Pathways significantly represented by all isolates as compared to mock.

<p>(A) Graphs represent the number of genes differentially up- or down- regulated for each isolate compared to mock. Red represents the number of genes up-regulated, green represents the number of genes down-regulated, and white represents the number of genes that are not significantly different from mock. (B) Graphs represent the fold change expression of significant DEGs within these pathways.</p

Seasonal and pandemic IAV isolates used in this study.

<p>Seasonal and pandemic IAV isolates used in this study.</p

Apical cytokine and chemokine production by wd-NHBE cells infected with seasonal and pandemic IAVs.

<p>After infection, the apical side of the culture insert was washed twice and harvested for Luminex multiplex analysis. The error bars indicate mean ± SEM from 3 replicates per isolate per time point from one representative experiment. A total of two experiments were conducted with two donors. Letters indicate significant differences between isolates (a- different from KY/180, b- different from KY/136, c- different from BN/59, and d- different from Mock).</p

Differentially expressed genes in IAV–infected NHBE cells at 36 hpi.

*<p>No. significant genes p<0.05, 2-fold cut-off.</p

Early Host Responses of Seasonal and Pandemic Influenza A Viruses in Primary Well-Differentiated Human Lung Epithelial Cells

<div><p>Replication, cell tropism and the magnitude of the host's antiviral immune response each contribute to the resulting pathogenicity of influenza A viruses (IAV) in humans. In contrast to seasonal IAV in human cases, the 2009 H1N1 pandemic IAV (H1N1pdm) shows a greater tropism for infection of the lung similar to H5N1. We hypothesized that host responses during infection of well-differentiated, primary human bronchial epithelial cells (wd-NHBE) may differ between seasonal (H1N1 A/BN/59/07) and H1N1pdm isolates from a fatal (A/KY/180/10) and nonfatal (A/KY/136/09) case. For each virus, the level of infectious virus and host response to infection (gene expression and apical/basal cytokine/chemokine profiles) were measured in wd-NHBE at 8, 24, 36, 48 and 72 hours post-infection (hpi). At 24 and 36 hpi, KY/180 showed a significant, ten-fold higher titer as compared to the other two isolates. Apical cytokine/chemokine levels of IL-6, IL-8 and GRO were similar in wd-NHBE cells infected by each of these viruses. At 24 and 36 hpi, NHBE cells had greater levels of pro-inflammatory cytokines including IFN-α, CCL2, TNF-α, and CCL5, when infected by pandemic viruses <i>a</i>s compared with seasonal. Polarization of IL-6 in wd-NHBE cells was greatest at 36 hpi for all isolates. Differential polarized secretion was suggested for CCL5 across isolates. Despite differences in viral titer across isolates, no significant differences were observed in KY/180 and KY/136 gene expression intensity profiles. Microarray profiles of wd-NHBE cells diverged at 36 hpi with 1647 genes commonly shared by wd-NHBE cells infected by pandemic, but not seasonal isolates. Significant differences were observed in cytokine signaling, apoptosis, and cytoskeletal arrangement pathways. Our studies revealed differences in temporal dynamics and basal levels of cytokine/chemokine responses of wd-NHBE cells infected with each isolate; however, wd-NHBE cell gene intensity profiles were not significantly different between the two pandemic isolates suggesting post-transcriptional or later differences in viral-host interactions.</p></div

Apical and basal secretion of cytokines and chemokines in wd-NHBE cultures infected with seasonal and pandemic IAV at 36 hpi.

<p>Culture supernatants were harvested from the apical and basal side of the culture inserts and screened for presence of protein using Luminex platform. The error bars indicate mean ± SEM from 3 replicates per isolate per time point from one representative experiment. The mean and SEM from 3 replicates per isolate per time point are shown. Asterisks indicate significance of p<0.05 (*), p<0.01 (**), and p<0.001 (***).</p

Notable genes upregulated in wd-NHBE cells infected with seasonal and pandemic IAV isolates at 36 hpi.

<p>Fold change values obtained by 1-way ANOVA analysis comparing gene expression intensities of seasonal and pandemic IAV-infected cells to mock. Analysis conducted using Ingenuity core analysis (p<0.05, 2-fold change cut-off).</p

Virus titer detected in supernatant from cells infected seasonal and pandemic IAVs.

<p>(A) wd-NHBE, (B) ud-NHBE, (C) A549, and (D) Calu-3 cells were infected with 3 MOI of seasonal (BN/59) or pandemic (KY/180, KY/136) viral isolates and apical wash from wd-NHBE cells and supernatants from ud-NHBE, A549, and Calu3 cells were collected at 8, 24, 36, 48, and/or 72 hpi. The virus titer was determined using a TCID₅₀ assay. In (E), the amount of viral HA RNA in cells was quantified by qRT-PCR wd-NHBE cells using the Ct method. Data are presented as the mean ± SEM of the virus titer pooled from 3 replicates from three independent experiments with 3 donors (A–D) or 1 donor (E). Asterisks indicate significance of p<0.05 (*), p<0.01 (**), and p<0.001 (***) respectively. The dotted line indicates the limit of detection of the TCID₅₀ assay.</p

Basal cytokine and chemokine production by wd-NHBE cells infected with seasonal and pandemic IAVs.

<p>After infection cell culture supernatants were harvested from the basal side of the culture insert and a multiplex analysis was performed using Luminex platform. A total of two experiments were conducted with two donors. The error bars indicate mean ± SEM from 3 replicates per isolate per time point from one representative experiment. Letters indicate significant differences between isolates (a- different from KY/180, b- different from KY/136, c- different from BN/59, and d- different from Mock).</p

Top five significant canonical pathways in IAV-infected NHBE cells at 36 hpi relative to mock.

<p>Rankings are listed based on statistical significance scored using Fischer's Exact Test (p-value <0.05). For each canonical pathway we report the p-value of Fisher's exact test to measure significance and the proportion of genes in the pathway that were actually significantly represented in the brackets.</p