Project description:We have performed analyses of murine primary bone marrow derived neutrophils challenged with either ultra-low dose or high dose of LPS. Neutrophils can be differentially programmed to distinct states by varying dosages of LPS. Purified bone marrow neutrophils were treated with PBS, 100 pg/ml LPS or 100 ng/ml LPS overnight, and harvested for scRNAseq analysis to examine their profiles of gene expression.
Project description:We have generated immune-enhancing neutrophils by culturing murine primary bone marrow derived neutrophils with either super-low dose of LPS. Immune-enhancing neutrophils preferentially express co-stimulatory molecules such as CD74, CD44 and CD86, and exhibit reduced expression of CD11b. Purified bone marrow neutrophils were treated with PBS or 100 pg/ml LPS overnight in the presence of GM-CSF, and harvested for scRNAseq analysis to examine their profiles of gene expression.
Project description:To determine how neutrophils are reprogrammed by lung CD140a+ MCs (mesenchymal cells), we employed lung CD140a+ MCs, and ex-vivo cultured with BM(bone marrow)-derived neutrophils. After the treatment, neutrophils were harvested for RNA extraction and the transcriptional profiles were analyzed by RNA sequencing (RNA-seq).
Project description:Neutrophil gene transcription following lipopolysaccharide exposure. Microarray analysis of lipopolysaccharide-treated human neutrophils. Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.
Project description:These data are used to determine whether extracellular RNase A treatment affects cellular RNA levels. GlycoRNAs were sequenced in both primary bone marrow neutrophils as well as an in vitro neutriphil differentiation cell line (HOXB8).
Project description:Neutrophils are known to be stimulated by different periodontal bacteria to produce reactive oxygen species and cytokines. It is inportant to investigate the gene changes made by bacteria of importance, of which, for periodontal disease, fusobaterium nucleatum is one. we used microarrays to investigate gene experssion changes in peripheral blood neutrophils werwhich e stimulated with or with out Fusobacterium Nucleatum (10953). Neutrophils from periodonatlly healthy individuals (n=4) were isolated and stimulated for 3hrs with or without fusobaterium nucleatum (10953). RNA was then extracted from these and pooled before hybridization on Affymetrix microarrays
Project description:The CCAAT/enhancer-binding proteins (CEBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated CEBP-beta (CEBPB) and CEBP-epsilon (CEBPE) double-knockout (bbee) mice and compared their phenotypes to those of single-deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2-3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(-)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct CEBP targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice. To rule out the regulatory influence of both CEBPB and CEBPE on macrophage-related genes, expression analysis of bone marrow-derived macrophages was performed. Macrophages were derived from murine bone marrow with the use of murine M-CSF. The macrophages were stimulated with both LPS (100 ng) and IFN-gamma (100 ng) for 24h, and RNA was extracted for array analysis. Overall, RNA was extracted from stimulated macrophages of one WT mouse, one CEBPB-KO mouse, one CEBPE-KO mouse and one double-KO mouse.