Human monocyte-derived DCs mature following exposure to LVS strain 13B47.

<p>DCs were stimulated with either LVS, 13B47, ΔcapC, 1664d, or <i>E. coli</i> for 24 hours (MOI = 10). Cells were harvested and analyzed for changes in surface expression of CD86, CD80, and HLA-DR. Cells were gated on CD1b-positive population. (A) Representative histograms for CD86, CD80, and HLA-DR expression on LVS-, 13B47-, and <i>E. coli</i>-treated DCs from one experiment. Histograms for ΔcapC- and 1664d-infected DCs were similar to LVS (data not shown). (B) Mean percentages of DCs with high CD86, CD80, and HLA-DR expression (± SEM) from three individual experiments with different donors. (C) Geometric mean fluorescence intensities (GMFI) of CD86, CD80, and HLA-DR (± SEM) on DCs from three individual experiments with different donors. Statistically significant differences in CD86, CD80, and HLA-DR expression by infected DCs were determined by one-way ANOVA, followed by Bonferroni comparison of means (*, p<0.05; **, p<0.01; ***, p<0.001).</p

<i>F. tularensis</i> LVS strain 13B47 stimulates human monocyte-derived DCs and macrophages to produce proinflammatory cytokines.

<p>LVS and LVS mutants, 13B47, ΔcapC, and 1664d, were cultured overnight in a chemically defined media (CDM) or Mueller-Hinton (MH) broth. The four bacterial cultures were used to inoculate macrophages (A, 1.5×10⁵ cells/well) and DCs (B and C, 5×10⁵ cells/well) at an MOI of 10. As a positive control, DCs and macrophages were cultured with <i>E. coli</i> strain sd-4 (MOI = 10). Supernatants were harvested after 24 hours (A–B) or at indicated times (C), and TNF-α, IL-6, and IL-12p40 were measured by ELISA. Data are expressed as the mean ± SEM of three individual experiments with different donors. The level of cytokine production from each group was compared by a one (A–B) or two-way ANOVA (C), followed by the Bonferroni comparison of means. ($$$, <i>p</i><0.001 for <i>E. coli</i> vs. all other groups). When comparing only the DCs infected with the <i>F. tularensis</i> strains, 13B47 elicited higher cytokine production than the uninfected group (A–B) or LVS cultured in the same media (C). *, p<0.05; **, p<0.01; ***, p<0.001. BLD = below limits of detection of the ELISA.</p

Survival of immunized mice following intratracheal Schu S4 challenge^a.

a<p>Mice were immunized with either LVS or ΔFTL_0883 at the indicated dose and then challenged with 100 CFU of Schu S4 i.t.</p>b<p>Significant difference p<0.005 by log rank test.</p

LVS strain 13B47 is attenuated for growth in human macrophages and replicates slowly in DCs.

<p>DCs or macrophages were infected in gentamicin protection assays (MOI = 500) with LVS or 13B47 and lysed at the indicated times post infection. Data shown are mean ± SEM from three individual experiments with different donors. Statistically significant differences in growth at 24 hours post infection were determined by Student's <i>t</i>-test (*, p<0.05).</p

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

Enhanced proliferation and IFN-γ production by CD4⁺ T cells stimulated with LVS strain 13B47-infected DCs.

<p>Purified CFSE-labeled CD4⁺ T cells from a single donor were co-cultured with either <i>E. coli</i>-infected, <i>F. tularensis</i> LVS-infected, or 13B47-infected DCs from a different donor at a ratio of 10∶1 (2×10⁵ T cells/2×10⁴ DCs/well) for 5 days. (A) Representative dot plots showing loss of CFSE fluorescence versus CD4 staining on day 5 for each group from one experiment. (B) The mean percentages of proliferating CD4⁺ T cells were calculated (± SEM) from five individual experiments with different donors. (C) IFN-γ levels were measured in day 5 supernatants by ELISA. Data are presented as the mean ± SEM from four individual experiments with different donors that were represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031172#pone-0031172-g004" target="_blank">Figure 4B</a>. BLD = below limits of detection of the ELISA. Statistically significant differences in mean percentages and GMFI for all groups were determined by one-way ANOVA, followed by Bonferroni comparison of means (*, p<0.05; **, p<0.01; ***, p<0.001).</p

FTL_0883 deletion mutant, ΔFTL_0883, elicits maturation of DCs and is attenuated for growth similar to 13B47.

<p>Human (A–E) and murine DCs (F–G) were cultured with either LVS, 13B47, ΔFTL_0883, or ΔFTL_0883::pJH1- FTL_0883 at an MOI of 10 (A–B, D, and F) or 500 followed by gentamicin treatment (C–E, and G). For cytokines, supernatants were harvested at 24 hours (A, C, and F) or the indicated time points (B), and IL-12p40 and TNF-α were measured by ELISA. For flow cytometry experiments (D), DCs were harvested 24 hours post infection and GMFIs for CD80 and CD86 were measured. For gentamicin protection assays (E, G), DCs were infected with LVS strains at an MOI of 500 and then lysed at indicated time points to enumerate intracellular bacteria. Data are presented as the mean ± SEM from at least two independent experiments. Statistically significant differences between groups were determined by one (A, C, D, and F) or two-way ANOVA (B, E, and G), followed by Bonferroni comparison of means (*, p<0.05; **, p<0.01; ***, p<0.001). BLD = below limits of detection of the ELISA.</p

ΔFTL_0883-vaccinated mice have reduced bacterial burdens.

<p>Mice were vaccinated i.t. (1.5×10³ CFU) with either LVS or ΔFTL_0883 (n = 4 mice/group/time point). At indicated time points (days 1, 3, and 6 for experiment 1; days 3, 6, and 10 for experiment 2), mice were sacrificed and the CFU/organ was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031172#s2" target="_blank">Materials and Methods</a>. The limits of detection for the lung, spleen, and liver are depicted by the black dashed lines and were 100, 100, and 200 CFU, respectively. Data are presented as mean ± SD for each individual experiment. Statistically significant differences between groups in each experiment were determined by two-way ANOVA, followed by Bonferroni comparison of means (*, p<0.05; ***, p<0.001).</p