Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4

Abstract

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

Figure 1. PLY enhances cytokine production by splenocytes independently of TLR4.

(A) Spleen cells from C3H/HeN and C3H/HeJ mice were incubated for 72 hours with PLY (0.32–200 ng/ml) (58,500 HU/mg) or W433F in the presence or absence of HkSp (1 bacterium:1 spleen cell). Splenocytes were then stimulated with PMA (5 µg/ml) and ionomycin (300 ng/ml) for a further 24 hours to activate the cells. IFN-γ concentrations were determined in supernatants. ** P<0.01 and *** P<0.001 vs. bacteria + PMA/ionomycin alone. (B) Spleen cells (1×10⁶/ml) from C3H/HeJ mice were incubated with plate-bound anti-CD3 (10 µg/ml) alone or with PLY (0.064–40 ng/ml) (58,500 HU/mg). IFN-γ, IL-5, IL-10 and IL-17A were measured in supernatants after 72 hours. * P<0.05 and *** P<0.001 vs. anti-CD3 alone. +P<0.05 vs. 1.6 ng/ml. All cytokine concentrations are presented as the mean (+ SEM) for triplicate samples.

Figure 2. PLY is required for IFN-γ and IL-17A induction following infection with S. pneumoniae.

(A) IFN-γ concentrations in lungs of infected mice in a model of acute pneumonia. MF1 mice were infected intranasally with wild-type (WT) or PLY-deficient (PLN-A) pneumococci. IFN-γ concentrations were determined in lung homogenates at 0 hours, 24 hours or 48 hours after infection. Results are mean cytokine concentration (+ SEM) for 5 mice per group. *** P<0.001 vs. WT. (B) IL-17A concentrations in lungs of infected mice in a model of resolving pneumonia. BALB/c mice were infected intranasally with WT or PLN-A pneumococci. IL-17A concentrations were determined in lungs at 0 hours, 24 hours or 48 hours after infection. Results are mean cytokine concentration (+ SEM) for 5 mice per group. ** P<0.01 and *** P<0.001 vs. WT. (C–E) Intracellular cytokine staining for IFN-γ and IL-17A in the lungs of S. pneumoniae infected mice. Lung cells were isolated from BALB/c mice 48 hours after intranasal infection with wild-type S. pneumoniae (D39). Cells were cultured for 5 hours with brefeldin A (GolgiPlug) in the presence of PMA and ionomycin followed by incubation with fluorescently-labelled antibodies against CD3e, CD4, CD8, NKp46, NK1.1, CD45, γδTCR, IFN-γ and IL-17A. Representative dot plots for IFN-γ production by NK cells (panel C; gated on CD4 and NKp46) and IL-17A production by γδ T cells (panel D; gated on side scatter and γδTCR) are shown for control mice (PBS) and pneumococcal-infected mice (D39). Numbers beside gated areas indicate the percentage of positive cells in that area. Histograms show the percentages (n = 6) of various CD45⁺ lung cell populations positive for intracellular IFN-γ or IL-17A (E).

Figure 3. Endotoxin-free PLY does not induce cytokine production by DC or macrophages but does enhance DC maturation in a TLR4-independent manner.

DC (A) or BMDM (B) from C57BL/6 mice were incubated with PLY (1 µg/ml or 0.5 µg/ml) or Pam3CSK (10 µg/ml) or LPS (500 pg/ml) for 24 hours. Supernatants were analyzed for IL-12p40, TNF-α and IL-6. Results are presented as mean cytokine concentrations (+ SEM) for triplicate samples. (C) DC from C3H/HeN or C3H/HeJ mice were incubated with medium, PLY (1 µg/ml) or LPS (500 pg/ml). After 24 hours, cells were washed and stained with antibodies specific for CD80, CD86, CD40 and MHC Class II. Immunofluorescence is shown for PLY- or LPS-treated DC (black line) compared to untreated cells incubated with medium (grey histograms). Plots are representative of three independent experiments. (D) Adoptive transfer of DC incubated with antigen and PLY promotes antigen-specific T cell responses. BALB/c mice were immunized subcutaneously in the footpad with DC (5×10⁵ cells/mouse) that had been incubated overnight with medium only as a control or with KLH antigen (10 µg/ml) in the presence or absence of PLY (1 µg/ml). 7 days later splenocytes were isolated and stimulated ex vivo with KLH (2 or 50 µg/ml). Proliferation was measured by [³H]-thymidine incorporation after 4 days of culture and is expressed as mean cpm (+ SEM; n = 5). *** P<0.001 vs. adoptive transfer of DC stimulated with KLH alone.

Figure 4. PLY synergizes with TLR agonists to enhance pro-inflammatory cytokine secretion by DC.

(A) DC from C3H/HeN or C3H/HeJ mice were incubated with PLY (1 µg/ml) for 1 hour before the addition of HkSp (10 bacteria:1 DC). After 24 hours supernatants were assayed for IL-12p40, IL-6, IL-23 and TNF-α. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P<0.001 vs. bacteria alone. (B) DC from C3H/HeJ mice were incubated with PLY (1 µg/ml) for 1 hour before stimulation with TLR or NLR agonists; HkSp (10 bacteria:1 DC), CpG (4 µg/ml), Pam3CSK (10 µg/ml), zymosan (20 µg/ml) or MDP (20 µg/ml). Supernatants were assayed for IL-1α and IL-1β after 24 hours. Results are mean cytokine concentrations (+ SEM) for triplicate samples. * P<0.05 and *** P<0.001 vs. agonist alone. (C) Haemolytic activity is required for the promotion of IL-1β secretion by PLY. DC from C57BL/6 mice were incubated with medium alone (control), or with PLY or W433F for 1 hour before stimulation with CpG (4 µg/ml) or HkSp (10 bacteria:1 DC). Following 24 hours incubation, supernatants were assayed for IL-1β. Results are mean cytokine concentrations (+ SEM) for triplicate samples. ***, P<0.001 vs. agonist alone, ### P<0.001 vs. PLY + agonist. (D) Activation of caspase-1 by PLY. DC (1×10⁶/ml) from C3H/HeN (represented as TLR4 +) or C3H/HeJ (represented as TLR4 −) mice were stimulated with PLY (2 µg/ml) (73,143 HU/mg) for 3, 6, 9, 12 or 24 hours or with medium (0 hours) as a control. Cell lysates were analysed for caspase-1 p10 expression by Western blot. (E) DC from C3H/HeJ mice were incubated with or without the caspase-1 inhibitor YVAD-fmk (40 µM) for 30 minutes before the addition of PLY (1 µg/ml) either alone or 1 hour before Pam3CSK (10 µg/ml). After 24 h supernatants were assayed for IL-1β, IL-1α and TNF-α by ELISA. *** P<0.001 vs. PAM3 + PLY alone.

Figure 5. The ability of PLY to enhance IL-1β secretion by DC is caspase-1 and NLRP3-dependent.

(A) DC from wild-type C57BL/6 or Caspase-1^−/− were incubated with PLY (0.5 µg/ml) for 1 hour before the addition of Pam3CSK (10 µg/ml) or HkSp (10 bacteria:1 DC). IL-1β, IL-1α and TNF-α concentrations were quantified in supernatants after 24 hours and are presented as mean values (+ SEM) from triplicate cultures. * P<0.05, ** P<0.01 and *** P<0.001 vs. agonist alone. (B) DC from wild-type C57BL/6 or NLRP3^−/− mice were incubated with PLY (0.5 µg/ml) for 1 hour before the addition of Pam3CSK (10 µg/ml) or HkSp (10 bacteria:1 DC). After 24 h supernatants were assayed for IL-1β, IL-1α and TNF-α by ELISA. *** P<0.001 vs. agonist alone.

Figure 6. In contrast to PLY-induced IL-1β secretion, the enhancement of TNF-α, IL-6, IL-10 and IL-12 by PLY is independent of NLRP3.

DC (6.25×10⁵/ml) from C57BL/6 or NLRP3^−/− mice were incubated with medium alone or with various concentrations of PLY (0.2–0.75 µg/ml) for 1 hour before the addition of CpG (4 µg/ml). After 24 hours supernatants were assayed for IL-1β, TNF-α, IL-6, IL-23, IL-10 and IL-12p70. Results are mean cytokine concentrations (+ SEM) for triplicate samples. ** P<0.01 and *** P<0.001 vs. CpG alone.

Figure 7. Live S. pneumoniae promotes IL-1β secretion by DC in a NLRP3-dependent manner and this requires PLY.

DC (6.25×10⁵/ml) from C57BL/6 or NLRP3^−/− mice were incubated for 24 hours with medium alone, with wild-type S. pneumoniae (D39; 10 bacteria:1DC) or with PLY-deficient bacteria (PLN; 10 bacteria:1 DC). As a positive control for IL-1β secretion and NLRP3 inflammasome activation, DC were primed with LPS (100 ng/ml) for 23 hours prior to one hour stimulation with ATP (2.7 mg/ml). Following incubation, supernatants were removed and assayed for IL-1β, IL-1α, TNF-α and IL-6. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P<0.001, NLRP3^−/− vs. C57BL/6 DC stimulated with the same treatments, ### P<0.001, D39 vs. PLN bacteria in C57BL/6 DC, ••• P<0.001, D39 vs. PLN bacteria in NLRP3^−/− DC.

Figure 8. PLY-induced IL-1β secretion by DC is dependent on potassium efflux and phagosomal rupture.

(A) DC from C3H/HeJ mice were incubated with medium alone or supplemented with KCl (50 mM) for 30 minutes before addition of PLY (1 µg/ml) either alone or 1 hour before Pam3CSK (10 µg/ml). After 24 hours, supernatants were assayed for IL-1β, IL-1α and TNF-α. ** P<0.01 and *** P<0.001 vs. PAM3 + PLY. (B) DC from C3H/HeJ mice were incubated with a cathepsin B inhibitor CA-074-Me (10 µM) or bafilomycin A (250 nM) for 30 minutes before addition of PLY (1 µg/ml) either alone or 1 hour before PAM3Csk (10 µg/ml). After 24 hours, supernatants were assayed for IL-1β, IL-1α and TNF-α. *** P<0.001 vs. PAM3 + PLY. All cytokine concentrations are presented as the mean (+ SEM) for triplicate samples.

Figure 9. The NLRP3 inflammasome is required for protection against S. pneumoniae infection.

C57BL/6 and NLRP3^−/− mice were nasally infected with 1×10⁶ CFU of wild-type S. pneumoniae (strain D39) or PLY-deficient S. pneumoniae (PLN-A). Mice were sacrificed at 0 and 24 hours post-infection and bacterial CFU in lung homogenates were assessed. Data are expressed as mean (+ SEM) CFU per mg of lung tissue (log₁₀) for five mice per group. * P<0.05 and ** P<0.01 vs. 0 hours p.i. in the same mouse strain, # P<0.05 and ## P<0.01 C57BL/6 24 hours p.i. vs. NLRP3^−/− 24 hours p.i. (A) Infection with wild-type bacteria. (B) Infection with PLY-deficient bacteria.