PMC

Differential BALF cell counts. Differential counts of macrophages (A), neutrophils (B), eosinophils (C) and lymphocytes (D) were assessed in all study groups. •: Significant changes (p < 0.05) when compared to non-SNP exposed animals, *: significant changes when compared to SAL-mice.

Expression of BALF cytokines and chemokines. Different BALF cytokine expression profiles were analyzed by an ELISA as for KC, MIP-1α, MCP-1, TNFα, IFNγ and IL1β, IL2, IL4, IL5, IL6, IL13, IL17A. •: Significant changes (p < 0.05) when compared to non- SNP exposed animals, *: significant changes when compared to non-allergic controls.

OVA specific IgE and IgG1 expression. Serum levels of OVA specific IgG1 (A) are detected by an ELISA and presented as total serum concentration. OVA-specific IgE levels (B) are measured as optical density (OD). •: Significant changes (p < 0.05) when compared to non-SNP exposed animals, *: significant changes when compared to non-allergic controls.

Study design and exposure scheme. Mice were sensitized intranasally (IN) on days 1, 3, 6 and 8 with 0.02% OVA or saline (SAL). SNP were co-administered at intranasal doses of 0, 10, 100, or 400 μg with OVA or saline. On days 22 and 23, OVA-mice were challenged intranasally with 0.5% OVA and SAL-mice with saline solution. Animals were sacrificed on day 24, 24 hours after the last intranasal challenge.

Heat map of gene expression array. Gene expression in lung tissue was analyzed with a 96-gene array. Relative increased gene expression towards control is indicated in red (≥2 fold expression) and relative decreased gene expression in green (≤2 fold). Black labels indicate no differences in gene expression. Increased gene expression in allergic and SNP exposed animals was found for various cytokines, chemokines and immune responsive genes as well as secretory mucus/surfactant genes. No changes greater than 2-fold were found for oxidative stress response, growth factors and different transcription factors (Additional file : Figure S5).

Immunohistochemistry of airway-associated eosinophils. Light photomicrographs of peri-bronchiolar and –vascular interstitium surrounding the proximal axial airway (AA) at generation 5. Tissues were immunohistochemically stained for eosinophils (murine-specific anti-major basic protein antibody; red chromagen; arrows) and counterstained with hematoxylin. OVA-induced inflammatory cell infiltrate composed of eosinophils and mononuclear cells (lymphocytes and plasma cells) is dose-dependently enhanced by SNP. Figures A-D are taken from OVA-treated mice that were co-exposed to 0 (saline control), 10, 100 and 400 μg SNP, respectively. bv: blood vessel; e: airway epithelium; a: alveolus; Scale bars = 50 μm.

Airway epithelial mucus production. Increase in airway epithelial mucus, as a feature of allergic airway disease, was analyzed on lung tissue at the 5th generation of the intraepithelial AB/PAS stained mucosubstances (arrows) in SNP/OVA-mice with increasing SNP exposure dose are shown in figure A (0 μg), B (10 μg), C (100 μg) and D (400 μg); AA = axial airway, e = airway epithelium, a = alveoli, bv = blood vessel. Morphometric measurement of intraepithelial AB/PAS mucosubstances are further shown in E and changes in Muc5ac and Clca3 gene expression in F. •: Significant changes (p < 0.05) when compared to non-SNP exposed animals, *: significant changes when compared to non-allergic controls.

Pulmonary histopathology. A diagram illustrates the locations of transverse tissue sections taken from the left lung lobe for microscopic examination (A). Light photomicrographs of representative lung sections taken at the level of the fifth axial airway (AA) generation and stained with hematoxylin and eosin (B-F). Representative light photomicrographs of a control animal (SAL-mouse; B), OVA-mouse (C) and SNP/OVA-mice with increasing SNP exposure doses (D-F) illustrating peribronchiolar and perivascular mixed inflammatory cell infiltration in OVA- and SNP/OVA-mice (arrows). Greater airway-associated inflammation is present in SNP/OVA-mice exposed to concentrations of 100 and 400 μg SNP (E and F, respectively) compared to the SAL- and OVA-mice. Scale bars = 1 mm.

Activation of immune cells from TBLN. Cells from TBLN of OVA- and SNP/OVA-mice (10 μg SNP) were stained for expression of surface molecules, including CD4, CD69, CD11c, CD11b, MHC II, and Gr-1. Cells were gated on singlets using FSC-A/FSC-H, lymphocytes, monocytes, or granulocytes using FSC-A/SSC-A, and individual cell populations, including CD4⁺ cells, CD11c⁺, Gr-1⁺, CD11c⁺Gr-1⁺CD11b^- pDC, CD11c⁺Gr-1^-CD11b^- AM. More details on the gating strategy are found in the supplementary information (Additional file : Figure S6). Expression of CD69 on CD4⁺ cells (A), Gr-1⁺ cells (B), CD11c⁺ cells (C), pDC (D), and AM (E), and expression of MHC II on CD11c⁺ cells (F) are presented in histograms, with X axis representing mean fluorescence intensity for CD69 or MHC II and Y for % of particular gated cell population. Six samples from OVA- (grey curve) or OVA/SNP- (black curve) mice were concatenated for flow cytometric analysis. Statistical analysis of percent values of CD69 expression from individual TBLN revealed a significant difference in CD11c⁺, AM, and pDC populations between OVA and SNP/OVA-mice.