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

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

<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

Course of infection caused by TIGR4 and SRL1.

<p>C57Bl/6 mice were infected with either TIGR4 (5 x 10⁶ cfu/mouse) or SRL1 (10⁶ cfu/mouse) by intranasal inoculation and culled at pre-determined intervals. Bacteria were enumerated in BALF (A), pleural aspirate (B) and blood (C). Concentration of IL-17A in BALF (D) and MPO in lung (E) were determined. (F) H&E tissue sections of mice infected for 48 hours at original magnification of x4. G. Immunohistochemistry using the neutrophil marker Ly-6G at an original magnification of x40. Points are means from three animals; bars show SEM. Representative of two independent experiments.</p

Nasal colonization with TIGR4 or SRL1 in IL-17RA^-/- mice.

<p>Bacterial counts in nasal wash enumerated 24 after inoculation. Line shows median value; differences are significant by Mann Whitney test, *, p < 0.05.</p

Infection with TIGR4 or SRL1 following depletion of neutrophils.

<p>C57Bl/6 mice (20 per group) were infected with 10⁶ cfu/mouse of TIGR4 (A-D) or SRL1 (E-F) by intranasal inoculation 24 hours after intraperitoneal inoculation of 500 μg antibody 1A8 (anti-Ly6G) or isotype matched control and observed for the course of the infection. (A) peripheral blood neutrophil counts following treatment with isotype control (filled circles) or anti-Ly6G (open circles) at indicated times following infection. Differences (Mann Whitney test) were not significant (NS) or significant (*, p < 0.05, ***, p < 0.001) (B) Isotype control treated mice (solid line) and neutrophil depleted mice (dashed line) had similar survival (A, P = 0.09, log rank test). (C) Median survival time among neutrophil depleted mice; each point is an individual animal, line is median. Differences are significant (**, Mann Whitney test, p = 0.01). (D) bacteremia and (E) clinical score between isotype treated mice (solid circles) and neutrophil depleted mice (open circles). Differences are not significant. (F) as (A) following infection with SRL1 (G) Outcome following SRL1 infection. Isotype control treated mice (solid line) had impaired survival compared to neutrophil depleted mice (dashed line) (p = 0.02, log rank test). (H) Clinical score in neutrophil depleted mice (open circles) compared to isotype treated mice (solid circles). Differences between the groups are significant (p = 0.01, 2-way ANOVA corrected for repeated measures).</p

Infection with SRL1 in <i>Il17ra</i> KO mice following depletion of neutrophils.

<p><i>Il17ra</i> KO mice were infected with 10⁶ cfu/mouse of SRL1by intranasal inoculation 24 hours after intraperitoneal inoculation of 500 μg antibody 1A8 (anti-Ly6G) or isotype matched control and observed for the course of the infection. (A) peripheral blood neutrophil counts following treatment with isotype control (filled circles) or anti-Ly6G (open circles) at 24 hours following infection. Each point is from an individual animal; line indicates median. Difference is significant (**, p < 0.01, Mann Whitney test) (B). Kaplan Meir plot of survival of infected mice. Isotype control treated mice (solid line) had improved survival compared to neutrophil depleted mice (dashed line) (p = 0.017, log rank test). (C) clinical score between isotype treated mice (solid circles) and neutrophil depleted mice (open circles) Differences are significant between the groups (p < 0.0001, 2-way ANOVA).</p

Infection of <i>Il17ra</i> KO mice with SRL1.

<p>C57Bl/6 mice (9–11 per group) were infected with 10⁶ cfu/mouse SRL1 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 < 0.01, log rank test). (B) Blood and (C) BALF bacterial counts 48 after infection. (D) BALF bacterial counts from mice which survived infection with SRL1 (pooled from multiple experiments). (E) Blood and (F) BALF neutrophils. (G) Weight of animals following infection; open circles IL-17RA KO and closed circles WT. Each point is the mean (n = 9–11); error bar is SEM. Difference between the groups is significant, (p < 0.001, two way ANOVA). (H) lung wet/dry mass ratio at 24 hours post infection. (I) Representative areas of inflamed lung from HE stained sections and neutrophil stained lung. (J) Lung inflammation measured by blinded assessment of HE stained sections. Significance levels as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007099#ppat.1007099.g002" target="_blank">Fig 2</a>. All data representative of two independent experiments.</p

<i>In vivo</i> imaging of MPO-specific bioluminescence in mice infected with <i>B</i>. <i>pahangi</i> L3 larvae.

<p>BALB/c mice were injected with 50 L3 of <i>B</i>. <i>pahangi</i> into the RHS footpad (<i>B</i>.<i>p</i>) and with HBSS into the LHS footpad. On day 4 (<b>A</b>) and day 11 (<b>B</b>) post-infection mice were imaged using an IVIS spectrum 20 minutes after subcutaneous injection of 200 mg/kg luminol. Representative images of 3 mice per group are shown. The colour scale indicates bioluminescence radiance in photons/second/cm²/steradian. Graphs show the bioluminescence total flux (in photons/second) within the same footpad region of interest (ROI, red ovals in images). Each symbol shows the total flux for a single mouse, lines indicate the means (n = 7 mice) and error bars show SD (**<i>p</i> < 0.01 using a Mann-Whitney test).</p