Cell proliferation of memory CD4⁺ T cells following co-culture with infected dendritic cells (DCs).

<p>(<b>A</b>) Memory CD4⁺ T cells loaded with proliferation dye were co-cultured with unstimulated DCs, DCs infected with PA103ΔUΔT MOI 30 (PA103), or DCs infected with Yorkhill 5 MOI 30 (Yorkhill 5). Panels show the distribution of the proliferation dye fluorescence in the cell population after 6-days; inset figures show the percentage of the initial population that has undergone one or more cell divisions. (<b>B</b>) Patterns of cytokine expression by non-proliferating CD4⁺ T cells following 6-days of culture with DCs infected with Yorkhill 5. (<b>C</b>) Patterns of cytokine expression by CD4⁺ T cells proliferating in response to culture with DCs infected with Yorkhill 5. Numbers in plot represent per cent cells in each quadrant. The experiment was repeated in 3 independent individuals with the same result.</p

Cytokine production by human memory CD4⁺ T cells in healthy controls and cystic fibrosis.

<p>Human memory CD4⁺ T cells were exposed to unstimulated DCs or DCs infected with the laboratory strains (PA103) or clinical <i>Pseudomonas aeruginosa</i> strains (Yorkhill). After 6 days of co-culture levels of (<b>A</b>) IL-17, (<b>B</b>) IL-22 and (<b>C</b>) IFN-γ were measured. Each point represents the mean of triplicate wells and is the result from one individual. The different bacterial strains used are: ♦, unstimulated; ○, PA103 ΔpcrV; •, PA103ΔUΔT; ▴, YH1; ▪, YH2; ×, YH5. The line indicates the median value. Differences between infection conditions (<b>A</b>–<b>C</b>) were evaluated by a Kruskal-Wallis test all of which had p values <0.0009). Differences between unstimulated and infected conditions were then tested using Dunn's multiple comparison test. ** significant p<0.01 and *** significant p<0.001.</p

DC Activation following infection with different strains of <i>P. aeruginosa</i>.

<p>Levels of CD86 (<b>A</b>) and CD40 (<b>B</b>) following DC activation 24 hours following treatment with the indicated microbes. The panels show representative plots of isotype staining (solid shading), staining of unstimulated DCs (solid line) and staining following microbial treatment (dotted line). <b>B</b> and <b>C</b>, mean fold-increase in mean fluorescence intensity (MFI) of CD86 and CD40 respectively in 5 independent individuals. This was calculated as the fold-increase in MFI in infected DCs versus uninfected controls. Columns show mean and SEM.</p

Tissue-homing characteristics of Pseudomonas aeruginosa-specific memory Th22 and Th17 cells.

<p><b>A–C</b> Human memory CD4⁺ T cells were sorted into CCR6-depleted (CCR6 negative) and CCR6-enriched (CCR6 positive) populations before being exposed to dendritic cells (DCs) infected with Yorkhill 5 MOI30 (<b>A</b>), DCs infected with PA103 ΔUΔT MOI30 (<b>B</b>), or stimulated with anti-CD3 and anti-CD28 which acted as a positive control by polyclonal T cell stimulation (<b>C</b>). Plots show CD4⁺ IFN-γ negative T cells after 6-days of co-culture. <b>D</b> Memory CD4⁺ T cells were cultured with DCs infected with PA103 ΔpcrV MOI30. Pseudomonas-specific memory CD4⁺ T cells were analysed for expression of intracellular IL-22 or IL-17 as shown together with the skin-homing chemokine receptors CCR4, CCR10 and CCR9 as indicated. Numbers in each plot represent per cent cells in each quadrant. Results are representative of those seen in three independent individuals.</p

Cytokine production of human memory CD4⁺ T cells in response to <i>P. aeruginosa</i>.

<p><b>A–C</b> Sorted human memory CD4⁺ T cells from healthy volunteers were co-cultured either with dendritic cells that had been infected with <i>P. aeruginosa</i> (DCs +) or with the supernatant from such infected DCs (DC −). Levels of IL-17A (<b>A</b>), IL-22 (<b>B</b>) and IFN-γ (<b>C</b>) were measured in supernatants after 6 days. DCs were infected at a MOI of 60 before culturing with T cells. Columns show the mean of triplicate determinations; error bars are ±1 standard error of mean. Results are representative of experiments in three separate individuals. <b>D–F</b>, secreted cytokine levels following DC infection at a variety of different MOIs. Each point represents a value from an individual healthy individual.</p

Memory CD4⁺ T cells subset response to different Pseudomonas strains in CF and controls.

<p>Following co-culture with dendritic cells infected with laboratory Pseudomonas aeruginosa (PA) strains (PA103) or clinical PA strains (Yorkhill), CD4⁺ T cells were classified into T helper cell subsets based on intracellular cytokine straining patterns, as shown for a representative patient in panel <b>A</b>. The Th1, Th17 and Th22 responses to both PA103 and Yorkhill PA strains is shown for healthy controls (<b>B</b>) and patients with CF (<b>C</b>), as a percentage of the total numbers of CD4⁺ memory cells analysed. The columns show the mean values +/− SEM; n = 8 for CF patients and 10 for healthy controls. Panel <b>D</b> shows the proportions of PA-specific Th17 cells in CF patients compared with controls. Each point represents the result from one individual; different bacterial strains are indicated by the symbols as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090263#pone-0090263-g004" target="_blank">Figure 4</a>. The line indicates the median value. Differences between controls and patients with CF (<b>D</b>) were evaluated by a Mann-Whitney test.</p

Infection of IL-17RA^-/- mice with SRL2.

<p>(A) Representative area of inflamed lung from H&E stained lung sections of mice infected with SRL2. (B) Kaplin-Meier plot of survival following infection of wild type (solid line) or IL-17RA^-/- (dashed line) mice with SRL2. Survival of IL-17RA^-/- mice was improved (logrank test, p = 0.03). (C) Weight following infection in IL-17RA^-/- mice (open circles) and control mice (closed circles). Points are means (n = 10); error bars are SEM. Differences between the groups were not significant (two-way ANOVA, p = 0.06). (D) as (C) but showing clinical score. Difference between the groups was significant (two-way ANOVA, p < 0.001). (E) peripheral blood neutrophil percentage in WT and IL-17RA^-/- mice 24 h after infection. Line shows median value. Closed symbols are animals that survived to the end of the 7 day experiment. Differences between the groups are significant (Mann Whitney test, p = 0.03). (F) BALF bacterial counts from mice that survived infection with SRL2. Line shows median value. Differences between the groups are significant (Mann Whitney test, p = 0.01). Animal shown in blue had mild clinical illness; the remainder had no clinical signs of infection.</p

Capsular size of bacterial isolates.

<p>Bacteria were visualized using electron microscopy and fluorescence microscopy with dextran exclusion. (A) Representative transmission electron microscopy images of SRL1 and TIGR4 following fixation using ruthenium red and lysine-acetate. (B) Capsular area of SRL1 and TIGR4 as visualized by electron microscopy. (C). Area of dextran exclusion per bacterial cell. (B-C) Line shows median. (Significant difference by Mann Whitney test, ** p < 0.01, ***, p < 0.001).</p

Infection of <i>Il17ra</i> KO mice with TIGR4.

<p>C57Bl/6 mice (10–16 per group) were infected with 10⁶ cfu/mouse TIGR4 by intranasal inoculation and either observed for the course of the infection or culled after 24 hours. (A) Kaplan Meier plot of survival for WT (solid line) or IL-17RA^-/- (dashed line), P < 0.001 (log rank test). (B) Blood and (C) BALF bacterial counts 24 h after infection. (D) blood and (E) BALF neutrophil counts 24 h after infection. (F) Lung inflammation measured by blinded assessment of hemotoxylin and eosin (HE) stained sections. (B-E), each point shows the value from an individual animal; line is median. Differences between groups were assessed by Mann Whitney test; * p <0.05, ** p< 0.01, ***, p < 0.001, ns, not significant. (G) Representative areas of inflamed lung from HE stained sections and neutrophil immunohistochemistry (Neutrophil IHC) stained lung at 24 h after infection. Original magnification x40. All data representative of two independent experiments.</p

<i>In vitro</i> neutrophil phagocytosis, killing and NET formation.

<p>(A) A constant of killing was calculated using the method described by Hampton (MOI 1). The paired constants for each strain derived from 3 independent experiments are shown; differences between the two strains were significant (paired t-test, p = 0.01). (B) Fluorescent pneumococci (MOI 10) were cultured with mouse neutrophils and visualized by flow cytometry at the indicated times after bacterial addition. (C) as (B) but associated bacteria (closed bars, SRL1, open bars TIGR4) enumerated by fluorescence microscopy. Bars are means of at least 50 determinations; error bars are SEM. Differences between the strains are significant, p < 0.001, two-way ANOVA). (D-F) NET formation by neutrophils was assessed by fluorescence microscopy (MOI 10). (D) Representative confocal images of neutrophils forming NETs (blue: <i>S</i>. <i>pneumoniae</i>, green: anti-neutrophil elastase, red: sytox orange). (E) Percentage of neutrophils in NETs following incubation with indicated strains or PMA. Bars are means of at least 4 separate low power fields; error bars are SEM. Significant differences between the groups were determined by t tests with Tukey’s post hoc correction; ** p <0.01, *** p< 0.001. (F) mean NET size formed by TIGR4 and SRL1. Bars as in (E). Difference between the strains is significant, p < 0.05, t test. B-F representative of two independent experiments.</p