Effect of HIF-1α overexpression on pneumococcal infection in unexposed and MS-WF exposed A) A549 and B) BEAS-2B cells.

<p>Cells were transfected with HIF-1α plasmid. Plasmid uptake was selected for using an antibiotic as described in the Methods section. The antibiotic was washed off and an infection assay was performed to measure CFU/ml. HIF-1α overexpression was protective against WF-stimulated infection in A) A549 cells (*p<0.05) and B) BEAS-2B cells (**p<0.01). MS-WF alone significantly increased pneumococcal infection in normal A) A549 (***p<0.001) and B) BEAS-2B cells (****p<0.0001). HIF-1α overexpression was achieved in all experiments. PAFR expression was, on average, 542% in all HIF-1α overexpression samples compared to controls. Each data point represents a single experiment. Data are analysed using 1 way ANOVA followed by Sidak’s multiple comparison test.</p

Effect of HIF-1α siRNA knockdown on pneumococcal infection in unexposed and MS-WF exposed A) A549 and B) BEAS-2B cells.

<p>Cells were transfected with either HIF-1α or negative control siRNA for 48h. An infection assay as described in the Methods section was then performed to measure CFU/ml. In A) A549 cells, HIF-1α knockdown significantly increased pneumococcal infection in unexposed cells and this was unaffected by MS-WF exposure (**p<0.01, **p<0.01 respectively). In B) BEAS-2B cells, HIF-1α knockdown significantly increased pneumococcal infection in unexposed cells and this was unaffected by MS-WF exposure (*p<0.05. **p<0.01 respectively). MS-WF alone significantly increased pneumococcal infection in A) A549 (*p<0.05) and B) BEAS-2B cells (**p<0.01). HIF-1α knockdown was achieved in all experiments. PAFR expression was, on average, 54% in all HIF-1α knockdown samples compared to controls. Data are from at least 3 separate experiments. Each data point represents the mean of 3 technical replicates within an experiment. Data are compared using 1 way ANOVA followed by Dunnett’s multiple comparison test.</p

Metal compositions of the WF samples used in the study by % weight per sample.

<p>MS-WF is our standard WF sample.</p

Effect of A) chromium-, B) iron-, C) manganese- and D) nickel-rich WF on pneumococcal infection in A549 cells.

<p>Cells were exposed to WF for 2h. The WF was then washed off and the cells infected with pneumococci at MOI 100. Loosely adherent bacteria were washed off and the cells lysed to analyse infection by measuring CFU/ml. All WF samples significantly increased pneumococcal infection of cells (*p<0.05, *p<0.05, ***p<0.001, *p<0.05 respectively). Data are from 3 separate experiments. Each data point represents the mean of 3 technical replicates within an experiment. Data are compared using t test.</p

Effect of A) chromium-, B) iron-, C) manganese- and D) nickel-rich WF on pneumococcal infection in BEAS-2B cells.

<p>Cells were exposed to WF for 2h. The WF was then washed off and the cells infected with pneumococci at MOI 100. Loosely adherent bacteria were washed off and the cells lysed to analyse infection by measuring CFU/ml. All WF samples significantly increased pneumococcal infection of cells (*p<0.05, *p<0.05, **p<0.01, *p<0.05 respectively). Data are from 3 separate experiments. Each data point represents the mean of 3 technical replicates within an experiment. Data are compared using t test.</p

Effect of MS-WF exposure +/- NAC treatment on HIF-1α levels in A) A549 and B) BEAS-2B cells.

<p>Cells were exposed to MS-WF +/- NAC as described previously, stained for HIF-1α and analysed by flow cytometry. In A) A549 cells, MS-WF exposure significantly increased HIF-1α levels compared to controls (***p<0.001) which were attenuated in NAC treated cells compared to untreated cells (***p<0.001). In B) BEAS-2B cells, MS-WF exposure significantly increased HIF-1α levels (****p<0.0001) which were attenuated by NAC treatment (***p<0.001). Data are from at least 3 separate experiments. Each data point represents a single experiment. Data are compared using 1 way ANOVA followed by Sidak’s multiple comparison tests.</p

% of HIF-1α knockdown and PAFR mRNA expression in A549 and BEAS-2B cells.

<p>HIF-1α knockdown and corresponding and PAFR mRNA % levels are presented in Table 2.</p

Constitutive expression of PAFR in nasal samples of welders vs controls.

<p>Nasal epithelial cells were obtained, stained for PAFR and analysed using flow cytometry. Constitutive expression of PAFR in the nasal epithelium of welders was significantly higher compared to controls (*p<0.05). Data are from 10 welders vs 10 controls and are normalised to isotype control for each sample. Data are compared using t test.</p

% of HIF-1α overexpression and PAFR mRNA expression in A549 and BEAS-2B cells.

<p>% of HIF-1α overexpression and PAFR mRNA expression in A549 and BEAS-2B cells.</p

Effect of chromium-, iron-, manganese- and nickel-rich WF samples on pneumococcal growth.

<p>Pneumococci were grown for 3h in WF and samples were taken every hour to measure CFU/ml. Pneumococci grown for 3h in A) chromium-rich WF had significantly lower CFU counts compared to controls (**p<0.01). Pneumococci grown for 3h in B) iron-rich WF, C) manganese-rich WF and D) nickel-rich WF did not have significant differences in CFU counts compared to controls. Data are from 3 separate experiments. Each data point represents one sample. Data are matched and compared using 2 way ANOVA.</p