). To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/-LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response. 2 pairs of THP-1 cells either stimulated with LPS or not. ChIP using either H3K9/K14Ac, RNA Pol II (phospho S5) or SP1 antibody. This submission represents chip-seq component of study.
Project description:Immortalized mouse macrophages (IMMs) were stimulated with 100 ng/ml lipopolysaccharide (LPS) for 0, 30, and 60 min. The samples were analyzed using LC-MS for global proteomics.