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Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus

ABSTRACT: RAW264.7 macrophages infected with MNV-1 and mock infected gene expression measured by microarray. To be published in Waugh, E. Chen, A. Baird, M. Fleming, S. Brown, C.M. and V K. Ward (2014) Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus. Virus Genes Four Samples, two mock and two MNV-1 infected.

ORGANISM(S): Mus musculus

SUBMITTER: Christopher Michael Brown Emily Waugh Chris Brown

PROVIDER: E-GEOD-50093 | ArrayExpress | 2013-12-22

SECONDARY ACCESSION(S): GSE50093PRJNA215999

REPOSITORIES: GEO, ArrayExpress

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Project description:Global, genomic responses of erythrocytes to infectious agents have been difficult to measure, because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of 35 genes, 9 of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data unexpectedly predict that blood stage P. falciparum may induce host responses common to infections of other pathogens. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. Human primary erythroid cells were differentiated from CD34+ hematopoietic stem cells isolated from growth factor-mobilized peripheral blood (ALL Cells, Inc.). Cells from five donors were cultured until polychromatophilic and orthochromatophilic stages of differentiation and served as uninfected control samples. Of the five donors, three were used to initiate Plasmodium falciparum (3D7) infection at a multiplicity of infection = 5. Infected cells were harvested 24 hours post-infection, and RNA was isolated with Trizol (Invitrogen) and purified with RNeasy columns (QIAGEN) according to manufacturer recommendations. Microarray labeling and hybridizations were done according to Affymetrix protocols using HG U133 plus 2.0 chips. For GenePattern analysis, all samples (5 control and 3 infected samples) were analyzed; for Dchip analysis, only three samples were analyzed (the same 3 donors served as control and infected samples).

Project description:Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated the kinetic transcriptional profile of gene expression in macrophages infected with Brucella melitensis strain 16M. A key aspect of Brucella virulence is their ability to proliferate within professional and nonprofessional phagocytic host cells, thereby successfully bypassing the bactericidal effects of phagocytes. Their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells. Defining the interaction between a host cell and Brucella is crucial to understanding the infectious process. Most researchers have studied the pathogens, but the host plays a very important role during infections. To date, relatively few host factors have been shown important in Brucella infections. However, little is known about the host networks that mediate infection. The objective of the study is to analyze the genes and cellular components related to the innate immunity response to determine the mechanisms through which Brucella avoids the host innate immunity. A total of 3576 and 3962 genes that are differentially expressed between 0 and 4 h and between 0 and 24 h were identified. The identified genes are related to immune processes, signal transduction, inflammation, apoptosis, cell membrane, transcriptional regulation, and intracellular trafficking. Our data have added to the current understanding of different host gene expressions during different infection phases by Brucella spp. The RAW264.7 cells were seeded into 24-well plates at 5-105 cells/well, incubated in 5% CO2 at 37 °C for 24 h, and then infected with Brucella at a multiplicity of infection of 200. To synchronize the infection, the infected plates were centrifuged at 200 ug for 5 min at room temperature and then incubated at 37 °C for 20 minutes. The infected cell monolayers were washed three times with PBS, overlaid with 0.5 ml of DMEM containing 100 mg/ml of ampicillin and 50 mg/ml of kanamycin at 37 °C for 0, 4, and 24 h. At the end of the incubation period, the culture medium was removed and centrifuged at 1500 rpm for 15 min. The cell pellet and adherent cells were resuspended in TRIzol. Total RNA were extracted from the cells at 0, 4 and 24 h post infection.

			Action	DRS
	E-GEOD-50093.idf.txt	Idf
	E-GEOD-50093.processed.1.zip	Processed
	E-GEOD-50093.raw.1.zip	Raw
	E-GEOD-50093.sdrf.txt	Txt

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Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus

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