Transcription profiling of mouse IgE/antigen-activated mast cells stimulated with TIM-3
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ABSTRACT: TIM-3 is known to be expressed on dendritic cells, monocytes, melanoma cells, mast cells and on activated Th1 cells. In activated Th1 cells, stimulating TIM-3 by one of its ligands, galectin-9, leads to apoptosis and thus it plays a central role in terminating Th1-type immune responses. Interestingly, in IgE/antigen-activated mast cells TIM-3 enhances the production of IL-13 and IL-4. To get a more complete picture about the gene expression changes induced by TIM-3 in mast cells, in vitro differentiated mouse immature mast cells were stimulated by an agonist anti-TIM-3 antibody and IgE-sensitized mouse immature mast cells were activated by antigen and an agonist anti-TIM-3 antibody for 2 or 16 hours (overnight). Experiment Overall Design: Bone marrow cells were differentiated in RPMI + 10% FCS + 5 ng/ml mouse IL-3 + 40 ng/ml mouse SCF for >4 weeks. The purity of the cell cultures was >90% at this time point (FcERIa+/c-kit+ cells). These in vitro-differentiated immature mast cells were then treated by either control goat IgG or an agonist anti-mouse TIM-3 antibody (RnD Systems, 15 ug/ml for 2 or 16 hours). For the IgE/antigen-activated mouse mast cells, these in vitro-differentiated immature mast cells were sensitized by 5 ug/ml anti-DNP IgE (Sigma) for 1 hour and then treated with 100 ng/ml DNP-HSA (antigen, Sigma) and either control goat IgG or an agonist anti-mouse TIM-3 antibody (RnD Systems, 15 ug/ml) for 2 or 16 hours. The anti-TIM-3 samples were labeled by Cy5 and they were compared to the Cy3-labeled, goat IgG controls in a dual-color, paired experimental setup. The Agilent Whole Mouse Genome 4x44K expression microarray kit and Dual-Color Protocol version 5.5 were used in the experiments.
Project description:Bone marrow-derived mast cells were differentiated over 4-6 weeks using bone marrow from Pac-1+/+ and Pac1-/- littermate mice. Cell purity was 99% c-kit and Fc epsilon receptor positive as assessed by flow cytometry. Cells were stimulated by Fc epsilon receptor crosslinking using IgE-DNP/HSA for sensitization for 18 hours and DNP-HSA antigen for crosslinking for 2 hours. Gene transcript abundance was determined and scaled to 150 using alogorithms in MicroArray Analysis Suite Software 5.0 (Affymetrix). Experiment Overall Design: Two genechips on independent mBMMC cultures was performed. The signal data of the .CEL files for the four Affymetrix 430A chips (2 Pac1+/+ and 2 Pac1â/â) were normalised using the RMA method 49. The ebayes function implemented in the version 1.3.12 of the Limma package (2003) of Bioconductor was used to analyse the data and P-values were adjusted for multiple testing. After conversion from logged values to the original intensities, the data was filtered to remove probe sets with mean, non-logged, intensities below 100. Transcripts with p-values less than 0.05 were considered different from Pac1+/+.
Project description:This SuperSeries is composed of the following subset Series:; GSE3993: Pac1+/+ versus Pac1-/- TG-macrophages_LPS 6h; GSE3994: Pac1+/+ versus Pac1-/- BMMCs_IgE-DNP 2 h Experiment Overall Design: Refer to individual Series
Project description:MicroRNA 155 (miR-155) has been shown to regulate the gene expression of important players of physiological and pathological processes, like hematopoietic lineage differentiation, immunity and inflammation, viral infections, cancer and cardiovascular diseases, among others. Degranulation is an event in which mast cells, upon activation of the FceRI, release their granule content rich in vasoactive amines, proteases and TNFa. Additionally activation of the receptor promotes de novo synthesis of cytokines, chemokines and growth factors. Analysis of bone marrow derived mast cells (BMMC) deficient in miR-155 showed a significant increase in FceRI mediated degranulation and in the release of cytokines like TNFa, IL-6 and IL-13. In addition miR 155-/- mice presented higher anaphylaxis reactions compared to WT mice. Gene expression analysis of BMMC was performed in order to identify intermediaries of FceRI mediated degranulation under the control of miR-155. The results indicate that miR-155 regulates negatively the expression of the regulatory subunits of the kinase PI3Kgamma, Pik3r5 (p101) and Pik3r6 (p84, p87PIKAP), involved in Ca+ influx and degranulation. Total RNA from 3 independent cultures of WT and miR-155-/- BMMC treated with anti-DNP IgE and 20 ng/ml DNP-HSA for 1hr were used to performed gene expression analysis using the Affymetrix GenechipM-BM-. Mouse Gene 1.0 ST
Project description:The effect of TIM-3 stimulation was studied on B16F10 mouse melanoma cells, in vitro. Experiment Overall Design: Cy3: B16F10 mouse melanoma cells, treated by unspecific goat IgG (control) for 2 hours. Experiment Overall Design: Cy5: B16F10 mouse melanoma cells, in vitro, treated by an agonist goat polyclonal anti-mouse TIM3 antibody (RnD Systems) for 2 hours. Experiment Overall Design: Four biological replicates were used (in this study, biological replicate means different passage numbers of the same cell line). In each case, the treated sample (Cy5) was compared to its control one (Cy3) in Agilent dual-color microarray experiments.
Project description:Best known for their roles in allergic diseases, the physiologic function of immunoglobulin E (IgE) and mast cells (the so-called allergy module of immunity) has been enigmatic. Recent evidence shows that allergic reactions can help to protect against the toxicity of venoms. As bacteria are a potent alternative source of toxins, we assessed the potential role of the allergy module in antibacterial host defense. We observed that the immune response against S. aureus skin infection includes specific IgEs and substantially improves systemic host defense against secondary S. aureus infections in mice. This acquired protection depends on functional IgE effector mechanisms and mast cells. Our results reveal the powerful antibacterial potential of the allergy module and therefore a novel physiologic function of IgEs and mast cells.
Project description:Cultured mature BMMCs were incubated overnight with anti-DNP IgE and stimulated with DNP-BSA. Then cells were collected for further analysis.
Project description:Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI3Kinase (PI3K) including p85α, p85β, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes. Two-condition experiment, wildtype vs. p85α-deficient mast cell progenitors stimulated with SCF. Biological replicates: 3 wildtype replicates, 3 p85α-deficient replicates.
Project description:Mast cells are known to be the key players in type I hypersensitivity reactions in humans and mice. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis. In this study we investigated the role of the transcriptional regulator MAZR in mast cells by comparing the expression profile of mast cells generated from wild-type (MazrF/F) and MAZR-deficient (MazrF/F x Vav-iCre) bone marrow cells. Our results from the array data demonstrate that MAZR acts preferentially as a transcriptional repressor in mast cells.
Project description:Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens. We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE levels in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Arrays were incubated with <25uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE levels.
Project description:The regulation of soluble N ethylmaleimide sensitive factor attachment receptor (SNARE) mediated membrane fusion by upstream signaling events is poorly understood. Here we show that the SNARE binding protein tomosyn-1 negatively regulates type I IgE Fc receptor (FcRI) induced degranulation of mast cells. After FcRI stimulation, tomosyn-1 (also STXBP5) was phosphorylated on serine and threonine residues and dissociated from the SNARE protein syntaxin 4 (STX4), followed by association with syntaxin 3 (STX3). Protein kinase A (PKA) and protein kinase C (PKC) prevented these activities. We identified PKC as the major kinase required for tomosyn-1 threonine phosphorylation and for the regulation of the interaction with STXs. Incubation with high IgE concentrations induced tomosyn-1 expression in cultured mast cells. In basophils from allergic patients with normal total IgE serum titers tomosyn-1 expression was lower than in patients with high IgE titers who expressed tomosyn-1 to the same extent as non-allergic subjects. Our findings identified tomosyn-1 as a negative regulator of mast cell degranulation that required PKC to switch its interaction with STX partners during fusion. The IgE-mediated upregulation of tomosyn-1 in allergic patients may represent a counter-regulatory mechanism to limit disease development.