Cytokine and chemokine secretion from mouse bone marrow derived macrophages after influenza A virus infection.

<p>(A) TNF-α (B) IP-10 protein secreted by the mouse bone marrow derived macrophages after influenza A viruses infection (as denoted in legend). Mean and standard error of duplicate assays are shown. All influenza A virus infected mouse macrophages secrete significantly higher concentration of TNF-α than mock infected cells (<i>p</i><0.05).</p

Viral matrix (M) gene expression copy number normalized to β-actin gene expression (10⁵ copies) by quantitative RT-PCR in influenza virus infected mouse bone marrow derived macrophages.

<p>Matrix gene mRNA copy number was assayed 3 h, 6 h and 24 h post-infection and normalized to those of β-actin mRNA in the corresponding sample. Means of triplicate assays are shown with standard error. Asterisk indicates statistical difference (<i>p</i><0.05).</p

Virus titer detected in the supernatant of influenza A virus infected mouse bone marrow derived macrophages.

<p>Virus titer of various (A) influenza H1N1, and (B) H5N1 influenza viruses was determined at 3, 24, 48 and 72 h post-influenza virus infection of mouse bone marrow derived macrophages. Means and standard error of triplicate assays were shown. Dotted line represents the lowest detection limit of the TCID₅₀ assay. The thermal inactivation (serial dilution of influenza virus was incubated in the cell-free culture medium alone at the corresponding time points) curves (dotted line) of influenza H1N1 and H5N1 viruses at 37°C were determined from culture wells without macrophages.</p

Cell characterization and lectin profile of the mouse bone marrow derived macrophages.

<p>Histogram showing the percentage of positive stained mouse bone marrow derived macrophages by flow cytometry (open peak-blue line). Isotype control (open peak-black line) and non-stained cells as negative control (shaded peak) of bone marrow derived macrophages stained with (A) CD14 and (B) F4/80. Lectin immune-staining assay to determine the sialic acid (SA) distribution on mouse bone marrow derived macrophages. (C) <i>Maackia amurensis</i> lectin (MAA) conjugated with FITC (the lectin that binds SA-α2,3Gal linked sialic acid) and (D) with <i>Sambucus nigra</i> lectin (SNA) conjugated with FITC (the lectin that binds SA-α2,6GalNAc).</p

Lectin binding: MAL-I binds to Sia-α2-3Galβ1-4GlcNAc (Panel A, D, G and J), MAL-II binds to Sia-α2-3Galβ1-3GalNAc (Panel B, E, H and K) and SNA binds to Sia-α2-6-linkage (Panel C, F, I and L) to determine the Sias distribution on the ud- and wd-NHBE cells.

<p>MAL-I, MAL-II and SNA bindings presented on the (A-C) <i>en face</i> staining of ud-NHBE, (D-F) cross-section staining of ud-NHBE, (G-I) cross-section staining of wd-NHBE cells <i>in vitro</i> cultures and (J-L) the human bronchial biopsy in reddish brown.</p

The (A) RANTES and (B) IP-10 protein of the supernatant collected at the apical compartment of the influenza HK98/H1N1 and VN04/H5N1 virus infected ud-NHBE (dark bars) and wd-NHBE (grey bars) cells at 24 h post infection.

<p>The chart showed the mean and the standard error from three independent experiments. Double asterisk indicated statistically significant difference of means with <i>p</i><0.001 and triple asterisks indicated statistically significant differences of means with <i>p</i><0.0001. UD indicated the protein concentration of the sample is below the detection limit.</p

Immunofluorescence staining of (A–C) ud-NHBE cells and (D–F) wd-NHBE cells at 16 h post infection with (A and D) mock infection, (B and E) HK98/H1N1 and (C and F) VN04/H5N1.

<p>Influenza nucleoprotein and matrix protein was stained in green with FITC-conjugated mouse antibody.</p

Wd-NHBE cells <i>in vitro</i> cultured in ALI (A) for 7 days and for (B) 21 days show the pseudostratified columnar type epithelium by H&E staining.

<p>(C) Positive staining of FITC-conjugated β-tubulin on apical surface of epithelium confirms the presence of cilia and (E) positive staining of biotinylated MUC5AC indicates the presence of mucin secreting goblet cells. Human bronchus stained with (D) FITC-conjugated β-tubulin and (F) biotinylated MUC5AC showed the presence of both ciliated and mucus secreting goblet cells along the epithelium. (G) Transepithelial resistance and (H) HAT mRNA expression by of the differentiating NHBE culture from D1 to D21 ALI culture.</p

Influenza matrix (M) gene expression after infection of influenza HK98/H1N1 virus (grey bars) and influenza VN04/H5N1 virus (black bars).

<p>Ud-NHBE supported M gene transcription from 3 h to 24 h post infection for both influenza viruses while wd-NHBE supported a better influenza HK98/H1N1 virus M gene transcription than influenza H5N1 virus. Bars represented the mean M gene expressed per 10⁵ β–actin house keeping gene and error bar represent the standard error of mean from three independent experiments. Asterisk indicates significant difference with <i>p</i><0.05.</p

Virus titer detected in the supernatant of influenza virus infected ud- and wd-NHBE cells.

<p>Virus titer of the (A) HK98/H1N1 and (B) VN04/H5N1 was determined after influenza viruses infected in the ud- and wd-NHBE cells from 1 h to 48 h post infection at MOI of 2. (C) The comparison of viral replication kinetic between influenza HK98/H1N1 and VN04/H5N1 viruses in ud- and wd-NHBE at 24 h post infection. The chart showed the mean and the standard error of the virus titer pooled from three independent experiments. Single asterisk indicated statistically significant difference of means with <i>p</i><0.05, double asterisks indicated statistically significant differences of means with <i>p</i><0.01. Dotted line represents the detection limit of the TCID₅₀ assay.</p