Unknown,Transcriptomics,Genomics,Proteomics

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Pilot study to identify technical variation in microarray data of mouse lungs in the context of inter-animal variation

ABSTRACT: Prior to generation of microarray data for a Top-down Systems analysis of influenza A infection, we evaluated the degree and origin of technical variation in gene expression microarray data from mouse lungs and compared this to inter-animal variation. Technical variation in these microarray data in the context of inter-animal variation supported a choice of biological rather than technical replicates. A) One mouse (260) was infected with 0.6LD50 of the mouse pathogenic H1N1 strain PR8. Lung tissues were collected at 72h post infection and processed to yield whole lung RNA that was used for microarray analysis. The RNA was extracted in 3 individual batches (I, II, III), each batch of RNA was amplified in 2 separate batches (1,2), and the individual batches of amplified product were again hybridized in 2 batches. This dataset contains 18 microarrays covering 1 experimental condition with 1 biological replicate and a matrix of technical replicates. B) 3 mice (245, 246, 247) were infected with 0.6LD50 of the mouse pathogenic H1N1 strain PR8. Lung tissues were collected at 72h post infection and processed to yield whole lung RNA that was used for microarray analysis. From each animal, RNA was extracted, the RNA was amplified in three separate batches (A, B, C), and the individual batches of amplified product were again hybridized in 2 batches. This dataset contains 24 microarrays covering 1 experimental condition with 3 biological replicates and a matrix of technical replicates. Datasets A) and B) are linked to two biological datasets - one 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) and one comprising whole lung microarrays from sham-infected or influenza H1N1-infected animals (contains 138 microarrays, 19 experimental conditions, 7 biological replicates).

ORGANISM(S): Mus musculus

SUBMITTER: Marlene Brandes

PROVIDER: E-GEOD-42521 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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).

Project description:Transcriptomic comparison of 5 cell types during lethal and 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 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 48h or 72h post infection. 5 different cell types were purified by flow sorting from lungs of individual animals and then processed to yield total RNA that was used for microarray analysis. The dataset contains 75 microarrays covering 25 experimental conditions with 3 biological replicates. This dataset is linked to a dataset containing 138 microarrays of whole lungs covering 20 experimental conditions.

Project description:Background: Pandemic H1N1 influenza A is a newly emerging strain of human influenza that is easily transmitted between people and has spread globally to over 116 countries. Human infection leads to symptoms ranging from mild to severe with lower respiratory complications observed in a small but significant number of infected individuals. Little is currently known about host immunity and Pandemic H1N1 influenza infections. Methods: We examined the pathogenic potential of the pandemic influenza A vaccine strain, A/California/07/2009 (H1N1), in ferrets, and characterized the host immune responses using microarray analysis. Gene expression profiles in lung tissue were compared with those from ferrets infected with A/Brisbane/59/2007. Results: Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of ISGs were expressed early, correlated to lung pathology, and abruptly decreased expression in 5 days. Interestingly, the drop in innate immune gene expression was replaced by a significant increase in the expression of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-pandemic influenza H1N1 antibodies were also observed on day 7, commensurate with the elimination of viral load. Conclusions: We propose that the innate phase of host immunity causes lung pathology and a delay or failure to effectively switch to the adaptive phase contributes to morbidity and mortality during severe human pandemic H1N1 influenza A infections. Keywords: influenza, immune response, cytokines, chemokines, lung infection, time course In the experiment with influenza A/California/07/2009 (H1N1),15 ferrets were randomly allocated to 5 groups: Day 0 (before infection), and Day 3, 5, 7 and 14 (post infection) with 3 biological replicates for each group. Likewise, a second experiment with A/Brisbane/59/2007 (H1N1) was carried out using the same experimental groups, except for a group in Day 2, instead Day 3. Ferrets were euthanized and lung tissue was excised for RNA purification on the scheduled date. The subsequent gene expression analysis was performed with Affymetrix GeneChip Canine Genome 2.0 Array. Day 0 groups were used as control.

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Pilot study to identify technical variation in microarray data of mouse lungs in the context of inter-animal variation

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