Scatter plots of different environmental parameters.

<p>A significant correlation was observed between the parameters urbanisation index (UI) and temperature (A; n = 16), (B) UI and NO₂ concentration (n = 40) and (C) temperature and NO₂ concentration (n = 16). Ozone was not correlated with either (D) UI (n = 40), (E) NO₂ (n = 40) or (F) temperature (n = 16); ***: p<0.001.</p

Content of PALMs in pollen samples plotted against different urbanisation-related environmental conditions.

<p>No correlation was seen between PALM_PGE2 and UI (A; n = 40), temperature (B; n = 16) or NO₂ concentration (C; n = 40). A significant association of high ozone concentrations and low PALM_PGE2 contents was observed (D; n = 40). PALM_LTB4 did not show any significant correlation. Neither UI (E; n = 40), nor temperature (F; n = 16), NO₂- (G; n = 40) and ozone (H; n = 40) were related to the content of PALM_LTB4. ***: p<0.001.</p

Bet v 1 content of birch pollen in trees against different, urbanisation-related environmental conditions.

<p>No significant correlation could be observed between Bet v 1 content and urbanisation index (A; n = 40) or NO₂ concentration (B; n = 40<b>)</b>. Bet v 1 showed a significant and negative correlation with temperature (C; n = 16) and was positively correlated with site-specific ozone levels (D; n = 40). *: p<0.05.</p

Immune stimulatory versus immune modulatory potential of pollen samples from higher- and lower-ozone-exposed birch trees.

<p>Aqueous extracts (APEs) of birch pollen sampled from high and low ozone exposed trees were chosen for neutrophil migration assays and stimulation of monocyte derived dendritic cells. APEs were applied in 3 concentrations and the AUC was calculated. Higher ozone-exposed pollen induced stronger neutrophil chemotaxis compared to pollen samples from lower ozone–exposed trees (<b>A</b>). In contrast, birch pollen from lower ozone-exposed trees were more potent in inhibiting the LPS-induced release of IL-12p70 from human monocyte-derived dendritic cells (<b>B</b>). APEs were prepared from birch pollen sampled from higher ozone-exposed trees (n = 2; mean ozone: 85 µg/m³) and from lower ozone-exposed trees (n = 2; mean ozone: 54 µg/m³). All APEs were tested in n = 3 patients. *: p<0.05 (Wilcoxon matched-pairs signed-ranks test).</p

Chemical and physical aerosol characterisation.

<p>(A) The ship diesel engine was operated for 4 h in accordance with the IMO-test cycle. (B) Approximately 28 ng/cm² and 56 ng/cm² were delivered to the cells from DF and HFO, respectively, with different size distributions. The HFO predominantly contained particles <50 nm, and the DF predominantly contained particles >200 nm, both in mass and number. (C) Number of chemical species in the EA particles. (D) Transmission electron microscope (TEM) images and energy-dispersive X-ray (EDX) spectra of DF-EA and HFO-EA; heavy elements (black speckles, arrow); and contributions of the elements V, P, Fe and Ni in the HFO particles using EDX (* = grid-material). (E) Exemplary EA concentrations (right) and concentration ratios (left) for particulate matter-bound species. For all experiments, n = 3.</p

Summary of the main HFO- and DF-particle exposure effects.

<p>The arrows indicate the direction of regulation for cellular functions derived from the most statistically significant enriched Gene Ontology terms from the transcriptome, proteome, and metabolome (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126536#pone.0126536.s012" target="_blank">S2 Table</a>).</p><p>^x BEAS-2B up, A549 down</p><p>* BEAS-2B down, A549 up</p><p>Summary of the main HFO- and DF-particle exposure effects.</p

Experimental set-up and global omics analyses.

<p>(A) An 80 KW common-rail-ship diesel engine was operated with heavy fuel oil (HFO) or refined diesel fuel (DF). The exhaust aerosols were diluted and cooled with clean air. On-line real-time mass spectrometry, particle-sizing, sensor IR-spectrometry and other techniques were used to characterise the chemical composition and physical properties of the particles and gas phase. Filter sampling of the particulate matter (PM) was performed to further characterise the PM composition. Lung cells were synchronously exposed at the air-liquid-interface (ALI) to aerosol or particle-filtered aerosol as a reference. The cellular responses were characterised in triplicate at the transcriptome (BEAS-2B), proteome and metabolome (A549) levels with stable isotope labelling (SILAC and ¹³C₆-glucose). (B) Heatmap showing the global regulation of the transcriptome, proteome and metabolome.</p

Effects of shipping particles on lung cells.

<p>The net effects from the particles were referenced against the gaseous phase of the emissions. (A) Number of the regulated components in the transcriptome shows more genes regulated by the DF than the HFO particles (in BEAS-2B cells). Similar results were observed for the proteome (B) and metabolome (C) (in A549 cells). (D) Meta-analyses for the transcriptome and proteome using the combined Gene Ontology (GO) term analysis of the 10% most regulated transcripts and proteins. Individual GO terms are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126536#pone.0126536.s012" target="_blank">S2 Table</a>; the hierarchical pathways are indicated on the right. (E) Gene regulation of Wiki-pathway bioactivation; (F) gene regulation of Wiki-pathway inflammation; g, secreted metabolites; and h, metabolic flux measurements using ¹³C-labelled glucose. For all experiments, n = 3.</p