Maximal Distance of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. C, D: Overlay of Round-shaped cell tracks after late PBS (C) and LPS injection (D), after aligning their first coordinates. One color by track. Values of black circle radii in µm, equal to average cell Maximal Distance, are indicated ± standard deviation. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p

Velocity of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p

Phenotype of CX₃CR1-GFP cell subsets in the lung.

<p>A: Total lung cells of CX₃CR1^+/gfp mice were gated on CX₃CR1 and analyzed for NK1.1, CD3e, CD11c, and CD11b expressions. B: Autofluorescence, CD80 and MHCII expressions on gate G1, G2 and G3 of panel A. Black histogram, isotype control; grey histogram, positive staining. C: Total lung cells were pre-gated on CD11c+ low autofluorecent cells and analyzed for the expression of CD11b and CD103. The expression of CX₃CR1 is shown on the left panel for gate G4 (CD11b⁻CD103⁺ DCs, grey histogram) and for gate G5 (CD11b⁺CD103⁻ DCs, black line). D: Total cells were pre-gated on CD45 cells and analyzed for the expression of F4/80 and CD11c. Expression of CX₃CR1 and CD11b is shown on the left panels for gate G6 (CD11c^lowF4/80^high), G7 (CD11c^highF4/80^high) and G8 (CD11c^highF4/80^low).Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data are representative of two distinct experiments.</p

Parameters used for individual cell analysis.

<p>A: Edge detection of two CX₃CR1+ pulmonary cells and their roundness coefficient. Scale bar  = 10 µm. B: The relevant parameters used in this work are: i) the Mean Roundness Coefficient (MRC), calculated for each cell by meaning Instantaneous Roundness Coefficient (IRC) at each consecutive observable time; ii) the Maximal Distance (MD) of a cell (red arrow) is the longest distance covered from the first position; iii) the Meandering Index (MI) is the final distance from the first position <i>D_n</i> divided by the total length covered.</p

Discrimination of two CX₃CR1-GFP cell subsets using flow cytometry or roundness.

<p>A: Expression of CX₃CR1 <i>vs</i> CD45 receptors in pulmonary cells and CD11c <i>vs</i> CD11b by CX₃CR1^high cells. Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data representative of two distinct experiments. B: Frequency distribution of Mean Roundness Coefficients (MRC) over 1h of CX₃CR1+ pulmonary cells in lung slices harvested from 6 mice, 1h30 (3 mice) and 5h (3 mice) after PBS injection. N = 406. Cells are followed for a mean time of 32 minutes, corresponding to 16 frames. Dashed line: sum of two Gaussian fitting the histogram. R² = 0.89.</p

Schematic representation of the drift correction strategy.

<p>A: Maximum intensity projection of a lung slice z-stack. Pulmonary CX₃CR1-GFP cells (green) and alveolar collagen mesh detected by collection of SHG signal (gray). Two-photon excitation wavelength  = 896 nm. B: Dashed red squares show the optic field imaged by the microscope (CX₃CR1-GFP in green and SHG in grey). The realignment phase consists in calculating the tissue drift using the maximization of SHG signal cross-correlation.</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

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

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

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