Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq influenza infected B6 & D2 mice

ABSTRACT: The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suitable to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilizing a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual from a single expression assay after H1N1 infection. We performed a comparative expression analysis to identify (i) correlations between host genes and the viral gene expression, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between the two mouse strains after infection. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection. Female, 10-12 weeks old mice were anesthetized by intra-peritoneal injection with Ketamine/Xylazine (85% NaCl (0.9%), 10% Ketamine, 5% Xylazine) with doses adjusted to the individual body weight. Mice were then intra-nasally infected with 20µl virus solution (2x10³ FFU PR8M) or mock-treated with PBS.

ORGANISM(S): Mus musculus

SUBMITTER: Robert Geffers

PROVIDER: E-GEOD-66040 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Alcoholism is a complex neurological disorder characterized by loss of control in limiting intake, and progressive compulsion to seek and consume ethanol. Prior studies have suggested that the characteristic behaviors associated with escalation of drug use are caused, at least in part, by ethanol-evoked changes in gene expression affecting synaptic plasticity. Implicit in this hypothesis is a dependence on new protein synthesis and remodeling at the synapse. It is well established that mRNA can be transported to neuronal distal processes, where it can undergo localized translation that is regulated in a spatially restricted manner in response to stimulation. It is unknown whether such modulation of the synaptic transcriptome might contribute to ethanol-induced synaptic plasticity. Using ethanol-induced behavioral sensitization as a model of neuroplasticity, here we investigated whether repeated exposure to ethanol altered the synaptic transcriptome, contributing to synaptic plasticity underlying a subsequent increase in the ethanol-evoked locomotor response. A synaptoneurosome preparation was utilized to enrich for RNAs trafficked to the synapse versus the proximal cellular fraction. Two complementary methods of genomic profiling, RNA-Seq and microarrays, were used to survey the synaptic transcriptome of DBA/2J mice subjected to ethanol-induced behavioral sensitization. Strikingly, a large number of genes regulated by acute ethanol showed adaptation to repeated ethanol exposure with sensitization. Genomic profiling showed distinct functional classes in RNA found to be enriched in the synaptoneurosome faction, consistent with their role in synaptic function. A subset of ethanol-responsive genes significantly enriched at the synapse were related to biological functions such as protein folding and extra-cellular matrix components, suggesting a role for local regulation of synaptic protein function by ethanol. In contrast, RNA classes regulated by ethanol in the cell soma included an over-abundance of RNA-binding and trafficking proteins, perhaps reflecting increased demand for dendritic RNA localization. These experiments document that both acute and repeated ethanol produce specific alterations in the complement of RNA at the synapse and lay the foundation for further investigations into the role of the synaptic transcriptome in behavioral adaptations to ethanol. 24 samples were analyzed from 3 treatment groups and 2 cell fractions with 4 biological replicates for each treatment/cell fraction.Cell fractionation was carried out on tissue from a frontal pole dissection of DBA2/J mice, isolating a cytosolic (S2) and synaptoneurosomal (P2) fraction. Treatment groups describe chronic treatment (10 daily injections) and treatment used on test day (single injeciion), producing saline-saline (SS), saline-ethanol (SE) and ethanol-ethanol (EE) groups. Analysis of RNA-seq compared similar treatments across cell fractions or different treatments within a cell fraction.

Project description:Identification of biological processes that distinguish lethal from non-lethal influenza infection and further use of these signatures in a top-down systems analysis investigating the relative pathogenic contributions of direct viral damage to lung epithelium vs. dysregulated immunity to during lethal influenza infection. For acutely lethal influenza infections, the relative pathogenic contributions of direct viral damage to lung epithelium vs. dysregulated immunity remain unresolved. Here, we take a top-down systems approach to this question. Multigene transcriptional signatures from infected lungs suggested that elevated activation of inflammatory signaling networks distinguished lethal from sublethal infections. Flow cytometry and gene expression analysis involving isolated cell subpopulations from infected lungs showed that neutrophil influx largely accounted for the predictive transcriptional signature. Automated imaging analysis together with these gene expression and flow data identified a chemokine-driven feed-forward circuit involving pro-inflammatory neutrophils potently driven by poorly contained lethal viruses. Consistent with these data, attenuation but not ablation of the neutrophil-driven response increased survival without changing viral spread. These findings establish the primacy of damaging innate inflammation in at least some forms of influenza-induced lethality and provide a roadmap for the systematic dissection of infection-associated pathology. Multiple mice were either sham infected, infected with the seasonal H1N1 influenza A virus TX91 (10^6PFU), or infected with various sublethal or lethal doses of the mouse pathogenic H1N1 strain PR8. Lung tissues were collected at various time points (24h, 48h, 72h and 240h post infection) and processed to yield whole lung RNA that was used for microarray analysis. The dataset contains 138 microarrays covering 20 experimental conditions with 7 biological replicates each. As an exception, the alternative non-infectious control condition (Alum treatment) contains 5 biological replicates. This dataset is linked to a dataset comparing the transcriptomes of 5 different cell types isolated from individual lungs of influenza A-infected or control animals (contains 75 microarrays covering 25 experimental conditions).

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RNA-seq influenza infected B6 & D2 mice

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