Effect of prior colonisation on cytokine levels during subsequent pneumonia.

<p>BALF samples were collected at either (A) 4 h or (B) 18 h and serum (C) at 18 h following pneumonia challenge of previously colonised (black bars) or control (white bars) CBA/Ca mice (n = 6 per group). Cytokine levels were measured by either Luminex bead assay or ELISA. Group mean + SEM are displayed. Dotted line represents limit of detection.</p

Role of CD4+ cells in the effect of prior colonisation on subsequent pneumonia.

<p>Cytokine levels (A) and bacterial CFU (B) recovered from colonised or control CBA/Ca mice 18 h following pneumonia challenge in the absence of CD4+ cells. Mice were depleted of CD4+ cells using GK1.5 prior to challenge. (A) Cytokine levels in BALF of colonised mice (black bars) or control mice (white bars) measured by Luminex or ELISA. Group mean + SEM are displayed. (B) Bacterial cfu in BALF, lung and blood of colonised or control CBA/Ca. Each symbol represents results for an individual mouse and bars the median for each group. The dotted line is the limit of detection. Significance of the difference in bacterial numbers recovered from lungs of colonised and control mice was assessed by Mann-Whitney U-test. Significance of differences in presence or absence of bacteraemia was assessed by Fisher exact test. Data are pooled from two experiments with similar results.</p

<i>S. pneumoniae</i> D39 colonises the nasopharynx of CBA/Ca mice.

<p>Bacterial cfu recovered from nasal washes and BALF 2, 11 and 28 days following intranasal inoculation in 10 µl of PBS with 10⁷ cfu D39 <i>S. pneumoniae</i>. Each dot represents results for an individual mouse and bars the median for each group. The dotted line is the limit of detection.</p

Role of antibody in the effect of prior colonisation on subsequent pneumonia.

<p>(A) Bacterial cfu in BALF, lung and blood of colonised or control μMT mice 18 h following pneumonia challenge. (B) Bacterial cfu in BALF, lung and blood of passively immunised CBA/Ca mice 18 h following pneumonia challenge. Mice received pooled serum collected from either colonised or control CBA/Ca mice prior to challenge. Each symbol represents results for an individual mouse and bars the median for each group. The dotted line is the limit of detection. Lung cfu are compared between groups by Mann-Whitney U-test. Presence or absence of bacteraemia was assessed by Fisher exact test.</p

Colonisation induces serum opsonophagocytic antibody.

<p>(A–B) Flow cytometry assays of neutrophil phagocytosis of FAM-SE labelled D39 after incubation in serum from either (A) colonised or control CBA/Ca mice or PBS, or (B) colonised or control μMT mice or PBS. A representative histogram is shown on the left (clear PBS, black 20% control sera, grey 20% colonised sera), and mean MFI ± SEM (n = 4 replicates) for each condition in the graph on the right. (C) MFI of alveolar macrophages present in BALF of previously colonised or control CBA/Ca mice 4 h following challenge with 10⁷ cfu FAM-SE-labelled D39 <i>S. pneumoniae</i>. Dots represent individual mice, bars represent medians. (D) Numbers of cfu of <i>S. pneumoniae</i> recovered from blood of colonised or control mice inoculated intravenously with 5×10⁶ cfu <i>S. pneumoniae</i> D39 (n = 6 per group) at 2 and 5 h. Dots represent data from individual mice, bars represent group medians.</p

Effect of prior colonisation on inflammation during subsequent pneumonia.

<p>(A–B) Numbers of neutrophils (hatched region) and macrophages (dotted region) present in BALF of previously colonised or control CBA/Ca mice at (A) 4 h and (B) 9 h following pneumonia challenge. Bars represent mean + SEM (n = 6 mice per group). (C) Lung inflammation scores assessed by blinded histological examination of lung sections collected 18 h following challenge of colonised or control mice. Dots represent individual mice, bars represent medians.</p

Effects of prior colonisation on progression of subsequent <i>S. pneumoniae</i> pneumonia.

<p>(A) Kaplan-Meier survival curves of previously colonised or control CBA/Ca mice following pneumonia challenge with 10⁷ cfu D39 <i>S. pneumoniae</i> in 50 µl PBS on day 28 (n = 18 or 19). (B–D) Bacterial cfu in BALF, lung or blood of colonised or control mice at (B) 4 h, (C) 9 h or (D) 18 h following pneumonia challenge. Each symbol represents results for an individual mouse and bars the median for each group. The dotted line is the limit of detection. Significance of the difference in bacterial numbers recovered from lungs of colonised and control mice was assessed by Mann-Whitney U-test. Significance of differences in presence or absence of bacteraemia was assessed by Fisher exact test. Data at 18 h are pooled from two experiments with similar results.</p

Systemic and mucosal antibody responses to colonisation.

<p>IgG (A and D), IgA (B and E) and IgM (C) responses to D39 in serum (A–C) or BALF (D–E) obtained from colonised or control mice measured using whole cell ELISAs.</p

Correlation of neutrophil association in 20% human serum measured using flow cytometry of clinical isolates of <i>S. pneumoniae</i> with the FI for C3b/iC3b deposition.

<p>Neutrophil association results are expressed as percentage of neutrophils associated with bacteria. Serotype 6A strains were excluded from this correlation due to their very high level of C3b/iC3b deposition results and their higher level of IgG binding (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024581#pone-0024581-g004" target="_blank">Figure 4</a>). <i>P</i> values and R² were obtained using Pearson's correlation test.</p

Correlations of C1q binding to <i>S. pneumoniae</i> clinical isolates in 25% human serum measured using flow cytometry.

<p>Correlations of C1q binding to <i>S. pneumoniae</i> clinical isolates in 25% human serum measured using flow cytometry to IgG binding (A) and C3b/iC3b deposition results (B). Serotype 6A strains were excluded from these correlations due to their very high level of C3b/iC3b deposition results and their higher level of IgG binding (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024581#pone-0024581-g004" target="_blank">Figure 4</a>). <i>P</i> values and R² were obtained using Pearson's correlation test.</p