Flow cytometry analysis of airway T cells at the peak of WT or ΔPT infection.

<p>(A) Percentage of T cells (defined as CD3⁺) in the BAL fluid from mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (B, C) Percentage of T cells that stained positive for IL-17 (B) and mean fluorescence intensity (MFI) of IL-17 staining in those cells (C) in the BAL fluid of mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (D, E) Representative contour plots of IL-17 staining in T cells in the BAL fluid of mice infected with either WT or ΔPT. Number in box denotes the percentage of IL-17⁺ T cells. SSC – side scatter. (F, G) Representative histograms of IL-17 staining of T cells in the BAL fluid (F) or lung tissue (G) of mice infected with either WT (open plot) or ΔPT (shaded plot).</p

Kinetics of neutrophil recruitment during <i>B. pertussis</i> WT and ΔPT infection.

<p>(A) Numbers of neutrophils in the airways and (B) bacterial loads in the respiratory tract of mice at the indicated times post-infection with 5×10⁵ CFU of WT (closed bars) or ΔPT (open bars) strains. <i>n</i> = 4 mice/treatment group. The data represent 1 of 3 separate experiments with similar results. * Significantly different from WT (P<0.05).</p

Kinetics of cytokine expression during <i>B. pertussis</i> WT and ΔPT infection.

<p>Fold increases in gene expression (relative to mock-infected mice) of the cytokines (A) IL-17, (C) IL-6, (E) IFN-γ, (G) TNF-α, and (H) IL-10, and corresponding protein levels of (B) IL-17, (D) IL-6 and (F) IFN-γ in lungs of mice at the indicated times post-infection with 5×10⁵ CFU of WT (black bars) or ΔPT (open bars) strains. <i>n</i> = 4 mice/treatment group. The data represent 1 of 2 separate experiments with similar results. *Significantly different from WT (P<0.05).</p

Kinetics of chemokine gene expression during <i>B. pertussis</i> WT, ΔPT and PT* infection.

<p>Fold increases in gene expression (relative to mock-infected mice) of the chemokines (A) KC, (B) MIP-2, and (C) LIX in lungs of mice at the indicated times post-infection with 5×10⁵ CFU of WT (black bars), ΔPT (open bars) or PT* (grey bars) strains. <i>n</i> = 4 mice/treatment group. The data represent 1 of 2 separate experiments with similar results. * Significantly different from WT (P<0.05).</p

Effect of α–IL-17 antibody treatment on <i>B. pertussis</i> WT infection.

<p>(A–C) Fold increases in gene expression (relative to mock-infected mice) of KC (A), numbers of airway neutrophils (B), and bacterial loads in the respiratory tract (C) in control or α–IL-17 antibody-treated mice on day 4 post-infection with 5×10⁵ CFU of WT <i>B. pertussis</i>. Mice were treated with antibody intranasally on day 3 post-infection. (D) Bacterial loads in the respiratory tract of control or α–IL-17 antibody-treated mice on day 8 post-infection with 5×10⁵ CFU of WT <i>B. pertussis</i>. Mice were treated with antibody intranasally on days 3, 5 and 7 post-infection. (E) Bacterial loads in the respiratory tract of control or α–IL-17 antibody-treated mice on days 14 and 21 post-infection with 5×10⁵ CFU of WT <i>B. pertussis</i>. Mice were treated with antibody intranasally on days 3, 6, 9, 12, 15 and 18 post-infection. *Significantly different from control mice (P<0.05).</p

Effect of equalizing bacterial loads of WT and ΔPT on IL-17 expression.

<p>(A, B) Bacterial loads in the respiratory tract of mice on the indicated days post-infection with (A) 5×10⁴ CFU of WT (black bars) versus 5×10⁵ CFU of ΔPT (open bars), or (B) 5×10⁵ CFU of WT versus 5×10⁶ CFU of ΔPT (compare to difference in bacterial loads when inoculum doses are equal in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007079#pone-0007079-g001" target="_blank">Fig. 1</a>). (C–E) Fold increases in gene expression (relative to mock-infected mice) of the cytokines IL-17 (C, D) or IFN-γ (E) in lungs of mice on the indicated days post-infection with the lower doses (C) or higher doses (D, E) of WT (black bars) or ΔPT (open bars) strains. <i>n</i> = 4 mice/treatment group. *Significantly different from WT (P<0.05).</p

Flow cytometry analysis of airway neutrophils and macrophages at peak WT or ΔPT infection.

<p>(A) Percentage of neutrophils (defined as Gr1^hi CD11b⁺) in the BAL fluid from mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (B, C) Percentage of neutrophils that stained positive for IL-17 (B) and mean fluorescence intensity (MFI) of IL-17 staining in those cells (C) in the BAL fluid of mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (D, E) Representative contour plots of IL-17 versus CD11b staining in neutrophils in the BAL fluid of mice infected with either WT (D) or ΔPT (E). Number in box denotes the percentage of IL-17⁺ neutrophils. (F) Percentage of macrophages (defined as CD11b⁻ CD11c⁺ Gr1^lo) in the BAL fluid from mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (G, H) Percentage of macrophages that stained positive for IL-17 (G) and MFI of IL-17 staining in those cells (H) in the BAL fluid of mice infected with 5×10⁵ CFU of <i>B. pertussis</i> WT (black bars) or ΔPT (grey bars). (I, J) Representative contour plots of IL-17 versus CD11c staining in macrophages in the BAL fluid of mice infected with either WT (I) or ΔPT (J). Number in box denotes the percentage of IL-17⁺ macrophages.</p

Systemic analyses of the protection afforded by FTC/TDF from rectal HIV-1 transmission.

<p>(A) Tissues from a representative non-treated control mouse (#1) showed the presence of productively HIV-1 infected cells expressing detectable viral RNA. (B) Tissues from a mouse receiving systemic PrEP (#25) demonstrated a complete lack of productively infected cells in any of the tissues analyzed. Black foci represent cells producing viral RNA (bar = 50 µm). (C) Tissues from infected non-treated control mice were positive for replication competent HIV-1 when co-cultured with activated allogeneic PBMC. Tissues from mice receiving systemic PrEP were consistently negative for the presence of HIV-1. Presence of replication competent virus was indicated by the detection of viral p24 in the culture supernatant. (D) Tissues from infected non-treated control mice were positive for HIV-1 DNA by real time PCR analysis. Tissues from mice that received systemic PrEP were consistently negative for the presence of HIV-1 DNA. Thin dashed lines represent the limit of detection for the respective assays.</p

Experimental design and reconstitution of BLT mice with human hematopoietic cells.

<p>(A) Systemic PrEP with FTC/TDF (daily administrations for 7 consecutive days) to prevent rectal, intravenous and vaginal HIV-1 transmission. Viral exposure was performed 3 hours following the third FTC/TDF dosing. (B) Peripheral blood human leukocytes (CD45⁺) levels in each of the groups of BLT mice used. (C) Peripheral blood human T lymphocytes (CD4⁺ and CD8⁺) levels in each of the groups of BLT mice used. Box-plot interpretation for this and subsequent figures: middle line is the median; box extends from the 25^th to the 75^th percentiles; error bars extend down to the lowest value and up to the highest value.</p

Systemic PrEP with FTC/TDF results in effective protection from intravenous HIV-1 transmission.

<p>(A) Kaplan-Meier plot of the time course to peripheral blood conversion following intravenous exposure to HIV-1 in BLT mice with or without systemic PrEP. (B) Seven (out of eight) mice receiving systemic PrEP were consistently negative for plasma viral RNA. Plasma viral RNA was detected in the systemic PrEP breakthrough mouse (#42) and the 6 non-treated control mice. Thin dashed line represents the limit of detection for this assay. (C) BLT mice receiving systemic PrEP were negative for PBMC-associated viral DNA by real time PCR. PBMC-associated viral DNA was detected in the systemic PrEP breakthrough mouse (#42) and the 6 non-treated control mice. (D) Average levels of human CD4⁺ T cells in peripheral blood showed loss of CD4⁺ T cells in the systemic PrEP breakthrough mouse (#42) and the 6 non-treated control mice. CD4⁺ T cells remained constant in the protected systemic PrEP treated BLT mice.</p