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. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: J Neurochem. 2011 Oct 11;119(4):736–748. doi: 10.1111/j.1471-4159.2011.07481.x

Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia

Richa Hanamsagar 1, Victor Torres 2, Tammy Kielian 3,*
PMCID: PMC3202981  NIHMSID: NIHMS324054  PMID: 21913925

Abstract

Microglia are important innate immune effectors against invading CNS pathogens, such as Staphylococcus aureus (S. aureus), a common etiological agent of brain abscesses typified by widespread inflammation and necrosis. The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing following exposure to both pathogen- and danger-associated molecular patterns. Although previous studies from our laboratory have established that IL-1β is a major cytokine product of S. aureus-activated microglia and is pivotal for eliciting protective anti-bacterial immunity during brain abscess development, the molecular machinery responsible for cytokine release remains to be determined. Therefore, the functional role of the NLRP3 inflammasome and its adaptor protein apoptosis-associated speck-like protein (ASC) in eliciting IL-1β and IL-18 release was examined in primary microglia. Interestingly, we found that IL-1β, but not IL-18 production, was significantly attenuated in both NLRP3 and ASC knockout (KO) microglia following exposure to live S. aureus. NLRP3 inflammasome activation was partially dependent on autocrine/paracrine ATP release and α- and γ-hemolysins produced by live bacteria. A cathepsin B inhibitor attenuated IL-β release from NLRP3 and ASC KO microglia, demonstrating the existence of alternative inflammasome-independent mechanisms for IL-1β processing. In contrast, microglial IL-18 secretion occurred independently of cathepsin B and inflammasome action. Collectively, these results demonstrate that microglial IL-1β processing is regulated by multiple pathways and diverges from mechanisms utilized for IL-18 cleavage. Understanding the molecular events that regulate IL-1β production is important for modulating this potent proinflammatory cytokine during CNS disease.

Keywords: inflammasome, microglia, IL-1β, IL-18, NLRP3, ASC, P2X7R, cathepsin B, hemolysins

INTRODUCTION

Staphylococcus aureus (S. aureus) is one of the main etiologic agents of brain abscesses in humans (Townsend and Scheld 1998). Accounting for 1 in 10,000 hospital admissions in the United States of an infectious disease nature, brain abscesses continue to be a considerable medical issue compounded by the increasing prevalence of antibiotic-resistant bacteria (Jones et al. 2004; Naesens et al. 2009). Microglial activation is evident within hours following CNS S. aureus infection and the immune response elicited during brain abscess development involves the rapid induction of several proinflammatory cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and IL-6 (Kielian et al. 2004a; Stenzel et al. 2005a). The production of these mediators is dependent on initial bacterial recognition within the CNS compartment, presumably via microglia triggered by MyD88-dependent pathways (Garg et al. 2009). However, additional molecular mechanisms that microglia utilize to elicit this widespread inflammatory response remain to be completely defined.

The inflammasome is an intracellular protein complex that serves as a platform for activating the proinflammatory cytokines IL-1β and IL-18 via caspase-1 action (Lamkanfi and Dixit 2009). Several inflammasome complexes have been identified, each named on the basis of the Nod-like receptor (NLR) that organizes the structure (Schroder and Tschopp 2010). Mutations in the NLRP3 (NALP3 or cryopyrin) inflammasome have been implicated in the pathogenesis of familial cold-induced autoinflammatory syndrome (Arostegui et al. 2004), Muckle-Wells syndrome (Hoffman et al. 2001; Agostini et al. 2004), and neonatal-onset multisystem inflammatory disease (Aksentijevich et al. 2002; Arostegui et al. 2010). This complex consists of NLRP3, apoptosis-associated speck-like protein (ASC), and pro-caspase-1. Together these proteins provide a platform for the proteolytic activation of pro-caspase-1 and subsequent processing of the inactive forms of IL-1β and IL-18 to their mature states (Mariathasan et al. 2004; Bryant and Fitzgerald 2009). The NLRP3 inflammasome can be activated by danger-associated molecular patterns (DAMPs) such as ATP signaling via P2X7R and K+ efflux as well as bacterial toxins that serve as a secondary signal to Toll-like receptor (TLR) activation by pathogens (Martinon et al. 2006; Petrilli et al. 2007; Qu et al. 2007). Indeed, prior work from our laboratory has established a critical role for TLR signaling in eliciting IL-1β production following S. aureus exposure (Kielian et al. 2007). However, the impact of P2X7R activity in response to pathogenic bacteria and potential interactions between the NLRP3 inflammasome and P2X7R in microglia remain to be defined. In macrophages and neutrophils, the NLRP3 inflammasome is critical for cytokine processing and its importance has also been established in response to various bacterial toxins (Craven et al. 2009; Netea et al. 2009). However, the molecular machinery regulating IL-β and IL-18 production in microglia and the functional role of the NLRP3 inflammasome in this process have not yet been investigated. This is important to establish since microglia exhibit several unique properties compared to macrophages, including low constitutive levels of major histocompatability complex and co-stimulatory molecule expression (Perry and Gordon 1988; Andersson et al. 1991; Lawson et al. 1994; Kreutzberg 1996; Perry 1998; Thrash et al. 2009). In addition, although microglia are capable of phagocytosis, their ability to clear pathogens is less pronounced compared to other professional phagocytes such as macrophages and neutrophils (Kreutzberg 1996).

To date, most studies examining inflammasome activity have utilized purified PAMPs in conjunction with extracellular ATP, which could conceivably deliver distinct signals to trigger IL-1β/IL-18 processing. In contrast, our approach was to examine inflammasome activity elicited by live S. aureus since this presents microglia with a complex milieu of PAMPs and secreted virulence factors, which is most reminiscent of what occurs during CNS infection in vivo. Here we show that the NLRP3 inflammasome plays a critical role in IL-1β production by microglia following exposure to live S. aureus. Microglial IL-1β release was also attenuated following exposure to S. aureus α- and γ-hemolysin mutants; however, residual IL-1β was still detected, suggesting the involvement of alternative toxins for maximal cytokine induction in response to live bacteria. S. aureus treatment induced extracellular ATP release, which acted as a DAMP in an autocrine/paracrine manner to augment microglial IL-1β production since cytokine release was significantly attenuated following P2X7R inhibition. Further, the ability of a P2X7R inhibitor to attenuate IL-1β production in NLRP3 KO and ASC KO microglia revealed a role for alternative inflammasome-independent but ATP-dependent pathways for cytokine processing. Similarly, attenuation of residual IL-1β release from NLRP3 and ASC KO microglia by caspase-1 inhibitor treatment further suggested the existence of an NLRP3/ASC-independent pathway for IL-1β maturation that eventually feeds into caspase-1. Surprisingly, neither NLRP3 nor ASC impacted microglial IL-18 production in response to S. aureus challenge, which differs from results previously reported for macrophages (Li et al. 2008). Collectively, these studies highlight the complexity of IL-1β regulation in microglia and emphasize the redundant mechanisms in place to ensure cytokine production from sensing both bacterial- and host-derived signals.

MATERIALS AND METHODS

Primary microglia culture

Mixed glial cultures were prepared from the cerebral cortices of neonatal NLRP3 and ASC KO (generously provided by Dr. Vishva Dixit, Genentech, San Francisco, California) and C57BL/6 WT mice (Charles River, Frederick, MD) (2-4 days of age) as previously described (Esen et al. 2004; Phulwani et al. 2008). Microglia were harvested from mixed glial cultures using a differential shaking technique, resulting in a purity of greater than 98%. The animal use protocol, approved by the University of Nebraska Medical Center Animal Care and Use Committee, is in accord with the National Institutes of Health guidelines for the use of rodents.

Bacterial strains

A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) isolate recovered from a patient with a fatal brain abscess (Sifri et al. 2007) was kindly provided by Dr. Costi Sifri (University of Virginia School of Medicine, Charlottesville, VA). The S. aureus Newman strain and its isogenic hla, hlgACB, lukAB, lukED mutants were described previously (Dumont et al. 2011). For bacterial treatments, each S. aureus strain was grown for 16 h in brain-heart infusion (BHI) broth at 37°C with constant agitation (250 rpm). The following day, cultures were further diluted 1:100 in BHI and grown to an OD of 6.0, whereupon each strain was diluted to 106 cfu/ml for microglial treatments. Bacterial titers were enumerated for each strain immediately after microglial stimulation to normalize cytokine levels and correct for potential differences in initial bacterial inoculums.

Microglia treatment paradigms

Microglia were plated in 96-well plates (2 × 105 cells/well) in antibiotic-free medium and incubated overnight at 37°C prior to bacterial exposure. In select experiments, microglia were pre-treated with an inhibitory ODN (ODN 2088; Invivogen, San Diego, CA) to block TLR9 signaling (Gurley et al. 2008) or various concentrations of the P2X7R inhibitor AZ11645373 (Tocris Biosciences, Ellisville, MO), caspase-1 inhibitor Z-WEHD-FMK (R&D Systems, Minneapolis, MN), or cathepsin B inhibitor CA-074 Me (Calbiochem, San Diego, CA) prior to live S. aureus exposure. None of the inhibitors tested had any impact on S. aureus growth in pilot studies (data not shown). In all experiments, microglia were treated with live S. aureus at a multiplicity of infection (MOI) of 1:1 for 6 h, whereupon conditioned supernatants were collected for determination of cytokine expression by ELISA. Later intervals were not examined to avoid bacterial overgrowth of microglial cultures and cellular toxicity. Because we utilized live bacteria to stimulate microglia, the absolute amount of IL-1β released between independent experiments was subject to slight variation, although the trends between WT, NLRP3 KO, and ASC KO microglia were consistent. This variability in absolute cytokine levels is influenced by several factors. First, although we propagate S. aureus for the same time period in culture and adjust to a constant OD for each experiment, this is only an estimate of bacterial numbers and hence, the exact bacterial inoculum used to treat WT, NLRP3 KO, and ASC KO microglia varied slightly between independent studies. Second, the rate of bacterial growth over the 6 h co-culture period with microglia cannot be controlled, and may also experience slight fluctuations between independent experiments. These caveats are not encountered in studies that utilize purified PAMPs such as LPS or PGN, which can be accurately titrated down to ng concentrations and elicit reproducible cytokine production between separate studies. The variability in IL-1β concentrations between independent experiments prevented us from pooling data from the 3-6 separate studies performed here. Therefore, we analyzed three independent replicates for each treatment group (i.e. WT, NLRP3 KO, and ASC KO) within each of our experiments and validated the significance and reproducibility of these findings in a minimum of 3 independent studies.

Enzyme-linked immunosorbent assay (ELISA)

Comparisons in IL-1β and TNF-α (BD Biosciences OptEIA; San Diego CA) and IL-18 (Bender Medsystems, San Diego, CA) expression between NLRP3 KO, ASC KO, and WT microglia were performed using standard sandwich ELISA kits according to the manufacturer’s instructions.

Cell Viability Assays

To assess whether live S. aureus strains or caspase-1 and P2X7R inhibitors exerted any toxic effects on microglia, LDH release assays were performed as described by the manufacturer (CytoTox; Promega, Madison, WI). In addition, as an independent assessment of cell viability, MTT assays were performed as previously described based on the mitochondrial conversion of (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT, Sigma St. Louis, MO) into formazan crystals (Kielian et al. 2004b). MTT values were elevated following S. aureus treatment compared to non-treated controls since live bacteria were also found to metabolize the MTT substrate. However, disparities in cell plating densities between WT, NLRP3 KO, and ASC KO microglia were ruled out since MTT values were nearly equivalent within treatment groups. In addition, visual inspection of microglial cultures following the 6 h treatment period with live bacteria also revealed no evidence of cell death (data not shown).

ATP measurements

ATP release from microglia at various time points following S. aureus exposure was evaluated using a luminescence-based ATP Detection Assay System (ATPlite; Perkin Elmer, Waltham, MA) according to the manufacturer’s instructions. Plates were evaluated using a Victor luminescence plate reader (VICTOR3V, Perkin Elmer, Shelton, CT).

Statistical Analysis

Significant differences in cytokine production between NLRP3 KO, ASC KO, and WT microglia across experimental groups were determined by one-way analysis of variance (ANOVA) followed by the Holm-Sidak method for pair-wise multiple comparisons using Sigma Stat (SPSS Science, Chicago, IL). For comparisons between two treatment groups a Student’s t-test was used. For all analyses, a p-value of less than 0.05 was considered statistically significant.

RESULTS

NLRP3 and ASC selectively regulate IL-1β but not IL-18 production by microglia upon exposure to intact S. aureus

One of the main cytokines produced by activated microglia upon stimulation with the Gram-positive bacterium S. aureus is IL-1β (Kielian et al. 2002; Esen and Kielian 2006), which is critical for mounting a protective anti-bacterial immune response during brain abscess development (Kielian et al. 2004a). Despite recent advances in defining the molecular events required for NLRP3 inflammasome activation and processing of pro-IL-1β and IL-18 into their active forms in other cell types (Cruz et al. 2007; Li et al. 2008; Kankkunen et al. 2010; Rajamaki et al. 2010), relatively little is known regarding the molecular pathways required for inflammasome activation in microglia. Our approach was to examine inflammasome activity elicited by live S. aureus, since this is most reflective of what occurs during CNS infection and importantly, the contribution of bacterial toxins can also be assessed. In contrast, most studies to date have utilized purified PAMPs (Craven et al. 2009) or bacterial supernatants (Munoz-Planillo et al. 2009), which require a second signal to induce mature IL-1β release and is arguably a more contrived system. Treatment of primary microglia with live S. aureus resulted in high levels of IL-1β production, whereas IL-18 release was less robust, suggesting that live bacteria provide the two requisite signals for inflammasome activation in microglia (Fig. 1). To determine whether microglial cytokine production was dependent on NLRP3 inflammasome activity, primary microglia isolated from WT, NLRP3 KO, or ASC KO mice were exposed to live S. aureus. NLRP3 KO microglia displayed significantly attenuated levels of IL-1β release in response to S. aureus; however, residual IL-1β production was still detectable, indicating that live S. aureus can also elicit cytokine release via a NLRP3-independent mechanism (Fig 2A). Surprisingly, although NLRP3 participates in IL-18 processing in other cell types (Li et al. 2008) no significant differences in IL-18 production were observed between NLRP3 KO and WT microglia (Fig 2B). As expected, TNF-α levels were not affected by NLRP3 loss since this cytokine does not require caspase-1-dependent processing prior to secretion (Fig 2C). Similarly, ASC KO microglia displayed reduced IL-1β expression in response to live S. aureus; however, residual IL-1β expression was less pronounced compared to NLRP3 KO microglia (100-200 pg/ml versus 400-800 pg/ml, respectively), suggesting the involvement of additional NLRP3-independent, ASC-dependent mechanisms for IL-1β processing (Fig. 3A). Similar to NLRP3 KO microglia, IL-18 and TNF-α levels remained unchanged between ASC KO and WT cells (Fig 3B, and C, respectively). The reduction in IL-1β secretion in NLRP3 and ASC KO microglia was not attributable to differences in cell numbers or toxicity, since viability assays revealed similar values between WT and KO microglia (Figs. 2D and 3D). Collectively, these results demonstrate that live S. aureus provides the two requisite signals to trigger NLRP3 inflammasome activation in microglia and subsequent IL-1β release. The residual IL-1β production observed in both NLRP3 and ASC KO microglia is indicative of cytokine processing pathways that are independent of both molecules. In contrast, IL-18 release is not influenced by either NLRP3 or ASC, which is a finding unique to microglia.

Figure 1. Microglial exposure to live S. aureus induces IL-1β and IL-18 production.

Figure 1

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Primary microglia were isolated from C57BL/6 mice and exposed to live S. aureus strain USA300 at a MOI of 1:1 for 6 h, whereupon supernatants were analyzed for IL-1β and IL-18 by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences in cytokine production between unstimulated versus S. aureus treated microglia are denoted with asterisks (*, p < 0.05; ***, p < 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 15 separate experiments. N.D. = not detected.

Figure 2. NLRP3 selectively regulates microglial IL-1β but not IL-18 secretion in response to intact S. aureus.

Figure 2

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NLRP3 WT and KO microglia were treated with live S. aureus strain USA300 at a MOI of 1:1 for 6 h, whereupon supernatants were evaluated for IL-1β (A), IL-18 (B), and TNF-α (C) expression by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Microglia viability was assessed by a standard MTT assay and the raw OD570 absorbance values are reported (D). Significant differences between S. aureus treated NLRP3 WT and KO microglia are denoted by asterisks (***, p < 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 10 separate experiments. N.D. = not detected.

Figure 3. ASC selectively regulates microglial IL-1β secretion in response to intact S. aureus.

Figure 3

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ASC WT and KO microglia were treated with live S. aureus strain USA300 at a MOI of 1:1 for 6 h, whereupon supernatants were evaluated for IL-1β (A), IL-18 (B), and TNF-α (C) expression by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Microglia viability was assessed by a standard MTT assay and the raw OD570 absorbance values are reported (D). Significant differences between S. aureus treated ASC WT and KO microglia are denoted by asterisks (***, p < 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 10 separate experiments. N.D. = not detected.

Microglial IL-1β release in response to live S. aureus is partially influenced by α- and γ-hemolysins

Recent studies have demonstrated that IL-1β processing is regulated by bacterial toxins that are presumed to influence K+ and ATP gradients across the mammalian cell membrane (Mariathasan et al. 2006; Petrilli et al. 2007). However, no studies to date have defined the array of virulence determinants responsible for IL-1β processing in microglia following pathogen exposure. To identify bacterial-derived factor(s) involved in inflammasome activation, we focused on known pore-forming toxins secreted by S. aureus using isogenic mutants that lack the ability to produce various toxins (Dumont et al. 2011). Initial studies confirmed bacterial hemolysin expression during the 6 h incubation period with microglia (data not shown). Microglial IL-1β production was significantly attenuated following exposure to either S. aureus α- (hla) or γ-hemolysin (hlgACB) mutants (Fig. 4); however, substantial IL-1β expression was still observed, indicating that other virulence determinants impact inflammasome activation. In contrast, IL-1β release was not significantly influenced by other S. aureus toxins, including the leukocidins lukAB and lukED. These studies are the first to define bacterial-derived factors responsible for IL-1β processing in microglia.

Figure 4. S. aureus α- and γ-hemolysins trigger IL-1β release from microglia.

Figure 4

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Primary microglia were isolated from C57BL/6 mice and exposed to S. aureus strain Newman or isogenic α-hemolysin (hla), γ-hemolysin (hlgACB), leukocidin AB (lukAB), or leukocidin ED (lukED) mutants for 6 h, whereupon supernatants were analyzed for IL-1β by ELISA, with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences between IL-1β induction by WT S. aureus Newman versus the various toxin mutant strains are denoted by asterisks (***, p ≤ 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 3 separate experiments.

S. aureus induces ATP release that stimulates P2X7R activation in an autocrine/paracrine manner to influence IL-1β release in microglia

Extracellular ATP has been shown to trigger NLRP3 inflammasome activation via the purinergic P2X7R (Mariathasan et al. 2006). However, the ability of bacteria to induce ATP release and subsequent inflammasome activation in microglia has not yet been examined. Exposure of microglia to live S. aureus led to significant ATP release over time, indicating evidence of a DAMP signal (Fig. 5). Pre-treatment of microglia with the P2X7R inhibitor, AZ11645373 significantly inhibited ATP levels, suggesting that ATP is released from microglia via the channel created following P2X7R activation (Fig. 5). To determine whether this bacterially-induced ATP release influenced inflammasome activation in microglia, cells were treated with various doses of AZ11645373. Indeed, P2X7R blockade significantly reduced IL-1β production in WT microglia following live S. aureus treatment in a dose-dependent manner (Fig 6A). Interestingly, P2X7R inhibition further attenuated IL-1β release in both NLRP3 and ASC KO microglia (Fig. 6B and C, respectively). However, AZ11645373 did not completely abolish IL-1β release, indicating the existence of alternative ASC- and P2X7R-independent mechanism(s) for IL-1β cleavage.

Figure 5. S. aureus treatment induces ATP release from microglia.

Figure 5

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Primary microglia isolated from C57BL/6 mice were exposed to live S. aureus strain USA300 for the indicated intervals, whereupon ATP release was determined using a luminescence-based assay. To evaluate whether P2X7R activation influenced ATP release, microglia were pre-treated with the P2X7R inhibitor AZ11645373 (AZ; 50 μM) for 30 min prior to S. aureus exposure. Significant differences in ATP release from microglia treated with S. aureus only versus S. aureus + AZ are denoted with asterisks (***, p ≤ 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 2 separate experiments.

Figure 6. S. aureus-induced IL-1β production is partially P2X7R-dependent.

Figure 6

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C57BL/6 WT (A), NLRP3 KO (B), or ASC KO (C) microglia were exposed to live S. aureus USA300 ± various concentrations of the P2X7R inhibitor AZ11645373 (AZ) for 6 h, whereupon IL-1β production was assessed by ELISA, with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences between microglia treated with S. aureus only versus S. aureus + AZ are denoted with asterisks (***, p < 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 5 separate experiments. N.D. = not detected.

One possibility to account for the residual IL-1β release observed in ASC KO microglia concomitant with P2X7R blockade is the involvement of alternative inflammasome components that are able to activate the final common enzyme, caspase-1, independent of the adaptor ASC. To examine the involvement of ASC-independent, caspase-1-dependent processing of IL-1β in microglia, caspase-1 activity was blocked with the specific inhibitor Z-WEHD-FMK. Treatment of WT, NLRP3 KO, and ASC KO microglia with the caspase-1 inhibitor led to a dose-dependent decrease in IL-1β expression (Fig. 7A and data not shown). Combinational treatment of microglia with both caspase-1 and P2X7R inhibitors nearly completely attenuated IL-1β release in response to live S. aureus in WT microglia (Fig. 7B). This finding indicates that the majority of IL-1β processing in microglia is attributable to autocrine/paracrine actions of ATP mediated by P2X7R, in addition to caspase-1 that is triggered via both ASC-dependent and –independent pathways. The low levels of residual IL-1β detected following caspase-1 and P2X7R blockade suggests the possibility of alternative enzymatic activities in microglia involved IL-1β cleavage apart from caspase-1.

Figure 7. The majority of IL-1β processing in microglia is attributable to autocrine/paracrine actions of ATP mediated by P2X7R in addition to caspase-1.

Figure 7

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C57BL/6 WT microglia were pretreated with various concentrations of the caspase-1 specific inhibitor Z-WEHD-FMK only (A) or Z-WEHD-FMK in combination with the P2X7R inhibitor AZ11645373 (AZ; B) for 30 min, whereupon microglia were exposed to live S. aureus USA300 for 6 h. IL-1β production was assessed by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences between microglia treated with S. aureus only versus S. aureus + inhibitors are represented by asterisks (**, p < 0.01; ***, p < 0.001; Student’s t-test). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 3 separate experiments. N.D. = not detected.

Cathepsin B contributes to IL-1β but not IL-18 processing in microglia

Recently, other enzymes have been identified that are capable of processing IL-1β besides caspase-1 (Netea et al. 2010). One example is cathepsin B, which is abundant in lysosomes of mononuclear phagocytes and neutrophils (Chu et al. 2009). However, few studies have examined the functional relevance of cathepsin B in proteolytic cleavage of pro-IL-1β following bacterial exposure (Duncan et al. 2009), although responses to DAMPs have been investigated (Halle et al. 2008; Terada et al. 2010). Blockade of cathepsin B activity with the inhibitor CA-074 Me significantly attenuated microglial IL-1β release in a dose-dependent manner from WT, NLRP3 KO, and ASC KO microglia (Fig. 8A-C, respectively), but had no effect on IL-18 secretion (Fig. 9). Although IL-18 release appeared elevated in ASC KO microglia following cathepsin B inhibitor treatment, this difference was not statistically significant and not observed in other independent experiments. A recent report demonstrated that CA074Me inhibited bacterial DNA recognition by TLR9 (Matsumoto et al. 2008), which may have impacted IL-1β expression in our studies by attenuating pro-IL-1β levels. However, treatment of microglia with an inhibitory ODN that blocks TLR9 signaling had no impact on the ability of CA074Me to attenuate IL-1β release (Supplemental Fig. 1), confirming that cathepsin B activity is partially responsible for IL-1β processing. To our knowledge, this is the first demonstration of inflammasome-independent cleavage of IL-1β in response to an infectious pathogen in microglia and demonstrates the numerous redundant mechanisms that microglia can utilize to elicit IL-1β release. This is perhaps not surprising given the importance of IL-1β in the host response to CNS bacterial infection (Kielian et al. 2004a; Garg et al. 2009).

Figure 8. Cathepsin B contributes to IL-1β activation in microglia.

Figure 8

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C57BL/6 WT (A), NLRP3 KO (B), or ASC KO (C) microglia were pretreated with the cathepsin B inhibitor CA-074 Me for 30 min, whereupon cells were exposed to live S. aureus USA300 for 6 h. IL-1β production was assessed by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences between microglia treated with S. aureus only versus S. aureus + cathepsin B inhibitor are denoted by asterisks (*, p < 0.05; **, p < 0.01; and ***, p < 0.001; One-way ANOVA). Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 3 separate experiments. N.D. = not detected.

Figure 9. Cathepsin B does not influence IL-18 processing in microglia.

Figure 9

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C57BL/6 WT (A), NLRP3 KO (B), or ASC KO (C) microglia were pretreated with the cathepsin B inhibitor CA-074 Me for 30 min, whereupon cells were exposed to live S. aureus USA300 for 6 h. IL-18 production was assessed by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Results are reported as the mean ± SD of three independent wells for each experimental treatment and were similar across 3 separate experiments. N.D. = not detected.

DISCUSSION

Numerous pathophysiological processes occur during brain abscess development, including necrosis, apoptosis, and robust parenchymal inflammation (Stenzel et al. 2005b; Esen and Kielian 2009). One of the main pro-inflammatory cytokines that is essential for generating a protective CNS anti-bacterial immune response is IL-1β (Kielian et al. 2004a). The importance of IL-1β in the experimental brain abscess model is further substantiated by our recent studies demonstrating enhanced mortality rates and bacterial burdens in IL-1R KO mice (Xiong and Kielian, unpublished observations). However, the molecular mechanisms leading to IL-1β production and the relative contributions of bacterial factors in this process have not yet been determined. Therefore, we employed a comprehensive approach to investigate triggers of IL-1β production from both the microglial and bacterial perspectives.

With regard to microglia, we first examined the cytoplasmic receptor NLRP3 since it has been implicated in mediating inflammasome activation in response to the largest array of PAMPs, including S. aureus as well as DAMPs in several cell types (Miller et al. 2007; Petrilli et al. 2007; Dostert et al. 2008; Craven et al. 2009; Gasse et al. 2009; Munoz-Planillo et al. 2009; Shimada et al. 2010). Microglia from NLRP3 KO mice produced significantly less IL-1β compared to WT cells, implicating a role for this cytoplasmic receptor in cytokine processing. However, since residual IL-1β was still detected, we next examined the downstream inflammasome adaptor ASC. Specifically, ASC contains a C-terminal pyrin domain, which serves to bridge NLRP3 and other NLRs that lack a caspase-recruiting domain (CARD) to caspase-1 (Mariathasan 2007). Therefore, the absence of ASC would impact additional cytoplasmic NLR sensors besides NLRP3 and potentially identify alternative routes for IL-1β activation. Indeed, IL-1β release was more substantially attenuated in ASC KO compared to NLRP3 KO microglia in response to S. aureus. This finding indicates that both NLRP3 and additional unidentified receptor(s) that utilize ASC to bridge caspase-1 are operative in microglia. Since IL-1β release was not completely ablated in ASC KO microglia following S. aureus exposure, this suggested that alternative ASC-independent pathways are also capable of eliciting caspase-1 activation and subsequent IL-1β processing.

Another potential mechanism that may influence IL-1β release is the induction of danger signals that have been implicated in NLRP3 inflammasome activation (Babelova et al. 2009; Gasse et al. 2009). One such danger signal is ATP, which we show here is released from primary microglia following exposure to live S. aureus. Previous studies with astrocytes have shown that P2X7R activation by extracellular ATP triggers further ATP release via the same receptor, resulting in an amplification loop (Anderson et al. 2004; Suadicani et al. 2006). To determine whether microglial ATP release impacted IL-1β processing in an autocrine/paracrine manner, microglia were treated with the irreversible P2X7R inhibitor AZ11645373 that effectively blocks K+ efflux from the cell, a trigger known to activate the NLRP3 inflammasome (Mariathasan et al. 2006; Petrilli et al. 2007). Microglia released ATP in a time-dependent manner in response to S. aureus treatment, which could be inhibited by P2X7R blockade. Furthermore, P2X7R blockade led to a dose-dependent inhibition of IL-1β release in WT microglia and was capable of further attenuating IL-1β production in ASC KO and NLRP3 KO cells. Activated P2X7Rs form pores that allow the passage of small molecules (i.e. < 1,200 Da) along with ions such as Ca2+ and K+ between the intracellular/extracellular milieus (Wang et al. 1996; Chen and Chen 1998; Gever et al. 2006; Suadicani et al. 2006). Therefore, theoretically any number of signals traversing this channel could be contributing to inflammasome activation. One such example is the ability of P2X7R activation to induce cathepsin B release in macrophages (Lopez-Castejon et al. 2010). Indeed, we have demonstrated that cathepsin B inhibition attenuated IL-1β release by WT, NLRP3 KO, and ASC KO microglia, identifying an inflammasome-independent mode of cytokine processing. It remains uncertain how cathepsin B impacts inflammasome activation since the two components are found in distinct intracellular compartments. To date, only two studies have examined the role of cathepsin B in NLRP3 inflammasome activation in microglia. One report suggests that cathepsin B is involved in IL-1β processing in response to chromogranin B stimulation (Terada et al. 2010). Furthermore, it has been shown that lysosomal damage elicited by β-amyloid leads to cathepsin B release and IL-1β processing via a caspase-1-dependent pathway (Halle et al. 2008). We expect that a similar mechanism could occur during S. aureus infection, in that phagocytosis of live bacteria induces lysosomal damage and cathepsin B release into the cytosol, where it is able to impact inflammasome activation. This explanation is further substantiated by recent studies demonstrating that S. aureus is capable of phagosomal escape and intracellular survival (Gresham et al. 2000; Watanabe et al. 2007; Kubica et al. 2008). However, this possibility currently remains speculative and additional studies are needed to evaluate the consequences of live S. aureus on lysosomal damage and/or cathepsin B release into the cytoplasm. Collectively, these findings demonstrate the existence of multiple mechanisms that can be employed for IL-1β processing in microglia (Fig. 10). The complexity of the system is not unexpected since rapid IL-1β induction is essential for an effective anti-bacterial immune response during CNS bacterial infection (Kielian and Hickey 2000; Kielian et al. 2004a).

Figure 10. Multiple pathways are involved in eliciting IL-1β secretion from microglia.

Figure 10

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Microglia utilize the NRLP3 inflammasome, in conjunction with the adaptor molecule ASC, to trigger IL-1β release. However, a component of IL-1β release is ASC-independent (indicated by “?”). In addition, live bacteria elicit ATP release from microglia, which acts as a “danger” signal to engage P2X7Rs contributing to IL-1β production. Alpha (α) and gamma (γ) toxin secretion by bacteria also impact IL-1β release, likely via their pore forming abilities. Proteolytic processing of IL-18 does not involve the NLRP3 inflammasome or any of the other pathways identified in this study for IL-1β.

One unexpected finding in the present study was that microglial IL-18 production was independent of NLRP3, ASC, caspase-1, P2X7R, and cathepsin B. This finding differs from what has been reported in macrophages, where both IL-1β and IL-18 are processed via caspase-1-dependent pathways (Mariathasan et al. 2004; Li et al. 2007; Li et al. 2008). The reasons for this are unclear; however, one explanation may be the use of live bacteria in the current study, whereas other reports utilized purified PAMPs in conjunction with ATP, which could conceivably deliver distinct signals to trigger IL-18 processing. In addition, recent studies have revealed that IL-1β and IL-18 are divergently processed in models of CNS inflammation (Gris et al. 2010; Jha et al. 2010) and similarly, we have found that IL-1β but not IL-18 levels are significantly attenuated in NLRP3 and ASC KO mice in our experimental brain abscess model (Hanamsagar and Kielian, unpublished observations). However, it is important to note that several studies did not evaluate IL-18 release in response to various inflammasome stimuli; therefore, the full impact of inflammasome activity on IL-18 processing cannot be appreciated (Franchi et al. 2007; Petrilli et al. 2007). We examined the role of cathepsin B as an alternative mechanism for IL-1β and IL-18 cleavage and found that cathepsin B inhibition could significantly attenuate IL-1β release from WT as well as NLRP3 and ASC KO microglia. However, IL-18 levels were not affected by the treatment of cathepsin B inhibitor, indicating the existence of novel mechanisms for IL-18 cleavage in microglia.

Recent studies have independently demonstrated the importance of S. aureus α-hemolysin in NLRP3 inflammasome activation (Mariathasan et al. 2006; Franchi et al. 2007; Craven et al. 2009). However, since these reports utilized either purified α-hemolysin or S. aureus supernatants, the physiological significance of these findings with regard to infection is uncertain. Our approach to utilize live S. aureus and isogenic toxin mutants presents microglia with the complex milieu of PAMPs and secreted virulence factors that is reminiscent of infection in vivo. Indeed, the fact that we identified numerous distinct pathways contributing to IL-1β processing in microglia was likely the result of our strategy to utilize live bacteria (Fig. 10). Both S. aureus α- and γ-hemolysins elicited IL-1β release from microglia; however, additional virulence determinants were also involved since significant amounts of IL-1β were still elicited by both Δhla and ΔhlgACB mutants. It is likely that multiple toxins exert additive/synergistic effects to regulate IL-1β processing in microglia, and testing this will require the generation of S. aureus strains that are deficient for multiple toxins. It is interesting that only α- and γ-hemolysins, but not the leukotoxins lukAB and lukED, impacted IL-1β processing since they all form pores in mammalian cell membranes (Bhakdi and Tranum-Jensen 1991; Menestrina et al. 1995; Gravet et al. 1998; Staali et al. 1998; Menestrina et al. 2003; Morinaga et al. 2003; Ventura et al. 2010; Dumont et al. 2011), which would be expected to induce K+ efflux, a known stimulus for inflammasome activation (Mariathasan et al. 2006; Petrilli et al. 2007). Interestingly, we were not able to detect secreted IL-1β by Western blotting in any treatment groups due to its rapid degradation by S. aureus proteases (data not shown), whereas the smaller cleavage forms of IL-1β were still detectable by ELISA.

A two signal model has been proposed to elicit IL-1β release, namely, transcriptional activation of the IL-1β gene must initially occur, followed by proteolytic processing of pro-IL-1β to its mature form (Tschopp and Schroder 2010). In terms of microglia, previous studies from our laboratory have identified TLR2 and MyD88 as important molecules leading to IL-1β expression in response to S. aureus (Kielian et al. 2005; Esen and Kielian 2006). In the current study, we extend these findings to identify the molecular machinery responsible for IL-1β secretion from microglia. To our knowledge, this study is the first to describe the complex pathways that microglia can utilize to regulate IL-1β processing. First, the cytokine can be induced by NLRP3-dependent pathways involving the adaptor ASC. Second, live bacteria induce ATP release, which acts via P2X7R to further trigger IL-1β production. Third, we have identified a novel pathway by which microglia can secrete IL-1β that is caspase-1-independent, although the contribution of this pathway is relatively minor compared to the other mechanisms characterized. Finally, we have identified bacterial toxins that are involved in triggering IL-1β release (Fig. 10). The multitude of pathways that can elicit IL-1β production by microglia reflects the fact that this cytokine is a key mediator of CNS inflammation. Studies are ongoing in our laboratory to further investigate the IL-1 system and the functional importance of inflammasome activation in vivo during CNS infection as well as identify inflammasome-independent modes of IL-18 processing.

Supplementary Material

Supp Fig S1

ACKNOWLEDGEMENTS

This work was supported by the NIH National Institute of Neurological Disorders and Stroke 2R01 NS055385 to T.K. We thank Amanda Angle for excellent technical assistance, Dr. Vishva Dixit for generously providing NLRP3 and ASC KO mice, and Dr. Costi Sifri for the USA300 CNS isolate.

Footnotes

The authors have no competing interests to declare.

REFERENCES

  1. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J. NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity. 2004;20:319–325. doi: 10.1016/s1074-7613(04)00046-9. [DOI] [PubMed] [Google Scholar]
  2. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, Stein L, Russo R, Goldsmith D, Dent P, Rosenberg HF, Austin F, Remmers EF, Balow JE, Jr., Rosenzweig S, Komarow H, Shoham NG, Wood G, Jones J, Mangra N, Carrero H, Adams BS, Moore TL, Schikler K, Hoffman H, Lovell DJ, Lipnick R, Barron K, O’Shea JJ, Kastner DL, Goldbach-Mansky R. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum. 2002;46:3340–3348. doi: 10.1002/art.10688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson CM, Bergher JP, Swanson RA. ATP-induced ATP release from astrocytes. J Neurochem. 2004;88:246–256. doi: 10.1111/j.1471-4159.2004.02204.x. [DOI] [PubMed] [Google Scholar]
  4. Andersson PB, Perry VH, Gordon S. The kinetics and morphological characteristics of the macrophage-microglial response to kainic acid-induced neuronal degeneration. Neuroscience. 1991;42:201–214. doi: 10.1016/0306-4522(91)90159-l. [DOI] [PubMed] [Google Scholar]
  5. Arostegui JI, Aldea A, Modesto C, Rua MJ, Arguelles F, Gonzalez-Ensenat MA, Ramos E, Rius J, Plaza S, Vives J, Yague J. Clinical and genetic heterogeneity among Spanish patients with recurrent autoinflammatory syndromes associated with the CIAS1/PYPAF1/NALP3 gene. Arthritis Rheum. 2004;50:4045–4050. doi: 10.1002/art.20633. [DOI] [PubMed] [Google Scholar]
  6. Arostegui JI, Lopez Saldana M. D., Pascal M, Clemente D, Aymerich M, Balaguer F, Goel A, del Castillo C. Fournier, Rius J, Plaza S, Robledillo J. C. Lopez, Juan M, Ibanez M, Yague J. A somatic NLRP3 mutation as a cause of a sporadic case of chronic infantile neurologic, cutaneous, articular syndrome/neonatal-onset multisystem inflammatory disease: Novel evidence of the role of low-level mosaicism as the pathophysiologic mechanism underlying mendelian inherited diseases. Arthritis Rheum. 2010;62:1158–1166. doi: 10.1002/art.27342. [DOI] [PubMed] [Google Scholar]
  7. Babelova A, Moreth K, Tsalastra-Greul W, Zeng-Brouwers J, Eickelberg O, Young MF, Bruckner P, Pfeilschifter J, Schaefer RM, Grone HJ, Schaefer L. Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors. J Biol Chem. 2009;284:24035–24048. doi: 10.1074/jbc.M109.014266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bhakdi S, Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991;55:733–751. doi: 10.1128/mr.55.4.733-751.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bryant C, Fitzgerald KA. Molecular mechanisms involved in inflammasome activation. Trends Cell Biol. 2009;19:455–464. doi: 10.1016/j.tcb.2009.06.002. [DOI] [PubMed] [Google Scholar]
  10. Chen WC, Chen CC. ATP-induced arachidonic acid release in cultured astrocytes is mediated by Gi protein coupled P2Y1 and P2Y2 receptors. Glia. 1998;22:360–370. doi: 10.1002/(sici)1098-1136(199804)22:4<360::aid-glia5>3.0.co;2-7. [DOI] [PubMed] [Google Scholar]
  11. Chu J, Thomas LM, Watkins SC, Franchi L, Nunez G, Salter RD. Cholesterol-dependent cytolysins induce rapid release of mature IL-1beta from murine macrophages in a NLRP3 inflammasome and cathepsin B-dependent manner. J Leukoc Biol. 2009;86:1227–1238. doi: 10.1189/jlb.0309164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Craven RR, Gao X, Allen IC, Gris D, Wardenburg J. Bubeck, McElvania-Tekippe E, Ting JP, Duncan JA. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells. PLoS One. 2009;4:e7446. doi: 10.1371/journal.pone.0007446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cruz CM, Rinna A, Forman HJ, Ventura AL, Persechini PM, Ojcius DM. ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. J Biol Chem. 2007;282:2871–2879. doi: 10.1074/jbc.M608083200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008;320:674–677. doi: 10.1126/science.1156995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dumont AL, Nygaard TK, Watkins RL, Smith A, Kozhaya L, Kreiswirth BN, Shopsin B, Unutmaz D, Voyich JM, Torres VJ. Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis. Mol Microbiol. 2011;79:814–825. doi: 10.1111/j.1365-2958.2010.07490.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duncan JA, Gao X, Huang MT, O’Connor BP, Thomas CE, Willingham SB, Bergstralh DT, Jarvis GA, Sparling PF, Ting JP. Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome. J Immunol. 2009;182:6460–6469. doi: 10.4049/jimmunol.0802696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Esen N, Kielian T. Central role for MyD88 in the responses of microglia to pathogen-associated molecular patterns. J Immunol. 2006;176:6802–6811. doi: 10.4049/jimmunol.176.11.6802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Esen N, Kielian T. Toll-like receptors in brain abscess. Curr Top Microbiol Immunol. 2009;336:41–61. doi: 10.1007/978-3-642-00549-7_3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Esen N, Tanga FY, DeLeo JA, Kielian T. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J Neurochem. 2004;88:746–758. doi: 10.1046/j.1471-4159.2003.02202.x. [DOI] [PubMed] [Google Scholar]
  20. Franchi L, Kanneganti TD, Dubyak GR, Nunez G. Differential requirement of P2X7 receptor and intracellular K+ for caspase-1 activation induced by intracellular and extracellular bacteria. J Biol Chem. 2007;282:18810–18818. doi: 10.1074/jbc.M610762200. [DOI] [PubMed] [Google Scholar]
  21. Garg S, Nichols JR, Esen N, Liu S, Phulwani NK, Syed MM, Wood WH, Zhang Y, Becker KG, Aldrich A, Kielian T. MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses. ASN Neuro. 2009;1 doi: 10.1042/AN20090004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gasse P, Riteau N, Charron S, Girre S, Fick L, Petrilli V, Tschopp J, Lagente V, Quesniaux VF, Ryffel B, Couillin I. Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis. Am J Respir Crit Care Med. 2009;179:903–913. doi: 10.1164/rccm.200808-1274OC. [DOI] [PubMed] [Google Scholar]
  23. Gever JR, Cockayne DA, Dillon MP, Burnstock G, Ford AP. Pharmacology of P2X channels. Pflugers Arch. 2006;452:513–537. doi: 10.1007/s00424-006-0070-9. [DOI] [PubMed] [Google Scholar]
  24. Gravet A, Colin DA, Keller D, Girardot R, Monteil H, Prevost G. Characterization of a novel structural member, LukE-LukD, of the bi-component staphylococcal leucotoxins family. FEBS Lett. 1998;436:202–208. doi: 10.1016/s0014-5793(98)01130-2. [DOI] [PubMed] [Google Scholar]
  25. Gresham HD, Lowrance JH, Caver TE, Wilson BS, Cheung AL, Lindberg FP. Survival of Staphylococcus aureus inside neutrophils contributes to infection. J Immunol. 2000;164:3713–3722. doi: 10.4049/jimmunol.164.7.3713. [DOI] [PubMed] [Google Scholar]
  26. Gris D, Ye Z, Iocca HA, Wen H, Craven RR, Gris P, Huang M, Schneider M, Miller SD, Ting JP. NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J Immunol. 2010;185:974–981. doi: 10.4049/jimmunol.0904145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gurley C, Nichols J, Liu S, Phulwani NK, Esen N, Kielian T. Microglia and Astrocyte Activation by Toll-Like Receptor Ligands: Modulation by PPAR-gamma Agonists. PPAR Res. 2008;2008:453120. doi: 10.1155/2008/453120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol. 2008;9:857–865. doi: 10.1038/ni.1636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat Genet. 2001;29:301–305. doi: 10.1038/ng756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jha S, Srivastava SY, Brickey WJ, Iocca H, Toews A, Morrison JP, Chen VS, Gris D, Matsushima GK, Ting JP. The Inflammasome Sensor, NLRP3, Regulates CNS Inflammation and Demyelination via Caspase-1 and Interleukin-18. J Neurosci. 2010;30:15811–15820. doi: 10.1523/JNEUROSCI.4088-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jones ME, Draghi DC, Karlowsky JA, Sahm DF, Bradley JS. Prevalence of antimicrobial resistance in bacteria isolated from central nervous system specimens as reported by U.S. hospital laboratories from 2000 to 2002. Ann Clin Microbiol Antimicrob. 2004;3:3. doi: 10.1186/1476-0711-3-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kankkunen P, Teirila L, Rintahaka J, Alenius H, Wolff H, Matikainen S. 1,3)-beta-glucans activate both dectin-1 and NLRP3 inflammasome in human macrophages. J Immunol. 2010;184:6335–6342. doi: 10.4049/jimmunol.0903019. [DOI] [PubMed] [Google Scholar]
  33. Kielian T, Hickey WF. Proinflammatory cytokine, chemokine, and cellular adhesion molecule expression during the acute phase of experimental brain abscess development. Am J Pathol. 2000;157:647–658. doi: 10.1016/S0002-9440(10)64575-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kielian T, Mayes P, Kielian M. Characterization of microglial responses to Staphylococcus aureus: effects on cytokine, costimulatory molecule, and Toll-like receptor expression. J Neuroimmunol. 2002;130:86–99. doi: 10.1016/s0165-5728(02)00216-3. [DOI] [PubMed] [Google Scholar]
  35. Kielian T, Esen N, Bearden ED. Toll-like receptor 2 (TLR2) is pivotal for recognition of S. aureus peptidoglycan but not intact bacteria by microglia. Glia. 2005;49:567–576. doi: 10.1002/glia.20144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kielian T, Bearden ED, Baldwin AC, Esen N. IL-1 and TNF-alpha play a pivotal role in the host immune response in a mouse model of Staphylococcus aureus-induced experimental brain abscess. J Neuropathol Exp Neurol. 2004a;63:381–396. doi: 10.1093/jnen/63.4.381. [DOI] [PubMed] [Google Scholar]
  37. Kielian T, McMahon M, Bearden ED, Baldwin AC, Drew PD, Esen N. S. aureus-dependent microglial activation is selectively attenuated by the cyclopentenone prostaglandin 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2) J Neurochem. 2004b;90:1163–1172. doi: 10.1111/j.1471-4159.2004.02579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kielian T, Phulwani NK, Esen N, Syed MM, Haney AC, McCastlain K, Johnson J. MyD88-dependent signals are essential for the host immune response in experimental brain abscess. J Immunol. 2007;178:4528–4537. doi: 10.4049/jimmunol.178.7.4528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 1996;19:312–318. doi: 10.1016/0166-2236(96)10049-7. [DOI] [PubMed] [Google Scholar]
  40. Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, Golda A, Maciag-Gudowska A, Brix K, Shaw L, Foster T, Potempa J. A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages. PLoS One. 2008;3:e1409. doi: 10.1371/journal.pone.0001409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lamkanfi M, Dixit VM. The inflammasomes. PLoS Pathog. 2009;5:e1000510. doi: 10.1371/journal.ppat.1000510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lawson LJ, Frost L, Risbridger J, Fearn S, Perry VH. Quantification of the mononuclear phagocyte response to Wallerian degeneration of the optic nerve. J Neurocytol. 1994;23:729–744. doi: 10.1007/BF01268086. [DOI] [PubMed] [Google Scholar]
  43. Li H, Nookala S, Re F. Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release. J Immunol. 2007;178:5271–5276. doi: 10.4049/jimmunol.178.8.5271. [DOI] [PubMed] [Google Scholar]
  44. Li H, Willingham SB, Ting JP, Re F. Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3. J Immunol. 2008;181:17–21. doi: 10.4049/jimmunol.181.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. J Immunol. 2010;185:2611–2619. doi: 10.4049/jimmunol.1000436. [DOI] [PubMed] [Google Scholar]
  46. Mariathasan S. ASC, Ipaf and Cryopyrin/Nalp3: bona fide intracellular adapters of the caspase-1 inflammasome. Microbes Infect. 2007;9:664–671. doi: 10.1016/j.micinf.2007.01.017. [DOI] [PubMed] [Google Scholar]
  47. Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S, Dixit VM. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature. 2004;430:213–218. doi: 10.1038/nature02664. [DOI] [PubMed] [Google Scholar]
  48. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature. 2006;440:228–232. doi: 10.1038/nature04515. [DOI] [PubMed] [Google Scholar]
  49. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–241. doi: 10.1038/nature04516. [DOI] [PubMed] [Google Scholar]
  50. Matsumoto F, Saitoh S, Fukui R, Kobayashi T, Tanimura N, Konno K, Kusumoto Y, Akashi-Takamura S, Miyake K. Cathepsins are required for Toll-like receptor 9 responses. Biochem Biophys Res Commun. 2008;367:693–699. doi: 10.1016/j.bbrc.2007.12.130. [DOI] [PubMed] [Google Scholar]
  51. Menestrina G, Serra M. Dalla, Pederzolli C, Bregante M, Gambale F. Bacterial hemolysins and leukotoxins affect target cells by forming large exogenous pores into their plasma membrane: Escherichia coli hemolysin A as a case example. Biosci Rep. 1995;15:543–551. doi: 10.1007/BF01204356. [DOI] [PubMed] [Google Scholar]
  52. Menestrina G, Serra M. Dalla, Comai M, Coraiola M, Viero G, Werner S, Colin DA, Monteil H, Prevost G. Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus. FEBS Lett. 2003;552:54–60. doi: 10.1016/s0014-5793(03)00850-0. [DOI] [PubMed] [Google Scholar]
  53. Miller LS, Pietras EM, Uricchio LH, Hirano K, Rao S, Lin H, O’Connell RM, Iwakura Y, Cheung AL, Cheng G, Modlin RL. Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo. J Immunol. 2007;179:6933–6942. doi: 10.4049/jimmunol.179.10.6933. [DOI] [PubMed] [Google Scholar]
  54. Morinaga N, Kaihou Y, Noda M. Purification, cloning and characterization of variant LukE-LukD with strong leukocidal activity of staphylococcal bi-component leukotoxin family. Microbiol Immunol. 2003;47:81–90. doi: 10.1111/j.1348-0421.2003.tb02789.x. [DOI] [PubMed] [Google Scholar]
  55. Munoz-Planillo R, Franchi L, Miller LS, Nunez G. A critical role for hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome. J Immunol. 2009;183:3942–3948. doi: 10.4049/jimmunol.0900729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Naesens R, Ronsyn M, Druwe P, Denis O, Ieven M, Jeurissen A. Central nervous system invasion by community-acquired meticillin-resistant Staphylococcus aureus. J Med Microbiol. 2009;58:1247–1251. doi: 10.1099/jmm.0.011130-0. [DOI] [PubMed] [Google Scholar]
  57. Netea MG, Simon A, van de Veerdonk F, Kullberg BJ, Van der Meer JW, Joosten LA. IL-1beta processing in host defense: beyond the inflammasomes. PLoS Pathog. 2010;6:e1000661. doi: 10.1371/journal.ppat.1000661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Netea MG, Nold-Petry CA, Nold MF, Joosten LA, Opitz B, van der Meer JH, van de Veerdonk FL, Ferwerda G, Heinhuis B, Devesa I, Funk CJ, Mason RJ, Kullberg BJ, Rubartelli A, van der Meer JW, Dinarello CA. Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages. Blood. 2009;113:2324–2335. doi: 10.1182/blood-2008-03-146720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Perry VH. A revised view of the central nervous system microenvironment and major histocompatibility complex class II antigen presentation. J Neuroimmunol. 1998;90:113–121. doi: 10.1016/s0165-5728(98)00145-3. [DOI] [PubMed] [Google Scholar]
  60. Perry VH, Gordon S. Macrophages and microglia in the nervous system. Trends Neurosci. 1988;11:273–277. doi: 10.1016/0166-2236(88)90110-5. [DOI] [PubMed] [Google Scholar]
  61. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 2007;14:1583–1589. doi: 10.1038/sj.cdd.4402195. [DOI] [PubMed] [Google Scholar]
  62. Phulwani NK, Esen N, Syed MM, Kielian T. TLR2 expression in astrocytes is induced by TNF-alpha- and NF-kappa B-dependent pathways. J Immunol. 2008;181:3841–3849. doi: 10.4049/jimmunol.181.6.3841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Qu Y, Franchi L, Nunez G, Dubyak GR. Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J Immunol. 2007;179:1913–1925. doi: 10.4049/jimmunol.179.3.1913. [DOI] [PubMed] [Google Scholar]
  64. Rajamaki K, Lappalainen J, Oorni K, Valimaki E, Matikainen S, Kovanen PT, Eklund KK. Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link between cholesterol metabolism and inflammation. PLoS One. 2010;5:e11765. doi: 10.1371/journal.pone.0011765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140:821–832. doi: 10.1016/j.cell.2010.01.040. [DOI] [PubMed] [Google Scholar]
  66. Shimada T, Park BG, Wolf AJ, Brikos C, Goodridge HS, Becker CA, Reyes CN, Miao EA, Aderem A, Gotz F, Liu GY, Underhill DM. Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion. Cell Host Microbe. 2010;7:38–49. doi: 10.1016/j.chom.2009.12.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Sifri CD, Park J, Helm GA, Stemper ME, Shukla SK. Fatal brain abscess due to community-associated methicillin-resistant Staphylococcus aureus strain USA300. Clin Infect Dis. 2007;45:e113–117. doi: 10.1086/522171. [DOI] [PubMed] [Google Scholar]
  68. Staali L, Monteil H, Colin DA. The staphylococcal pore-forming leukotoxins open Ca2+ channels in the membrane of human polymorphonuclear neutrophils. J Membr Biol. 1998;162:209–216. doi: 10.1007/s002329900358. [DOI] [PubMed] [Google Scholar]
  69. Stenzel W, Soltek S, Miletic H, Hermann MM, Korner H, Sedgwick JD, Schluter D, Deckert M. An essential role for tumor necrosis factor in the formation of experimental murine Staphylococcus aureus-induced brain abscess and clearance. J Neuropathol Exp Neurol. 2005a;64:27–36. doi: 10.1093/jnen/64.1.27. [DOI] [PubMed] [Google Scholar]
  70. Stenzel W, Dahm J, Sanchez-Ruiz M, Miletic H, Hermann M, Courts C, Schwindt H, Utermohlen O, Schluter D, Deckert M. Regulation of the inflammatory response to Staphylococcus aureus-induced brain abscess by interleukin-10. J Neuropathol Exp Neurol. 2005b;64:1046–1057. doi: 10.1097/01.jnen.0000189836.48704.ca. [DOI] [PubMed] [Google Scholar]
  71. Suadicani SO, Brosnan CF, Scemes E. P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci. 2006;26:1378–1385. doi: 10.1523/JNEUROSCI.3902-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Terada K, Yamada J, Hayashi Y, Wu Z, Uchiyama Y, Peters C, Nakanishi H. Involvement of cathepsin B in the processing and secretion of interleukin-1beta in chromogranin A-stimulated microglia. Glia. 2010;58:114–124. doi: 10.1002/glia.20906. [DOI] [PubMed] [Google Scholar]
  73. Thrash JC, Torbett BE, Carson MJ. Developmental regulation of TREM2 and DAP12 expression in the murine CNS: implications for Nasu-Hakola disease. Neurochem Res. 2009;34:38–45. doi: 10.1007/s11064-008-9657-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Townsend GC, Scheld WM. Infections of the central nervous system. Adv Intern Med. 1998;43:403–447. [PubMed] [Google Scholar]
  75. Tschopp J, Schroder K. NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? Nat Rev Immunol. 2010;10:210–215. doi: 10.1038/nri2725. [DOI] [PubMed] [Google Scholar]
  76. Ventura CL, Malachowa N, Hammer CH, Nardone GA, Robinson MA, Kobayashi SD, DeLeo FR. Identification of a novel Staphylococcus aureus two-component leukotoxin using cell surface proteomics. PLoS One. 2010;5:e11634. doi: 10.1371/journal.pone.0011634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Wang Y, Roman R, Lidofsky SD, Fitz JG. Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. Proc Natl Acad Sci U S A. 1996;93:12020–12025. doi: 10.1073/pnas.93.21.12020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Watanabe I, Ichiki M, Shiratsuchi A, Nakanishi Y. TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity. J Immunol. 2007;178:4917–4925. doi: 10.4049/jimmunol.178.8.4917. [DOI] [PubMed] [Google Scholar]

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