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

Enteral Immunization with Attenuated Recombinant Listeria monocytogenes as a Live Vaccine Vector: Organ-Dependent Dynamics of CD4 T Lymphocytes Reactive to a Leishmania major Tracer Epitope

Listeria monocytogenes is considered as a potential live bacterial vector, particularly for the induction of CD8 T cells. The CD4 T-cell immune response triggered after enteral immunization of mice has not yet been thoroughly characterized. The dynamics of gamma interferon (IFN-γ)- and interleukin-4 (IL-4)-secreting CD4 T cells were analyzed after priming through intragastric delivery of an attenuated ΔactA recombinant L. monocytogenes strain expressing the Leishmania major LACK protein; a peptide of this protein, LACK(158-173) peptide (pLACK), is a well-characterized CD4 T-cell target in BALB/c mice. Five compartments were monitored: Peyer's patches, mesenteric lymph nodes (MLN), spleen, liver, and blood. A single intragastric inoculation of ΔactA-LACK-LM in BALB/c mice led to colonization of the MLN and spleen at a significant level for at least 3 days. Efficient priming of IFN-γ-secreting pLACK-reactive CD4 T cells was observed in all tested compartments. Interestingly, IL-4-secreting pLACK-reactive CD4 T cells were detectable at day 6 or 7 only in blood and liver. The absence of translocation of viable bacteria through the intestinal epithelium after further ΔactA-LACK-LM inoculations was concomitant with the absence of an increase in the level of IFN-γ secreted by the MLN, blood, and splenic pLACK-reactive Th1 T cells, although the levels remained significantly above the basal level. No change in this population size was detected in the spleen. However, an increase in the number of intragastric inoculations had a clinical beneficial effect in L. major-infected BALB/c mice. L. monocytogenes thus presents the potential of an efficient vector for induction of CD4 T cells when administered by the enteral route

Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming.

International audienceIt has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent

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

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

Preexisting BCG-specific T cells improve intravesical immunotherapy for bladder cancer

Therapeutic intravesical instillation of bacillus Calmette-Guérin (BCG) is effective at triggering inflammation and eliciting successful tumor immunity in patients with non-muscle invasive bladder cancer, with 50 to 70% clinical response. Therapeutic success relies on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection; however, the precise mechanisms remain unclear. Using an experimental model, we demonstrate that after a single instillation, BCG could disseminate to bladder draining lymph nodes and prime interferon-γ-producing T cells. Nonetheless, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Parenteral exposure to BCG before instillation overcame this requirement; after the first intravesical instillation, BCG triggered a more robust acute inflammatory process and accelerated T cell entry into the bladder, as compared to the standard protocol. Moreover, parenteral exposure to BCG before intravesical treatment of an orthotopic tumor markedly improved response to therapy. Indeed, patients with sustained preexisting immunity to BCG showed a significant improvement in recurrence-free survival. Together, these data suggest that monitoring patients' response to purified protein derivative, and, in their absence, boosting BCG responses by parenteral exposure before intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses