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Comaprison of the phagosome proteome of bone marrow-derived macrophages and RAW264.7

ABSTRACT: Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterisation of phagosomes from RAW 264.7 and bone marrow-derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicates that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: Matthias Trost

LAB HEAD: Matthias Trost

PROVIDER: PXD001293 | Pride | 2015-02-10

REPOSITORIES: Pride

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			Action	DRS
	BMDMRAWcomparison.zip	Other
	BMDM_1.raw	Raw
	BMDM_2.raw	Raw
	BMDM_3.raw	Raw
	RAW_1.raw	Raw

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Publications

High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages.

Guo Manman M Härtlova Anetta A Dill Brian D BD Prescott Alan R AR Gierliński Marek M Trost Matthias M

Proteomics 20150205 18

Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal prote ...[more]

PMID: 25504905

Similar Datasets

Comaprison of the phagosome proteome of bone marrow-derived macrophages and RAW264.7

Project description:Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterisation of phagosomes from RAW 264.7 and bone marrow-derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicates that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1.

2020-02-08 | MSV000084938 | MassIVE

Temporal and ligand effects on phagosome maturation

Project description:Systems biology analysis of temporally-resolved phagosome proteomes following uptake via key phagocytic receptors Macrophages operate at the forefront of innate immunity, and their discrimination of pathogenic versus “self” particles is critical for a number of responses including efficient pathogen killing, antigen presentation and cytokine induction. In order to efficiently phagocytose and destroy the particles, macrophages express an array of receptors to sense and internalise prey particles. In this manuscript, particles conjugated to seven ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers were inoculated to murine bone marrow-derived macrophages (BMDMs) and proteomes of isolated phagosomes were accurately quantified among conditions and across a timecourse. While we identified a clear functional differentiation over the three timepoints, only subtle differences were detected between certain ligand-phagosomes, suggesting that triggering of receptors through a single ligand has only mild effects on phagosome proteome and function. This data shows for the first time a systematic time-course analysis of BMDM phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

2015-09-10 | PXD000772 | Pride

Pacific oyster phagosome LC-MSMS

Project description:Phagosomes are task-force organelles of innate immune systems responsible for the recognition, processing, and ultimately destruction of invading microorganisms. Among invertebrate and vertebrate species, evolutionary diversity and continuity abound in the protein machinery executing this coordinately regulated process, though its dynamics and full scope of regulators remain incompletely understood. In order to clarify molecular mechanisms underlying phagocytosis, we studied phagocyte response to beads and Vibrio species, using hemocytes of the Pacific oysters (Crassostrea gigas) as a marine invertebrate model. Phagosomes from different stages of phagocytosis were isolated by density-gradient centrifugation, and more than 400 phagosome-associated proteins were subsequently identified via high-throughput quantitative proteomics. In modeling key networks of phagosomal protein-protein interactions, our results support the essential roles of several processes driving phagosome formation and maturation, including actin cytoskeleton remodeling, and signal transduction by myosin and Rab proteins. In further quantitative proteomic analysis, trends of both upregulation and downregulation of protein expression were observed proteins during phagosome formation and maturation. Notably, the signal transducers CgRhoGDI and CgPI4K were implicated. In addition, six Rab proteins including Rab1, Rab2, Rab7, Rab11, RaB21, and Rab33 were also identified as active regulators or mediators in hemocyte phagocytosis. Our current work illustrates the diversity and dynamic interplay of phagosomal proteins, providing a framework for better understanding host-microbe interactions during phagosome formation and maturation in under-examined invertebrate species.

2020-04-27 | PXD015810 | Pride

The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to infection

Project description:Phagocytosis is an essential process for the microbicidal function of professional phagocytes, in which phagosome maturation plays a critical role. Macrophage activation by interferons (IFN) increases their microbicidal activity, but delays phagosomal maturation. Here, we investigated the role of ubiquitylation in phagosome functions. We show that phagosomal proteins are ubiquitylated and that phagosomes contain diverse ubiquitin chain types, including atypical ubiquitin chains. IFN-γ activation of macrophages substantially enhanced phagosomal ubiquitylation of both innate immune response proteins and vesicle trafficking proteins. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ubiquitin ligase activity facilitated enhanced phagosomal maturation, and increased cytokine responses to Staphylococcus aureus. Our data suggests that both innate immune signalling and phagolysosomal trafficking are controlled through ubiquitylation. In conclusion, we identify RNF115 and ubiquitylation as important regulators of innate immune signalling from the phagosome during bacterial infections.

2022-07-20 | PXD026843 | Pride

Expression Profiles of 15(S)-HETE treated RAW 264.7 cells

Project description:Hemozoin phagocytosis results in immunomodulation. This study was designed to explore gene expression responses to 15(S)-HETE in LPS-stimulated RAW 264.7 cells.

2009-12-17 | GSE15070 | GEO

CstF-64 Influenced Gene Expression

Project description:Triplicate samples of RAW 264.7 murine macrophages either untreated, stimulated with 100 ng/ml LPS for 18 hours, or constituitively over-expressing CstF-64 were analyzed by microarray using Affymetrix murine gene chip 430A. Keywords = RAW 264.7 macrophages Keywords = LPS Keywords = CstF-64 Keywords: repeat sample

2005-01-01 | GSE2002 | GEO

Expression data from BNL CL.2 liver cells when cultured with iron, RAW 264.7 macrophages or both

Project description:We found that co-culturing BNL CL.2 liver cells with RAW 264.7 macrophages increased IRP binding in the first. To further investigate this modulation we investigated the gene expression profile in BNL CL.2 cells cultured alone, with iron, with RAW 264.7 macrophages or in the presence of both iron and macrophages. This novel reconstituted liver cell-macrophage communication pathway with the present gene expression data provides a platform for addressing how macrophages participate in the iron homeostasis of liver cells and, ultimately, in systemic iron homeostasis. We used microarrays to determine the gene expression modulation in BNL CL.2 cells in response to 24h culture with 100 micromolar ferric ammonium citrate (FAC), co-culture with RAW 264.7 macrophages or both

2016-10-22 | E-GEOD-32090 | biostudies-arrayexpress

Comparative characterization of osteoclasts derived from murine bone marrow macrophages and RAW 264.7 cells using quantitative proteomics

Project description:Osteoclasts (OCs) are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to M-CSF and RANKL. In vitro models are principally based on primary bone marrow macrophages, but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form OCs in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in OCs derived from bone marrow macrophages and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different; including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major differences between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line when studying M-CSF-dependent signaling and functions.

2018-11-27 | PXD009610 | Pride

Effect of LPS and LPS-binding protein treatment for 48 hours on RAW 264.4 cells

Project description:All samples were gathered from mouse RAW 264.7 cells (macrophages). Control total RNA was extracted from untreated RAW 264.7 cells cultured for 48 hours. Test total RNA was extracted from lipopolysaccharide (100ng/ml) and lipopolysaccharide-binding protein (100pM) treated RAW 264.4 cells cultured for 48 hours. Experiment design included three control vs test arrays and three dye swap arrays. Keywords: other

2004-03-05 | GSE1104 | GEO

Effect of LPS and LPS-binding protein treatment for 1 hour on RAW 264.4 cells

Project description:All samples were gathered from mouse RAW 264.7 cells (macrophages). Control total RNA was extracted from untreated RAW 264.7 cells cultured for 1 hour. Test total RNA was extracted from lipopolysaccharide (100ng/ml) and lipopolysaccharide-binding protein (100pM) treated RAW 264.4 cells cultured for 1 hour. Experiment design included three control vs test arrays and three dye swap arrays. Keywords: other

2004-03-05 | GSE1099 | GEO