Dataset Information

Spatiotemporal changes of the phagosomal proteome in dendritic cells in response to LPS stimulation

ABSTRACT: Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. Nonetheless, the phagosomal proteome of DCs is only partially studied, in particular in response to inflammatory cues. The phagosomal proteome is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions. In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of the proteasome and MHC I molecules, tapasin etc., confirming increased antigen presentation efficacy in those cells. In addition, we identified several phagosomal proteins that were not previously associated with phagosomal functions and reveal a novel role for immune-responsive gene 1 (IRG1) in phagocytic uptake of particles in resting DCs.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Dendritic Cell

SUBMITTER: Matthias Trost

LAB HEAD: Pr Matthias Trost

PROVIDER: PXD012155 | Pride | 2019-02-28

REPOSITORIES: Pride

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Spatiotemporal Changes of the Phagosomal Proteome in Dendritic Cells in Response to LPS Stimulation.

Pauwels Anne-Marie AM Härtlova Anetta A Peltier Julien J Driege Yasmine Y Baudelet Griet G Brodin Priscille P Trost Matthias M Beyaert Rudi R Hoffmann Eik E

Molecular & cellular proteomics : MCP 20190226 5

Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when p ...[more]

PMID: 30808727

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Project description:The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions. All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 Âµm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record. http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

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	20170123_DCs_Phag_A1.raw	Raw
	20170123_DCs_Phag_A2.raw	Raw
	20170123_DCs_Phag_B1.raw	Raw
	20170123_DCs_Phag_B2.raw	Raw
	20170123_DCs_Phag_C1.raw	Raw