Effect of miR-182 on macrophage infection with Francisella tularensis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: David Gregory
PROVIDER: E-MTAB-4551 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2024-06-20 | E-MTAB-13676 | biostudies-arrayexpress
Project description:Foam cell formation from monocyte-derived macrophages is a hallmark of atheroscle-rotic lesions. Aspects of this process can be recapitulated in vitro by exposing MCSF-induced or platelet factor4 (CXCL4)-induced macrophages to oxidized (ox) or minimally modified (mm) low density lipoprotein (LDL). We measured gene expression in periph-eral blood mononuclear cells (PBMCs), monocytes and macrophages treated with CXCL1 (GRO-α) or CCL2 (MCP-1) as well as foam cells induced by native LDL, mmLDL or oxLDL using 22 Affymetrix gene chips. Using an advanced Bayesian error-pooling approach and a heterogeneous error model (HEM) with a false discovery rate (FDR) <0.05, we found 5,303 of 22,215 probe sets to be significantly regulated in at least one of the conditions. Among a subset of 917 candidate genes that were preselected for their known biological functions in macrophage foamcell differentiation, we found that 290 genes met the above statistical criteria for significant differential expression patterns. While many expected genes were found to be upregulated by LDL and oxLDL, very few were induced by mmLDL. We also found induction of unexpected genes, most strikingly MHC-II and other dendritic cell markers such as CD11c. The gene expression patterns in response to oxLDL were similar in MCSF-induced and CXCL4-induced macrophages. Our findings suggest that LDL and oxLDL, but not mmLDL, induce a dendritic cell-like phenotype in macrophages, suggesting that these cells may be able to present antigens and support an immune response. Experiment Overall Design: Human blood was drawn from the antecubital veins of healthy blood donors and provided as buffy coats by the Virginia Blood Services (Richmond, VA). The mononuclear fractions were pooled from four unidentified donors to decrease individual variations in monocytes. Mixed peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque 1.077 (Sigma Diagnostics, Inc., St. Louis, MO). Following centrifugation, the mononuclear layer was removed and washed with PBS containing 0.02% ethylenediaminetetraacetate (EDTA). The pellet was resuspended in 1X H-lyse Buffer (R&D Systems Inc., Minneapolis, MN), and washed with wash buffer. PBMCs contain mainly monocytes and lymphocytes as well as platelets that tend to be associated with blood monocytes. From these PBMCs, monocytes were isolated using a negative selection monocyte isolation kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated fraction was > 97% as estimated by flow cytometry using anti-CD14 (data not shown). Although these cells are often called âuntouchedâ monocytes and thought to show little activation, the gene chip analysis conducted on these cells shows massive changes in gene expression compared to PBMCs (see below). Experiment Overall Design: Monocytes were cultured in Macrophage Serum-Free Medium (MSFM, Invitro-gen, Carlsbad, CA) in the presence of 1% media supplement nutridoma-HU (Roche Molecular Biochemicals, Indianapolis, IN) and 100 nM M-CSF for 6 days, after which the cells showed the expected morphological signs of macrophage differentiation. These macrophages were incubated either with 100 nM CCL2 or CXCL1 for 5 hours. CXCL1 was selected because we have previously found that it is an important arrest chemokine for monocytes in vitro and in atherosclerotic arteries in vivo. CCL2 was chosen because mice lacking CCL2 or its receptor CCR2 are relatively resistant to atherosclerosis, suggesting a role in macrophage recruitment, differentiation and/or survival. Human monocyte-derived macrophages (MDM) were also incubated with native LDL, oxidized LDL (oxLDL) or minimally modified LDL (mmLDL) (each at a concentration of 100 ug/ml) for 2 days to induce foam cell formation. Foam cell formation was verified by oil red O staining (figure 1) and by determining their cholesterol and cholesterol ester content. OxLDL and mmLDL were prepared from the same native LDL for each experiment as described. Control experiments were conducted on macrophages cultured in M-CSF without LDL for an additional 2 days. Two separate sets of monocytes were incubated with CXCL4 (100 nM) for 6 days, another procedure known to induce macrophage differentiation (29), with and without oxLDL to induce foam cell formation. RNA was extracted from cells in all 11 conditions (table 1) and gene expression was measured in duplicates at the University of Virginia Gene Expression Core Facility using Affymetrix equipment.
2008-06-15 | E-GEOD-7138 | biostudies-arrayexpress
Project description:Since macrophage activation can now be studied at a global level using modern microarray and proteomic analyses, discovery of novel macrophage activation genes is inevitable and important for understanding HIV-associated dementia (HAD). We isolated two different types of primary human macrophages, microglia and monocyte-derived macrophages (MDM) from brain tissue and whole blood respectively. The microarray analysis of differentially regulated macrophage activation genes reported here supports our previous assertions that the mixed glia (MIX) cultured in starvation conditions (DMEM alone) are a non-activated, or quiescent, tissue culture model for studying macrophage activation in the brain. Transcript levels from these quiescent cultures provided a background level of gene expression, and allowed for the identification of upregulated macrophage activation genes in the MIX brain cultures upon treatment with an array of soluble activation factors: serum components, cytokines, and growth factors. We found that 914 genes in the MIX cultures and 734 genes in the MDM cultures had a greater than 2-fold increase in expression. We discovered 180 genes that were increased greater than 2 fold in both culture types. Microarray-specific statistical analyses were performed to complement fold change analysis: Significance Analysis of Microarrays (SAM), and Partek Pro. In the MIX cultures, we detected over a hundred fold increase in IL-1? and TIMP1 transcription; Caspase 9, S100A8 and 9, MMP-12, IL-8, MCP-1, MRC-1, and IL-6 were also upregulated. Activation of starved MDM cultures resulted in fewer upregulated genes compared to MIX cultures. Genes upregulated in both MIX and MDM included CCL2 (MCP1), CCL7, CXCL5, TNFSF14, kinases, and phosphatases. These microarray data may provide leads for identifying previously unknown neurotoxins, disease biomarkers, and pathways responsible for the neuronal apoptosis observed in HAD, and for the eventual identification of therapeutic targets and treatments. For each gene, we performed a per-gene normalization, whereby an expression ratio was calculated by dividing the raw data from the activated media samples by the raw signal for the same gene from the non-activated media samples (Fig. 1). The same microarray data were also analyzed again using the very common per-chip normalization method recommended by the manufacturer where raw signals for each chip were multiplied by a scaling factor such that the trimmed mean for each chip became an arbitrary number (150) (Affymetrix, 2001). The per-chip normalization method reduces person-to-person biological variability and potentially hides important changes in gene expression (Hill et al., 2001),(Bolstad et al., 2003). Therefore, we report our data using a per-gene normalization method, which yielded higher signal and low noise (uninteresting non-biological variability caused by chips, and/or experimental sample preparation), and given our experimental design, reduced person-to-person variability that these experiments cannot address directly. For later statistical analyses, the data were log transformed in order to obtain a normal distribution of the expression values of the >12,625 genes (Bolstad et al., 2003)
2007-08-29 | E-GEOD-1910 | biostudies-arrayexpress
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2018-04-02 | E-MTAB-5894 | biostudies-arrayexpress
Project description:Patients diagnosed with coronavirus disease 2019 (COVID-19) mostly become critically ill around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific IgG in serum of critically ill COVID-19 patients induces hyper-inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more pro-inflammatory because of different glycosylation, particularly low fucosylation, of the Fc tail. Notably, low anti-spike IgG fucosylation normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ Receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Finally, we demonstrate that the hyper-inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small molecule inhibitor of the kinase, Syk.
2021-04-30 | E-MTAB-10431 | biostudies-arrayexpress
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2014-10-01 | E-GEOD-60824 | biostudies-arrayexpress
Project description:There is currently no human macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (for example THP-1 cells) or ex vivo maturation of circulating primary monocytes. We have derived a unique sub-clone of the THP-1 cell line capable of spontaneous and perpetual differentiation into alveolar macrophage-like cells. Here we used the Agilent G4851C (v3) array to look at the gene expression of the new cell line which can be compared it with THP-1 cells and PMA stimulate THP-1 cells (Accession number E-MTAB-4153). In addition the new human alveolar macrophage like cell line has been treated with unmodified lipid to look at lipid uptake by these cells and the effect on gene expression.
2020-01-31 | E-MTAB-7169 | biostudies-arrayexpress
Project description:Human blood monocytes were differentiated over six days with either 100 ng/ml M-CSF or 1 umol/l CXCL4 In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4. two MCSF samples and two CXCL4 samples
2010-03-07 | E-GEOD-20484 | biostudies-arrayexpress
Project description:We measured TNFα activity on the transcriptional level by identifying TNFα-inducible genes in human blood monocyte-derived macrophages (MDM). On the basis that TNFα does not act in isolation, we used bacterial lipopolysaccharide (LPS) as a prototypic model of a stimulus to invoke secretion of a wide range of immunologically active factors, including TNFα. In order to identify gene expression attributable to LPS-induced TNFα, we compared the transcriptome of MDM 24 hours after stimulation with 100 ng/ml ultra-pure LPS in the presence and absence of the soluble TNF receptor fusion protein Etanercept (10 µg/ml).
2017-08-04 | E-MTAB-5095 | biostudies-arrayexpress