In mice, the cytokines IL-1β 
, IFN-γ and IL-17A 
have been shown to play important protective roles in immunity against pneumococci. Here we show that PLY strongly enhances the secretion of IFN-γ by splenocytes in vitro
and is required for IFN-γ and IL-17A responses during pneumococcal infection in vivo
. NK cells are the principal cellular source of IFN-γ in the lungs following infection while γδ T cells are the major producers of IL-17A. This supports recent findings that γδ T cells are a key source of IL-17A production in vitro
and in vivo
in response to IL-1 stimulation 
and pathogen-derived products 
. The latter study found that IL-17-producing γδ T cells share a number of characteristic features with Th17 cells. IL-1 and IL-23 play key roles in the induction of IL-17A production by γδ T cells and in the differentiation and expansion of Th17 cells 
. Other cytokines, including IL-6 and TNF-α, have also been shown to be important in Th17 cell differentiation 
. Importantly, our data show that PLY can synergize with TLR agonists to enhance the secretion of IL-1α, IL-1β, IL-23, TNF-α and IL-6 by DC, which can induce IL-17A production by γδ T cells and promote the differentiation and expansion of Th17 cells. The ability of Freund's complete adjuvant or LPS to induce antigen-specific Th17 cell responses in vivo
requires IL-1R1, indicating a key role for IL-1 in adjuvant-driven Th17 cell responses 
Pneumolysin also strongly enhanced TLR agonist-induced IL-12 secretion, which together with cytokines such as IFN-γ and IL-18, is important for Th1 differentiation and stabilization 
. Like IL-1β, active IL-18 also requires cleavage of its precursor form by caspase-1 and enhanced levels have been reported in macrophages co-stimulated with recombinant PLY and PLY-deficient S. pneumoniae 
. Since IFN-γ produced by NK cells is important for the instruction of Th1 responses 
, our demonstration of NK cells producing IFN-γ in the lungs of infected mice could suggest subsequent promotion of pathogen-specific Th1 responses. Likewise, it has been suggested that IL-17A derived from γδ T cells can promote Th17 responses 
and we demonstrate that there are IL-17A-producing γδ T cells in the lungs of mice infected with pneumococcus. Both of these effects were strongly dependent on pneumolysin, suggesting that the toxin is a key factor in pneumococcus-induced IFN-γ secretion by NK cells and IL-17A production by γδ T cells. Furthermore, since IL-23R signalling promotes the expansion and maintenance of γδ T cells in response to intracellular bacterial infection 
, the ability of pneumolysin to promote both IL-1 and IL-23 production is likely to contribute strongly to these γδ T cell responses.
During pneumococcal infection, PLY may synergize with pneumococcal or endogenous danger signals to induce the secretion of inflammatory cytokines. Reduced concentrations of plasma IL-6 have been reported from mice infected with PLY-deficient S. pneumoniae
compared to isogenic wild-type pneumococci 
. In addition, IL-1 and TNF-α are induced during murine pneumococcal disease 
and elevated levels of both have been reported in patients with pneumococcal meningitis. Notably, studies using mice deficient in IL-1 receptor type 1 (IL-1R1−/−
or IL-1β 
have shown the importance of IL-1 in conferring resistance to pneumococcal meningitis and pneumonia respectively. The latter study found that IL-1β, but not IL-1α, plays a major role in resistance to pneumococcal infection. Therefore, the inflammasomes, which are required for IL-1β processing and secretion, are likely to be crucial components of the host defense to S. pneumoniae
. Shoma et al
. recently reported that recombinant PLY stimulated the secretion of IL-1α, IL-1β and IL-18 in a caspase-1 dependent manner in macrophages and that this was augmented by co-stimulation with pneumococci 
. Importantly, we show here for the first time that the enhancement of IL-1β secretion by PLY is NLRP3-dependent. The ability of live pneumococci to promote IL-1β secretion by DC is also strongly dependent on NLRP3. One of the key observations in this study is that NLRP3 is required for protective immunity against pneumococcal infection. The first demonstration of a role for NLRP3 in defence against a Gram-negative pathogen, Salmonella typhimurium
, was recently described 
, but our study is the first report to show that NLRP3 is required for protection against a Gram-positive bacterium.
NLRP3 activation by PLY required lysosomal damage and the release of cathepsin B, as inhibitors of these processes reduced PLY-induced IL-1β secretion. Phagosomal rupture has previously been shown to be important in NLRP3 inflammasome activation by other compounds including silica crystals, aluminium salts and microparticles 
and a role for cathepsin B was recently reported in the promotion of IL-1β secretion by the pore-forming toxin tetanolysin O 
. Furthermore, the ability of PLY to activate NLRP3 was dependent on the haemolytic activity of the toxin. Taken together, our data suggest that PLY-induced pore formation results in K+
efflux from DC and intracellular changes including lysosomal destabilization. The release of lysosomal products, such as cathepsin B, into the cytosol may promote the generation of danger signals, which are detected by NLRP3 or intermediary factors, resulting in inflammasome assembly and caspase-1 activation. Active caspase-1 could then process pro-IL-1β, which is generated in response to a second stimulus such as a TLR/NLR ligand, into mature IL-1β which is released from the cell.
Although a direct role for PLY in the induction of IL-1 or TNF-α during pneumococcal disease has not been established, the reduced inflammation and pathology in the lungs of mice infected with PLY-deficient pneumococci compared to wild type strains 
suggests a diminished inflammatory cytokine response. In particular, lower levels of T cell infiltration and neutrophil recruitment into the lungs have been reported from mice infected with PLY-deficient S. pneumoniae
compared to wild-type pneumococci 
. TNF-α and IL-1β are two of the key cytokines involved in neutrophil recruitment in the lungs of mice infected intratracheally with wild-type S. pneumoniae 
. Additionally, IL-17A plays a key role in neutrophil recruitment and its induction in vivo
is strongly dependent on IL-1 
An additional major finding of this study is that the stimulatory effects of PLY on DC and splenocyte cytokine secretion are independent of TLR4. While we demonstrate that PLY can promote expression of costimulatory molecules and proinflammatory cytokines in DC, these effects are independent of TLR4. We also show that PLY exerts adjuvant effects, promoting antibody responses against a co-administered antigen in both wild-type and TLR4-defective mice, indicating that the immunostimulatory effects of PLY in vivo do not require TLR4. Therefore, further studies into the involvement of pathogen recognition receptors in sensing PLY in innate cells are warranted.
Streptococcus pneumoniae is a pathogen of significant clinical importance and understanding its interaction with the immune system is crucial. We propose that upon infection with S. pneumoniae, PLY, in synergy with pneumococcal PAMPs promotes the secretion of proinflammatory cytokines, particularly IL-1β, that promote an inflammatory response and mediate protective immunity. We identify PLY as a novel NLRP3 inflammasome activator and show that the ability of the live bacterium to promote IL-1β secretion is also strongly NLRP3-dependent. More importantly, NLRP3 is required for protective immunity against respiratory pneumococcal infection.