Unknown,Transcriptomics,Genomics,Proteomics

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H1N1 influenza A/New Caledonia/20/1999 time series in monocyte-derived dendritic cells

ABSTRACT: Genome-wide gene expression patterns were measured in human monocyte-derived dendritic cells (DCs) infected in vitro with seasonal H1N1 influenza A/New Caledonia/20/1999. To provide a mechanistic explanation for the timing of gene expression changes over the first 12 hours post-infection, we developed a statistically rigorous enrichment approach integrating genome-wide expression kinetics and time-dependent promoter analysis. Our approach, TI me-Dependent Activity Linker (TIDAL), generates a regulatory network that connects transcription factors associated with each temporal phase of the response into a coherent linked cascade. TIDAL infers 12 transcription factors and 32 regulatory connections that drive the antiviral response to influenza. To demonstrate the generality of this approach, TIDAL was also used to generate a network for the DC response to measles infection. Monocyte-derived DCs were obtained from healthy human blood donors following a standard protocol. The recent seasonal H1N1 influenza virus A/New Caledonia/20/1999 (NC) virus was titrated by immunofluorescence 18 hours after infection of MDCK cell plates using monoclonal antibodies specific for Influenza-NP protein generated by the Mount Sinai Hybridoma Core Facility followed by addition of anti-mouse IgG-FITC and visualization using fluorescent microscopy. For infection of naive DCs, NC stocks were appropriately diluted in DulbeccoM-bM-^@M-^Ys Modified Eagle Medium (DMEM) and added directly into pelleted DCs at a multiplicity of infection (MOI) of 1 After incubation for 40 minutes at 37 M-bM-^WM-&C, fresh DC growth medium (without GMCSF and IL-4) was added back to the infected cells (1 106 cells/ml) for the remainder of the infection. The reaction was stopped at 1, 2, 4, 6, 8, 10, and 12 hours after infection by fixing the cells with RNAprotect Cell Reagent (Qiagen, Duesseldorf Germany). Naive non-infected DCs underwent the same experimental procedure as infected DCs in the absence of virus to ensure that mechanical manipulations could not be responsible for differences in experimental readouts. All time points and controls were performed in triplicates. Cells were homogenized by using QIAshredder microcentrifuge spin-columns (Qiagen, Duesseldorf Germany) and RNA was isolated from cells using Qiagen Micro RNeasy plus kit following the manufactures protocol (Qiagen, Duesseldorf Germany). RNA quality was assayed by determination of the RNA integrity number using the 2100 Bioanalyzer (Agilent). RNA samples were processed and hybridized to HumanHT-12 v4 Expression BeadChip Kit (Illumina San Diego, CA) by the Mount Sinai Genomics Institute following the manufacturerM-bM-^@M-^Ys instructions, and raw expression data were output by the Illumina GenomeStudio software.

ORGANISM(S): Homo sapiens

SUBMITTER: Juilee Thakar

PROVIDER: E-GEOD-41067 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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H1N1 influenza A/New Caledonia/20/1999 time series in monocyte-derived dendritic cells

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