Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Single cell analysis of macrophages exposed to beads coated with LPS from Salmonella enterica subsp. typhimurium

ABSTRACT: We present a detailed single cell analysis of the macrophage response to LPS from Salmonella enterica. By combining single cell transcriptional analysis, fluorescently labeled, LPS-coated beads, and cytometry we are able to distinguish the responses of macrophages that have internalized LPS-coated beads and those that have not. Analysis of 96 single macrophages that were either: left untreated, were exposed to but did not internalize uncoated beads, were exposed to and internalized uncoated beads, were exposed to but did not internalize LPS-coated beads, or were exposed to and did internalize LPS-coated beads.

ORGANISM(S): Mus musculus

SUBMITTER: Deborah Hung

PROVIDER: E-GEOD-65529 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.

Avraham Roi R Haseley Nathan N Brown Douglas D Penaranda Cristina C Jijon Humberto B HB Trombetta John J JJ Satija Rahul R Shalek Alex K AK Xavier Ramnik J RJ Regev Aviv A Hung Deborah T DT

Cell 20150903 6

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macr ...[more]

PMID: 26343579

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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

Dataset Information

Single cell analysis of macrophages exposed to beads coated with LPS from Salmonella enterica subsp. typhimurium

Publications

Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.

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