Impairment of IFN-γ production by LT in purified NK cells, contrasting with absence of effect by ET.

<p>(<b>A,B</b>) IFN-γ production by purified CD49b⁺ cells pre-treated for 1 h with PA and increasing concentrations of LF or EF and then stimulated for the whole incubation time with rIL-12 and rIL-18 in the presence of toxins. Data are mean ± SD of triplicates and are representative of one experiment of three performed; SD values are hidden by symbol size. T test; *, <i>P</i><0.05 compared with the group incubated with PA only. (<b>C</b>) Inhibition of p38, JNK and ERK phosphorylation by LT in purified CD49b+ cells activated by rIL-12 and rIL-18 for 10 min; total ERK1/2 was used as loading control. Data represent one of at least two independent experiments. (<b>D</b>) NK cell viability (left panel; Live/Dead Cell Staining) and metabolic activity (right panel; MTS assay) after 18 h-incubation with LT. *, <i>P</i><0.05 compared to the untreated group. (<b>E</b>) Intracellular cAMP production by purified CD49b⁺ cells treated with ET for 1 h. Data are mean ± SD of triplicates per condition and are representative of one experiment out of three. T test; *, <i>P</i><0.05 compared with the untreated group.</p

Differential inhibition by ET and LT of the spore-induced IL-12 and IFN-γ production by splenocytes.

<p>IL-12p40/p70 (<b>A</b>) and IFN-γ (<b>B</b>) production by splenocytes pre-incubated for 1 h with PA and increasing concentrations of LF or EF; spore stimulation was then performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002481#ppat-1002481-g001" target="_blank">Figure 1A</a> in the presence of toxins. (<b>C</b>) Similar incubation conditions as in (<b>A,B</b>) with either addition of rIL-18 or rIL-12p70, or IL-12 neutralization. The data represent mean cytokine concentrations of triplicates in culture supernatants (± SD) representative of three independent experiments. T test; *, <i>P</i><0.05 compared with the group incubated with FIS without toxins.</p

Network of cytokine dependence of NK cell activation by <i>B. anthracis</i> spores.

<p>Effect of neutralization of IL-12, IL-18 or IL-15Rα on (<b>A</b>) IL-12p40/p70 concentration in culture supernatants of FIS-stimulated BMDMs or (<b>B</b>) IFN-γ production by splenocytes (SPL; left panel), or purified CD49b⁺ cells co-cultured with BMDMs (right panel). (<b>C</b>) Splenocytes (SPL) from wild-type (WT), IL-12R^−/− or IL-12^−/− C57BL/6 mice were stimulated with FIS, or ConA as a positive control. (<b>D</b>) CD49b⁺ cells from WT C57BL/6 mice were co-cultured with BMDMs from IL-12R^−/− or IL-12^−/− C57BL/6 mice in the presence of FIS. (<b>E</b>) CD49b⁺ cells from WT or IL-12R^−/− C57BL/6 mice were co-cultured with BMDMs from WT C57BL/6 mice in the presence of FIS with or without IL-18 neutralizing antibody. (<b>F</b>) Effect of short-term priming with IL-12 or IL-18 on spore-stimulation of splenocytes; corresponding cytokine neutralization was maintained for the remainder of the assay. (<b>G</b>) Purified CD49b⁺ cells from WT or MyD88^−/− C57BL/6 mice were co-cultured with BMDMs from WT C57BL/6 in the presence of FIS. (<b>H</b>) Purified CD49b⁺ cells from C57BL/6 WT mice were co-cultured with BMDMs from WT or MyD88^−/− C57BL/6 mice in the presence of FIS; IL-12 (left panel), or IFN-γ (right panel) production. For all experiments with purified CD49b⁺ cells, no IFN-γ was detected after direct stimulation with spores (<b>D, E, G, H</b>; data not shown). For all experiments, values are mean ± SD for at least three measurements and are representative of at least three independent experiments. Significant differences between experimental conditions are indicated with asterisks (t test; *, <i>P</i><0.05; **, <i>P</i><0.01).</p

ET efficiently inhibits NK cell cytotoxic activity <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Pre-incubation of purified CD49b⁺ cells with ET and LT inhibits lysis of YAC-1 target cells. Data represent mean ± SD (n = 3) of one of at least three independent experiments. T test; *, <i>P</i><0.05, ** <i>P</i><0.01 as compared with the no-toxin group. (<b>B</b>) <i>In vivo</i> effect of ET and LT on the natural cytotoxic activity of NK cells: C57BL/6 wild-type and syngeneic MHC class I-deficient β2m−/− splenocytes were differentially labeled with CFSE and adoptively transferred intravenously in equal number (“injected mix”) into C57BL/6 syngeneic wild-type recipients; elimination of the MHC class I-deficient cells (CFSE high) was quantified 16–20 h later in the spleen and confirmed to be mediated by the NK cell population of the recipients, either after <i>in vivo</i> NK cell activation by poly:(IC) injection, or after <i>in vivo</i> NK cell depletion through injection of anti-NK1.1 antibodies (experiment 1). The effect on elimination of the MHC class I-deficient cells of ET, LT (experiments 1 to 3) or the toxin CyaA of <i>Bordetella pertussis</i> (or its inactive mutant CyaE5) (experiment 3) was then quantified: all toxins were injected intravenously 8 h prior CFSE-labeled mixed cell inoculation. Controls were injected with PA, EF, or LF only; MHC class I-deficient cells were eliminated as in the non-treated recipients (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002481#ppat.1002481.s001" target="_blank">Figure S1D</a>). Data represent histogram plots from three independent experiments showing relative percentages of the high (MHC class I-deficient) and low (normal) CFSE cell populations. (<b>C</b>) Mean percent specific lysis of MHC class I-deficient cells of 3 independent assays performed. The percent specific lysis was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002481#s4" target="_blank">Materials and Methods</a>. T test; *, <i>P</i><0.05 compared to the untreated group.</p

Recruitment and role of NK cells during <i>B. anthracis</i> infection and impact of <i>in vivo</i> toxin production.

<p>(<b>A</b>) Circulating NK cells 5 h post-inoculation, viewed by biphoton imaging; dermal collagen in blue (SHG), vascular flow in red (rhodamine B) and NK cells in green (CFSE); scale bar = 20 µm; time-scale in milliseconds indicated on each image. (<b>B</b>) Adherent, then rolling NK cell 5 h post-inoculation; scale bar = 20 µm; time-scale in seconds indicated on each image. (<b>C</b>) Extravasated NK cells at 18 h post-inoculation; scale bar = 10 µm (top), 40 µm (bottom). (<b>D</b>) Subcapsular NK cell in the cervical lymph node draining the infected ear 18 h post-inoculation; NK cells in green (CFSE) and capsular collagen in blue (SHG); scale bar = 20 µm. Data representative of 3 mice. (<b>E</b>) Absolute numbers of CD49b⁺ (left panel) and F4/80⁺ cells (right panel) in the cervical lymph node draining the site of cutaneous infection with spores of the 9602P(PA−EF+LF+), 9602L(PA+EF+LF−), 9602C(PA+EF−LF+) strains 24 h post-inoculation (2.91±0.03 log₁₀ CFU per mouse). Controls were injected with PBS. Each symbol represents the value for an individual mouse; horizontal lines indicate the mean value for each group. Data are pooled from two independent experiments. T test; **<i>P</i><0,01 as compared with the 9602P-injected group. (<b>F</b>) <i>In vivo</i> effect of NK cell depletion on systemic bacterial dissemination in the spleen. Bacterial load was determined 18 h after infection into the ear with spores of the 9602P strain (3.05±0.29 log₁₀ CFU per mouse). Data are pooled from two independent experiments.T test; *, <i>P</i><0.05; **, <i>P</i><0,01 as compared with the non-treated group.</p