A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury
2015; Springer Nature; Volume: 7; Issue: 4 Linguagem: Inglês
10.15252/emmm.201404883
ISSN1757-4684
AutoresChristopher Whidbey, Jay Vornhagen, Claire Gendrin, Erica Boldenow, Jenny Mae Samson, Kenji Doering, Lisa Ngo, Ejiofor A.D. Ezekwe, Jens H. Gundlach, Michal A. Elovitz, Denny Liggitt, Joseph A. Duncan, Kristina M. Adams Waldorf, Lakshmi Rajagopal,
Tópico(s)Neonatal Health and Biochemistry
ResumoResearch Article6 March 2015Open Access A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury Christopher Whidbey Christopher Whidbey Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Jay Vornhagen Jay Vornhagen Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Claire Gendrin Claire Gendrin Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Erica Boldenow Erica Boldenow Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Jenny Mae Samson Jenny Mae Samson Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Kenji Doering Kenji Doering Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Lisa Ngo Lisa Ngo Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Ejiofor A D Ezekwe Jr. Ejiofor A D Ezekwe Jr. Department of Medicine, Division of Infectious Diseases and Pharmacology, School of Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jens H Gundlach Jens H Gundlach Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Michal A Elovitz Michal A Elovitz Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Denny Liggitt Denny Liggitt Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Joseph A Duncan Joseph A Duncan Department of Medicine, Division of Infectious Diseases and Pharmacology, School of Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Kristina M Adams Waldorf Kristina M Adams Waldorf Department of Obstetrics and Gynecology, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Lakshmi Rajagopal Corresponding Author Lakshmi Rajagopal Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Christopher Whidbey Christopher Whidbey Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Jay Vornhagen Jay Vornhagen Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Claire Gendrin Claire Gendrin Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Erica Boldenow Erica Boldenow Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Jenny Mae Samson Jenny Mae Samson Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Kenji Doering Kenji Doering Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Lisa Ngo Lisa Ngo Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Search for more papers by this author Ejiofor A D Ezekwe Jr. Ejiofor A D Ezekwe Jr. Department of Medicine, Division of Infectious Diseases and Pharmacology, School of Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Jens H Gundlach Jens H Gundlach Department of Physics, University of Washington, Seattle, WA, USA Search for more papers by this author Michal A Elovitz Michal A Elovitz Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Denny Liggitt Denny Liggitt Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Joseph A Duncan Joseph A Duncan Department of Medicine, Division of Infectious Diseases and Pharmacology, School of Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Kristina M Adams Waldorf Kristina M Adams Waldorf Department of Obstetrics and Gynecology, School of Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Lakshmi Rajagopal Corresponding Author Lakshmi Rajagopal Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA Department of Global Health, University of Washington, Seattle, WA, USA Search for more papers by this author Author Information Christopher Whidbey1,2, Jay Vornhagen1,2,‡, Claire Gendrin1,‡, Erica Boldenow1,‡, Jenny Mae Samson3,‡, Kenji Doering3, Lisa Ngo1, Ejiofor A D Ezekwe4, Jens H Gundlach3, Michal A Elovitz5, Denny Liggitt6, Joseph A Duncan4, Kristina M Adams Waldorf7 and Lakshmi Rajagopal 1,2 1Department of Pediatric Infectious Diseases, University of Washington and Seattle Children's Research Institute, Seattle, WA, USA 2Department of Global Health, University of Washington, Seattle, WA, USA 3Department of Physics, University of Washington, Seattle, WA, USA 4Department of Medicine, Division of Infectious Diseases and Pharmacology, School of Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 5Maternal and Child Health Research Program, Department of Obstetrics and Gynecology, Center for Research on Reproduction and Women's Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 6Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA, USA 7Department of Obstetrics and Gynecology, School of Medicine, University of Washington, Seattle, WA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 206 884 7336; Fax: +1 206 884 7311; E-mail: [email protected] EMBO Mol Med (2015)7:488-505https://doi.org/10.15252/emmm.201404883 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K+ efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth. Synopsis During human pregnancy, infection of the amniotic fluid can lead to in utero fetal injury. This study provides novel mechanistic insights into how a lipid toxin produced by group B streptococci (GBS) induces membrane permeabilization, inflammation, cell death, and fetal injury. The hemolytic pigment produced by GBS induces membrane defects in lipid bilayers, without the formation of discrete pores. Membrane defects induced by the GBS pigment triggers potassium efflux and osmotic lysis of red blood cells. The potassium efflux leads to NLRP3 inflammasome-dependent pyroptosis in macrophages. NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent mechanisms contribute to GBS pigment-mediated fetal injury. Introduction Preterm birth and early onset neonatal infections are estimated to cause approximately 1.4 million neonatal deaths annually (Weston et al, 2011). Currently, there is no effective therapy for prevention of in utero infections, preterm births, and stillbirths. An important pathogen that causes perinatal and neonatal infections is Group B streptococci (GBS) or Streptococcus agalactiae. GBS are β-hemolytic, Gram-positive bacteria that are typically found as recto-vaginal colonizers in healthy adult women (Badri et al, 1977; Dillon et al, 1982). However, ascending in utero GBS infection increases the risk of preterm, premature rupture of membranes (PPROM), fetal injury, and preterm birth (Matorras et al, 1989). As ascending infections cannot be treated by intrapartum antibiotic prophylaxis, new strategies are needed to more effectively treat and prevent in utero infections and early onset GBS disease. To develop such strategies, it is paramount to gain a better understanding of GBS virulence factors and how they impact the host immune response. An important virulence determinant of GBS is the toxin known as β-hemolysin/cytolysin (hereafter referred to as the hemolysin). This toxin is responsible for the characteristic zone of β-hemolysis exhibited by GBS, and hemolytic strains are associated with virulence (Liu et al, 2004; Fettucciari et al, 2006; Costa et al, 2012). Also, hyperhemolytic GBS such as those deficient in the two component system CovR/S (due to absence of repression of the hemolysin biosynthesis operon) are significantly more pathogenic, while non-hemolytic GBS are severely attenuated (Sendi et al, 2009; Lembo et al, 2010). Despite these advances, studies aimed at understanding the mechanism of action of the GBS hemolysin were confounded by difficulties associated with purifying the toxin. Although previously suggested to be a protein toxin (Marchlewicz & Duncan, 1981; Pritzlaff et al, 2001), we recently showed that the molecule responsible for hemolytic activity of GBS is the ornithine rhamnopolyene lipid/ pigment (Whidbey et al, 2013) also known as granadaene (Rosa-Fraile et al, 2006). With the identification of the GBS hemolysin as a lipid, understanding how the toxin itself contributes to inflammation, cytotoxicity, and preterm birth is critical for development of neutralizing strategies against the lipid toxin. Previous studies that were performed with hemolytic extracts of GBS had contaminating proteins (Marchlewicz & Duncan, 1981); consequently, the exact mechanisms of pigment-induced cytotoxicity are unclear. Mechanisms that promote ascending GBS infection and the immune responses invoked during this process also remain poorly defined. Studies using pregnant animal models have shown that intrauterine inflammation caused by bacterial infection triggers disruption of placental membranes leading to fetal injury and preterm birth (Elovitz & Mrinalini, 2004; Equils et al, 2009; Vanderhoeven et al, 2014). A study using intraperitoneal injection of heat-killed GBS showed that a pan-caspase inhibitor was able to delay, but not prevent, preterm birth in a murine model (Equils et al, 2009). Although hemolytic GBS strains have been described to activate the NLRP3 inflammasome in murine dendritic cells and macrophages (Costa et al, 2012; Gupta et al, 2014), whether the hemolytic pigment/lipid toxin is sufficient for inflammasome activation and whether this leads to pyroptosis are not known. Recently, when exogenous GBS RNA was transfected into murine macrophages, the NLRP3 inflammasome was described to associate with GBS RNA, but inflammasome activation required the presence of hemolytic GBS (Gupta et al, 2014); however, the relevance of these findings to GBS infection in vitro and in vivo is unclear. Because NLRP3 activation occurs only in the presence of hemolytic GBS (Costa et al, 2012; Gupta et al, 2014), we aimed to understand how the purified GBS pigment activates the inflammasome, induces cell death, and establish the consequence on fetal injury. Here, we demonstrate that the purified GBS pigment/lipid toxin induces membrane permeabilization and the efflux of intracellular potassium which triggers osmotic lysis in red blood cells and NLRP3 inflammasome and caspase 1 activation leading to pyroptosis in macrophages. In a pregnant murine model of intrauterine infection, hyperhemolytic/hyperpigmented GBS strains increased the incidence of preterm birth and in utero fetal death (IUFD) in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. Collectively, these findings provide novel insight into how a bacterial lipid toxin/pigment mediates cell death and demonstrates its relevance to bacterial infection and preterm birth. Results The GBS lipid toxin lyses red blood cells using a colloidal osmotic mechanism Previous work from our group demonstrated that hyperhemolytic GBS strains penetrate human placenta and can be associated with women in preterm labor (Whidbey et al, 2013). We further showed that hemolytic activity of GBS is due to the ornithine rhamnolipid pigment (see structure in Fig 1A, (Rosa-Fraile et al, 2006)) and not due to any protein toxin (Whidbey et al, 2013). Despite these findings, the mechanism of how the hemolytic pigment/lipid toxin causes hemolysis, cytolysis, and inflammation-mediated cell death was not known. To understand how the pigment/lipid toxin lyses host cells, we first examined pigment-mediated lysis of human red blood cells (RBCs). We hypothesized that the pigment may lyse RBC either by the mechanism of direct lysis where the lipid itself dissolves the membrane as observed with detergents or by the mechanism of colloidal osmotic lysis where the lipid forms pores or causes membrane perturbations and lysis occurs via osmotic pressure. To determine how the GBS pigment induces cell lysis, we first measured the kinetics of both K+ and hemoglobin (Hb) release from RBC treated with 400 nM pigment. As a control, an equal amount of extract from a non-hemolytic strain of GBS (∆cylE) was included. The results shown in Fig 1B indicate that while the pigment induced the release of both K+ and Hb from RBC, efflux of the smaller K+ ion was faster than efflux of the larger Hb, as measured by time to 50% release (Fig 1B; 4.8 min versus 8.4 min; P < 0.0001, extra sum-of-squares F test). These results suggest that the pigment induces membrane permeabilization that allows K+ ions to efflux, followed by the release of Hb. The slight lag in release of Hb versus K+ suggests a colloidal osmotic mechanism of lysis by the GBS pigment, rather than rapid dissolution of the membrane by direct lysis wherein no lag is expected between K+ and Hb. A similar lag between K+ and Hb release was observed during hemolysis mediated by Staphylococcus aureus α-toxin (Supplementary Fig S1A), whereas 100% release of both K+ and Hb occurred instantly with direct lysis mediated by Triton X-100 (Supplementary Fig S1B). Figure 1. Colloidal osmotic lysis and membrane permeabilization caused by the GBS pigment/lipid toxin The GBS pigment also known as granadaene is an ornithine rhamnopolyene (Rosa-Fraile et al, 2006). Human red blood cells (RBCs) were incubated with 400 nM pigment or control ΔcylE extract, and kinetics of K+ and Hb release was monitored. Data shown are the average and SEM of six independent experiments. The time to 50% K+ and 50% Hb release with pigment was 4.8 min and 8.4 min, respectively; n = 6, P < 0.0001, extra sum-of-squares F test. Role of osmoprotectants in pigment-treated RBC. Human RBCs were pre-incubated with GBS pigment for 2 min at RT, centrifuged, and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0.40 nm (PEG200), 0.56 nm (PEG400), 0.89 nm (PEG1000), 1.1 nm (PEG1500), or 1.6 nm (PEG3000), respectively. Release of Hb was measured after 1 h of incubation at 37°C. Data shown are the average and SEM of three independent experiments. Characteristics of membrane permeabilization by the GBS pigment in artificial lipid bilayers. Lipid bilayers were generated using diphytanoylphosphatidylcholine (DPhPC) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract. In the pigment-treated sample, channel conductance indicating disruption of the membrane is seen within 45 s. Erratic and non-discrete fluctuations in current are observed, suggesting the formation of multiple, small membrane defects. The bilayer eventually breaks at 120 s. In lipid bilayers treated with the control ΔcylE extract, the mean ionic current trace remains constant at 0 pA, showing no membrane disruption. Data shown are representative of three independent experiments. Download figure Download PowerPoint We also performed protection assays with osmoprotectants of various sizes ranging from a hydrodynamic radius of 0.40 nm (PEG200) to 1.6 nm (PEG3000). To this end, human RBCs were pre-treated with the GBS pigment for 2 min and the RBCs were pelleted to remove any unincorporated pigment. Pigment-treated RBCs were then resuspended in PBS or PBS containing 30 mM osmoprotectant, and hemolytic activity was measured (for details, see 4). The results shown in Fig 1C indicate that smaller osmoprotectants such as PEG200 and PEG400 did not protect RBC from pigment-mediated hemolysis, whereas complete protection from hemolysis was observed in the presence of the larger osmoprotectants such as PEG1500 and PEG3000. In comparison, minimal protection was observed with SDS, which causes direct and instant lysis of RBC (Supplementary Fig S2). The GBS hemolytic lipid induces membrane permeabilization of artificial lipid bilayers To determine if membrane permeability observed with the GBS pigment requires the active cellular response of host cells, we tested the ability of the pigment to disrupt artificial lipid bilayers using model black lipid membranes (BLMs). BLMs mimic membrane lipid bilayers but lack the active cellular responses of host cells. To test our hypothesis, BLM composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was established across an aperture separating two chambers of a U-tube as previously described Butler et al (2008). A voltage was applied, and current was measured between the two chambers; an increase in current corresponds to a compromise in membrane integrity. We observed that treatment of BLMs with pigment resulted in an increase in measured current, while treatment with the control ΔcylE extract resulted in no change (data from 2 μM are shown in Fig 1D and from 75 nM are shown in Supplementary Fig S3). Interestingly, jumps were erratic in both frequency and magnitude. The initial compromised bilayer area was in the order of 1 nm2. Subsequently, the size of the compromise in the bilayer fluctuated frequently before the bilayer finally ruptured at either 120 s (Fig 1D) or 345 s (Supplementary Fig S3), depending on pigment concentration. The increase in conductance observed in the lipid bilayers due to the GBS pigment is not canonical with either the formation of discrete protein pores that are usually marked by well-defined jumps in membrane conductance or detergent-mediated bilayer solubilization, which is marked by a rapid increase in conductance followed by spontaneous disappearance of the entire membrane (Supplementary Fig S3). The changes in conductance fluctuations are consistent with the pigment intercalating into and perforating the bilayer; this appears to be a dynamic process in which channel conductance appears and disappears. Taken together, these data demonstrate that the membrane permeability observed in pigment-treated cells occurs independently of a cellular response and is unique in its mode of action by neither conforming to a typical pore-forming protein toxin nor inducing instant lysis as observed with detergents. Purified GBS lipid toxin/pigment is sufficient for induction of IL-1β release and cytolysis To understand how the hemolytic pigment triggers host cell death, we examined the pro-inflammatory and cytotoxic properties elicited by the purified hemolytic pigment. In these experiments, we also included GBS strains with differences in hemolytic activity such as GBS WT A909, isogenic hyperhemolytic/hyperpigmented strain ∆covR (lacking the two component regulator CovR/S that represses biosynthesis of the hemolytic pigment), and non-hemolytic ∆cylE, ∆covR∆cylE strains that were derived from WT and ∆covR but lack the cylE gene important for pigment biosynthesis (for details, see (Whidbey et al, 2013)). Given that macrophages are important for defense against GBS infections, we utilized macrophages derived from M-CSF-treated human peripheral blood mononuclear cells (PBMC) as well as differentiated THP-1 cells as models of human macrophage-like cells. Human PBMC and THP-1-derived macrophages were treated with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcylE and ΔcovRΔcylE at a multiplicity of infection (MOI) of 1 for 4 h, and cytotoxicity was measured by LDH release. The results shown in Figs 2A and 3A show that LDH release indicative of cell death was significantly higher in macrophages treated with hyperhemolytic GBS when compared to non-hemolytic strains. We also observed that hyperhemolytic GBS induced significantly more IL-1β release when compared to the isogenic non-hemolytic strains (Figs 2B and 3B). Figure 2. The GBS pigment toxin/lipid toxin is pro-inflammatory and cytotoxic to primary human macrophages A, B. PBMC-derived macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by ELISA (B). C, D. PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBS pigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). Data information: Data shown are the average of four independent experiments performed in triplicate, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 4, *P = 0.021 for WT versus ΔcovR; *P = 0.01 for ΔcovR versus ΔcovRΔcylE. (B) n = 4, *P = 0.031 for WT versus ΔcovR; *P = 0.036 for ΔcovR versus ΔcovRΔcylE. (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005. Download figure Download PowerPoint Figure 3. The GBS pigment toxin/lipid toxin is pro-inflammatory and cytotoxic to immortalized THP-1 monocyte-derived macrophages A, B. WT THP-1 macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). C, D. WT THP-1 macrophages were incubated with various concentrations of GBS pigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01. Download figure Download PowerPoint To determine the importance of the pigment/lipid toxin in the pro-inflammatory and cytotoxic nature of GBS, the human PBMC- and THP-1-derived macrophages were incubated with various concentrations of purified pigment or control ΔcylE extract for 4 h, and cytotoxicity was measured as the loss of metabolic activity as measured by a redox dye, alamar blue. Cytotoxicity due to GBS pigment was dose dependent and 50% cell death was observed at approximately 1–2 μM (Figs 2C and 3C), which is noticeably higher than the EC50 for RBC (< 0.1 μM, Fig 1C). This observation is likely due to the membrane turnover that occurs in macrophages and not RBC, and has also been observed with other bacterial exotoxins (Keyel et al, 2011). To identify the pathways activated by the purified GBS pigment/lipid toxin, we measured cytokine levels in the supernatants of pigment-treated macrophages. Interestingly, levels of IL-1β were significantly increased in pigment-treated PBMC and THP-1 cells compared to control ΔcylE extract-treated cells (Figs 2D and 3D). Consistent with the above observations, IL-18 levels were also increased in pigment-treated THP-1 macrophages, but other pro-inflammatory cytokines such as TNFα, IL-6, and IFN-γ were not significantly increased (Supplementary Fig S4). Taken together, these data show that the purified hemolytic GBS pigment is pro-inflammatory and cytotoxic. Pigment-induced cytotoxicity and immune response are NLRP3 inflammasome dependent The increase in secretion of IL-1β and IL-18 observed in human macrophages treated with GBS pigment suggests that the pigment can trigger activation of the inflammasome. The inflammasome is a cytosolic complex, which mediates cleavage of pro-caspase 1 to active caspase 1, which in turn cleaves pro-IL-1β and pro-IL-18 into their active forms. One major inflammasome comprises the NLR (nucleotide binding, leucine-rich repeat containing) protein known as NLRP3, which associates with the adaptor ASC (apoptosis-associated speck-like protein containing the caspase recruitment domain, CARD) (Taxman et al, 2010). To determine if the GBS pigment/lipid toxin activates NLRP3, we exposed previously characterized THP-1 human macrophage cell lines that were constitutively knocked down for expression of NLRP3 or the adapter protein ASC ((Willingham et al, 2007), Supplementary Fig S5) to GBS strains (WT, isogenic ΔcovR, ΔcylE, ΔcovRΔcylE) or purified pigment. As controls, THP-1 macrophages transfected with empty vector or a shRNA of scrambled ASC sequence were included. The results shown in Fig 4A indicate that GBS induced significant cell death in macrophages containing the NLRP3 inflammasome in a hemolysin-dependent manner. Notably, cell death was significantly decreased in macrophage cell lines knocked down for expression of NLRP3 or ASC (Fig 4A). Similarly, hemolytic and hyperhemolytic GBS strains induced increased IL-1β secretion in macrophages in an NLRP3 inflammasome-dependent manner (Fig 4B). Consistent with these observations, we observed that increasing concentrations of the purified GBS hemolytic pigment induced cell death and IL-1β secretion in an NLRP3 inflammasome-dependent manner (Fig 4C and D). These results demonstrate that in macrophages, the GBS hemolytic pigment primarily induces an NLRP3 inflammasome-dependent programmed cell death. The cell death observed in NLRP3-deficient macrophages with hyperpigmented GBS∆covR (25–40%, see THP-1/shASC, THP-1/shNLRP3 in Fig 4A) could not be prevented by the addition of osmoprotectants such as PEG1500 or by the caspase 3/7 inhibitor Z-DEVD-FMK (Supplementary Fig S6). Also, levels of IL-1β released by THP-1/shASC and THP-1/shNLRP3 cells exposed to GBS∆covR were not significantly different from cells exposed to GBS WT, ∆cylE, or ∆covR∆cylE (Fig 4B). Based on these observations, we predict that the residual cell death observed in inflammasome-deficient cells due to hyperpigmented GBS can be attributed to an inflammasome- and caspase 3/7-independent pathway, as suggested previously with rat neuronal cells (Reiss et al, 2011). Figure 4. The GBS pigment induces NLRP3 inflammasome-dependent cell death in human macrophages A, B. THP-1 macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incu
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