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Originally published online as doi:10.1189/jlb.1105626 on May 26, 2006

Published online before print May 26, 2006
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(Journal of Leukocyte Biology. 2006;80:267-277.)
© 2006 by Society for Leukocyte Biology

Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response through Toll-like receptors 2, 4, and 9 in species-specific patterns

Trine H. Mogensen*,1, Søren R. Paludan{dagger}, Mogens Kilian{dagger} and Lars Østergaard*

* Department of Infectious Diseases, Skejby Hospital, Aarhus, Denmark; and
{dagger} Institute of Medical Microbiology and Immunology, University of Aarhus, Denmark

1Correspondence: Department of Infectious Diseases, Skejby Hospital, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. E-mail: trine.mogensen{at}dadlnet.dk

ABSTRACT

Toll-like receptors (TLRs) are pattern recognition receptors (PRR) that recognize molecular structures on pathogens and activate host defenses. Although much is known about specific bacterial components that activate TLRs, few studies have addressed the question of which TLRs are involved in immune activation by live bacteria. Here, we demonstrate that live Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, the three principal causes of bacterial meningitis, use distinct sets of TLRs to trigger the inflammatory response. Using human embryonic kidney 293 cell lines, each overexpressing one type of TLR, we found that S. pneumoniae triggered activation of the transcription factor nuclear factor-{kappa}B and expression of interleukin-8, only in cells expressing TLR2 or -9. The same response was evoked by H. influenzae in cells expressing TLR2 or -4 and by N. meningitidis in cells expressing TLR2, -4, or -9. It is interesting that the ability of S. pneumoniae and N. meningitidis to activate TLR9 was severely attenuated when bacteria had been heat-inactivated prior to stimulation of the cells. In human peripheral blood mononuclear cells, we blocked TLR2, -4, or -9 and confirmed the essential role of these TLRs and also identified differential functions of TLRs in activation of the inflammatory response. Collectively, we here demonstrate that S. pneumoniae, H. influenzae, and N. meningitidis each activate several TLRs in species-specific patterns and show that infection with live pathogens may lead to activation of PRR not targeted by inactivated bacteria.

Key Words: inflammation • bacteria • NF-{kappa}B

INTRODUCTION

The inflammatory immune response is of crucial importance for the early containment of infection but at the same time has the potential to result in immunopathology [1 ]. The final outcome of infection therefore depends on an intricate balance between the pathogen and the host response. One of the central components of the innate immune system is the family of Toll like receptors (TLRs). These pattern recognition receptors (PRR) recognize evolutionarily conserved pathogen-associated molecular patterns (PAMPs) present on most types of microorganisms [2 ]. Once TLRs are activated, they signal "danger" to the host and trigger signaling cascades, leading to antimicrobial and inflammatory responses involving innate and adaptive immunity [2 ]. To date, 10 TLRs have been identified in humans, and they each recognize different microbial structures. TLR1, -2, -4, -5, -6, and -10 are located on the cell surface and mainly recognize bacterial products unique to the invading organism. TLR2 recognizes bacterial lipoproteins [3 ], peptidoglycan, and lipoteichoic acids [4 ]; TLR4 recognizes lipopolysaccharide (LPS) [5 ]; and TLR5 detects bacterial flagellin [6 ]. In contrast, TLR3, -7, and -8, which together with TLR9, are located within endosomal compartments, are specialized primarily in viral detection and more generally, in recognition of RNA [7 ]. Double-stranded RNA is recognized by TLR3 [8 ] and single-stranded RNA by TLR7 and -8 [9 , 10 ]. TLR9 recognizes unmethylated CpG DNA [11 ], which is a component of viruses and bacteria, implying that this TLR may play a role in a broader range of bacterial and viral infections [12 ]. TLRs are expressed by most cell types, although clear differences between cell populations are apparent. However, the most important TLR-expressing cell types are believed to be dendritic cells (DC), macrophages, and B lymphocytes [2 ].

TLR ligand engagement results in intracellular signal transduction, including activation of nuclear factor (NF)-{kappa}B and mitogen-activated protein kinases. The TLR-activated signaling pathways proceed through an adaptor protein [most importantly, myeloid differentiation primary-response protein 88 (MyD88)]; members of the interleukin-1 (IL-1) receptor-associated kinase family; tumor necrosis factor (TNF) receptor-associated factor 6; and transforming growth factor-ß-activated protein kinase-1, which activates the inhibitory protein {kappa}B (I{kappa}B) kinase (IKK) complex. Finally, IKK phosphorylates the I{kappa}B protein and targets it for degradation, hence liberating NF-{kappa}B, which migrates to the nucleus and activates transcription of target genes [13 14 15 16 ]. Therefore, it appears that NF-{kappa}B plays a pivotal role in TLR-induced proinflammatory signaling by up-regulating the expression of a wide range of cytokines, cell adhesion molecules, major histocompatibility complex molecules, and antiapoptotic proteins [17 , 18 ]. In addition, a set of TLRs (TLR3, -4, -7, -8, and -9) can activate signaling to the family of interferon (IFN) regulatory factors (IRFs) and hence, induce IFN-{alpha}/ß expression and other IFN-regulated, immunomodulating genes [7 ].

During recent years, the involvement and function of TLRs in the pathogenesis of bacterial infection, including bacteriemia and meningitis, have been studied extensively [19 20 21 ]. Several different bacterial components have been demonstrated to specifically activate individual TLRs. However, the picture that emerges is increasingly complex, and recent data indicate more complexity and cross-talk than initially anticipated, as different components of the same organism can activate several different TLRs and lead to individual signaling cascades and different patterns of gene expression [2 , 16 ]. Whereas much is now known about mechanisms of TLR activation by purified bacterial components, only few studies have addressed the question of how entire live bacteria activate the inflammatory response. In this study, we have investigated the pattern of TLR activation by Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, three principal causes of bacterial meningitis and septicemia. We compared the pattern of TLR activation induced by each of these three bacteria in human embryonic kidney (HEK)293-derived cell lines transfected with human TLRs and in human peripheral blood mononuclear cells (PBMC). Here, we describe the use of distinct, yet overlapping, sets of TLRs by live S. pneumoniae, H. influenzae, and N. meningitidis for activation of the inflammatory response and also report that only live bacteria triggered a response through TLR9. Our data thus point out the relevant TLRs involved in the pathogenesis of meningitis induced by each of these bacteria and imply that proper activation of the host immune response to infection requires infection with a live pathogen.

MATERIALS AND METHODS

Cell culture
PBMC were isolated from blood obtained from healthy adult donors by Isopaque-Ficoll separation. The blood was diluted and laid on top of the Ficoll Paque (Amersham Biosciences, UK) and centrifuged at 600 g for 30 min at room temperature. The PBMC-containing interphase was isolated, and the cells were washed in phosphate-buffered saline (PBS) containing 100 µg heparin per ml. Subsequently, the cells were centrifuged at 200 g for 15 min at room temperature and resuspended in RPMI-1640 medium containing 5% heat-inactivated fetal calf serum (FCS). The cells were seeded in 96- and six-well tissue plates at a density of 2.0 x 105 and 4.0 x 106 cells per well, respectively, and left overnight to settle before further treatment. Transfected HEK293 cells (HEK293-pcDNA3, HEK293-TLR2, HEK293-TLR3, HEK293-TLR4/MD2, HEK293-TLR9 [22 ]) were generously donated by Dr. Katherine A. Fitzgerald (Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester) or were purchased from InvivoGen (San Diego, CA; HEK293-TLR7, HEK293-TLR8). The transfected cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, antibiotics, and 500 µg/ml G418 (Roche, Indianapolis, IN) or 10 µg/ml Blasticidin (InvivoGen). For experiments, transfected cells were seeded in 96- and six-well tissue plates at a density of 1.5 x 104 and 5.0 x 105 cells per well, respectively, in DMEM supplemented with 10% FCS and left overnight to settle before further treatment.

Bacteria and reagents
The bacteria used were N. meningitidis strains NGO93 serogroup B, serotype 15:P1.2, MLEE cluster I1,ET76 and BZ139 serogroup B, serotype 2b:P1.2, MLEE cluster A4, ET7, S. pneumoniae strains SK1013 serotype 4 (TIGR4) and SK1025 [immunoglobulin A (IgA) protease-negative mutant of SK1013], and the H. influenzae serotype b strains HK707 (Division I) and HK715 (Division II). The bacteria were grown overnight in brain heart infusion broth with 10% Levinthal broth (Statens Serum Institute, Copenhagen), reaching a concentration of 18.0 ± 2.2 x 108 bacteria per ml as determined in a Thoma counting chamber. For stimulation, 100 µl and 10 µl from this stock were added to the cell cultures in six- and 96-well plates, respectively, reaching final volumes of 2 ml and 200 µl, respectively. Heat-inactivated bacteria were obtained by incubation of the cultures for 30 min at 65°C. For stimulations with cytokines and pure TLR ligands, we used TNF-{alpha} (R&D Systems, Minneapolis, MN), Pam3CSK4, polyIC, LPS, R848, oligodeoxynucleotide (ODN)2006, and ODN M362 (all InvivoGen). For inhibition of TLR2, -4, and -9, we used azide-free mouse monoclonal anti-TLR2 (clone TL2.1), mouse monoclonal anti-TLR4 (clone HTA125; both ImmunoKontakt, Switzerland), and ODN TTAGGG (InvivoGen), respectively.

Purification of genomic bacterial DNA
For purification of protein- and RNA-free genomic bacterial DNA, we used freshly grown suspensions of bacteria and the DNA isolation kit for cells and tissue (Roche).

Enzyme-linked immunosorbent assay (ELISA) and Luminex technology
Human IL-8 and IFN-{gamma}-inducible protein (IP)-10 were detected by ELISA. Maxisorp plates were coated overnight at 4°C with primary antibody [4 µg/ml anti-IL-8, 2 µg/ml anti-IP-10 (both R&D Systems) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 0.02% sodium azide, pH 9.6)]. After blocking for 2 h at 37°C or 3 h at room temperature with PBS, pH 7.4, containing 1% w/v bovine serum albumin and 0.05% sodium azide, samples and standard dilutions of cytokine (concentration range: 3.9–2000 pg/ml) were added to the wells, and the plates were incubated at room temperature for 2 h or overnight at 4°C. Subsequently, the wells were incubated for 2 h at room temperature with a biotin-labeled detection antibody [0.02 µg/ml anti-IL-8, 0.1 µg/ml anti-IP-10 (both R&D Systems)] in blocking buffer. Finally, horseradish peroxidase (HRP)-conjugated streptavidin diluted in blocking buffer was added and incubated for 20 min at 20°C, and the result was visualized by the tetramethylbenzidine system (R&D Systems). After 10 min, the color reaction was stopped by addition of 5% H2SO4. Between each step, the plates were washed three to four times with PBS containing 0.05% v/v Tween 20. The results were quantified by reading at 450 nm. Human IL-6, IL-8, and TNF-{alpha} were measured by Luminex technology, using a custom-made three-plex kit, purchased from Bio-Rad (Hercules, CA), following the instructions of the manufacturer.

Preparation of nuclear extracts
To isolate nuclear proteins we used a nuclear extraction kit (Active Motif, Carlsbad, CA). Briefly, cells were washed twice with ice-cold PBS supplemented with phosphatase inhibitors, scraped off the plate, and spun down (2000 g for 1 min) before resuspension in 250 µl 1x hypotonic buffer and 15 min on ice. The supplied detergent (25 µl) was added, and the mixture was vortexed 10 s and centrifuged at 14,000 g for 30 s. The supernatants were removed, and 25 µl Complete lysis buffer was added to the nuclei and incubated 30 min at 4°C with rocking. The samples were vortexed and centrifuged at 14,000 g for 10 min at 4°C. Supernatants containing nuclear proteins were harvested and transferred to new, prechilled tubes.

NF-{kappa}B DNA-binding activity
ELISA-based measurement of DNA-binding activity of the nuclear NF-{kappa}B subunit p65 was performed according to the manufacturer’s protocol (Active Motif). Briefly, 5 µg nuclear extract was used per sample in duplets in a 96-well plate precoated with consensus oligonucleotides for NF-{kappa}B (5'-GGGACTTTCC-3'). After washing to remove nonspecific binding, a specific antibody to the NF-{kappa}B subunit p65 was added. After antibody binding, the plate was washed again before adding a HRP-conjugated secondary antibody. The peroxidase substrate was added, and colorimetric change was measured at an optical density of 450 (OD450).

Statistical analysis
The data are presented as means ± SEM. The statistical significance was estimated with the Wilcoxon rank sum test. P values of less than 0.05 were considered statistically significant.

RESULTS

S. pneumoniae, H. influenzae, and N. meningitidis activate NF-{kappa}B and induce proinflammatory cytokines in PBMC
It is well established that infection with S. pneumoniae, H. influenzae, or N. meningitidis evokes inflammatory responses and that this contributes to the pathogenesis of the diseases caused by these bacteria [23 24 25 26 27 ]. We initially looked at the pattern of some central proinflammatory cytokines secreted by isolated human PBMC after 24 h of stimulation with live S. pneumoniae, H. influenzae, or N. meningitidis. As demonstrated in Figure 1A 1B 1C , all three types of bacteria induced IL-8, TNF-{alpha}, and IL-6, but whereas TNF-{alpha} was secreted in similar amounts by all bacteria, N. meningitidis induced significantly higher levels of IL-8 (N. meningitidis vs. H. influenzae and N. meningitidis vs. S. pneumoniae: both P<0.05) and also, higher levels of IL-6 (N. meningitidis vs. S. pneumoniae: P<0.05). With respect to IP-10, this cytokine was only induced by S. pneumoniae and N. meningitidis but not by H. influenzae (Fig. 1D) . We next harvested nuclear extracts from PBMC incubated for 2 h in the presence of live S. pneumoniae, H. influenzae, or N. meningitidis and found that NF-{kappa}B was activated by all bacteria to an almost similar extent, as measured by DNA binding of p65. Earlier studies by others have demonstrated the requirement of NF-{kappa}B activation for induction of IL-8, TNF-{alpha}, IL-6, and IP-10 [28 , 29 ], and our data thus confirm these findings but further show that for IP-10, which is activated differentially by the three bacteria, additional signals are required.


Figure 1
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  		Figure 1. Induction of cytokine expression and NF-{kappa}B activation in PBMC during bacterial infection. Blood was isolated, and PBMC were purified and seeded in culture. The cells were treated with 9 x 107 live bacteria per ml (S. pneumoniae, SK1013; H. influenzae, HK707; N. meningitidis, NGO93), control medium, or LPS. (A–D) The supernatants were harvested 24 h post-treatment, and IL-8 (A), TNF-{alpha} (B), IL-6 (C), and IP-10 (D) were measured by ELISA and Luminex as described. The results are shown as mean ± SEM. (E) Nuclear extracts were isolated 2 h post-treatment, and NF-{kappa}B p65 DNA-binding activity was measured by TransAM. The results are shown as mean ± SEM. Similar results were obtained in three independent experiments.

Use of TLR2, -4, and -9 for activation of NF-{kappa}B and IL-8 by live bacteria
As we wished to study the involvement of various specific TLRs in the generation of an inflammatory response induced by live bacteria, we decided to use HEK293 cell lines stably overexpressing different TLRs. We first looked at cell lines expressing TLR2, -4, or -9, as these receptors have been linked to signaling by Gram-positive bacteria, Gram-negative bacteria, and bacterial DNA, respectively [3 4 5 , 11 ]. Following stimulation of HEK293 cell lines with live bacteria, NF-{kappa}B activation was measured on nuclear extracts by TransAM, and IL-8 secreted in the supernatants was determined by ELISA. The control cell line HEK293-pcDNA3, harboring the empty vector used for transfection of the stable cell lines, displayed no NF-{kappa}B activation or IL-8 production, apart from a small induction consistently observed with N. meningitidis, possibly a result of a low degree of TLR-independent signaling, the nature of which remains unknown (Fig. 2A and 2B ). When TLR2 was overexpressed, all bacteria activated NF-{kappa}B with H. influenzae and N. meningitidis triggering the largest response. To our surprise, only low amounts of IL-8 were induced by S. pneumoniae after 20 h incubation, despite a marked NF-{kappa}B activation within 2 h (Fig. 2C and 2D) . This is different from the results obtained with PBMC shown in Figure 1 , where IL-8 was indeed induced by live S. pneumoniae. However, when we looked at the HEK293 cells under the microscope, we observed extensive cell lysis and death after 20 h of incubation, which probably explains the lack of IL-8 production. In support of this, RNA yield analysis showed reduced recovery (data not shown), and Trypan blue staining revealed extensive cell death of HEK293 cells (85–95%) but not PBMC (5–15%) 20–24 h after treatment with live S. pneumoniae. The same figures for HEK293 after treatment with live H. influenzae and N. meningitidis were 7–13% and 9–17%, respectively. Figure 2E and 2F , shows that in cell lines overexpressing TLR4, only H. influenzae and N. meningitidis activated NF-{kappa}B and IL-8, demonstrating signaling through TLR4, which is particularly pronounced for N. meningitidis. It is interesting that when we examined cells overexpressing alternative TLRs, we found that S. pneumoniae and N. meningitidis but not H. influenzae activated TLR9 (Fig. 2G and 2H) , a receptor previously associated with bacterial-unmethylated DNA [11 ]. Centrifugation of the bacterial culture for 15 min at 4500 g showed that the TLR9-stimulating activity was mainly associated with the bacteria, but a significant fraction could be recovered from the supernatant (data not shown). Again, the reason why only low amounts of IL-8 were produced by S. pneumoniae may be a result of cell lysis, which was observed under the microscope after 20 h incubation. As compared with the cells expressing TLR2 or TLR4, the bacteria induced relatively low levels of IL-8 in the HEK293-TLR9 cells but activated comparable levels of NF-{kappa}B. The reason for this discrepancy between TLR9-induced IL-8 and NF-{kappa}B is presently not known. Finally, doing similar experiments with cell lines overexpressing each of TLR3, -7, or -8, we found that none of these TLRs was activated by the three bacteria, as revealed by IL-8 production (data not shown). Altogether, these data demonstrate that live S. pneumoniae signals through TLR2 and -9, and H. influenzae activates TLR2 and -4, whereas N. meningitidis uses TLR2, -4, and -9.


Figure 2
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  		Figure 2. Induction of NF-{kappa}B activation and IL-8 expression by live bacteria in HEK293 cells transfected with TLR2, -4, or -9. HEK293 cells were stably transfected with a control plasmid (pcDNA3) or plasmids encoding human TLR2, -4, or -9. The cells were seeded and treated with 9 x 107 live bacteria per ml bacteria (S. pneumoniae, SK1013; H. influenzae, HK707; N. meningitidis, NGO93), control medium, or positive controls (A, B: TNF-{alpha}, 25 ng/ml; C, D: Pam3Csk4, 200 ng/ml; E, F: LPS, 100 ng/ml; H: ODN2006, 3 µM). (A, C, E, G) For measurement of NF-{kappa}B activity, cells were lysed 2 h post-treatment, nuclear proteins were isolated, and p65 DNA-binding activity was measured by TransAM. (B, D, F, H) For measurement of IL-8 expression, supernatants were harvested 20 h post-treatment, and cytokine levels were determined by ELISA. The results are shown as mean ± SEM. Similar results were obtained in three to six independent experiments.

Bacterially induced TLR9-dependent signaling is reduced by heat inactivation
To examine if TLR-mediated signaling is dependent on the presence of live bacteria, we next set up similar experiments using bacteria that had been previously heat-inactivated at 65°C for 30 min. Under these conditions, N. meningitidis was still able to induce a low degree of NF-{kappa}B activation and IL-8 secretion in the control cell line in the absence of TLRs (Fig. 3A and 3B ). The response induced through TLR2 and -4 was largely identical to the picture previously observed using live bacteria, except that heat-inactivated S. pneumoniae was now able to induce NF-{kappa}B and IL-8 (Fig. 3C and 3D) . This phenomenon seems to be a result of the inability of heat-inactivated streptococci to mediate cell lysis, as cells were intact after 20 h incubation with heat-inactivated bacteria. As shown in Figure 3G and 3H , signaling through TLR9 was weak when bacteria were heat-inactivated, contrasting with results obtained with live bacteria (Fig. 2G and 2H) . Although minor amounts of NF-{kappa}B and IL-8 were induced through TLR9 by heat-inactivated N. meningitidis, these two mediators were only slightly more increased than in the parental cell line not expressing TLR9 (Fig. 3A and 3B) . Taken together, our data, using heat-inactivated bacteria, reveal a consistent difference from the situation with live bacteria at the level of TLR9 signaling, whereas the results obtained with TLR2 and -4-overexpressing cells were largely identical to the pattern observed with live bacteria.


Figure 3
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  		Figure 3. NF-{kappa}B activation and IL-8 expression by heat-inactivated bacteria in HEK293 cells transfected with TLR2, -4, or -9. HEK293 cells were stably transfected with a control plasmid (pcDNA3) or plasmids encoding human TLR 2, -4, or -9. The cells were seeded and treated with 9 x 107 heat-inactivated bacteria per ml (S. pneumoniae, SK1013; H. influenzae, HK707; N. meningitidis, NGO93), control medium, or positive controls (A, B: TNF-{alpha}, 25 ng/ml; C, D: Pam3Csk4, 200 ng/ml; E, F: LPS, 100 ng/ml; H: ODN2006, 3 µM). (A, C, E, G) For measurement of NF-{kappa}B activity, cells were lysed 2 h post-treatment, nuclear proteins were isolated, and p65 DNA-binding activity was measured by TransAM. (B, D, F, H) For measurement of IL-8 expression, supernatants were harvested 20 h post-treatment, and cytokine levels were determined by ELISA. The results are shown as mean ± SEM. Similar results were obtained in three to six independent experiments.

TLR activation patterns are preserved within each bacterial serotype/-group and are observed over a broad range of concentrations
Having examined the pattern of TLR activation induced by live versus heat-inactivated bacteria, we were intrigued to know whether the observed pattern would be preserved within each bacterial serotype/-group or whether individual strains within the same serotype/-group would differ. We therefore included other strains of N. meningitidis, H. influenzae, and S. pneumoniae and measured NF-{kappa}B activation and IL-8 production. The two N. meningitidis strains included in the study express the same serogroup B capsular polysaccharide but are phylogenetically distinct and express different outer membrane proteins. The two H. influenzae serotype b strains represent two distinct evolutionary lineages (Divisions I and II), which differ significantly in pathogenic potential [30 ]. The two S. pneumoniae strains represent the genome-sequenced serotype 4 strain, TIGR4. SK1013 is the wild-type strain, whereas SK1025 is an isogenic IgA1 protease-deficient mutant [31 ]. Previous studies demonstrated that the Neisseria gonorrhoeae IgA1 protease is a potent inducer of proinflammatory cytokines [32 ]. In spite of these differences, the results for the additional strains were similar to those shown in Figures 2 and 3 , thus suggesting that our results reveal response patterns that are conserved within the examined serogroups/-types of N. meningitidis, H. influenzae, and S. pneumoniae (Fig. 4 ).


Figure 4
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  		Figure 4. Conservation of the pattern of TLRs activated by S. pneumoniae, H. influenzae, and N. meningitidis within bacterial serotypes/-groups. HEK293 cells stably transfected with TLR2, TLR4/MD2, or TLR9 were treated with 9 x 107 live or heat-inactivated bacteria per ml of the strains indicated in the figure. Twenty hours later, supernatants were harvested, and IL-8 levels were measured by ELISA. The results are shown as mean ± SEM. Similar results were obtained in three independent experiments.

To examine if the observed phenomena were seen over a broad range of conditions, we performed dose-response experiments in the cell lines after stimulation with varying amounts of live and heat-inactivated bacteria. As seen in Figure 5A 5B 5C 5D 5E 5F , expression of IL-8 was induced in a dose-dependent manner by the bacteria, and the differential use of TLRs by S. pneumoniae, H. influenzae, and N. meningitidis was also observed throughout the range of bacterial concentrations used.


Figure 5
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  		Figure 5. Dose-response dependency and kinetics of the IL-8 response in HEK293-TLR2, HEK293-TLR4/MD2, and HEK293-TLR9 cells. The cells were seeded and treated with live (A–C) or heat-inactivated bacteria (D–F) in the doses indicated. Twenty hours later, supernatants were harvested. (G–I) The cell lines were treated with 9 x 107 live or heat-inactivated bacteria per ml, and supernatants were harvested at the indicated time-point post-treatment. Levels of IL-8 were measured by ELISA. The results are shown as mean ± SEM. Similar results were obtained in two independent experiments.

To characterize the system further, we also performed kinetics on the cytokine response induced by N. meningitidis in the cell lines, as these bacteria activated TLR2, -4, and -9. In the TLR2- and TLR4-expressing cell lines, live and heat-inactivated bacteria induced detectable levels of IL-8 4 h post-treatment, and plateau levels were reached after 12 h (Fig. 5G and 5H) . In the TLR9-expressing cell line, the response was slightly delayed, and live bacteria induced significantly more IL-8 than heat-inactivated bacteria at all time-points (Fig. 5I) .

Thus, the TLR activation patterns for S. pneumoniae, H. influenzae, and N. meningitidis are preserved within each bacterial serotype/-group and are observed over a broad range of bacterial concentrations.

Genomic DNA of S. pneumoniae, H. influenzae, and N. meningitidis stimulates cytokine expression
To examine if the differential ability of the three bacteria to activate signaling and cytokine expression through TLR9 was a result of different stimulatory potency of the bacterial DNA, we purified genomic DNA from the bacteria and stimulated HEK293-TLR9 cells. Two hours later, nuclear extracts were purified, and NF-{kappa}B activity was measured. As seen in Figure 6 , DNA purified from all three bacteria was able to stimulate cells through TLR9, whereas only live S. pneumoniae and N. meningitidis stimulated the response when whole bacteria were used. Thus, our data suggest that the differential ability of S. pneumoniae, H. influenzae, and N. meningitidis to activate TLR9 may not be a result of differences in the intrinsic ability of their genomic DNA to signal though this TLR but rather suggest that differences in the availability of DNA for TLR9 interaction account for the observed phenomenon.


Figure 6
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  		Figure 6. NF-{kappa}B activation by purified bacterial DNA in HEK293-TLR9 cells, which were treated with 6 µg/ml purified DNA or 9 x 107 live or heat-inactivated bacteria per ml. The cells were lysed 2 h post-treatment, nuclear proteins were isolated, and p65 DNA-binding activity was measured by TransAM. The results are shown as mean ± SEM. Similar results were obtained in two independent experiments.

TLR antagonists differentially inhibit bacterially induced cytokine expression in PBMC
To extend our findings from the TLR-overexpressing HEK293 cell lines to a more physiological situation, we again turned to human PBMC. As a measure of the induction of a proinflammatory response, we measured IL-6, IL-8, TNF-{alpha}, and IP-10 production after 20 h stimulation of PBMC with live S. pneumoniae, H. influenzae, or N. meningitidis (Fig. 7A 7B 7C 7D 7E 7F 7G 7H ). Subsequently, we used TLR antagonists to examine if we could inhibit bacterially induced cytokine production. Data from negative control experiments showed that neither anti-TLR2 and -TLR4 antibodies nor inhibitory ODN, by themselves, induced cytokine production (Fig. 7A , 7C , 7E and 7G ). When we looked at the efficacy of the anti-TLR2 and -TLR4 antibodies, we found that they strongly inhibited TLR activation, although they did not abrogate the response completely (Fig. 7B , 7D , 7F and 7H ). We obtained 50–75% inhibition, and slightly better results were obtained with the anti-TLR4 antibody than with the anti-TLR2 antibody. An even more substantial inhibition was observed with the TLR9 antagonistic ODN, where a 60–80% reduction in the response was measured. For S. pneumoniae, we knew from previous experiments (Figs. 2 and 3) that TLR2 and TLR9 were activated, and we therefore added anti-TLR2 antibodies and inhibitory ODN to cell cultures prior to stimulation with S. pneumoniae. We found that the expression of IL-6, IL-8, and TNF-{alpha} was inhibited by anti-TLR2 antibodies to a larger extent than by inhibitory ODN, demonstrating that TLR2 contributes more to production of these cytokines than does TLR9. In contrast, inhibitory ODN completely abrogated IP-10 production, whereas anti-TLR2 had a more modest inhibitory effect on the production of this cytokine. With respect to H. influenzae, we used anti-TLR2 and -TLR4 antibodies based on our previous findings that these two receptors were relevant (Fig. 2D and 2F) . In agreement with earlier data, H. influenzae did not activate IP-10 production (Fig. 7A) . As shown in Figure 7C , 7E and 7G , anti-TLR2 and -TLR4 antibodies inhibited IL-6, IL-8, and TNF-{alpha} production equally, and none of the antibodies alone was capable of totally inhibiting the response, confirming that TLR2 and -4 contribute to H. influenzae-induced cytokine production. Finally, for N. meningitidis, we found that expression of IL-6, IL-8, and TNF-{alpha} was strongly inhibited by anti-TLR4 antibodies and only marginally by anti-TLR2 antibodies and inhibitory ODN, whereas expression of IP-10 was reduced in the presence of either TLR antagonist. These data therefore confirm observations from the HEK293 cell lines that N. meningitidis activates TLR2, -4, and -9 and also shows distinct roles of these TLRs in regulation of cytokine expression.


Figure 7
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  		Figure 7. Inhibition of cytokine expression induced by live bacteria in PBMC by antagonists of TLR2, -4, and -9. Blood was isolated, and PBMC were purified and seeded in culture. The cells were treated with neutralizing antibodies against TLR2 and -4 (each 10 µg/ml) or inhibitory ODN (5 µM) 15 min prior to infection with 9 x 107 live bacteria per ml (S. pneumoniae, SK1013; H. influenzae, HK707; N. meningitidis, NGO93) or treatment with Pam3Csk4 (200 ng/ml), LPS (100 ng/ml), or ODN M362 (2 µM). Twenty-four hours later, supernatants were harvested, and levels of IL-8 (A, B), TNF-{alpha} (C, D), IL-6 (E, F), and IP-10 (G, H) were measured by Luminex (A–F) and ELISA (G, H). The results are shown as mean ± SEM. Similar results were obtained in two independent experiments.

DISCUSSION

In the present study, we have investigated the pattern of TLR activation by three leading causes of bacterial meningitis, i.e., S. pneumoniae, H. influenzae type b, and N. meningitidis. Our results demonstrate that these three bacteria use different sets of TLRs and that each bacterium can activate several TLRs. It is important that we describe the pattern of TLR used by intact, live and heat-inactivated bacteria, which has not been done previously. We demonstrate that S. pneumoniae activates TLR2 and -9, and H. influenzae uses TLR2 and -4, whereas N. meningitidis is able to activate TLR2, -4, and -9.

We found that S. pneumoniae induced NF-{kappa}B activation through TLR2 and -9 and that the ability to stimulate through the latter was dependent on the viability of the bacteria. The ability of S. pneumoniae to activate TLR2 is well-described [33 ] and involves recognition of the cell wall components peptidoglycan and lipoteichoic acid [34 ]. In addition, there is evidence that pneumolysin is able to stimulate cells through TLR4, although this has not been found in all studies [33 , 35 , 36 ]. Our data did not reveal activation of TLR4 by S. pneumoniae and is thus in agreement with some reports [33 ] and in contrast to others [35 , 36 ]. When we looked at IL-8 production induced by lysed S. pneumoniae, only minor amounts were detected. We believe that the inability of live S. pneumoniae to induce IL-8 may be a result of the activity of bacterial pneumolysin inducing lysis of target cells and thereby preventing cytokine production. Similar results were described by Fowler et al. [37 ], who examined cytokine secretion by human menigeoma cells infected with meningeal pathogens and observed a selective inability of pneumococci to induce a cytokine response as a result of cell death compared with N. meningitidis, H. influenzae, and Escherichia coli. However, it should be noted that there appear to be some differences with respect to the ability of S. pneumoniae to lyse different cell types, as PBMC had normal appearance under the microscope after 24 h in the presence of live pneumocci and were able to secrete large amounts of IL-6, IL-8, TNF-{alpha}, and IP-10. The finding that live S. pneumoniae stimulate TLR9 identifies a novel PRR for this bacterium. Previous work by others in MyD88 knockout and TLR2/TLR4 double-knockout mice has demonstrated that additional TLRs must be involved in recognition of S. pneumoniae [38 , 39 ], and our data strongly indicate a role for TLR9.

When examining H. influenzae-mediated TLR activation, we found involvement of TLR2 and -4, but in contrast to the other bacteria investigated, no TLR9 activation was detected. Previous reports have demonstrated that H. influenzae activates TLR2 through outer membrane proteins, including porin [40 ] and lipoprotein P6 [41 , 42 ], whereas TLR4 is activated via lipooligosaccharide (LOS) [43 , 44 ]. Latz et al. [45 ], who found that a H. influenzae type b-outer membrane protein complex glycoconjugate vaccine requires the presence of TLR2 for obtaining optimal immunogenicity, further illustrated the importance of TLR2. It is interesting that a recent report suggested the existence of a close interaction between TLR2 and TLR4, based on findings that changes in LOS of H. influenzae can favor increased signaling through TLR2 and that TLR4 activation induces TLR2 expression and vice versa [44 ].

The inflammatory response induced by N. meningitidis was robust and involved TLR2, -4, and -9. Previous reports that have linked individual bacterial components to TLR2 and -4 activation include the demonstration that the outer membrane protein porin stimulates cells via TLR2 [46 , 47 ] and that the Neisseria Lip lipoprotein induces NF-{kappa}B in TLR2-transfected HEK293 cells [48 ]. With respect to TLR4, meningococcal LOS-induced activation of macrophages is CD14/TLR4-MD2-dependent [49 ], typical of the pattern of TLR4 engagement by Gram-negative bacteria. The idea that TLR4 is of major importance in the recognition of N. meninigitidis is supported by the description of an association between rare TLR4 mutations and an increased susceptibility to meningococcal disease [1 ]. However, our finding that N. meningitidis serogroup B also activated TLR9 has not been described previously. TLR9-induced NF-{kappa}B activation and IL-8 production were more pronounced for live N. meningitidis, whereas heat-inactivated meningococci elicited only a weak response. This difference may be a result of the active liberation of bacterial DNA by live meningococci, which takes place during meningococcal infection and can be correlated directly with plasma LPS levels [50 ]. Such bacterial DNA is a strong stimulus for TLR9 activation and may play a role during induction of the overwhelming inflammatory response, sometimes observed during fulminant meningococcal disease. Finally, we consistently observed a two- to threefold NF-{kappa}B activation and IL-8 production in the parental HEK293 cells, devoid of any TLR, thereby implying that a contribution from TLR-independent signaling induced by meningococci cannot be excluded entirely.

The key finding of the present study is the use by the host of multiple TLRs to recognize the invading pathogens and initiate the inflammatory response. This observation poses the question as to why several TLRs are involved, as each alone seems capable of triggering an inflammatory response. First, as cells express TLRs in cell type-specific patterns, activation of multiple TLRs allows for stimulation of more cell types. For instance, human TLR9 is expressed abundantly on plasmacytoid DC and B cells but not on macrophages, whereas the two former cell types do not express TLR2, which, however, is expressed on macrophages [2 , 51 , 52 ]. Therefore, in the case of S. pneumoniae, TLR2 and TLR9 activate different cell types, which together mount an inflammatory response. Second, it has been reported that different TLRs act sequentially during the innate phase of the immune response. Weiss et al. [53 ] showed that TLR4 is crucial for the early cytokine response against Salmonella, whereas TLR2, but not TLR4, appears to be required for retaining this response at later time-points [53 ]. The idea of TLRs acting sequentially is further supported by the fact that some TLRs are constitutively expressed only at low levels but are induced during infection and inflammation [52 ]. Third, engagement of more than one TLR may also be a means of the host to avoid immune evasion by the infectious agent. Although the PAMPs recognized by the TLRs are generally essential for pathogenicity and therefore, rarely altered, it has been shown recently that Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis encode flagellins with atypical amino acid sequences in the region of the molecule recognized by TLR5 and therefore, are not recognized by this receptor [54 ]. The presence of multiple TLRs for a given microbe hence secures recognition of the invading pathogen and enhances the chances of eradication. Fourth, although all TLRs trigger similar responses, there are clear differences between the TLRs, wherefore engagement of multiple TLRs may be important for the host to fine-tune the response against the invading pathogen.

When we measured cytokine production by PBMC, we observed that S. pneumoniae and N. meningitidis, but not H. influenzae type b, induced expression of IP-10, which is a potent chemoattractant for activated T cells [55 ]. This induction correlated with the ability of these bacteria to stimulate TLR9, and accordingly, blocking of this receptor strongly reduced expression of IP-10 by PBMC. At the mechanistic level, this observation can be explained by the fact that IP-10 gene expression is regulated by NF-{kappa}B and members of the IRF family [29 ] and that TLR9 potently activates IRF-7 [7 ]. Regardless of the underlying mechanism, there is now evidence demonstrating that activation of multiple TLRs is required to mount an efficient response against microorganisms. For instance, although TLR2 and TLR4 have been shown to be involved in recognition of mycobacterial infections in mice, TLR2- and TLR4-deficient mice are only partially and minimally compromised, respectively, in terms of resistance against Mycobacterium avium, whereas MyD88–/– mice display a severely reduced capacity to control the infection [56 ]. These observations suggest that several TLRs may cooperate in activating an inflammatory response in a synergistic manner.

Collectively, in this work, we have examined the pattern of TLR activation by three principal causes of bacterial meningitis. We demonstrate that live S. pneumoniae activates TLR2 and TLR9. H. influenzae type b activates TLR2 and TLR4, whereas N. meningitidis activates TLR2, TLR4, and TLR9. Thus, distinct, yet overlapping, sets of TLRs are used to mount the inflammatory response. These findings may suggest that establishment of an efficient immune response is dependent on orchestrated activation of specific TLRs and could thus have implications for future design of adjuvants in vaccine development. Conversely, as some TLRs have been reported to contribute to immunopathology rather than host defense [57 ], it is important to understand individual roles of the different TLRs during specific infections and hence, potentially define TLRs, which may be blocked therapeutically in patients with meningitis to dampen the inflammatory response and thus, improve the prognosis of this serious disease.

ACKNOWLEDGEMENTS

The work was supported by grants from LEO Pharma Research Foundation, Kathrine og Vigo Skovgaards Fond, Kong Christian IX og Dronning Louises Jubilæumslegat, Direktør Jacob Madsen og Hustru Olga Madsens Fond, and The Danish Medical Research Council (Grant No. 22-03-0183). The authors thank Jonna Guldberg, Tove Findahl, and Kirsten Stadel Petersen for excellent technical assistance.

Received November 3, 2005; revised March 29, 2006; accepted March 30, 2006.

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