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. 2019 Aug 27:10:2030.
doi: 10.3389/fimmu.2019.02030. eCollection 2019.

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages

Affiliations

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages

Michiel M Kroes et al. Front Immunol. .

Abstract

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1β and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.

Keywords: Bordetella pertussis; NLRP3; crosstalk; human; inflammasome; innate immunity; interferon-gamma; interleukin-18.

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Figures

Figure 1
Figure 1
B. pertussis induces NLRP3 inflammasome activation in human MΦ-like THP-1 cells. MΦ-like THP-1 cells were stimulated with B. pertussis (Tohama I, MOI = 100, 10 or 1) or left untreated for 22 h in the presence (dashed bars) or absence (clear bars) of the caspase inhibitor, Z-VAD-FMK. (A) IL-1β (n = 5) and (C) IL-6 (n = 3) were measured in the supernatant of at least three independent experiments. (B) LDH release (n = 3) was determined with a Cytotoxicity Assay. LDH release is shown as a percentage of the LDH released relative to the percentage of LDH released in the positive control, LPS + nigericin (100% cell death), for NLRP3 activation. (D–F) MΦ-like NLRP3 deficient THP-1 cells (horizontal lines) were incubated with B. pertussis (Tohama I, MOI = 10) for 22 h. (D) IL-1β (n = 3) and (F) IL-6 (n = 3) levels were measured in the supernatant using ELISAs. (E) The LDH released by MΦ-like NLRP3 deficient THP-1 cells (n = 4) was shown as relative to the LDH release from fully lysed cultures. Results are expressed as medians with interquartile range from at least three independent experiments. Black dots represent the average values from each experiments.
Figure 2
Figure 2
B. pertussis induces inflammasome activation in primary human mo-MΦ. (A) Mo-MΦ were stimulated with B. pertussis (Tohama I, MOI = 100) for 6 h after which the transcription levels of inflammasome associated genes were determined using reverse transcriptase qPCR. Data is expressed as mean fold change of three donors calculated as the transcription levels relative to the transcription levels in untreated mo-MΦ. (B) The levels of IL-1β (n = 7) and (C) IL-18 (n = 6) released into the supernatant by mo-MΦ stimulated with B. pertussis for 22 h in the presence (red squares) or absence (black dots) of a caspase inhibitor (Tohama I, MOI = 10). (D) IL-1β secretion of mo-MΦ stimulated with a clinical B. pertussis strain (B4393, MOI = 10) in de presence (red squares) or absence (black dots) of a caspase inhibitor. Black dots and red squares represent values of individual donors. (E) Representative images of the cellular ASC (green) distribution as determined by flow imaging of untreated mo-MΦ or mo-MΦ stimulated with a clinical B. pertussis strain (B4393, MOI = 100). *p < 0.05, **p < 0.01.
Figure 3
Figure 3
IL-18 primes NK cells to produce IFNγ in response to B. pertussis. CD56+CD3 NK cells were incubated with medium (clear bars) or B. pertussis (B4393, MOI = 10, dashed bars) in the presence or absence of 5 ng/ml rhIL-18 for 18 h after which Brefeldin A was added for 4 h to inhibit cytokine secretion. (A) Stimulated NK cells were intracellularly stained for IFNγ and the percentage of IFNγ+CD56+CD3 NK cells was analyzed using flow cytometry (n = 10). Results are expressed as medians with interquartile range. Black dots represent values of individual donors. (B) Results are expressed as a histogram of IFNγ+CD56+CD3 NK cells from one representative donor. *p < 0.05, **p < 0.01.
Figure 4
Figure 4
Increased proinflammatory cytokine secretion in B. pertussis-stimulated mo-MΦ/NK co-culture. Mo-MΦ and NK cell single cultures and mo-MΦ/NK co-cultures were stimulated with B. pertussis (B4393, MOI = 10, dashed bars) or left untreated (clear bars) for 22 h. Secreted levels of (A) IL-1β, (B) IL-18, (C) GM-CSF, (D) IL-23, (E) TNFα, and (F) IL-10 were measured in the supernatant (n = 7). Results are expressed as medians with interquartile range. Black dots represent values of individual donors. *p < 0.05.
Figure 5
Figure 5
B. pertussis-stimulated mo-MΦ activate human NK cells. Mo-MΦ and NK cell single cultures and mo-MΦ/NK co-cultures were stimulated with B. pertussis (B4393, MOI = 10, dashed bars) or left untreated (clear bars) for 22 h. Secreted levels of (A) IFNγ and (B) Granzyme B were measured in the supernatant (n = 7). NK cells were stained for (C,D) IL-2Rα (n = 4) and (E,F) HLA-DR (n = 6) and the expression of these markers was analyzed on CD56+CD3 NK cells. (A–C,E) Results are expressed as medians with interquartile range. Black dots represent values of individual donors. (D,F) Results are expressed as dot plots of one representative donor. *p < 0.05.
Figure 6
Figure 6
IL-18 contributes to NK cell activation and IFNγ secretion in a B. pertussis-stimulated mo-MΦ/NK co-culture. (A,B) Mo-MΦ/NK co-cultures were stimulated with B. pertussis (B4393, MOI = 10, dashed bars) for 22 h in the presence of a caspase inhibitor (squares). (A) IFNγ was measured in the supernatant and (B) IL-2Rα expression was determined on CD56+CD3 NK cells using flow cytometry. Data is shown as relative to the cultures stimulated with B. pertussis in the absence of the caspase inhibitor (dots). (C,D) Mo-MΦ/NK co-cultures were stimulated with B. pertussis (B4393, MOI = 10, dashed bars) for 22 h in the presence of IL-18 blocking antibodies (triangles) or isotype control hIgA2 (dots). (C) IFNγ was measured in the supernatant and (D) IL-2Rα expression was determined on CD56+CD3 NK cells using flow cytometry. Data is shown as relative to the cultures stimulated with B. pertussis in the presence of hIgA2 (n = 4). Results are expressed as medians with interquartile range. Black dots, squares and triangles represent values of individual donors.
Figure 7
Figure 7
Interplay between human mo-MΦ and NK cells in the presence of B. pertussis (graphics). B. pertussis activates the NLRP3 inflammasome in human macrophages resulting in the secretion of, amongst others, IL-18 and IL-1β. IL-18 primes the NK cells to produce IFNγ and express IL-2Rα and HLA-DR in response to B. pertussis. Inflammasome activation and the crosstalk between human macrophages and NK cells results in an enhanced proinflammatory response to this pathogen (Made with illustrations from: https://smart.servier.com/).

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