Experimental design used to study the effect of nasal exposure to ovalbumin in mice.

<p>BALB/c mice (11–13 per group) were sensitized by intranasal administration of 100 µg of ovalbumin in 20 µl PBS in the absence of adjuvants 5 times a week for 2 weeks. After that, mice were challenged by intranasal administration of same dose of ovalbumin two times a week for 6 weeks. As a control, mice were treated with PBS with the same protocol. Two days after the last challenge, mice were sacrificed for obtaining the NAL fluids and tissue samples.</p

Histology of allergic rhinitis and <i>mev</i> mice.

<p>(A) Hematoxylin and eosin stains of the nasal cavity from controls, allergic rhinitis (OVA) and <i>mev</i> mice. (B) In allergic and <i>mev</i> mice, there is clearly increased nasal inflammation with eosinophilia, as compared to controls. (C) Epithelial thickness significantly increased in allergic mice compared with controls and <i>mev</i> mice (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001).</p

Relative mRNA expression of Th2 cytokines (IL-4 and IL-13) in controls, allergic rhinitis (OVA), and <i>mev</i> mice (*<i>p</i><0.05 and ***<i>p</i><0.001).

<p>IL-4 is significantly up-regulated in both allergic rhinitis and <i>mev</i> mice compared with controls. However, IL-13 significantly increased only in OVA model.</p

The method of NAL technique used in this study.

<p>(A) Photograph of the trans-pharyngeal approach to obtain nasal lavage samples from mouse models. (B) Comparison of yields in the recovered fluid volume after the NAL between trans-tracheal and trans-pharyngeal approaches (***<i>p</i><0.001).</p

Mucus metaplasia in the nasal airways of <i>mev</i> mice.

<p>(A) Alcian blue staining of the nasal airway from <i>WT</i>, <i>mev</i>, <i>mev x IL-4 KO</i>, <i>mev x IL-13 KO</i>, <i>mev x IFN-γ KO</i>, and <i>IFN-γ KO</i> mice. Representatives of multiple samples are shown.</p

Chemokine production in the nasal lavage fluids of <i>mev</i> mice.

<p>ELISA showed proteins concentrations of (A) Eotaxin, (B) CXCL1, and (C) TNF-α from <i>WT</i>, <i>mev</i>, <i>mev x IL-4 KO</i>, <i>mev x IL-13 KO</i>, <i>mev x IFN-γ KO</i>, and <i>IFN-γ KO</i> mice (n = 6 each, and *P<0.05, **P<0.01, and ***P<0.001).</p

Th2 and Th1 cytokine profiles in the nasal airways of the <i>mev</i> mice.

<p>Real-time PCR showed mRNA expression of (A) IL-4, (B) IL-5, (C) IL-13, and (D) IFN-γ from <i>WT</i>, <i>mev</i>, <i>mev x IL-4 KO</i>, <i>mev x IL-13 KO</i>, <i>mev x IFN-γ KO</i>, and <i>IFN-γ KO</i> mice (n = 6 each). The results were normalized to GAPDH (as reference gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103685#pone.0103685-Lee2" target="_blank">[28]</a>) and presented as the difference of Ct values (ΔCt) between them (n.d.: not detected; *P<0.05, **P<0.01, and ***P<0.001).</p

Targeted gene depletion of Th1 cytokine IFN-γ resulted in a severely Th2-skewed inflammation in the nasal airway of the <i>mev</i> mice.

<p>Histology and the nasal lavage (NAL) cytology of <i>mev</i> (n = 7), <i>mev with IFN-γ KO</i> (n = 9), and <i>IFN-γ KO</i> (n = 12) mice. (A) Total and differential cell counts in NAL samples were determined for each group of mice (*P<0.05, **P<0.01, and ***P<0.001). (B) Representative H&E-stained nasal sections from each group.</p

Gelatin zymography for MMP-2 and MMP-9 activities in the NAL fluids of <i>mev</i> mice.

<p>(A) MMP activity was visualized as clear bands against a dark blue background of gelatin substrate in gel. Molecular weight markers were used to estimate the molecular masses of the pro and active forms of MMP-2 and MMP-9. Expression of MMP-9 was identified as mainly pro-forms instead of minor active forms. Expression of MMP-2 was seen as active form without pro-form. MMP-9 expression was more prominent than MMP-2 in the NAL fluids. (B) Blots were scanned and densitometry was performed. Density of each band expressed as an arbitrary unit. This gel is a representative of two independent experiments.</p

Chemokine expression in the nasal tissues of the <i>mev</i> mice.

<p>Real-time PCR showed mRNA expression of (A) CCL2 (MCP-1), (B) CCL5 (RANTES), and (C) GM-CSF from <i>WT</i>, <i>mev</i>, <i>mev x IL-4 KO</i>, <i>mev x IL-13 KO</i>, <i>mev x IFN-γ KO</i>, and <i>IFN-γ KO</i> mice (n = 6 each). The results were normalized to GAPDH (reference gene) and presented as the difference of Ct values (ΔCt) between them (*P<0.05, **P<0.01, and ***P<0.001).</p