Sequencing of LPS-stimulated immune cells from chicken
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ABSTRACT: 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:RNAs were analysed from a pool of day 7 chicken embryos, the DF-1 chicken fibroblast cell line and chicken bone marrow-derived macrophages (stimulated with CSF-1).
Project description:Urea can serve as nitrogen source for coral holobionts and plays a cruscial role in coral calcification, although the degradation of urea by coral symbionts is not fully understood. In this study, we investigated the urea utilized pathway and the responses of the Symbiodiniaceae family to urea under high temperature conditions. Genome screening revealed that all Symbiodiniaceae species contain the urease (URE) and DUR2 subunit of urea amidolyase (UAD) system. However, only three speciesCladocopium goreaui, Cladopium c92, and Symbiodinium pilosum possess a complete UAD system, including both DUR1 and DUR2. Phylogentic analyses revealed that the UAD system in Symbiodiniaceae clusters more closely with symbiotic bacteria, indicating that horizontal gene transfer of UAD system has occured in coral symbionts. Physiology analysis showed that the symbiodiniacean species Cladocopium goreaui, which containing both URE and UAD, grew better under urea than ammonium conditions, as indicated by higher maximum specific growth rates. Furthermore, most genes of Symbiodiniaceae involved in urea utilization appeared to be stable under various conditions such as heat stress (HS), low light density, and nitrogen deficiency, wheras in ammonium and nitrate transporters were significantly regulated. These relatively stable molecular regulatory properties support sustained urea absorption by Symbiodiniaceae, as evidenced by an increase in δ15N2-urea absorption and the decreases in δ5N-NO3-, and δ15N-NH4+ from cultural environment to Symbiodiniaceae under HS conditions. Token together, this study reveals two distinct urea utilization systems in coral ecosystem and highlights the importance of the urea cycle in coral symbionts when facing fluctuating nitrogen environment in future warming ocean.
Project description:Langerhans cells (LCs) are antigen presenting cells residing in the epidermis. Due to the difficulties with obtaining sufficient quantities of LCs for functional studies, many controversies exist about their origin and function. To gain insights into the molecular mechanisms underpinning LC biology and to elucidate how similar they are to a classical tissue resident DCs of myeloid origin, we undertook a whole transcriptome analysis of human skin migratory CD1a+ LCs and CD11c+ DDCs, stimulated with an epidermal pro-inflammatory cytokine, TNF-α over a time course of 24h. RNA was extracted from 250,000 human skin migratory CD1a+ LCs and CD11c+ DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. The interactive 3D diagram presenting the transcriptomic networks in human skin DCs can be viewed at: http://www.macrophages.com/LC_vs_DC RNA was extracted from 250,000 human skin migratory CD1a+ LCs and DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. *submitter cannot locate the CEL files
Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.
Project description:For this study, thymic transcriptome responses to an acute heat stress and/or lipopolysaccharide (LPS) were investigated in a broiler line (heat and disease susceptible) and an inbred Fayoumi line (heat and disease resistant) of chickens. In a 2 x 2 design, 22 day-old birds were exposed to heat stress (35°C for 7 hours), lipopolysaccharide (100 µg/kg average body weight per line), or both stressors. Thermoneutral temperature (25°C) and phosphate buffered saline were used as the respective controls. Tissue samples were collected from the thymus and used to isolate high quality RNA. cDNA libraries (n = 31) were constructed and sequenced on the HiSeq 2500.
Project description:scRNAseq of monocytes from in vitro Trained immunity experiments stimulated by β-glucan (BG), uric acid (UA), muramyl dipeptide (MDP), oxidized low-density lipoprotein (oxLDL), or RPMI-Control, and respective samples restimulated with Lipopolysaccharide (LPS).
Project description:For this study, bursal transcriptome responses to an acute heat stress and/or lipopolysaccharide (LPS) were investigated in a broiler line (heat and disease susceptible) and an inbred Fayoumi line (heat and disease resistant) of chickens. In a 2 x 2 design, 22 day-old birds were exposed to heat stress (35°C for 7 hours), lipopolysaccharide (100 µg/kg average body weight per line), or both stressors. Thermoneutral temperature (25°C) and phosphate buffered saline were used as the respective controls. Tissue samples were collected from the bursa of Fabricius and used to isolate high quality RNA. cDNA libraries (n = 31) were constructed and sequenced (2 technical replicates per library; 62 total datasets) on the HiSeq 2500.
Project description:Host defense against bacterial and fungal infections diminishes with age. In humans, this is thought to be caused in part by a decline in neutrophil responses. However, it remains unclear whether a similar decline in neutrophil function occurs in mice. Here, we show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment to the peritoneum was elevated during lipopolysaccharide (LPS)-induced septic peritonitis but not aseptic peritonitis. Neutrophils from old mice showed reduced chemoattractant-induced reactive oxygen species (ROS) production upon priming with LPS but not GM-CSF/TNF. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas NETosis was impaired independently of LPS. The chemoattractant-stimulated production of PIP3 was reduced upon priming with LPS but not GM-CSF/TNF, whereas PI(4,5)P2 levels were constitutively low. Unexpectedly, chemotaxis was normal regardless of priming pathway, as were the chemoattractant-stimulated activities of Rac1 and Rac2. The expression of 5% of neutrophil proteins was deregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as toll-like receptor (TLR) pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. Upregulation of Cd180 and downregulation of MyD88 may contribute to the impaired LPS priming and LPS-dependent PIP3 production. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS-TLR4 pathway dependence resolves some of the controversy regarding the effects of age on murine neutrophils and confirms mice are an appropriate model for the declining human neutrophil function.
Project description:This experiment was performed to estimate the difference between DCs stimulated with intracellular ROS or LPS. Total RNA obtainded from DCs stimulated with intracellular ROS or LPS was analyzed for transcriptome
Project description:BMDMs or iMACs were generated via differentiation of bone marrow cells or immortalized precursors, respectively, in culture media containing m-CSF for 7 day. Upon differentiation, BMDMs or iMacs were left untreated or stimulated as described below. 1) BMDM WT were stimulated with LPS, PGE2, IL-4, IL-10, LPS+PGE2, LPS+IL-4, LPS+IL-10 for 4 hours. Fragmented chromatin was immuno-precipitated for H3K27Ac (each condition at least in duplicate). These experiments were performed to investigate the cross-antagonism in the concomitant presence of pro-inflammatory (LPS) and anti-inflammatory stimuli (IL-4, IL-10, or PGE2). 2) BMDM WT were stimulated with LPS, PGE2, LPS+PGE2 for either 2 or 4 hours. Fragmented chromatin was immuno-precipitated for IRF1(4h), STAT1 (4h), PU.1 (2h, also for IL-10 and LPS+IL-10 stimulation), NFkB (2h), MEF2A (2h), MEF2D (2h), MEF2C (2h), JUNB (2h) (each condition in single). These experiments were performed to assess TFs occupancy at previously defined PGE2-sensitive and PGE2-resistant enhancers. 3) MEF2A-deficient iMac clones (D7, A7, A8, C7) and MEF2A-proficient (referred to as wild-type) iMac clones (NE, B3 and D10) were generated via CRISPR/Cas9 and stimulated or not with LPS for either 2 or 4 hours: each condition in single. Fragmented chromatin was immuno-precipitated for H3K27Ac (4h) or for PU1 (2h). These experiments were performed to investigate the role of MEF2A on LPS-induced chromatin remodeling.