Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms

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

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

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

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