Effect of H3N2 (X-31) influenza A virus infection on BALF cellularity in wild type (WT) and Nox2^−/y mice.

<p>Mice were treated with 1×10⁴ PFU of the low virulence H3N2 strain of influenza A virus and the number of (A) total cells, (B) macrophages and (C) neutrophils counted in the BALF 3 and 7 days post infection. Data are shown as mean ± SD for 6–8 mice per group. *<i>P</i>&lt;0.05 vs respective no virus group, #<i>P</i>&lt;0.05 vs D3 X-31-treated WT mice mice, ∧<i>P</i>&lt;0.05 vs D7 X-31-treated WT mice (ANOVA and Dunnett's <i>post hoc</i> test).</p

Number of the CD8+ T cell response.

<p>Enriched CD8+ T cells from BALF (A–C) or spleen (D–F) were obtained from influenza A virus (X-31)-infected WT and Nox2^−/y mice 7 days post infection. These were then stained with fluorescently labelled tetramer complexes specific for the two immunodominant CD8+ T cell epitopes (D^bNP_366–372 and D^bPA_224–232) and data analysed by flow cytometry. Data are shown as mean ± SD of 4 individual mice.</p

Effect of H3N2 (X-31) influenza A virus infection on viral titer and body weight in wild type (WT) and Nox2^−/y mice.

<p>Mice were treated with 1×10⁴ PFU of the low virulence H3N2 strain of influenza A virus and (A) viral titer determined 3 days post infection and (B) body weight recorded for up to 7 days post infection. Data are shown as mean ± SD for 6–8 mice per group. *<i>P</i>&lt;0.05 vs WT mice (Students' unpaired <i>t</i> test).</p

Nox2β and native Nox2 protein sequence alignment and bioinformatics.

<p>(a) The predicted amino acid sequence of the splice variant protein is aligned with the amino acid sequence of native Nox2 (Acc: BAE31076). Hyphens indicate region of spliced sequence. Asterisks indicate histidine residues reported to be important in heme binding. Underlined amino acids specify putative NADPH binding sites and hatched boxes signify predicted p47phox binding regions. Spotted boxes indicate the putative FAD binding motifs and the hash indicate predicted glycosylation sites. Alignment was achieved using DNASIS MAX software. (b) Hydrophobicity plot generated by “<b><i>The Molecular Toolkit:</i></b><i> Colorado State University”.</i> Red boxes indicate two regions of significant hydrophobicity.</p

Superoxide production from BALF cells using L-O12-enhanced chemiluminescence.

<p>BALF cells obtained from H3N2 (X-31) influenza A virus-infected WT and Nox2^−/y mice 3 days post infection. Data are shown as mean ± SD relative light units per second (RLU/s) for 6–8 mice per treatment group. *<i>P</i>&lt;0.05 vs WT no virus mice, #<i>P</i>&lt;0.05 vs respective WT mice (ANOVA and Dunnett's <i>post hoc</i> test).</p

Peroxynitrite production in mouse lung infected with H3N2 (X-31) influenza A virus using 3-nitrotyrosine (3-NT) immunofluorescence.

<p>Representative sections of lung tissue obtained from WT (A) and Nox2^−/y mice (B) infected with X-31 were incubated with mouse monoclonal anti-3-nitrotyrosine antibody (1∶50) followed by biotinylated anti-mouse IgG reagent. (C and D) The same sections showing corresponding light microscope images. WT mice lung sections displayed strong immunofluorescence for 3-NT in the inflammatory cells that infiltrated the airways and in the alveolar tissue. In contrast, Nox2^−/y mice displayed markedly less immunofluorescence for 3-NT.</p

Expression of truncated transcripts of Nox2 in macrophages using RT-PCR and macrophage cDNA templates.

<p>(a) 1 µl of primary peritoneal macrophage cDNA template was amplified using a primer set including exon 3 forward and exon 13 reverse. (B) Re-amplification and nested PCR reactions were run using either 1 µl or 2 µl of cDNA isolated from the ∼400 base pair bands observed in (a) and with primer sets containing exon 3 forward and exon 13 reverse. (c) 0.5–4.0 µl of RAW264.7 cDNA was amplified using a primer set including exon 1 forward and exon 13 reverse. (D) Nested PCR reactions using either 0.5 or 1 µl cDNA eluted from the 500 bp band observed in (c) and with primer sets including an exon 3 forward and exon 13 reverse. Solid red arrows indicate bands, which were eluted and sequenced while broken arrows indicate eluted bands for nested PCR reactions. Note all samples were separated by running on 3% agarose gels.</p

Superoxide generation by Nox2β.

<p>(a) Representative RT-PCR reaction employing the splice variant specific primers (SS - 3′UTR) and 3 µl RAW264.7 cell cDNA templates following either vehicle, siRNA or scrambled siRNA treatments and (b) the corresponding histogram of densitometric analysis of cDNA bands (n = 3). Representative Western blot images showing the protein expression of (c) native Nox2 or (e) Nox2β in RAW264.7 cells following either vehicle or siRNA treatment and (d) and (f) their respective histograms showing densitometric analysis normalized to β-actin expression (n = 3). Superoxide production in the presence of PDB following either (g) vehicle or siRNA treatment (n = 6) or (h) scrambled siRNA or siRNA treatment. Statistical analyses were conducted using either a 1-way ANOVA with a Bonferroni multiple comparisons post-hoc test for PCR bands or a two-tail paired t-test for Western blotting and superoxide data. Statistical significance was taken when the P&lt;0.05.</p

Expression of Nox2βin atherosclerotic arteries.

<p>(a) Western blots images and (b) corresponding densitometric analysis showing the expression of Nox2β and native Nox2 in mouse aorta from age-matched wild type mice and APOE^−/− mice on a high fat diet for 19–26 weeks. (C) Western blot image showing the lack of expression of Nox2β in aorta taken from the novel double APOE^−/−/Nox2^−/y mouse.</p

Cleaved caspase 3 immunofluorescence to assess lung alveolar epithelial apoptosis.

<p>Representative sections of lung tissue obtained from WT (A) and Nox2^−/y mice (B) infected with influenza A virus (H3N2; X-31) were incubated with rabbit polyclonal anti-cleaved caspase 3 antibody (1∶250) followed by goat anti-rabbit Alexa fluor 488 (Invitrogen; 1∶500) secondary antibody. (C and D) The same sections showing corresponding light microscope images. WT mice lung sections displayed strong immunofluorescence for cleaved caspase 3 in the alveolar tissue. In contrast, Nox2^−/y mice displayed markedly less immunofluorescence for cleaved caspase 3.</p