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IL-1β produced by phagocytes is important for protection against Staphylococcus aureus. Secretion of this cytokine requires both activation of a transcriptional signal to stimulate production of pro-IL-1β, and a second signal to stimulate processing by inflammasome complexes and release of the mature cytokine. We show here that phagocytosis and lysozyme-based degradation of bacterial cell walls are functionally coupled to activation of NLRP3 inflammasomes and secretion of IL-1β in response to live S. aureus and to S. aureus peptidoglycan. Further a S. aureus enzyme, peptidoglycan O-acetyl transferase A, previously demonstrated to make cell wall peptidoglycan resistant to lysozyme strongly suppresses inflammasome activation and inflammation in vitro and in vivo. This is the first demonstration of a case whereby a bacterium specifically subverts IL-1β secretion through chemical modification of its cell wall peptidoglycan.
Although Staphylococcus aureus permanently colonizes mucosal tissues in at least 20% of individuals, it can also cause pathological conditions ranging from minor skin and soft tissue infections to life-threatening invasive diseases (Foster, 2005). To survive in the host, S. aureus must evade immune defenses. Recent reports suggest several mechanisms by which staphylococci protect themselves including resistance to specific antimicrobial peptides, neutralization of reactive oxygen species (ROS), inactivation of complement, inhibition of neutrophil migration, and evasion of phagocytosis (reviewed in (Foster, 2005; Rooijakkers et al., 2005)).
IL-1β is a key cytokine in orchestrating host defense against S. aureus (Hultgren et al., 2002; Kielian et al., 2004; Miller et al., 2006; Miller et al., 2007; Verdrengh et al., 2004), yet little is known about how the organism counters IL-1β-mediated immunity. Miller et al. reported that mice genetically deficient in production of IL-1β or unable to respond to the cytokine are exquisitely sensitive to cutaneous infection with the bacteria (Miller et al., 2006; Miller et al., 2007). Others have similarly shown that IL-1 signaling is important for defense against systemic and brain infections and in a septic arthritis model (Hultgren et al., 2002; Kielian et al., 2004; Verdrengh et al., 2004). The primary role for IL-1β in host defense against S. aureus appears to be in regulating neutrophil recruitment to sites of infection. Although some reports suggest that S. aureus can respond to the presence of IL-1β with upregulation of virulence traits (Kanangat et al., 2007; McLaughlin and Hoogewerf, 2006), no studies to date have explored the capacity of S. aureus to limit production of IL-1β by the host immune system.
Secretion of IL-1β is tightly controlled. While release of many inflammatory cytokines and chemokines requires a single signal such as that from a Toll-like receptor, secretion of IL-1β requires at least two specific signals. First, a transcriptional response is activated leading to production of pro-IL-1β. A second signal causes pro-IL-1β to be processed by caspase-1 and released from cells. In the context of anti-bacterial host defense, the first signal may be provided by Toll-like receptors such as TLR2 or TLR4. Typically, activation of these receptors alone is not sufficient to trigger release of mature IL-1β. The second signal stimulates assembly of a multi-protein complex called the inflammasome which regulates activation of caspase-1. One of the most well characterized inflammasomes consists of NLRP3 (also called Cryopyrin or Nalp3), ASC, and caspase-1, although other complexes based on Nalp1 or Ipaf have been reported (Martinon and Tschopp, 2004).
Cell walls of gram-positive bacteria contain many components that are recognized by the innate immune system. These rigid structures are macromolecular assemblies of cross-linked peptidoglycan (PGN), polyanionic teichoic acids, and surface proteins. PGN, the core component of virtually all bacterial cell walls, is a polymer of β(1–4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crosslinked by short peptides. The crosslinking peptides contain m-diaminopimelic acid in all Gram-negative bacteria and in Gram-positive bacilli (DAP-type PGN). Most other Gram-positive bacteria, including S. aureus, have L-lysine instead (Lys-type PGN) (Dziarski, 2004). Muramyl dipeptide (MDP), one MurNAc linked to two peptides of this polymer, is the minimal essential structure of bacterial PGN required for adjuvant activity (Inohara et al., 2003). MDP is present in practically all Gram-positive and Gram-negative bacteria, and is detected by Nod2, a cytosolic receptor that activates NF-κB (Inohara et al., 2005). Polyanionic teichoic acids are composed of wall teichoic acid (WTA) and lipoteichoic acid (LTA). WTA is covalently linked to the PGN, whereas LTA is a macroamphiphile with its glycolipid anchored in the membrane and its poly(glycerophosphate) chain extending into the wall (Neuhaus and Baddiley, 2003). LTA is sensed by TLR2 (Morath et al., 2002; Schwandner et al., 1999). It is not clear that any of these cell wall components are sufficient to activate inflammasomes and induce IL-1β secretion from mouse macrophages.
Certain inert inflammatory stimuli such as aluminum salt crystal (alum), silica, and asbestos can trigger inflammasome activation in macrophages, and growing evidence suggests that the particulate nature of these stimuli is important for this activity (Dostert et al., 2009; Dostert et al., 2008; Eisenbarth et al., 2008; Franchi and Nunez, 2008; Hornung et al., 2008; Li et al., 2008; Sharp et al., 2009). It is not clear how these diverse, essentially indigestible particles are sensed and trigger inflammasome activation. Two recent reports have suggested that particle-induced production of ROS may be involved (Cassel et al., 2008; Dostert et al., 2008). Alternatively, Hornung et al. have suggested that such particles cause lysosomal swelling and rupture, and that release of lysosomal enzymes into the cytoplasm triggers activation of the Nalp3 inflammasome (Hornung et al., 2008).
Less is currently known about how extracellular microbial stimuli activate IL-1β release, or whether the physical nature of the exposure (soluble vs particulate delivery) influences this inflammatory response. Mariathasan et al. have previously demonstrated that exposure to S. aureus can activate the NLRP3 inflammasome in LPS pre-treated mouse macrophages and trigger release of some IL-1β (Mariathasan et al., 2006). Similarly, in human cells, antibiotic-killed S. aureus and purified cell wall peptidoglycan (PGN) have been demonstrated to induce IL-1β secretion (Wang et al., 2000). In this study, we demonstrate that S. aureus PGN must be particulate to activate NLRP3 inflammasomes and IL-1β secretion in mouse macrophages and that the particles must be internalized via phagocytosis. Furthermore, although this phagocytic process triggers production of ROS by the NADPH phagocyte oxidase, ROS production is not required. Instead, we show that lysozyme-dependent degradation of PGN within phagolyosomes is necessary to induce IL-1β secretion from macrophages. We show that live S. aureus strongly suppresses this response by specifically making its PGN resistant to lysozyme. While live S. aureus can stimulate some inflammasome activation, mutant bacteria unable to block lysozyme induce much more inflammasome activation and inflammation in vitro and in vivo. The study shows that bacterial cell wall degradation in macrophage phagosomes is functionally linked to activation of the NLRP3 inflammasome and IL-1β secretion to mount an efficient immune response against extracellular bacterial infection.
In order to examine the mechanisms by which S. aureus induces IL-1β secretion from macrophages, we stimulated mouse bone marrow derived macrophages (BMDMs) with S. aureus PGN for 18 hours. Consistent with these previous reports, we observed release of exclusively the mature processed 17 kDa form of IL-1β into the supernatant as determined by immunoblotting (Figure 1A) and ELISA (Figure 1B). A combination of Pam3CSK4 lipopeptide and muramyl dipeptide (MDP) activated cellular production of the 35 kDa pro-IL-1β, but did not trigger processing and release of the mature p17 form. PGN-induced IL-1β was due to PGN and not contaminants such as bacterial toxins since boiling, trypsin-treatment, SDS treatment, and phenol extraction (Dziarski and Gupta, 2005) did not ablate cytokine production (Supplementary Figure 1A).
The structure of PGN depends on the organism from which it is derived (Supplementary Figure 2A). Martinon et al. have demonstrated that the DAP-type PGN from E. coli is sensed by the NLRP3 inflammasome resulting in activation of caspase-1 causing maturation and secretion of IL-1β (Martinon et al., 2004). S. aureus produces Lys-type PGN, and we therefore determined whether this type of PGN is also sufficient to activate the NLRP3 inflammasome. Macrophages from animals deficient in NLRP3, ASC or caspase-1 failed to produce IL-1β in response to S. aureus PGN (Figure 1C), although IL-1β secretion was normal in Ipaf−/− macrophages. TNF-α induction was unaffected (Figure 1D).
In addition to the core peptide cross-linked carbohydrate, PGN preparations typically contain lipoteichoic acid (LTA) which, via Toll-like receptor 2 (TLR2), would be sufficient to provide the first signal for pro-IL-1β production. Indeed, S. aureus PGN-induced IL-1β production is inhibited in macrophages from TLR2-deficient mice (data not shown). However, the source of the second signal is less clear. Muramyl dipeptide (MDP, Supplementary Figure 2A) is the minimal essential structure of bacterial PGN required for adjuvant activity and is detected by the cytosolic receptor Nod2 (Inohara et al., 2005). There are conflicting reports as to whether Nod2 is able to activate caspase-1 and IL-1β maturation and release in response to MDP (Hsu et al., 2008; Kanneganti et al., 2007a; Maeda et al., 2005; Marina-Garcia et al., 2008; Netea et al., 2005; Pan et al., 2007), and it is unknown whether Nod2 is responsible for PGN-induced IL-1β secretion. We therefore compared IL-1β secretion from wild-type and Nod2−/− macrophages in response to S. aureus PGN. Nod2−/− cells produced large amounts of IL-1β in response to PGN, indicating that inflammasome activation was intact. The overall levels of IL-1β (Figure 1E) and TNF-α (Figure 1F) produced were reduced slightly in Nod2−/− cells, indicating that Nod2 participates in PGN sensing but is not required for inflammasome activation. Further NOD2−/− cells produce mature IL-1β equally as well as wild type cells in response to PGN when the cells are primed with LPS to stimulate pro-IL-1β (Supplementary Figure 1B). Consistent with these data, we observed that no combination of pure TLR2 and Nod2 agonists is sufficient for inflammasome activation and secretion of mature IL-1β (Figure 2A). All combinations tested, except MDP on its own, triggered secretion of TNF-α (Figure 2B). When macrophages treated overnight with TLR2/Nod2 agonists were pulsed for 2 hours with ATP, we observed IL-1β secretion that paralleled TNF-α production (Figure 2C), directly demonstrating that these agonists are sufficient to stimulate pro-IL-1β accumulation, but not processing and secretion of the mature cytokine.
Unlike soluble stimuli such as MDP, Pam3CSK4, and LTA, PGN is an insoluble particle. Macrophages bind these particles and internalize them via phagocytosis after which the particles reside in an acidified phagolysosome for many hours (Supplementary Figure 3A & B). To determine whether the particulate form of PGN is important for IL-1β releasing activity, we treated PGN with mutanolysin or lysostaphin to solubilize the material (Fournier and Philpott, 2005). These enzymes cleave different parts of PGN without degrading the MDP component (Supplementary Figure 2A & B). Macrophages stimulated for 18 hours with even very high doses of the mutanolysin-digested PGN or lysostaphin-digested PGN failed to secrete IL-1β (Figure 2D). In contrast, solubilization of the particles had little or no effect on the production of TNF-α (Figure 2E). Similarly, production of pro-IL-1β in the cells was unaffected by solubilization since ATP treatment of the stimulated cells triggered inflammasome activation and release of mature IL-1β for all stimuli (Figure 2F). Collectively these results indicate that the particulate nature of PGN is essential for its IL-1β releasing activity.
Because PGN must be particulate to induce IL-1β release, we hypothesized that phagocytosis is required. Fluorescently labeled PGN is internalized by macrophages, as illustrated by flow cytometry (Figure 3A), and this internalization is completely blocked in the presence of cytochalasin D. Thus the particles are internalized via an actin-dependent phagocytic process. We found that PGN-induced IL-1β and IL-18 secretion were blocked in the presence of cytochalasin D (Figure 3B, C and D). Secretion of TNF-α was not affected by cytochalasin D (Figure 3E), and immunoblotting revealed that pro-IL-1β production was not affected (Figure 3B). Cytochalasin D treatment did not block ATP-induced release of IL-1β from cells exposed to PGN (Figure 3F) or to mutanolysin-treated PGN (Figure 3G), indicating both that the drug did not interfere with accumulation of pro-IL-1β and that the mechanism of ATP-induced inflammasome activation is actin-independent. Taken together, these data demonstrate that phagocytosis is specifically important for PGN particles to activate the inflammasome.
Recent reports have suggested that ROS produced during phagocytosis may be an important unifying mechanism for inflammasome activation induced by diverse stimuli including ATP, asbestos, and silica particles (Cassel et al., 2008; Cruz et al., 2007; Dostert et al., 2008). During phagocytosis, the NADPH phagocyte oxidase assembles on phagosome membranes and produces large amounts of ROS, although it is not yet clear how many of these experimental inflammasome stimuli actually activate the NADPH phagocyte oxidase. Importantly, macrophages lacking the essential NADPH phagocyte oxidase component gp91phox produce IL-1β normally in response to silica particles, suggesting that this source of ROS may not be critical (Dostert et al., 2009). PGN particles induce robust NADPH phagocyte oxidase-dependent ROS production in mouse BMDMs (Supplementary Figure 3C). However, PGN-induced IL-1β production is unaffected in gp91phox-deficient macrophages even though chemical inhibitors of ROS suppress cytokine secretion (Supplementary Figures 3D & E). Together these data indicate that while some oxidative process may play a role, NADPH phagocyte oxidase activation is not a viable mechanism by which phagocyotis is coupled to inflammasome activation. Similar to ROS production, intracellular rupture of phagolysosomes has been postulated to be a unifying mechanism by which diverse particles activate inflammasomes (Halle et al., 2008; Hornung et al., 2008). We observed that S. aureus PGN particles remain quantitatively within intact acidified compartments after internalization, suggesting that this mechanism cannot account for PGN-induced IL-1β secretion (Supplementary Figures 3B).
Since neither NADPH phagocyte oxidase nor lysosomal rupture seemed to account for PGN-induced IL-1β secretion, we explored whether other aspects of phagosome activity were required. We treated macrophages with bafilomycin A1, a specific inhibitor of the vacuolar proton pump(Herskovits et al., 2007). Bafilomycin A1 treatment significantly suppressed PGN-induced IL-1β secretion but did not affect TNF-α production (Figure 4A and B), indicating that phagosome acidification is required for PGN-induced IL-1β secretion. The most abundant enzyme responsible for PGN digestion in phagolysosomes is lysozyme, and we therefore hypothesized that PGN degradation by lysozyme might be the key to PGN-induced inflammasome activation. We found that N,N′,N″-Triacetylchitotriose (triNAG), an inhibitor of lysozyme(Dahlquist et al., 1966; Fukamizo et al., 1992; Laible and Germaine, 1985; Lehrer and Fasman, 1967), strongly inhibited IL-1β release from macrophages stimulated with PGN (Figure 4C), while induction of TNF-α secretion was not affected (Figure 4D). Moreover, triNAG did not have any effect on IL-1β release from macrophages stimulated with either ATP or alum after Pam3CSK4 priming (Figure 4E and F), indicating that triNAG does not non-specifically block IL-1β release or caspase-1 activation. Further, we observed that triNAG inhibits IL-1β production induced by peptidoglycans from other of bacteria including Bacillus subtilis and Pseudomonas aeruginosa (Figure 4G). Collectively these results demonstrate that lysozyme activity towards PGN particles is essential for inflammasome activation.
These data are intriguing since the cell wall of pathogenic S. aureus is known to be highly resistant to lysozyme (Bera et al., 2005). However, most methods for preparing purified PGN from S. aureus generate a product that is sensitive to lysozyme (Gelius et al., 2003; Mellroth et al., 2003; Park et al., 2007). Recently Bera et al. reported that O-acetylation of MurNAc of S. aureus PGN, a process catalyzed by O-acetyl transferase A (oatA), is the specific modification that makes this PGN and the bacterial cell wall lysozyme resistant (Bera et al., 2005). PGN from S. aureus mutants lacking oatA (ΔoatA) is more sensitive to lysozyme. We therefore hypothesized that if lysozyme-based degradation is required for PGN-induced IL-1β secretion, wild type S. aureus should trigger significantly less IL-1β release from macrophages than mutants with a lysozyme-sensitive PGN. We infected macrophages with live wild-type and ΔoatA mutant bacteria and monitored both intracellular survival and IL-1β secretion. Macrophages more efficiently kill the ΔoatA mutant (Figure 5A), however despite the decreased bacterial load in ΔoatA-treated cells, more IL-1β (Figure 5B) and more IL-18 (Figure 5C) were secreted. Enhanced IL-1β and IL-18 release occurred under conditions where no significant changes in TNF-α were observed (Figure 5C). ΔoatA bacteria stimulated activation of caspase-1, as indicated by the appearance of the cleaved p10 subunit, whereas wild type bacteria did not (Figure 5D).
The enhanced IL-1β produced in response to ΔoatA infection is largely dependent on the NLRP3 inflammasome (Figure 5E). Consistent with these data, PGN freshly purified from wild type S. aureus induces less IL-1β secretion than PGN purified from ΔoatA bacteria, while they induced similar amounts of TNF-α (Supplementary Figure 4A). Further, as previously reported, the ΔoatA PGN was significantly more sensitive to lysozyme in vitro than wild type PGN (data not shown). Enhanced inflammasome activation has been associated with caspase-1 dependant cell death, or pyroptosis (Bergsbaken et al., 2009). We therefore examined whether ΔoatA bacteria induce significant macrophage cell death. Infection of macrophages with wild type or ΔoatA bacteria did not induce any cell death for up to 24 hours after infection (Supplementary Figure 4B).
Our in vitro results demonstrated that the lysozyme-resistance of wild-type S. aureus contributes not only to intracellular survival but also to evasion of IL-1β secretion from macrophages. To assess the significance of these observations to disease pathogenesis, we used a murine subcutaneous challenge model. In these studies, individual animals were injected simultaneously in one flank with the wild-type S. aureus strain and in the opposite flank with the ΔoatA mutant. At the site of ΔoatA injection, mice developed sizeable abscess lesions reaching an average size of 11 mm2 by day 1, but injection of an equivalent inoculum of the wild type bacteria on the contralateral flank failed to produce visible lesions in most mice (Figure 6A and B). Analysis of bacterial load in lesions after two days revealed that wild type bacteria survived better (Figure 6C), suggesting that reduced inflammation correlates with enhanced bacterial survival. Histological analysis confirmed that both strains of bacteria induce formation of multiple abscesses under the skin, but there were no clear differences in the morphology of the abscesses (Supplementary Figure 5A). To determine whether the increased inflammatory response elicited by the oatA mutant is due solely to enhanced inflammasome activation, we treated mice with anti-IL-1β/IL-1β8 antibodies and repeated the infection. In this case, the wild type and ΔoatA bacteria induced identical amounts of inflammation (Figure 6D), while control antibody-treatment did not affect the difference between wild type and ΔoatA bacteria (Supplementary Figure 5B). Thus the data collectively demonstrate that S. aureus evades inflammasome activation and subsequent mucosal inflammation through chemical modification of its cell wall peptidoglycan.
In this study, we have shown that bacterial cell wall degradation in macrophage phagosomes is functionally linked to activation of the inflammasome and IL-1β secretion. We demonstrated that the particulate nature of cell wall PGN is essential for activation of NLRP3 inflammasomes. Muramidase digestion of PGN solubilized the particles and destroyed their ability to activate inflammasomes, even though this treatment preserved the MDP motif and the capacity to stimulate production of pro-IL-1β and other cytokines. Further, phagocytosis and lysozyme-mediated degradation of PGN is required to induce IL-1β secretion. Modification of PGN by S. aureus to become resistant to lysozyme not only protects the organism from lysozyme-based killing in macrophages, it also strongly inhibits production of IL-1β in response to infection in vitro and in vivo. This is the first demonstration of a case whereby a bacterium specifically subverts IL-1β secretion through chemical modification of its cell wall peptidoglycan.
The most common clinical manifestation of S. aureus is cutaneous infection, a highly localized infection in which IL-1β is particularly important for orchestrating host defense. Miller et al. demonstrated that neutrophil recruitment to sites of cutaneous infection is impaired in mice deficient in IL-1R, IL-1β, or ASC and that the bacteria grow significantly faster at these sites (Miller et al., 2006; Miller et al., 2007). Our data demonstrate that phagosomal degradation of S. aureus cell wall PGN is a specific trigger for NLRP3 inflammasome activation and that direct inhibition of lysozyme in macrophages with triNAG strongly inhibits IL-1β secretion. A variety of additional bacteria including certain strains of E. coli, Salmonella, and Pseudomonas have been reported to possess different mechanisms for suppressing lysozyme activity, and it is possible that such mechanisms may also influence host IL-1β production and local inflammation. It is widely appreciated that during serious infections, both the pathogen and the host inflammatory response contribute significantly to pathology. While treatments such as antibiotics can rapidly control bacterial burden, clinical options for dampening inflammatory immune responses are limited. Our findings suggest that novel therapeutic approaches to control unwanted inflammation might be developed by targeting lysozyme-based PGN degradation in host immune cells.
A growing number of NLRP3 inflammasome activators have been reported (Kanneganti et al., 2007b; Petrilli et al., 2007; Sutterwala et al., 2006; Ye and Ting, 2008), and these are generally particulate or crystalline in nature. These include monosodium urate crystals (Martinon et al., 2006), calcium pyrophosphate dihydrate crystals (Martinon et al., 2006), silica (Cassel et al., 2008; Dostert et al., 2008; Hornung et al., 2008), asbestos (Cassel et al., 2008; Dostert et al., 2008), alum(Eisenbarth et al., 2008; Franchi and Nunez, 2008; Hornung et al., 2008; Kool et al., 2008; Li et al., 2008), and hemozoin (Dostert et al., 2009; Tiemi Shio et al., 2009). The mechanisms by which phagocytosis of these diverse particles is coupled to inflammasome activation are not yet clear. Using anti-oxidants and gene knockdown, some studies have demonstrated that ROS production is important for silica and asbestos particles to activate NLRP3 inflammasomes raising the intriguing possibility that phagosome-associated respiratory burst activity might be the link (Cassel et al., 2008; Dostert et al., 2008). However, it is not clear whether all of these model particles induce phagocyte oxidase activity or whether all particles that strongly activate the phagocyte NADPH oxidase would be expected to activate the NLRP3 inflammasome. Subsequent studies, in agreement with our results for peptidoglycan and alum, indicate that inflammasome activation in response to silica, uric acid and hemozoin does not seem to involve the phagocyte NADPH phagocyte oxidase that is responsible for phagosome-associated ROS production (Dostert et al., 2009). Further, the model particle zymosan, which potently activates the phagocyte NADPH oxidase, does not activate the inflammasome in bone marrow macrophages. Taken together, the data may support a role for oxidases that are not associated with phagocytosis in regulation of the inflammasome, but ROS production cannot explain the association of phagocytosis with inflammasome activation.
Two recent reports have suggested that particulate inflammasome stimuli act through destabilization of phagosomal membranes and release of phagosomal contents into the cytosol (Halle et al., 2008; Hornung et al., 2008). Hornung et al. proposed that silica, monosodium urate crystals, and alum particles are internalized into macrophage phagosomes and cause swelling and rupture of internal compartments leading to IL-1β secretion (Hornung et al., 2008). This model is attractive in that it provides a general mechanism by which diverse particles can activate the same inflammatory response from macrophages. In a related report, Halle et al. demonstrated that fibrillar amyloid-β is phagocytosed by microglia and activates NLRP3 inflammasomes by phagosomal destabilization (Halle et al., 2008). It has been reported that fibrillar amyloid-β is digested relatively poorly by microglia (Frackowiak et al., 1992), and Halle et al. suggest that resistance to degradation may contribute to the loss of lysosomal integrity and subsequent inflammasome activation (Halle et al., 2008). Although phagosomal destabilization may be possible for PGN, it seems unlikely that loss of phagosomal integrity is a normal part of phagocytic attack and sensing of live microorganisms. Indeed, we observe that peptidoglycan particles remain fully sequestered in acidic intracellular compartments following phagocytosis. The simplest explanation is that peptidoglycan products are released upon degradation and sensed, either within the phagosome or after transport into the cytosol. Indeed, both NLRP3 and NLRP1 have been reported to directly sense MDP (Faustin et al., 2007; Martinon et al., 2004), and it is possible that release of MDP specifically in phagosomes permits its delivery into the cytosol in a way than cannot be mimicked by simply adding extracellular soluble MDP to cells. Alternately, it is possible that phagosomes have a generic mechanism of sensing degradation of polymers. Further studies will be necessary to establish this part of the underlying mechanism.
Caspase-1–deficient mice (Kuida et al., 1995) (kindly provided by Richard Flavell, Yale University, New Haven, CT), NLRP3-deficient (Mariathasan et al., 2006), Ipaf-deficient (Mariathasan et al., 2004), and ASC-deficient mice (Mariathasan et al., 2004) (kindly provided by Vishva Dixit, Genentech, South San Francisco, CA) were maintained in specific pathogen-free conditions at the Institute for Systems Biology (Seattle, WA). C57BL/6, CD-1, Nod2-deficient mice, and gp91phox-deficient mice were obtained from the Jackson Laboratory and were maintained in specific pathogen-free conditions at Cedars–Sinai Medical Center. All mice were cared for under institutional IACUC approval. Bone marrow-derived macrophages from wild-type or mutant mice backcrossed onto the C57BL/6 background were cultured in RPMI 1640 media containing 10% L929 cell supernatant, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine for 6 days before use. Wild type SA113 and isogenic ΔoatA S. aureus were as previously described (Bera et al., 2005).
Pam3CSK4, muramyl dipeptide, and purified lipoteichoic acid from Staphylococcus aureus were obtained from InvivoGen. PGNs from Staphylococcus aureus and Bacillus subtilis were from Sigma-Aldrich/BioChemika. Cytochalasin D, ATP, zymosan, and N,N′,N″-triacetylchitotriose were from Sigma-Aldrich. Alum (Imject Alum) was from Thermo scientific.
Intracellular survival assays were performed according to a previous report (Liu et al., 2005). Wild type SA113 and ΔoatA bacteria were grown for 18 hours to 24 hours to reach stationary phase. Concentration was checked by spectrophotometer and confirmed by counting CFU on LB-agar plates. Bacterial cultures were washed in PBS. Serial dilutions of bacteria were made for several multiplicities of infection (M.O.I.) in RPMI 1640 + 10% FCS. Diluted bacteria cultures were added onto 1 × 105 BMDMs /100 μl/well in the same media (M.O.I.= from 50 to 3), centrifuged at 515 × g for 4 min, then incubated at 37°C in a 5% CO2 incubator for 30 min. Wells were washed three times with PBS, and Gentamicin (final concentration 300 μg/ml GIBCO BFL) was added in the fresh media to kill extracellular bacteria. After one hour, culture media was replaced with fresh media containing 100 μg/ml Gentamicin to avoid killing of intracellular bacteria by high concentration of the antibiotic. At specified time points, BMDMs were washed three times with PBS, lysed in 0.02% Triton X-100, and CFU were calculated by plating on LB-agar. Culture supernatants were collected after 20 hours for ELISA.
10–16-wk-old male CD-1 mice were injected subcutaneously in one flank (chosen randomly) with (5 × 107 CFU) wild type S. aureus and simultaneously in the opposite flank with the same dose of ΔoatA mutant strain for direct comparison. Bacterial cultures were washed, diluted, and resuspended in PBS, following an established protocol for generating localized S. aureus subcutaneous infection (Nizet et al., 2001). Lesion size, as assessed by the maximal length × width of the developing ulcers, was recorded daily. Where indicated, mice were injected i.p. with 12.5 μg each of anti-IL-1β and anti-IL-18 antibodies (R&D systems) five hours prior to infection. Control mice were injected with 25 μg of rat IgG.
For detection of pro-IL-1β and processed IL-1β, BMDMs were resususpended in Opti-MEM media seeded into 6-well plates and were stimulated for 18 h as indicated in figure legends. After clarification by centrifugation, protein in the 1.5 ml supernatants were precipitated with 20 μl Strataclean resin (Stratagene) and resuspended in sample buffer. Cells were lysed in SDS-PAGE sample buffer with protease inhibitor cocktail (Sigma). Samples were boiled for 5 min, proteins were separated by SDS-PAGE and transfered to immobilon-P membrane, and IL-1β protein was detected by immunoblot with monoclonal antibody 3ZD (obtained from Biological Resources Branch of National Cancer Institute at Frederick, MD).
Macrophages were preincubated as indicated with cytochalasin D (5 μM) or vehicle control (0.1 % DMSO) for 30 min, and were subsequently incubated with FITC-labeled PGN (10 μg/ml) for 4 hours. Cells were washed and lifted in PBS containing 1 mM EDTA, 1 mM sodium azide and 2.4 U/ml proteinase K (to remove bound but uninternalized PGN particles) prior to analysis by flow cytometry.
IL-1β (eBiosciences), IL-18 (Medical & Biological Laboratories), and TNF-α (BD Biosciences) levels in culture supernatants were assayed using ELISA kits according to the manufacturers' instructions.
In vitro data are typically presented as mean +/− standard deviation of a single experiment and are representative of three or more independent experiments. For in vivo experiments, lesion sizes were compared using the paired Student's t test.
We thank Richard Flavell for Caspase-1–deficient mice(Kuida et al., 1995); Vishva Dixit and Genentech for Ipaf-, NLRP3-, and ASC-deficient mice. This work was supported by grants to DMU from the National Institutes of Health (GM085796) and the American Heart Association (0640100N).
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