SEARCH

SEARCH BY CITATION

Keywords:

  • AIM2;
  • ASC ;
  • caspase 1;
  • inflammasome;
  • interleukin-1;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
Thumbnail image of graphical abstract

Interleukin-1β (IL-1β) is essential for eliciting protective immunity during the acute phase of Staphylococcus aureus (S. aureus) infection in the central nervous system (CNS). We previously demonstrated that microglial IL-1β production in response to live S. aureus is mediated through the Nod-like receptor protein 3 (NLRP3) inflammasome, including the adapter protein ASC (apoptosis-associated speck-like protein containing a caspase-1 recruitment domain), and pro-caspase 1. Here, we utilized NLRP3, ASC, and caspase 1/11 knockout (KO) mice to demonstrate the functional significance of inflammasome activity during CNS S. aureus infection. ASC and caspase 1/11 KO animals were exquisitely sensitive, with approximately 50% of mice succumbing to infection within 24 h. Unexpectedly, the survival of NLRP3 KO mice was similar to wild-type animals, suggesting the involvement of an alternative upstream sensor, which was later identified as absent in melanoma 2 (AIM2) based on the similar disease patterns between AIM2 and ASC KO mice. Besides IL-1β, other key inflammatory mediators, including IL-6, CXCL1, CXCL10, and CCL2 were significantly reduced in the CNS of AIM2 and ASC KO mice, implicating autocrine/paracrine actions of IL-1β, as these mediators do not require inflammasome processing for secretion. These studies demonstrate a novel role for the AIM2 inflammasome as a critical molecular platform for regulating IL-1β release and survival during acute CNS S. aureus infection.

The AIM2 inflammasome is protective during acute CNS bacterial infection. A disconnect in phenotypes between the inflammasome sensor Nod-like receptor protein 3 (NLRP3) and its adaptor ASC (apoptosis-associated speck-like protein containing a caspase-1 recruitment domain) during acute CNS Staphylococcus aureus (S. aureus) infection led to the discovery of absent in melanoma 2 (AIM2) as a critical inflammasome sensor. The AIM2 inflammasome is potentially triggered by dsDNA in cells harboring intracellular S. aureus, leading to ASC and caspase 1 recruitment, resulting in pro-IL-1β processing and cytokine secretion. This cascade, in turn, is protective to the host during acute infection. The NLRP3 inflammasome is also activated in response to S. aureus challenge by α-hemolysin (hla); however, it is not critical for host survival. ASC also regulates the production of other inflammatory mediators, presumably via indirect effects mediated by IL-1β action.

Abbreviations used
AIM2

absent in melanoma 2

ASC

apoptosis-associated speck-like protein containing a caspase-1 recruitment domain

MRSA

methicillin-resistant Staphylococcus aureus

NLRP3

Nod-like receptor protein 3

ROS

reactive oxygen species

Staphylococcus aureus (S. aureus) is a serious human health threat because of its ability to cause a wide array of diseases, ranging from skin and soft tissue infections to more life-threatening conditions such as sepsis, endocarditis, and brain abscess (Lowy 1998). Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a frequent cause of hospital- and community-associated infections, which complicates treatment (Drago et al. 2007) and MRSA isolates have been recovered from brain abscesses (Sifri et al. 2007; Naesens et al. 2009; Arora et al. 2012; David et al. 2012). Brain abscesses can originate from many sources, including: (i) subacute or chronic otitis media, mastoiditis, frontal or ethmoid sinusitis, or dental infection; (ii) hematogenous dissemination from peripheral sites of infection, including bacterial endocarditis or lung abscesses (Madsen 1983; Kassis et al. 2010; Wadhwa et al. 2012); (iii) immunocompromised patients with HIV infection, organ transplantation, chemotherapy, or steroid usage (Young and McGwire 2005; Muzumdar et al. 2011); (iv) penetrating trauma to the calvarium; or (v) infectious complications following neurosurgical procedures. Without timely treatment intervention, a brain abscess can rupture, which is associated with a high mortality rate. Therefore, elucidating mechanisms of protection as well as pathophysiology elicited by S. aureus after CNS colonization may unveil novel therapeutic targets.

Numerous proinflammatory cytokines are elicited during acute CNS S. aureus infection, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) (Kielian et al. 2004, 2007b; Stenzel et al. 2005; Garg et al. 2009; Xiong et al. 2012). Our prior studies utilizing IL-1β and IL-1RI knockout (KO) mice demonstrated a critical role for IL-1 signaling during early brain abscess pathogenesis, as both mouse strains displayed marked susceptibility to infection and elevated bacterial burdens compared to wild-type (WT) animals (Kielian et al. 2004; Xiong et al. 2012). Our recent report showed that microglial IL-1β production in response to live S. aureus was mediated by the Nod-like receptor protein 3 (NLRP3) inflammasome (Hanamsagar et al. 2011), which has been studied extensively for its role in IL-1β secretion in response to diverse pathogenic signals, including S. aureus α-hemolysin (Craven et al. 2009). However, little is known about the functional importance of inflammasomes during CNS bacterial infection, with only one report documenting that IL-1β release during S. pneumoniae meningitis is controlled via the NLRP3 inflammasome (Hoegen et al. 2011). To further dissect the underlying mechanisms mediating IL-1β release during early CNS infection, we focused on the NLRP3 inflammasome utilizing a well-established mouse model of S. aureus-induced brain abscess that recapitulates various aspects of human disease. We found that ASC (apoptosis-associated speck-like protein containing a caspase-1 recruitment domain) and caspase 1/11 were critical for survival during the first 24 h after infection, whereas NLRP3 was dispensable, revealing the involvement of a NLRP3-independent, ASC-dependent inflammasome during acute CNS S. aureus infection. In addition to NLRP3, other ASC-dependent inflammasomes have been described including absent in melanoma 2 (AIM2), NLRC4, and NLRP6 (Fernandes-Alnemri et al. 2009; Hornung et al. 2009; Broz et al. 2010; Rathinam et al. 2010; Elinav et al. 2011). When considering alternative inflammasomes that could play a role during CNS S. aureus infection, AIM2 seemed the most plausible since intracellular survival of S. aureus has been reported in phagocytes enabling the detection of cytoplasmic dsDNA, whereas NLRC4 and NLRP6 recognize motifs that are unrelated to S. aureus (Gresham et al. 2000; Watanabe et al. 2007). Subsequent experiments identified this upstream adaptor as AIM2, which was unexpected, since prior to our study, AIM2 had been implicated in sensing dsDNA in macrophages following viral or L. monocytogenes infection (Fernandes-Alnemri et al. 2010; Rathinam et al. 2010; Warren et al. 2010). To our knowledge, this is the first study to examine the functional role of inflammasome components during CNS S. aureus infection and reveals a unique role for AIM2 in S. aureus detection in the brain.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

Animals

NLRP3 and ASC KO mice were generously provided by Dr Vishva Dixit (Genentech, San Francisco, CA, USA) and AIM2 and caspase 1/11 KO mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All KO mice were on a C57BL/6 background. Age- and sex-matched C57BL/6 mice were used as WT controls. Studies were performed with mice between 8 and 12 weeks of age.

Experimental brain abscess model

Brain abscesses were induced in mice as previously described by the intracerebral injection of a MRSA USA300 isolate (CAV1002) recovered from an otherwise healthy individual who succumbed to a brain abscess (Kielian et al. 2001, 2007a; Sifri et al. 2007). 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. This study was performed in accordance with the ARRIVE guidelines.

Quantitation of bacterial titers and proinflammatory mediator expression

Mice were euthanized at the indicated intervals after infection and perfused transcardially with ice-cold phosphate-buffered saline. Brain abscesses were dissected within 1–2 mm of the lesion margins and immediately disrupted in 500 μl of homogenization buffer [1X phosphate-buffered saline supplemented with a protease inhibitor cocktail tablet (Roche, Mannheim, Germany) and RNase inhibitor (Promega, Madison, WI, USA)]. Serial 10-fold dilutions of brain abscess homogenates were plated on trypticase soy-agar plates supplemented with 5% sheep blood (Hemostat Laboratories, Dixon, CA, USA) to determine bacterial titers (expressed as log10 CFU/g wet tissue weight). To compare proinflammatory mediator expression profiles between KO versus WT mice, a multianalyte microbead array was utilized according to the manufacturer's instructions (MILLIPLEX; Millipore, Billerica, MA, USA) that detects IL-1α, IL-1β, TNF-α, IFN-γ, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CCL2, CCL3, CCL4, and CCL5. Results were analyzed using a Bio-Plex workstation (Bio-Rad, Hercules, CA, USA) and adjusted based on the amount of total protein extracted from brain abscess homogenates for normalization using a colorimetric Bio-Rad DC Protein Assay kit (Bio-Rad). IL-1β was also measured by sandwich ELISA (BD OptIEA, San Diego, CA, USA).

Statistical analyses

Significant differences between experimental groups were determined by one-way anova followed by Pairwise Multiple Comparison (Tukey Test) or a Student's t-test to compare between two groups. For both analyses, p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

ASC but not NLRP3 KO mice show enhanced susceptibility to CNS S. aureus infection

Our recent study revealed that the NLRP3 inflammasome was critical for microglial IL-1β secretion in response to live S. aureus (Hanamsagar et al. 2011). To determine whether these findings extended to in vivo infection, NLRP3 and ASC KO mice were challenged intracerebrally with S. aureus and disease progression was monitored. Survival rates between NLRP3 KO and WT mice were not significantly different (Fig. 1a), whereas ASC KO animals displayed increased susceptibility to CNS S. aureus infection, evidenced by a 50% reduction in survival within the first 24 h compared with WT mice (Fig. 1b). This was unexpected, as numerous studies have reported that NLRP3 and ASC KO mice display comparable phenotypes in several disease models (Gris et al. 2010; Jha et al. 2010; Zaki et al. 2010; Hoegen et al. 2011). To eliminate potential confounds from survival bias, ASC KO animals were evaluated at 12 and 18 h post infection in all subsequent experiments, whereas NLRP3 KO animals were assessed at 3 and 7 days. NLRP3 KO mice were examined at later intervals, since early experiments did not reveal any significant differences in survival or inflammation at earlier time points (data not shown). Interestingly, bacterial burdens were similar between ASC KO and WT mice at both 12 and 18 h after infection (Fig. 1e), demonstrating that the enhanced susceptibility of ASC KO mice was not attributable to unchecked bacterial growth. Likewise, S. aureus burdens were similar between NLRP3 KO and WT animals at day 3 after infection; however, bacterial clearance was impaired in NLRP3 KO mice at later intervals (i.e., day 7; Fig. 1d). The dichotomy between NLRP3 and ASC KO phenotypes suggested either the involvement of an alternative NLR sensor that is ASC-dependent or that ASC assumes a non-canonical role during acute CNS S. aureus infection. The former possibility appeared more plausible, since caspase 1/11 KO animals displayed a similar sensitivity to CNS S. aureus infection as ASC KO mice, with no changes observed in bacterial burdens (Fig. 1c and f).

image

Figure 1. The inflammasome molecules ASC and caspase 1/11, but not NLRP3, are critical for survival during acute CNS S. aureus infection. C57BL/6 wild-type (WT) and NLRP3 knockout (KO) (a and d), ASC KO (b and e), or caspase 1/11 KO (c and f) mice were injected intracranially with 104 CFU of S. aureus USA300 and assessed for survival (a–c; n = 7–16/group from 2–6 independent experiments). Bacterial burdens from NLRP3 KO mice (d) were assessed at days 3 and 7, whereas ASC and caspase 1/11 KO mice (e and f, respectively) were determined at 12 and 18 h after infection, to avoid potential confounds from survival bias (n = 7–12/group). Significant differences in survival and bacterial burdens between groups were determined by a log-rank test and Student's t-test, respectively (*< 0.05; **< 0.01).

Download figure to PowerPoint

ASC loss has a broad impact on proinflammatory mediator production in the infected CNS

Based on the profound sensitivity of ASC KO mice to CNS S. aureus infection, we next evaluated whether this translated into altered proinflammatory mediator expression profiles. As expected, IL-1β production was significantly attenuated in ASC KO animals (Fig. 2a). In addition, several other cytokines and chemokines, including IL-6, CCL2, and CXCL10 were also significantly reduced in ASC KO mice (Fig. 2b–d). This was intriguing, as these mediators do not rely on inflammasome processing for their secretion, suggesting that IL-1β influences cytokine/chemokine production in an autocrine/paracrine manner during acute CNS S. aureus infection. Interestingly, IL-18 expression was equivalent between ASC KO and WT mice at all intervals examined (data not shown), corroborating our prior microglial data where IL-18 was processed in an inflammasome-independent manner (Hanamsagar et al. 2011). Other cytokines, such as IL-10, TNF-α, and IFN-γ, and innate immune cell infiltrates (i.e. neutrophils, monocytes, and macrophages) were not significantly different between WT and ASC KO animals at either time point examined (data not shown).

image

Figure 2. ASC loss affects several inflammatory mediators besides interleukin-1β (IL-1β). C57BL/6 wild-type (WT) and ASC knockout (KO) mice (7–8/group) were injected intracranially with 104 CFU of S. aureus USA300, whereupon abscess homogenates were collected at the indicated time points after infection for quantitation of IL-1β (a), IL-6 (b), CCL2 (c), and CXCL10 (d) expression by MILLIPLEX multianalyte bead arrays. Results were normalized to total protein content for each sample and significant differences in mediator expression were determined by Student's t-test (*< 0.05; **< 0.01; and ***< 0.001).

Download figure to PowerPoint

AIM2 is a critical inflammasome sensor during acute S. aureus CNS infection

Based on the observed dichotomy in survival patterns between NLRP3 KO versus ASC and caspase 1/11 KO mice following CNS S. aureus infection, the involvement of an alternative ASC-dependent inflammasome sensor was investigated. Several inflammasomes including AIM2, NLRC4, and nod-like receptor protein 6 are known to recruit ASC, leading to downstream caspase 1 activation (Hornung et al. 2009; Broz et al. 2010; Elinav et al. 2011). Based on its ability to recognize bacterial dsDNA in the cytoplasm of infected cells and the fact that intracellular survival of S. aureus has been reported (Gresham et al. 2000; Watanabe et al. 2007; Hornung et al. 2009; Fernandes-Alnemri et al. 2010; Kim et al. 2010), we examined disease pathogenesis in AIM2 KO mice. Interestingly, AIM2 KO animals showed enhanced susceptibility to S. aureus infection in a manner that was similar to ASC and caspase 1/11 KO animals (Fig. 3a), suggesting that AIM2 is a critical molecule during acute S. aureus infection in the brain. In addition, the expression of the same proinflammatory mediators that were decreased in ASC KO mice (Fig. 2) was also significantly inhibited in AIM2 KO animals (Fig. 3c–f), providing further support for an AIM2/ASC pathway in sensing CNS S. aureus infection. To our knowledge, this is the first report implicating AIM2 in protective immunity to S. aureus as well as its action within the CNS in any infectious disease model.

image

Figure 3. Absent in melanoma 2 (AIM2) is critical for survival and inflammatory mediator induction during acute CNS infection. C57BL/6 wild-type (WT) and AIM2 knockout (KO) mice (7–8/group) were injected intracranially with 104 CFU of S. aureus USA300, whereupon survival (a), bacterial burdens (b), and interleukin-1β (IL-1β) (c), IL-6 (d), CCL2 (e), and CXCL10 (f) levels in abscess homogenates were quantified by MILLIPLEX multianalyte bead arrays. Results were normalized to total protein content for each sample and significant differences in mediator expression were determined by Student's t-test (*< 0.05).

Download figure to PowerPoint

image

Figure 4. The absent in melanoma 2 (AIM2) inflammasome is protective during acute CNS S. aureus infection. A disconnect in phenotypes between the inflammasome sensor NLRP3 and its adaptor ASC during acute CNS S. aureus infection led to the discovery of AIM2 as a critical inflammasome sensor. The AIM2 inflammasome is potentially triggered by dsDNA in cells harboring intracellular S. aureus, leading to ASC and caspase 1 recruitment, resulting in pro-IL-1β processing and cytokine secretion. This cascade, in turn, is protective to the host during acute infection that is independent of controlling bacterial burdens. The NLRP3 inflammasome is also activated in response to S. aureus challenge by α-hemolysin (hla); however, it is not critical for host survival. Furthermore, the cross-talk between AIM2 and NLRP3 inflammasomes is also a possibility as described in recent studies (Kim et al. 2010; Wu et al. 2010). ASC also regulates the production of other inflammatory mediators, presumably via indirect effects mediated by interleukin-1β (IL-1β) action.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

We previously established a pivotal role for the NLRP3 inflammasome in mediating IL-1β release from microglia in response to live S. aureus (Hanamsagar et al. 2011). In the current report, we have extended these findings to identify the molecular composition of the inflammasome that is critical for protection during acute CNS bacterial infection in vivo. Prior work from our laboratory demonstrated that IL-1RI KO mice succumb to CNS S. aureus infection within the first 24 h following bacterial exposure (Xiong et al. 2012); therefore, we predicted that similar results would be observed for both NLRP3 and ASC KO animals, since this inflammasome platform has the largest range of molecular triggers leading to IL-1β processing and release. Unexpectedly, ASC, but not NLRP3 KO mice were exquisitely sensitive to CNS S. aureus infection. The observed disconnect between ASC and NLRP3 phenotypes differs from most of the current inflammasome literature (Gris et al. 2010; Jha et al. 2010; Zaki et al. 2010; Hoegen et al. 2011), although divergent involvement of NLRP3 versus ASC has been reported in models of experimental autoimmune encephalomyelitis and bacterial pneumonia (Shaw et al. 2010; van Lieshout et al. 2014). Differential requirements for NLRP3 versus ASC in eliciting IL-1β production following S. aureus infection could be one explanation, since cytokine levels were significantly reduced in ASC KO mice but not NLRP3 KO animals (data not shown). The inability to elicit wild-type IL-1β production in ASC KO mice functionally equates with the inability of the cytokine to signal via the IL-1RI, which agrees with the finding that both ASC KO and IL-1RI KO animals in our previous study (Xiong et al. 2012) displayed a similar heightened sensitivity to CNS S. aureus infection, with approximately 33–50% of mice succumbing within the first 24 h. Collectively, these findings highlight the essential role of IL-1 signaling for protection after acute CNS bacterial exposure. IL-1β exerts numerous functions within the CNS, including modulation of blood–brain barrier permeability, activation of resident glia, and indirectly affecting leukocyte recruitment by augmenting chemokine and adhesion molecule expression at the blood–brain barrier (Gosselin and Rivest 2007). In the context of CNS S. aureus infection, IL-1β is critical for survival and bacterial containment, and plays an indirect role in dictating leukocyte influx by augmenting chemokine expression (Kielian et al. 2004; Xiong et al. 2012). However, we did not detect any significant differences in peripheral leukocyte infiltrates or bacterial burdens between WT and ASC or AIM2 KO mice in this study, which may result from the combined actions of IL-1β and IL-1α loss in IL-1RI KO animals in our previous report (Xiong et al. 2012), as both cytokines have overlapping biological activities. In addition, it is likely that IL-1β exerts beneficial effects during acute CNS infection that remain to be defined. A similar protective role for IL-1β has been reported in West Nile encephalitis (Ramos et al. 2012). Despite the marked reduction in IL-1β in ASC KO mice, residual cytokine remained, suggesting that pro-IL-1β may also be processed by currently unknown alternative mechanisms, albeit relatively minor. For example, caspase 1 may be activated via other inflammasomes responsive to S. aureus-related triggers or danger-associated molecular patterns released from necrotic cells (Willingham et al. 2009; Lamkanfi et al. 2010; Davis et al. 2011). Alternatively, other caspases or enzymes (i.e., cathepsins) may be involved in pro-IL-1β processing (and potentially IL-18 cleavage) in this model (Terada et al. 2010).

One unexpected finding during our in vivo studies was the distinct outcomes in NLRP3 and ASC KO mice following CNS S. aureus infection, which differs from other inflammatory models that generally reported similar phenotypes in both strains (Gris et al. 2010; Jha et al. 2010; Zaki et al. 2010; Hoegen et al. 2011). This raised the possibility that alternative ASC-dependent inflammasome sensor(s) were likely upstream effectors for triggering IL-1β release and host survival during CNS S. aureus infection. Other inflammasomes known to recruit ASC for caspase 1 activation include AIM2, NLRC4, and nod-like receptor protein 6 (Hornung et al. 2009; Broz et al. 2010; Elinav et al. 2011). The AIM2 inflammasome senses cytosolic dsDNA following bacterial or viral infections (Hornung et al. 2009). Phagocytosis and intracellular killing of S. aureus by host cells may lead to the release of bacterial DNA into the cytoplasm and subsequent AIM2 inflammasome activation. Alternatively, numerous reports have demonstrated intracellular survival of S. aureus (Gresham et al. 2000; Watanabe et al. 2007), where the organism may be capable of triggering cytosolic AIM2. Indeed, here we show that AIM2 KO mice displayed a phenotype that closely paralleled that of ASC KO animals in terms of survival after infection and impaired proinflammatory mediator production. Collectively, these findings reveal a novel role for AIM2, and not the NLRP3 inflammasome, for host survival following S. aureus infection in the CNS. However, another layer of complexity is that AIM2 can also act independent of caspase 1, by recruiting caspase 8 and inducing caspase 8-dependent apoptosis, as shown in a study of Francisella infection (Pierini et al. 2012). In addition, several inflammasomes, including NLRP3, AIM2, and NLRC4 are known to act cooperatively, likely as an evolutionary mechanism to ensure maximal pathogen eradiation or as a means to add multiple layers of regulatory control over cytokine secretion (Kim et al. 2010; McCoy et al. 2010; Wu et al. 2010; Coll and O'Neill 2011). Therefore, it is possible that the concerted action of several ASC-dependent inflammasomes is elicited following CNS S. aureus infection, although this remains to be determined (Fig. 4). Assessing the cellular localization and spatial distribution patterns of these inflammasome molecules may provide insights into their role in disease pathogenesis.

Although we predict that AIM2 and ASC are primarily active in microglia during acute CNS infection, it remains possible that astrocytes and/or infiltrating monocytes/macrophages may also play a role. However, unpublished data from our laboratory show that highly purified astrocytes do not secrete IL-1β in response to live S. aureus challenge in vitro. It is possible that in an in vivo setting, astrocytes may interact with microglia to secrete IL-1β, which is supported by a recent report (Holm et al. 2012); however, any such release might be small and insignificant in comparison with microglia. Macrophages and monocytes express both AIM2 and ASC (Mariathasan et al. 2005; Fernandes-Alnemri et al. 2009; Rathinam et al. 2010) and are activated during bacterial infection. However, we previously showed that CNS intrinsic cells were critical for the initial sensing of CNS S. aureus infection (Garg et al. 2009) and the rapid kinetics of susceptibility of AIM2 and ASC KO mice to intracerebral S. aureus challenge suggests the involvement of CNS intrinsic sensors of infection. Therefore, although it is possible that the lack of AIM2 or ASC in infiltrating monocytes/macrophages is involved in regulating IL-1β release and infection outcome at later stages in the disease process, the sum of currently available evidence points to a key role for CNS intrinsic cells during the early phase of infection. However, this prediction remains to be tested and would require the use of Cre-specific mice to ablate AIM2 or ASC expression in astrocytes or microglia/macrophages combined with the use of radiation bone marrow chimeras to discriminate between the involvement of the latter two populations.

Reactive oxygen species (ROS) have been shown to be an important upstream activator of the NLRP3 inflammasome in response to numerous stimuli (Martinon 2010; Zhou et al. 2011). However, we have previously demonstrated that ROS does not induce IL-1β secretion in the context of live S. aureus challenge in microglia (Hanamsagar et al. 2011). Similarly, a recent study has shown that mycobacterium-induced AIM2 inflammasome activation is not ROS dependent (Yang et al. 2013). Therefore, we do not anticipate a role of ROS in the observed pathology of AIM2 or ASC KO mice reported here; however, this remains to be examined.

In conclusion, our studies reveal the existence of a complex regulatory network for inflammasome activation and cytokine secretion during CNS S. aureus infection that is primarily AIM2/ASC-dependent and NLRP3-independent. To our knowledge, a role for the AIM2 inflammasome during S. aureus infection either in the CNS or the periphery has not yet been demonstrated. Although sustained inflammation following CNS infection can be detrimental, it is clear that the early release of proinflammatory cytokines, such as IL-1β, is essential for host survival. Therefore, it will be critical to design future studies to understand the temporal aspects of inflammasome function to develop therapeutic interventions that are safe and effective based on the nature of the insult.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References

This work was supported by NIH National Institute of Neurological Disorders and Stroke (NINDS) 2R01 NS040730 to T.K. The authors thank Dr Vishva Dixit at Genetech for providing NLRP3 and ASC KO mice, Debbie Vidlak for performing the MILLIPLEX analysis, Amanda Angle for excellent technical assistance, and Dr Charles Kuszynski and Victoria Smith in the UNMC Cell Analysis Facility for assistance with FACS analysis. The authors thank Ms. Kari Nelson, supported by NIH/NIAID P01 AI083211, for editorial assistance and Dr Paul Drew for critical review of the manuscript. All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  • Arora P., Kalra V. K. and Pappas A. (2012) Multiple brain abscesses in a neonate after blood stream infection with methicillin-resistant Staphylococcus aureus. J. Pediatr. 161, 563563.e1.
  • Broz P., Newton K., Lamkanfi M., Mariathasan S., Dixit V. M. and Monack D. M. (2010) Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J. Exp. Med. 207, 17451755.
  • Coll R. C. and O'Neill L. A. (2011) The cytokine release inhibitory drug CRID3 targets ASC oligomerisation in the NLRP3 and AIM2 inflammasomes. PLoS ONE 6, e29539.
  • Craven R. R., Gao X., Allen I. C., Gris D., Bubeck Wardenburg J., McElvania-Tekippe E., Ting J. P. and Duncan J. A. (2009) Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells. PLoS ONE 4, e7446.
  • David M. Z., Medvedev S., Hohmann S. F., Ewigman B. and Daum R. S. (2012) Increasing burden of methicillin-resistant Staphylococcus aureus hospitalizations at US academic medical centers, 2003-2008. Infect. Control Hosp. Epidemiol. 33, 782789.
  • Davis B. K., Roberts R. A., Huang M. T. et al. (2011) Cutting edge: NLRC5-dependent activation of the inflammasome. J. Immunol. 186, 13331337.
  • Drago L., De Vecchi E., Nicola L. and Gismondo M. R. (2007) In vitro evaluation of antibiotics' combinations for empirical therapy of suspected methicillin resistant Staphylococcus aureus severe respiratory infections. BMC Infect. Dis. 7, 111.
  • Elinav E., Strowig T., Kau A. L. et al. (2011) NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145, 745757.
  • Fernandes-Alnemri T., Yu J. W., Datta P., Wu J. and Alnemri E. S. (2009) AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509513.
  • Fernandes-Alnemri T., Yu J. W., Juliana C. et al. (2010) The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. Immunol. 11, 385393.
  • Garg S., Nichols J. R., Esen N. et al. (2009) MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses. ASN Neuro, 1, 7790.
  • Gosselin D. and Rivest S. (2007) Role of IL-1 and TNF in the brain: twenty years of progress on a Dr. Jekyll/Mr. Hyde duality of the innate immune system. Brain Behav. Immun. 21, 281289.
  • Gresham H. D., Lowrance J. H., Caver T. E., Wilson B. S., Cheung A. L. and Lindberg F. P. (2000) Survival of Staphylococcus aureus inside neutrophils contributes to infection. J. Immunol. 164, 37133722.
  • Gris D., Ye Z., Iocca H. A. et al. (2010) NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J. Immunol. 185, 974981.
  • Hanamsagar R., Torres V. and Kielian T. (2011) Inflammasome activation and IL-1beta/IL-18 processing are influenced by distinct pathways in microglia. J. Neurochem. 119, 736748.
  • Hoegen T., Tremel N., Klein M. et al. (2011) The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release. J. Immunol. 187, 54405451.
  • Holm T. H., Draeby D. and Owens T. (2012) Microglia are required for astroglial Toll-like receptor 4 response and for optimal TLR2 and TLR3 response. Glia 60, 630638.
  • Hornung V., Ablasser A., Charrel-Dennis M., Bauernfeind F., Horvath G., Caffrey D. R., Latz E. and Fitzgerald K. A. (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514518.
  • Jha S., Srivastava S. Y., Brickey W. J. et al. (2010) The inflammasome sensor, NLRP3, regulates CNS inflammation and demyelination via caspase-1 and interleukin-18. J. Neurosci. 30, 1581115820.
  • Kassis H., Marnejon T., Gemmel D., Cutrona A. and Gottimukkula R. (2010) Streptococcus sanguinis brain abscess as complication of subclinical endocarditis: emphasizing the importance of prompt diagnosis. South. Med. J. 103, 559562.
  • Kielian T., Barry B. and Hickey W. F. (2001) CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses. J. Immunol. 166, 46344643.
  • Kielian T., Bearden E. D., Baldwin A. C. and Esen N. (2004) 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. 63, 381396.
  • Kielian T., Esen N., Liu S. et al. (2007a) Minocycline modulates neuroinflammation independently of its antimicrobial activity in staphylococcus aureus-induced brain abscess. Am. J. Pathol. 171, 11991214.
  • Kielian T., Phulwani N. K., Esen N., Syed M. M., Haney A. C., McCastlain K. and Johnson J. (2007b) MyD88-dependent signals are essential for the host immune response in experimental brain abscess. J. Immunol. 178, 45284537.
  • Kim S., Bauernfeind F., Ablasser A., Hartmann G., Fitzgerald K. A., Latz E. and Hornung V. (2010) Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome. Eur. J. Immunol. 40, 15451551.
  • Lamkanfi M., Sarkar A., Vande Walle L., Vitari A. C., Amer A. O., Wewers M. D., Tracey K. J., Kanneganti T. D. and Dixit V. M. (2010) Inflammasome-dependent release of the alarmin HMGB1 in endotoxemia. J. Immunol. 185, 43854392.
  • van Lieshout M. H., Scicluna B. P., Florquin S. and van der Poll T. (2014) NLRP3 and ASC differentially affect the lung transcriptome during pneumococcal pneumonia. Am. J. Respir. Cell Mol. Biol. 50, 699712.
  • Lowy F. D. (1998) Staphylococcus aureus infections. N. Engl. J. Med. 339, 520532.
  • Madsen S. T. (1983) Sepsis, endocarditis, and brain abscess. Scand. J. Gastroenterol. Suppl. 85, 4854.
  • Mariathasan S., Weiss D. S., Dixit V. M. and Monack D. M. (2005) Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J. Exp. Med. 202, 10431049.
  • Martinon F. (2010) Signaling by ROS drives inflammasome activation. Eur. J. Immunol. 40, 616619.
  • McCoy A. J., Koizumi Y., Higa N. and Suzuki T. (2010) Differential regulation of caspase-1 activation via NLRP3/NLRC4 inflammasomes mediated by aerolysin and type III secretion system during Aeromonas veronii infection. J. Immunol. 185, 70777084.
  • Muzumdar D., Jhawar S. and Goel A. (2011) Brain abscess: an overview. Int. J. Surg. 9, 136144.
  • Naesens R., Ronsyn M., Druwe P., Denis O., Ieven M. and Jeurissen A. (2009) Central nervous system invasion by community-acquired meticillin-resistant Staphylococcus aureus. J. Med. Microbiol. 58, 12471251.
  • Pierini R., Juruj C., Perret M., Jones C. L., Mangeot P., Weiss D. S. and Henry T. (2012) AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages. Cell Death Differ. 19, 17091721.
  • Ramos H. J., Lanteri M. C., Blahnik G. et al. (2012) IL-1beta signaling promotes CNS-intrinsic immune control of West Nile virus infection. PLoS Pathog. 8, e1003039.
  • Rathinam V. A., Jiang Z., Waggoner S. N. et al. (2010) The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat. Immunol. 11, 395402.
  • Shaw P. J., Lukens J. R., Burns S., Chi H., McGargill M. A. and Kanneganti T. D. (2010) Cutting edge: critical role for PYCARD/ASC in the development of experimental autoimmune encephalomyelitis. J. Immunol. 184, 46104614.
  • Sifri C. D., Park J., Helm G. A., Stemper M. E. and Shukla S. K. (2007) Fatal brain abscess due to community-associated methicillin-resistant Staphylococcus aureus strain USA300. Clin. Infect. Dis. 45, e113e117.
  • Stenzel W., Soltek S., Miletic H., Hermann M. M., Korner H., Sedgwick J. D., Schluter D. and Deckert M. (2005) An essential role for tumor necrosis factor in the formation of experimental murine Staphylococcus aureus-induced brain abscess and clearance. J. Neuropathol. Exp. Neurol. 64, 2736.
  • Terada K., Yamada J., Hayashi Y., Wu Z., Uchiyama Y., Peters C. and Nakanishi H. (2010) Involvement of cathepsin B in the processing and secretion of interleukin-1beta in chromogranin A-stimulated microglia. Glia 58, 114124.
  • Wadhwa R., Thakur J. D., Nanda A. and Guthikonda B. (2012) Sterile hemorrhagic brain abscess in infective endocarditis. Neurol. India 60, 240242.
  • Warren S. E., Armstrong A., Hamilton M. K., Mao D. P., Leaf I. A., Miao E. A. and Aderem A. (2010) Cutting edge: Cytosolic bacterial DNA activates the inflammasome via Aim2. J. Immunol. 185, 818821.
  • Watanabe I., Ichiki M., Shiratsuchi A. and Nakanishi Y. (2007) TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity. J. Immunol. 178, 49174925.
  • Willingham S. B., Allen I. C., Bergstralh D. T., Brickey W. J., Huang M. T., Taxman D. J., Duncan J. A. and Ting J. P. (2009) NLRP3 (NALP3, Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways. J. Immunol. 183, 20082015.
  • Wu J., Fernandes-Alnemri T. and Alnemri E. S. (2010) Involvement of the AIM2, NLRC4, and NLRP3 inflammasomes in caspase-1 activation by Listeria monocytogenes. J. Clin. Immunol. 30, 693702.
  • Xiong J., Burkovetskaya M., Karpuk N. and Kielian T. (2012) IL-1RI (interleukin-1 receptor type I) signalling is essential for host defence and hemichannel activity during acute central nervous system bacterial infection. ASN Neuro, 4, 175185.
  • Yang Y., Zhou X., Kouadir M. et al. (2013) The AIM2 inflammasome is involved in macrophage activation during infection with virulent Mycobacterium bovis strain. J. Infect. Dis. 208, 18491858.
  • Young J. D. and McGwire B. S. (2005) Infliximab and reactivation of cerebral toxoplasmosis. N. Engl. J. Med. 353, 15301531. discussion 1530–1531.
  • Zaki M. H., Boyd K. L., Vogel P., Kastan M. B., Lamkanfi M. and Kanneganti T. D. (2010) The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 32, 379391.
  • Zhou R., Yazdi A. S., Menu P. and Tschopp J. (2011) A role for mitochondria in NLRP3 inflammasome activation. Nature 469, 221225.