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Infection and Immunity, November 2005, p. 7428-7435, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7428-7435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess

Tammy Kielian,* Anessa Haney, Patrick M. Mayes, Sarita Garg, and Nilufer Esen

Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Received 21 June 2005/ Returned for modification 19 July 2005/ Accepted 16 August 2005


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ABSTRACT
 
Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that plays an important role in innate immune recognition of conserved structural motifs on a wide array of pathogens, including Staphylococcus aureus. To ascertain the functional significance of TLR2 in the context of central nervous system (CNS) parenchymal infection, we evaluated the pathogenesis of S. aureus-induced experimental brain abscess in TLR2 knockout (KO) and wild-type (WT) mice. The expression of several proinflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor alpha, and macrophage inflammatory protein-2, was significantly attenuated in brain abscesses of TLR2 KO mice compared to WT mice during the acute phase of infection. Conversely, interleukin-17 (IL-17), a cytokine produced by activated and memory T cells, was significantly elevated in lesions of TLR2 KO mice, suggesting an association between innate and adaptive immunity in brain abscess. Despite these differences, brain abscess severity in TLR2 KO and WT animals was similar, with comparable mortality rates, bacterial titers, and blood-brain barrier permeability, implying a role for alternative PRRs. Expression of the phagocytic PRRs macrophage scavenger receptor type AI/AII and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was increased in brain abscesses of both TLR2 KO and WT mice compared to uninfected animals. However, LOX-1 induction in brain abscesses of TLR2 KO mice was significantly attenuated compared to WT animals, revealing that the TLR2-dependent signal(s) influence LOX-1 expression. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition in the CNS which occurs, in part, through engagement of TLR2 and highlight the importance of receptor redundancy for S. aureus detection in the CNS.


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INTRODUCTION
 
Brain abscesses develop in response to a parenchymal infection with pyogenic bacteria, beginning as a localized area of cerebritis and evolving into a suppurative lesion surrounded by a well-vascularized fibrotic capsule. Despite recent advances made in detection and therapy, brain abscess remains a serious central nervous system (CNS) infectious disease that can lead to long-term complications, including seizures, loss of mental acuity, and focal neurological defects that are lesion site dependent (32, 47). The most common etiologic agents of brain abscess are Streptococcus strains and Staphylococcus aureus (32, 47). On the basis of its prevalence in human CNS infection, our laboratory has utilized S. aureus to establish an experimental brain abscess model in the mouse that accurately reflects the course of disease progression in humans, providing an excellent model system to identify critical molecules responsible for the establishment of CNS antibacterial immunity (2, 18, 19).

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) expressed on cells of the innate immune system that allow for the recognition of conserved structural motifs on a wide array of pathogens referred to as pathogen-associated molecular patterns (PAMP) (17, 29). To date, 11 TLRs have been identified, with Toll-like receptor 2 (TLR2) playing a pivotal role in recognizing structural components of various gram-positive bacteria, fungi, and protozoa (29). Relevant to brain abscess, we have demonstrated that both S. aureus and peptidoglycan (PGN) lead to significant increases in TLR2 expression in both microglia and astrocytes, which may enhance cell sensitivity to bacteria during the course of brain abscess development (8, 22, 25). In addition to resident glial activation, peripheral immune cell infiltrates are a hallmark of evolving brain abscesses, with neutrophils and macrophages representing the major infiltrating cell types (2, 18). Both neutrophils and macrophages express TLR2 (10, 34, 45, 48) and represent the primary bactericidal effector populations in the CNS parenchyma during brain abscess (18, 19). Previous studies have revealed that TLR2 is essential for S. aureus recognition by macrophages and the establishment of protective immunity in response to systemic gram-positive bacterial infections (7, 44, 45, 50). Collectively, these findings indicate that TLR2 may be important for inducing effective antibacterial immune responses in the CNS parenchyma towards brain abscess containment and resolution.

To assess the relative function of TLR2 in brain abscess, we compared disease pathogenesis in TLR2 knockout (KO) and wild-type (WT) mice. TLR2 was found to influence the expression of several proinflammatory mediators, including inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-{alpha}), and macrophage inflammatory protein-2 (MIP-2/CXCL2), during the acute stage of infection. In addition, a role for TLR2 in regulating the adaptive immune response in brain abscess was demonstrated by the finding that interleukin-17 (IL-17) levels were significantly elevated in TLR2 KO animals compared to WT animals. Despite these differences, TLR2 did not play a significant role in controlling the extent of infection in brain abscess with similar bacterial titers and changes in blood-brain barrier permeability observed in TLR2 KO and WT animals. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition which occurs, in part, through engagement of TLR2 and highlight the importance of receptor redundancy for S. aureus recognition in the CNS.


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MATERIALS AND METHODS
 
Mice. Toll-like receptor 2 knockout mice (45) backcrossed for five generations (C57BL/6 background) were generously provided by Shizuo Akira (Osaka University, Japan) and distributed in the United States by Douglas Golenbock (University of Massachusetts, Worcester, MA). Age- and sex-matched C57BL/6 mice (Harlan Labs, Indianapolis, IN) were used as wild-type controls. For all brain abscess studies, TLR2 KO and WT mice were used between 6 to 8 weeks of age.

Generation of experimental brain abscesses. Live S. aureus (strain RN6390 generously provided by Ambrose Cheung, Dartmouth Medical School) was encapsulated in agarose beads prior to implantation in the brain as previously described (19, 21). Previous studies from our laboratory have established that the introduction of sterile agarose beads does not induce detectable inflammation or peripheral immune cell infiltrates (2, 19). To induce brain abscesses, mice were anesthetized with 2.5% avertin intraperitoneally and a 1-cm longitudinal incision was made along the vertex of the skull extending from the ear to the eye. A rodent stereotaxic apparatus equipped with a Cunningham mouse adaptor (Stoelting, Kiel, WI) was used to implant S. aureus-encapsulated beads into the caudate putamen using the following coordinates relative to bregma: +1.0 mm rostral, +2.0 mm lateral, and –3.0 mm deep from the surface of the brain. A burr hole was made, and a 5-µl Hamilton syringe fitted with a 26-gauge needle was used to slowly deliver 2 µl beads (104 CFU) into the brain parenchyma. The needle remained in place for 2.5 min following injection to minimize bead efflux and potential leakage into the meninges. The burr hole was sealed with bone wax, and the incision was closed using surgical glue. Animals were closely monitored over the course of each study to quantitate changes in body weight.

Simultaneous collection of RNA and protein from brain abscesses. To collect brain abscess extracts for analysis, lesion sites were visualized by the stab wound created during injections and sectioned within 1 to 2 mm on all sides. Upon recovery, brain abscess samples were homogenized in 500 µl of phosphate-buffered saline (PBS) supplemented with a Complete protease inhibitor cocktail tablet (Roche, Indianapolis, IN) and 160 U/ml of RNase inhibitor (Promega, Madison, WI) using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). At this point, a 20-µl aliquot of abscess homogenate was removed for quantitative culture of viable bacteria described below. Subsequently, homogenates were centrifuged at 14,000 rpm for 15 min at 4°C to pellet membrane material, and supernatants were removed and stored at –70°C until enzyme-linked immunosorbent assay (ELISA) and Bio-Plex analysis as described below. The pellet fraction was divided into two aliquots for extraction of total RNA and protein for subsequent quantitative real-time reverse transcription-PCR (qRT-PCR) and Western blotting, respectively.

Quantitation of viable bacteria from brain abscesses. To quantitate the numbers of viable bacteria associated with brain abscesses in vivo, serial 10-fold dilutions of brain abscess homogenates were plated onto blood agar plates (Becton Dickinson). Titers were calculated by enumerating colonies and are expressed as CFU per milliliter of homogenate.

In vivo blood-brain barrier permeability assay. Comparisons of blood-brain barrier permeability in TLR2 KO and WT mice were performed using an Evans blue dye extravasation assay as previously described with minor modifications (2). Briefly, mice were anesthetized with 2.5% avertin and subsequently administered 100 µl of a solution of 2.0% Evans blue dye (Sigma) in PBS via the tail vein. Animals were euthanized 60 min following Evans blue injection and perfused transcardially with PBS to remove residual dye from circulation. The brain was immediately removed, and the abscess site was dissected away from healthy tissue and weighed. Tissues were homogenized in 500 µl of 50% trichloroacetic acid (wt/vol) with a Polytron homogenizer and then centrifuged at 12,500 rpm for 15 min at 4°C. Supernatants were collected and read using a fluorescence spectrophotometer (Molecular Dynamics, Sunnyvale, CA) at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. Dye concentrations associated with the brain abscesses of TLR2 KO and WT mice were calculated on the basis of a standard curve of Evans blue (10 to 500 ng/ml in 50% trichloroacetic acid). Results are expressed as nanograms of Evans blue per gram of tissue weight to normalize values for differences in tissue sampling size.

qRT-PCR. Total RNA from brain abscesses of TLR2 WT and KO mice was isolated using the TriZol reagent and treated with DNase I (both from Invitrogen, Carlsbad, CA) prior to use in qRT-PCR studies. The experimental procedure was performed as previously described (22). Briefly, CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and 6-carboxytetramethylrhodamine (TAMRA) TaqMan probes were designed as previously described (8) and synthesized by Applied Biosystems (ABI, Foster City, CA). ABI Assays-on-Demand Taqman kits were utilized to examine macrophage scavenger receptor (MSR) type AI/AII, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and TNF-{alpha} expression. The levels of gene expression between abscesses from TLR2 WT and KO mice were calculated after normalizing cycle thresholds against the "housekeeping" gene GAPDH and are presented as the change in induction or reduction (2–{Delta}{Delta}Ct) value relative to TLR2 WT mice.

ELISA. Protein levels of murine TNF-{alpha} (OptiEIA; BD PharMingen, Carpenteria, CA) and IL-17 and MIP-2 (DuoSet; R&D Systems, Minneapolis, MN) were quantified in brain abscess homogenates using ELISA kits according to the manufacturer's instructions. Results were normalized to the amount of total protein extracted from tissues to correct for differences in sampling size as previously described (2, 20).

Multianalyte microbead array to detect proinflammatory mediator expression. To expand the analysis of inflammatory mediators differentially expressed in TLR2 KO and WT mice, a mouse 18-plex cytokine microbead suspension array system was used according to the manufacturer's instructions (Bio-Plex; Bio-Rad, Hercules, CA). This microbead array allows for the simultaneous detection of 18 individual inflammatory molecules in a single 50-µl sample, including IL-1{alpha}, IL-1ß, TNF-{alpha}, gamma interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 p40 and p70, IL-17, MIP-1{alpha}, RANTES, KC, granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor. Results were analyzed using a Bio-Plex Workstation (Bio-Rad) and adjusted on the basis of the amount of total protein extracted from abscess tissues for normalization. The level of sensitivity for each microbead ELISA was 1.95 pg/ml.

Western blotting. Differences in iNOS and CD14 expression in brain abscesses of TLR2 KO and WT mice were evaluated by Western blot analysis as previously described (26). Blots were probed using rabbit anti-mouse iNOS (NOS-2; Santa Cruz, San Diego, CA) or rat anti-mouse CD14 (clone 4C1; BD PharMingen) antibody, followed by horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Jackson Immunoresearch, West Grove, PA). Blots were stripped and reprobed with a rabbit antiactin polyclonal antibody (Sigma, St. Louis, MO) to verify uniformity in gel loading and developed using the ChemiGlow West substrate (Alpha Innotech, San Leandro, CA) followed by exposure to X-ray film.

Statistics. Significant differences between experimental groups were determined using the unpaired Student's t test at the 95% confidence interval with Sigma Stat (SPSS Science, Chicago, IL). This analysis was determined to be most appropriate, because although we were evaluating changes in proinflammatory mediator expression over time, repeated measurements were not made on the same animal (mice were sacrificed to collect abscess homogenates at each time point), precluding analysis of variance and posthoc analysis of the data.


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RESULTS
 
Induction of proinflammatory mediator expression is attenuated in brain abscesses of TLR2 KO mice compared to WT mice. Toll-like receptor 2 has been shown to play an important role in the host immune response to gram-positive bacterial infections in the periphery (44), and to some extent, this receptor dictates the ensuing host antibacterial response in Streptococcus pneumoniae meningitis (7, 28). However, the functional importance of TLR2 in the context of a CNS parenchymal infection, such as brain abscess, has not yet been examined and may differ from meningitis on the basis of the highly focal nature of lesions in the former. We first evaluated the impact of TLR2 on the expression of proinflammatory mediators previously determined to be pivotal during the acute phase of brain abscess to ascertain whether defects in CNS bacterial recognition were evident (19, 20). The kinetics of TNF-{alpha} induction was altered in TLR2 KO mice compared to WT animals, with lower levels of cytokines in the former during the acute phase of disease (Fig. 1). A similar reduction in the production of MIP-2, a potent neutrophil chemokine, was observed in TLR2 KO mice compared to WT animals, with significantly reduced MIP-2 expression evident at day 1 following bacterial exposure (Fig. 2). Previous studies from our laboratory have established that the intracerebral inoculation of sterile agarose beads into WT mice does not induce detectable TNF-{alpha} or MIP-2 expression (2).



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  		FIG. 1. TLR2 is important for regulating TNF-{alpha} production during the acute phase of experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for TNF-{alpha} mRNA (A) and protein (B) expression by qRT-PCR and ELISA, respectively. For qRT-PCR, the level of TNF-{alpha} expression was calculated after normalizing signals against the "housekeeping" gene GAPDH and is presented in relative mRNA expression units (mean ± standard deviation [SD] [error bar] of four independent experiments). ELISA results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of TNF-{alpha} (in picograms) per milligram of protein (mean ± SD; representative of four independent experiments). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05; **, P < 0.001).



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  		FIG. 2. MIP-2 expression is attenuated during the acute stage of brain abscess development in TLR2 KO mice compared to WT mice. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for MIP-2 protein expression by ELISA. Results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of MIP-2 (in picograms) per milligram of protein (± standard deviations [error bars]). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of four independent experiments.

Nitric oxide is a reactive nitrogen intermediate with potent antimicrobial and immune modulatory effects. It is produced by any one of three enzyme isoforms of NOSs. iNOS expression is stimulated in glia and mononuclear phagocytes by a variety of inflammatory cytokines and bacterial products, including S. aureus lipopolysaccharide and PGN (8, 30). Quantitation of iNOS levels is routinely used to reflect NO production in vivo, since the free radical itself is highly unstable and difficult to measure accurately in tissue extracts. To determine whether iNOS expression in brain abscess is regulated by TLR2-dependent signaling, we compared iNOS levels in lesions from TLR2 KO and WT mice. The induction of iNOS expression was delayed in TLR2 KO mice compared to WT animals, with detectable protein levels not observed in the former until day 3 following S. aureus infection (Fig. 3). In addition, similar to the other proinflammatory mediators examined, iNOS levels were attenuated in brain abscesses of TLR2 KO mice compared to WT animals. These results suggest that TLR2 is important for regulating the induction of iNOS expression in acute brain abscess, with alternative receptors substituting for TLR2 during later stages of infection.



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  		FIG. 3. TLR2 regulates iNOS induction in experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU), whereupon brain abscess protein extracts (40 µg per sample) were prepared at the indicated day postinfection (day 1 [D1] or day 3 [D3] postinfection) and analyzed for iNOS expression by Western blotting as described in Materials and Methods. Blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading. Results are presented from two or three individual animals per group and are representative of three independent experiments.

Collectively, these findings reveal that the expression of a subset of proinflammatory mediators, specifically TNF-{alpha}, MIP-2, and iNOS, is regulated by TLR2 in the context of brain abscess, suggesting that this receptor is capable of influencing the inflammatory milieu during the acute stage of infection.

Brain abscesses of TLR2 KO mice are associated with elevated levels of IL-17. To broaden our analysis of inflammatory mediators regulated by TLR2-dependent activation in brain abscess, a multianalyte suspension microbead array was utilized. This approach led to the novel finding that IL-17 expression was significantly elevated in brain abscesses of TLR2 KO mice compared to WT animals from days 3 to 7 postinfection (Fig. 4). IL-17 is primarily produced by activated and memory CD4+ and CD8+ T cells, whereas its receptor is ubiquitously expressed (1, 52). These results provide evidence to suggest an association between TLR2-driven innate immune signals and the induction of adaptive immunity in brain abscess.



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  		FIG. 4. The absence of TLR2 signaling leads to augmented IL-17 expression throughout brain abscess development. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods. Brain abscess homogenates from TLR2 KO or WT animals (n = 4 to 6 per group for each time point) were prepared at the indicated time points and analyzed for IL-17 protein expression by ELISA. Results were normalized to the amount of total protein recovered from abscesses and reported as the mean values of IL-17 (in picograms) per milligram of protein (± standard deviations [SD] [error bars]). Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of two independent experiments.

TLR2 influences CD14 expression in evolving brain abscesses. Since recent studies indicate that the PRR CD14 is capable of recognizing PGN from the cell walls of gram-positive bacteria, effectively augmenting TLR2-dependent signal transduction (6, 41, 55), the consequences of TLR2 loss on CD14 expression in brain abscess was examined. CD14 levels were below the limit of detection in both uninfected TLR2 KO and WT mice (Fig. 5B). Within 1 day following S. aureus infection, CD14 expression was induced in both TLR2 KO and WT mice; however, CD14 mRNA and protein levels were attenuated in brain abscesses of TLR2 KO animals compared to WT mice (Fig. 5), suggestive of a defect in either microglial activation and/or influx of neutrophils and macrophages into the CNS, since all of these cell populations express CD14. The identity of CD14-expressing cells as either CNS or bone marrow derived is currently under investigation in our laboratory using radiation bone marrow chimeric mice. These findings reveal that a TLR2-dependent signal(s) is important for modulating the expression of additional PRRs, such as CD14, that may function together in a multireceptor complex to maximize pathogen recognition in the CNS.



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  		FIG. 5. The expression of the pattern recognition receptor CD14 is attenuated in TLR2 KO mice. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods, whereupon brain abscess homogenates were prepared (n = 4 to 6 animals per group for each time point) at the indicated intervals and analyzed for CD14 mRNA and protein expression by qRT-PCR (A) and Western blotting (B), respectively. (B) Western blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading with results from two or three individual mice per group presented at either day 1 (D1) or 3 (D3) postinfection as well as uninfected animals. Significant differences between TLR2 KO and WT mice are denoted with asterisks (*, P < 0.05). Results are representative of three independent experiments.

Brain abscess pathogenesis is qualitatively similar in TLR2 KO and WT mice. Toll-like receptor 2 has been shown to play a pivotal role in controlling the extent of gram-positive bacterial replication in both the periphery (44) and CNS in experimental models of S. pneumoniae meningitis (7, 28). However, it is not known whether this receptor is important for pathogen containment in a parenchymal infection, such as brain abscess, which lacks a systemic infection component. To assess the importance of TLR2 in brain abscesses, we compared the course of disease progression in TLR2 KO and WT mice. Despite its role in influencing the nature of the early proinflammatory milieu in response to S. aureus, TLR2 was not essential for regulating brain abscess severity, with comparable mortality rates, weight loss, and bacterial burdens observed for TLR2 KO and WT mice throughout the course of disease (Fig. 6 and data not shown). Similar findings were obtained with a 0.5-log-unit-higher inoculum of bacteria (i.e., 5 x 104 CFU [data not shown]). In addition, the permeability of the blood-brain barrier was quantitatively similar in TLR2 KO and WT animals (data not shown). Collectively, these results indicate that TLR2 does not play a major role controlling bacterial burdens in brain abscesses, suggesting that alternative phagocytic PRRs are responsible for pathogen containment.



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  		FIG. 6. TLR2 is not pivotal for bacterial containment in experimental brain abscess. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods. Animals (n = 4 to 6 per group) were euthanized at the indicated time points, and the number of viable organisms associated with brain abscesses was determined by quantitative culture. Titers are expressed as the mean log10 CFU per milliliter of brain abscess homogenate (± standard deviation [SD] [error bar]). Results are representative of eight independent experiments.

Expression of the phagocytic PRRs macrophage scavenger receptor type AI/AII and LOX-1 is augmented in brain abscesses. Our results indicate that a receptor(s) other than TLR2 is responsible for regulating bacterial replication in brain abscesses, since bacterial burdens were not significantly different in TLR2 KO and WT mice. This finding led us to initiate studies to examine the expression of the phagocytic PRRs MSR and LOX-1 during the course of brain abscess development. Expression of both MSR and LOX-1 mRNAs was elevated in brain abscesses of TLR2 KO and WT mice compared to uninfected animals at the majority of time points examined (Fig. 7). However, a role for TLR2-dependent signal(s) in inducing maximal LOX-1 expression was revealed by the finding that LOX-1 levels were significantly attenuated in brain abscesses of TLR2 KO mice than in infected WT animals (Fig. 7B). In contrast, MSR expression was induced to similar extents in both TLR2 KO and WT mice (Fig. 7A). Collectively, these findings suggest that MSR and/or LOX-1 may play an important role in dictating the course of brain abscess progression by regulating bacterial replication and spread following S. aureus infection.



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  		FIG. 7. Expression of the phagocytic PRR LOX-1 is influenced by TLR2-dependent signal(s) in experimental brain abscesses. TLR2 KO and WT mice were injected with S. aureus-encapsulated agarose beads (104 CFU) as described in Materials and Methods, whereupon total RNA was extracted from brain abscess tissues (n = 4 animals per group for each time point) at the indicated intervals and analyzed for macrophage scavenger receptor 1 (MSR) (A) and LOX-1 (B) expression by qRT-PCR. Gene expression levels were calculated after normalizing target signals (MSR or LOX-1) against the "housekeeping" gene GAPDH and are presented as the change in mRNA induction compared to uninfected animals. Significant differences between uninfected versus S. aureus-infected TLR2 WT and KO mice (*, P < 0.05; **, P < 0.001) and significant differences between brain abscesses of TLR2 WT and KO animals (#, P < 0.05) are also indicated. Results are representative of three independent experiments.


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DISCUSSION
 
The receptor(s) responsible for recognizing gram-positive bacterial pathogens associated with brain abscess leading to the subsequent release of proinflammatory mediators in the CNS parenchyma remains undefined. Although TLR2 does not appear to dictate the extent of bacterial replication in brain abscesses, this receptor does influence the induction of proinflammatory mediator expression during the acute stage of infection.

Identifying the signals, effectors, and sequence of innate immune responses in the experimental brain abscess model has been a focus of our laboratory in recent years (2, 18-21, 23). However, despite these efforts, a great deal of information regarding the roles of host immunity and bacterial virulence factors in disease pathogenesis remains to be elucidated. On the basis of the results obtained to date using the experimental brain abscess model, the following sequence of events in disease evolution is proposed and discussed in the context of the potential functional role of TLR2. First, the initial bacterial recognition event in the CNS parenchyma is thought to be mediated by resident microglia and astrocytes. Indeed, we have demonstrated that both glial cell types are capable of recognizing intact S. aureus, in part, via a TLR2-dependent pathway (8, 22) and respond with the robust elaboration of numerous proinflammatory mediators that likely participate in amplification of the innate immune response and recruitment of peripheral immune cells into evolving abscesses (8, 22, 25, 26). Additional evidence to support a role for resident glia in mediating the initial responses to S. aureus infection in the CNS is provided by the finding that proinflammatory mediator production is evident as early as 1 h following bacterial exposure, which precedes the significant accumulation of neutrophils into the CNS, which occurs around 24 h following infection (23, 24; T. Kielian, unpublished observations). Neutrophils represent the first innate immune effector cell from the periphery to enter the CNS in response to parenchymal S. aureus infection; neutrophils are followed by macrophages, which accumulate to significant levels within 1 and 3 days postinfection, respectively (9, 19, 24). These professional phagocytes are thought to play an essential role in pathogen containment, and we have demonstrated that neutrophil depletion significantly increases S. aureus burdens and leads to enhanced mortality in the experimental brain abscess model (19). It is likely that TLR2 expression on these peripheral immune cell populations is critical in dictating pathogen containment and brain abscess evolution, and studies designed to directly assess the functional importance of TLR2 in the CNS versus peripheral compartments are under way in our laboratory using radiation bone marrow chimeric mice. Recent evidence indicates that lymphocytes are associated with developing brain abscesses within 3 days following S. aureus infection (42), and we have independently confirmed this finding and identified enhanced T-cell infiltrates associated with lesions of TLR2 KO mice (T. Kielian, unpublished observations). Relative to the functional significance of proinflammatory mediators elicited in response to S. aureus infection in the brain abscess model, we have reported that IL-1, TNF-{alpha}, and CXCR2 ligands play a pivotal role in controlling bacterial burdens during the acute phase of experimental brain abscess development (19, 20). Importantly, in the current study we have evidence to suggest that TLR2-dependent signals influence the expression of these mediators during the acute stage of disease, as evident by the reduction in TNF-{alpha}, IL-1ß, and MIP-2 levels in lesions of TLR2 KO mice compared to WT animals. On the basis of the work presented here in conjunction with recent studies revealing that the responses of CD14-deficient mice are nearly identical to those observed in TLR2 KO animals (T. Kielian, unpublished data), we propose that these two PRRs are involved in a multireceptor complex to regulate proinflammatory mediator release during the acute stage of brain abscess. However, our findings also revealed that additional PRRs are responsible for bacterial containment since S. aureus burdens were not affected in TLR2 KO mice. Logical candidates include the phagocytic PRRs MSR and LOX-1 that have recently been shown to collaborate with TLR2 to regulate innate immune responses to various pathogens (16, 49). Interestingly, we have found that TLR2-dependent signals influence the extent of LOX-1 induction in the brain abscess model, suggesting possible receptor cross talk. We are currently initiating studies to directly examine the potential roles of MSR and LOX-1 in regulating pathogen containment in the brain abscess model using receptor KO mice. Collectively, these studies have illuminated an important point, namely, that the development of antibacterial immune responses in the CNS parenchyma cannot be accounted for by the activity of a single receptor, a concept that has emerged over recent years (11, 12, 28, 33).

Innate and adaptive immunity are linked, and recent evidence demonstrates that TLR-dependent signaling leads to the initiation of adaptive immune responses (13, 36). Evaluation of inflammatory mediator release by multianalyte suspension microbead arrays revealed novel molecules produced during brain abscess that are likely to influence the nature and course of disease development. Of particular interest was the significant induction of the T-cell-derived cytokine IL-17 in brain abscesses of TLR2 KO mice compared to WT animals. Since we detected elevated IL-17 levels in TLR2 KO mice during the early stages of primary infection, attributing cytokine production to activated and memory T cells appears unusual. However, we have previously reported that "naïve" mice under nonspecific pathogen-free conditions harbor a population of S. aureus-reactive T cells in the spleen (2). The origin of these S. aureus-specific lymphocytes is not known but may have arisen from routine pathogen exposure, since S. aureus is ubiquitous in nature or from the restimulation of cross-reactive lymphocytes that are specific for a highly conserved epitope expressed on another bacterial pathogen. Therefore, it is conceivable that "endogenous" S. aureus-reactive memory T cells are recruited into the CNS during the early stages of brain abscess development and are responsible for the subsequent production of IL-17. Indeed, preliminary studies from our laboratory have demonstrated that the numbers of both CD4+ and CD8+ T-cell infiltrates are enhanced in brain abscesses of TLR2 KO mice compared to WT animals (T. Kielian, unpublished observations). It is intriguing to speculate that elevated IL-17 production in TLR2 KO animals represents a compensatory mechanism to counteract the observed delay in the production of neutrophil-attracting chemokines, since IL-17 is a potent stimulus for the production of these chemoattractants (31, 40, 53). In addition, IL-17 has been shown to be pivotal in the establishment of antimicrobial immunity, since IL-17 KO mice are more susceptible to systemic bacterial infections (3, 4, 54). Currently, the biological implications of elevated IL-17 levels in brain abscesses of TLR2 KO mice and how the loss of TLR2 leads to elevated cytokine expression are not known, but these issues represent areas of active investigation in our laboratory.

The relative importance of TLR2 in brain abscess pathogenesis was not as dramatic compared to recent reports examining the role of this receptor in Streptococcus pneumoniae meningitis (7, 28). For example, both meningitis studies revealed that TLR2 regulated bacterial burdens in the CNS, whereas we did not observe a critical role for TLR2 in pathogen containment in brain abscess. This finding could be explained by the differential extent of infection in both models, where bacteremia occurs in conjunction with CNS infection during meningitis, whereas brain abscess is typified by a focal infection confined to the CNS parenchyma. Indeed, studies documenting a critical role for TLR2 in the pathogenesis of gram-positive bacterial infections have been conducted using disseminated systemic infectious disease models (5, 7, 44, 51). The role of TLR2 on proinflammatory cytokine expression within the CNS during bacterial meningitis is unclear, since both published studies have reported conflicting results. For example, Echchannaoui et al. demonstrated that TNF-{alpha} expression is elevated in the CNS of TLR2 KO mice during late-stage meningitis (7), whereas Koedel et al. reported no significant differences in TNF-{alpha} levels in TLR2 KO and WT animals (28). We find that TLR2-dependent signaling in brain abscesses regulates TNF-{alpha} release during the acute phase of disease; however, it is important to note that TNF-{alpha} is still produced in the CNS of KO animals, albeit at lower levels, indicating that alternative receptors are capable of signaling cytokine release in response to S. aureus. Additional evidence to support that, in addition to TLR2, alternative PRRs are involved in S. aureus recognition and containment in brain abscesses is demonstrated by the inability of TLR2 to impact bacterial burdens or alter blood-brain barrier permeability. Nonetheless, it appears that TLR2 plays a role in the host antibacterial immune response in both bacterial meningitis and brain abscess, although it is apparent that additional, as-yet-undefined PRRs contribute to pathogen recognition.

Indeed, recent evidence from other models of systemic infectious disease support the concept that multiple PRRs act in concert to induce protective antibacterial immune responses (11, 27, 28, 35, 39). This concept of receptor redundancy is not unexpected, since bacterial pathogens, such as S. aureus, have the potential to elicit devastating consequences in a tissue that has limited regenerative capacity, such as the CNS. Therefore, the host repertoire of available PRRs should be substantial, ensuring that an effective antibacterial immune response will be rapidly elicited upon infection of the CNS parenchyma. In considering the myriad of antigens that S. aureus harbors, additional candidate PRRs that may participate in bacterial recognition in concert with TLR2 during brain abscess evolution include the phagocytic scavenger receptors. Indeed, recent studies have demonstrated that TLR-dependent signaling and phagocytosis are functionally linked and one pathway can potentiate the other, resulting in the amplification of proinflammatory activities (49).

Members of the scavenger receptor family are promiscuous receptors involved in the nonopsonic receptor-mediated phagocytosis of several polyanionic acids, such as lipoteichoic acid of S. aureus, modified low-density lipoproteins, and apoptotic cells (14, 37, 38). Scavenger receptors are expressed on a variety of cell types, including activated microglia and astrocytes in the CNS as well as macrophages and neutrophils in the periphery (14, 37). To our knowledge, this study provides the first report of elevated LOX-1 expression in a CNS infectious disease. In addition, LOX-1 induction is dependent, in part, on TLR2, since LOX-1 levels were significantly attenuated in brain abscesses of TLR2 KO mice compared to WT animals. This apparent regulation of LOX-1 by TLR2 is reminiscent of what we have recently reported in primary microglia, where the S. aureus-dependent induction of LOX-1 was significantly diminished in TLR2 KO cells compared to WT microglia (22). Interestingly, MSR expression is also augmented during brain abscess development; however, TLR2-driven signals do not appear to be essential for gene expression, since MSR levels were equivalent in TLR2 KO and WT mice. Evidence to substantiate an important role for scavenger receptors in bacterial infections is provided by studies documenting that disease pathogenesis is significantly altered in mice with a target deletion of macrophage scavenger receptor types AI and AII (15, 43, 46); however, the role of scavenger receptors in S. aureus-induced brain abscesses has yet to be examined.

In summary, although TLR2 regulates proinflammatory mediator expression during the acute stage of brain abscess and appears to influence the development of adaptive immune responses, additional PRRs are involved in eliciting an effective CNS antibacterial immune response. The requirement of a multireceptor complex, including TLR2, for effective microbial recognition is logical, given the potential for a pathogen, such as S. aureus, to cause significant damage in a tissue that has limited regenerative capacity. Importantly, the results presented here are, to our knowledge, the first to demonstrate an association between TLR2 and the adaptive immune response in CNS infectious disease.


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ACKNOWLEDGMENTS
 
We thank Shizuo Akira for generously providing the TLR2 KO mice, Paul Drew for critical review of the manuscript, and Gail Wagoner and Napoleon Phillips for excellent technical assistance.

This work was supported by the NIH National Institute of Mental Health (RO1 MH65297) to T.K. and the National Institute of Neurological Disorders and Stroke-supported core facility at UAMS (P30 NS047546).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 846, Little Rock, AR 72205. Phone: (501) 526-6348. Fax: (501) 526-6756. E-mail: KielianTammyL{at}uams.edu. Back

Editor: J. D. Clements


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Infection and Immunity, November 2005, p. 7428-7435, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7428-7435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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