Up-regulated pneumococcal genes following adherence to human Met-5A PMC cells.

<p>Differential gene expression was analyzed by microarray in pneumococci adherent to Met-5A cells at 2h post challenge. Genes demonstrating ≥ mean 2-fold changes in expression in all (n = 3) replicates with significance (t-Test p-value) are ordered by <i>sp</i> number.</p><p>Up-regulated pneumococcal genes following adherence to human Met-5A PMC cells.</p

Interactions of <i>Streptococcus pneumoniae</i> with pleural mesothelial cells <i>in vitro</i>.

<p>A) Association: monolayers of Met-5A cells were infected with various MOI of <i>Streptococcus pneumoniae</i> strain D39. Total associated (defining adherent and internalised bacteria combined) bacteria were counted following saponin lysis of infected monolayers over time. The data are representative of >3 independent experiments; the symbols represent mean adherence and the error bars the standard deviations of wells infected in triplicate. B) Invasion: monolayers of Met5A cells were infected for 9h with MOI 0.025 of D39 and intracellular bacteria were recovered after gentamicin treatment and saponin lysis. Monolayers were also pre-treated with cytochalasin D prior to bacterial infection. The bars represent the mean bacterial cfu of three independent experiments and the error bars the standard error of the means.</p

RT-pPCR validation of microarray gene expression changes in human Met-5A pleural mesothelial cells following infection with D39 <i>Streptococcus pneumoniae</i>.

<p>Met-5A cells were infected with D39 (MOI 200 bacteria/cell) for 2h. RNA was extracted from pleural cells and analyzed on human microarrays. Mean fold expression changes were calculated for each gene from n = 5 experiments and a ≥ 2-fold increase/decrease was considered significant. RT-qPCR validation was done from n = 3 independent infection experiments in triplicate. The 10 most differentially up- and down-regulated genes are shown, as derived from IPA analysis, and the remainder can be seen in the gene datasets in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142773#pone.0142773.s005" target="_blank">S3A and S3B Table</a>. C, cytoplasm, Cy, cytosol; ES, extracelular space; PM, plasma membrane; N, nucleus; R, ribosomes.</p><p>* denotes statistical significance (Ct values) <0.05.</p><p>RT-pPCR validation of microarray gene expression changes in human Met-5A pleural mesothelial cells following infection with D39 <i>Streptococcus pneumoniae</i>.</p

mRNA transcription by RT-qPCR for selected pneumococcal genes.

<p>Using the random stratification method, differentially expressed genes were stratified and genes then randomly selected from each stratum for validation. Both up- and down-regulated genes lists were divided into strata containing three genes and random number generating software (Apple Inc.) was used to select the gene for validation. The data are from five independent infection experiments and three control samples, i.e. pneumococci grown without adherence to cells. RT-qPCR was done in triplicate and fold expression changes for each gene by microarray analysis are shown alongside. To demonstrate that possible contamination with host RNA did not impact on pneumococcal RT-qPCR mRNA expression data, as a control, RT-qPCR was also done using Met-5A PMC uninfected cDNA; for all genes examined, the level of non-specific amplification of Met-5A cell cDNA was <0.005% of the specific amplification of control pneumococcal cDNA.</p><p>* denotes statistical significance (Ct values) <0.05.</p><p>mRNA transcription by RT-qPCR for selected pneumococcal genes.</p

Differentially expressed pneumococcal genes identified by the application of data filtering algorithms.

<p>Gene lists generated by GeneSpring GX software were initially filtered using the students’ t-Test analysis of variance to yield only those genes with a p-value <0.05. Of these genes, only those displaying a mean ≥ 2-fold expression change were selected. Finally, only those genes meeting the filtering criteria in all biological replicates (n = 3) for a particular experimental condition were selected.</p

Down-regulated pneumococcal genes following adherence to human Met- cells.

<p>Differential gene expression was analyzed by microarray in pneumococci adherent to Met-5A cells at 2h post challenge. Genes demonstrating ≥ mean 2-fold changes in expression in all (n = 3) replicates with significance (t-Test p-value) are ordered by <i>sp</i> number.</p><p>Down-regulated pneumococcal genes following adherence to human Met- cells.</p

Analysis of data obtained from human microarray experiments using Ingenuity Pathways Analysis (IPA).

<p>A) IPA software v7.6 (Ingenuity® Systems, USA) was used to identify the top canonical pathways in PMC that were most affected by D39 infection. This was determined by calculating the ratio of the total number of differentially expressed genes in each pathway as a proportion of the total number of genes involved in that pathway. A minimum threshold of 5% gene perturbations was then imposed on pathways and those meeting these inclusion criteria were then ranked according to the proportion of gene perturbations. The 20 most highly ranked pathways by P-value and ratio are shown. B) Network map showing the distributions of specific genes that overlap between the top canonical pathways. The reader is referred to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142773#pone.0142773.s005" target="_blank">S3B Table</a> for a complete listing of canonical pathways.</p

Inflammatory cytokine responses of Met-5A PMC.

<p>A) Met-5A cell monolayers (n = 3 wells) were infected in triplicate wells with live D39 (MOI 1–200), stimulated with pure TNF-α (100ng/ml) or Nm-OM (100ng/ml) and also pre-stimulated with TNF-α (100ng/ml) for 4h and then infected with various MOI (1–200) of live D39. Control cells were left with medium alone. Cells were also treated with live D39 without any other stimulation (no cytokine production at any of the concentrations tested between MOI 1–200). After 24h, supernatants were removed and production of IL-6 and IL-8 quantified by ELISA. The bars represent mean cytokine levels and the errors bars the standard deviation from triplicate wells from a representative experiment (n = 2). * denotes statistically significant reduction in IL-6 or IL-8 compared to cells treated with TNFα only (p<0.05). B) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and NFkB p65 protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05). C) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and p38 MAP Kinase protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05).</p

Differentially expressed genes in 4 biopsies from duodenum with multiple plumes seen by confocal laser endomicroscopy compared to 4 with minimal plumes.

<p>Differentially expressed genes in 4 biopsies from duodenum with multiple plumes seen by confocal laser endomicroscopy compared to 4 with minimal plumes.</p

Clinical characteristics of study participants.

<p>Clinical characteristics of study participants.</p