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International Immunology Advance Access originally published online on February 16, 2007
International Immunology 2007 19(4):375-389; doi:10.1093/intimm/dxm003
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TLR2-dependent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macrophage responsiveness

Richard Graveline1,*, Mariela Segura2,*, Danuta Radzioch2 and Marcelo Gottschalk1

1 Groupe de Recherche sur les Maladies Infectieuses du Porc and Centre de Recherche en Infectiologie Porcine, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, Québec, J2S 2M2, Canada
2 Centre for the Study of Host Resistance, Research Institute of the McGill University Health Centre, McGill University, Montréal, Québec, Canada

Correspondence to: M. Gottschalk; E-mail: marcelo.gottschalk{at}umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Streptococcus suis capsular type 2 is an important swine pathogen and an agent of zoonosis. Although meningitis is the most common form of disease, septicemia and septic shock are also frequently reported. Despite reports that CD14 is involved in the recognition of encapsulated S. suis by host cells, the mechanisms underlying exacerbated release of pro-inflammatory cytokines, which may have a negative impact on disease outcome, are unclear. Here, we demonstrated that stimulation of human monocytes by whole encapsulated S. suis or its purified cell wall components influences the relative expression of Toll-like receptor (TLR)-2 and CD14 mRNA. Moreover, this stimulation triggered the release of cytokines (tumor necrosis factor-{alpha}, IL-1ß and IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1), which was significantly reduced by antibody-mediated blocking of TLR2 but not TLR4. Mouse macrophages deficient in TLR2 also showed impaired cytokine responses to encapsulated bacteria. Given that this response was completely abrogated in myeloid differentiation factor 88 (MyD88)-deficient macrophages, other TLRs might also be involved. Furthermore, we demonstrated that the presence of capsular polysaccharide (CPS)-modulated S. suis interactions with TLRs. In the absence of CPS, uncovered cell wall components induced cytokine and chemokine production via TLR2-dependent as well as -independent pathways, whereas CPS contributes to MCP-1 production in a MyD88-independent manner. Overall, this study contributes to a better understanding of the inflammatory processes induced by an encapsulated pathogen and suggests that the relative expression of CPS, known to be modulated during bacterial invasion and dissemination in the host, might alter interactions with host cells and, consequently, the outcome of the inflammatory response.

Keywords: capsule, cell wall, inflammation, meningitis, TLR


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Streptococcus suis, a Gram-positive encapsulated coccus, is considered to be a global agent of infection in swine, as well as in humans who work in close contact with pigs (1). To date, 35 capsular types have been identified and S. suis capsular type 2 is regarded as the serotype most frequently associated with disease in both humans and pigs (2). Recently, an unprecedented outbreak in China resulted in >200 human cases that were directly linked to a concurrent outbreak of S. suis infection in pigs. Of these human cases, ~20% were fatal (3–5). The clinical presentation of S. suis may vary from asymptomatic bacteremia to fulminant systemic disorder. Meningitis is the most striking feature, and the presence of fibrin, oedema and cellular infiltrates in the meninges and choroids plexus are the histopathological features most frequently observed. Symptoms reported in the latest outbreak in China include high fever, malaise, nausea and vomiting, followed by meningitis, subcutaneous hemorrhage, toxic shock and coma in severe cases (4, 5). This increased severity of S. suis infection in humans, such as a shorter incubation time, more rapid disease progression and higher rate of mortality, underscores the critical need to better understand the factors associated with pathogenesis of S. suis infection (1).

Indeed, our knowledge of S. suis virulence factors is still limited. Although several molecules have been proposed, only the capsular polysaccharide (CPS) has been proven so far to be critical to the pathogenesis of S. suis infections (6–8). Among other putative factors, a hemolysin belonging to the cholesterol-binding toxin family, virulence-related proteins, a fibronectin-binding protein, different proteases and enzymes have been reported in S. suis (1, 9–13). Although the pathogenesis of meningitis caused by S. suis capsular type 2 remains poorly elucidated, some mechanisms have been proposed. Streptococcus suis is often transmitted via the respiratory route and remains localized in the palasile tonsils. From this site, bacteria may gain access to the bloodstream, where they persist until they reach the central nervous system (CNS). Although there are some controversies regarding the mechanisms used by S. suis to disseminate in the blood, it is believed that S. suis avoids clearance by professional phagocytes via its anti-phagocytic CPS (6–8, 14–16).

Mechanisms used by S. suis to cross the blood–brain barrier (BBB) are poorly understood. Recently, it has been shown that S. suis is able to adhere to and invade endothelial cells of porcine brain origin (17, 18). However, other mechanisms, such as the up-regulation of pro-inflammatory mediators and increased leukocyte trafficking, may contribute to the breakdown of the BBB (11, 19–21). All these processes may, therefore, have a direct impact on the development of meningitis. Indeed, studies from our laboratory have shown that S. suis is able to induce the release of cytokines and chemokines by human brain microvascular endothelial cells (20), mouse and human phagocytes (22, 23) as well as porcine blood leukocytes (24). In addition, S. suis was shown to up-regulate the expression of adhesion molecules on human monocytes (25). Therefore, the capacity of S. suis to induce a strong inflammatory response in the CNS may be the leading cause of increased intracranial pressure, which is responsible for the clinical signs of meningitis.

Our understanding of the pathogenesis of disease caused by S. suis has been further increased by the observation that S. suis mediates CD14-dependent and -independent cytokine and chemokine production by human monocytes (23). CD14 has been shown to be important in the recognition of LPS, the major component of the outer membrane of Gram-negative bacteria (26) and also other cell wall constituents of Gram-positive bacteria (27, 28). The CD14 receptor is known to interact with members of the Toll-like receptor (TLR) family, which share common signaling pathways that trigger the association of adaptor molecules, such as the myeloid differentiation factor 88 (MyD88), with several kinases and lead to the nuclear translocation of nuclear factor-kappa B (NF-{kappa}B), which induces the expression of target genes (29–31). TLRs have been shown to recognize and mediate signaling for a wide range of microbial components. For example, TLR2, in combination with TLR1 or TLR6, recognizes bacterial lipoprotein, lipoteichoic acid (LTA) and peptidoglycans (PGNs), as well as yeast lipoarabinomannan and zymosan (30, 32). Furthermore, both TLR2 and TLR4 have been reported to interact with CD14 and to mediate the recognition of bacterial cell wall components from Gram-negative and Gram-positive bacteria (31, 33, 34). Although a common feature of Gram-positive bacteria is the characteristic presence of PGN and LTA in their cell walls, the expression of these pathogen-associated molecular patterns does not necessarily lead to similar activation through TLRs, as notably reported for Group B Streptococcus (GBS) (28, 33, 35–37). In addition, the presence of an external layer of CPS in some bacterial species further modifies the interaction with TLRs and might give rise to differential activation responses (37–39). Thus, the purpose of this work is to evaluate the interaction of S. suis capsular type 2 and its surface components with TLR2, TLR4 and CD14 on phagocytic cells and the consequent cell activation through these receptors and their common downstream adaptor molecule, MyD88. The modulating role of the CPS is addressed to better understand the interactions of this encapsulated bacterium with cells of the innate immune system.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
Cell culture media, fetal bovine serum (FBS), penicillin G and streptomycin were purchased from GIBCO (Burlington, VT), 2ß-mercaptoethanol from Bio-Rad (Mississauga, Ontario, Canada), ultra-purified Escherichia coli O55:B5 LPS and Mycoplasma fermentans 2-kDa macrophage-activating lipopeptide from Apotech Corporation (Epalinges, Switzerland), recombinant human IFN-{gamma} from Cedarlane Laboratories (Hornby, Ontario, Canada) and polymyxin B sulfate from Sigma–Aldrich (Oakville, Ontario, Canada).

Bacterial strains and growth conditions
The S. suis type 2 virulent strain 31533, originally isolated from a case of porcine meningitis (40) and previously used for studies of cytokine production by both murine and human cells, was used as the reference strain in comparison with its isogenic non-encapsulated mutant B218 (22–24). Mutant B218, which was produced in our laboratory by allelic exchange and corresponds to a previously reported transposon-derived mutant (8), has been used in previous studies (16, 17, 24). Bacteria were grown overnight on bovine blood agar plates at 37°C, and isolated colonies were used as inocula for Todd–Hewitt broth (THB) (Difco Laboratories, Detroit, MI, USA), which were incubated for 18 h at 37°C. Working cultures for cell stimulation assays were produced by inoculating 10 ml of the overnight culture into 200 ml THB with agitation at 37°C for 6 h until they reached the mid-log phase (540 nm, optical density of 0.4–0.5). Bacteria were washed twice in PBS, pH 7.4, and diluted to ~2 x 109 colony-forming unit (CFU) ml–1 in PBS. The final suspension was plated onto THB agar in order to accurately determine CFU ml–1. Bacteria were then heat-killed by incubating organisms at 60°C for 45 min [minimal experimental conditions required for killing S. suis (22)]. Killed cultures were subcultured onto blood agar plates at 37°C for 48 h to confirm the absence of viable organisms. Killed bacteria preparations were stored at 4°C and re-suspended in cell culture media immediately before stimulation assays. Heat-killed bacteria were used since live bacteria were previously reported to induce cell death under these assay conditions (23).

Purified bacterial components
Purified CPS of S. suis capsular type 2 was prepared as previously reported (41). Purified cell wall was produced using a modified protocol adapted from Tuomanen et al. (42) and Heumann et al. (43) as previously reported (22).

Cell lines and cell culture
THP-1 human monocytic cell line, derived from an acute monocytic leukemia (American Type Culture Collection TIB-202, Rockville, MD, USA), was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin G (100 U ml–1), streptomycin (100 µg ml–1) and 2ß-mercaptoethanol (50 mM). Cells were incubated at 37°C with 5% CO2. Bone marrow-derived TLR2–/– and MyD88–/– mouse macrophage cell lines were generated by immortalization with a recombinant retroviral J2 construct, as previously described (44, 45). The bone marrow cells were obtained from tlr2 (46) and MyD88 gene knockout mice (47). As wild type (WT) counterpart for TLR2+/+ cell line, we used a macrophage cell line derived from bone marrow of C57BL/6J mice that shared the same genetic background as the TLR knockout mice. This WT control cell line had previously been characterized in detail (48) and is, hereafter, referred as TLR2WT. The MyD88+/+ cell line was derived from bone marrow of MyD88+/+ mice (44, 45, 47). Macrophages were grown at 37°C with 5% CO2 in Dulbecco's modified eagle medium supplemented with penicillin G and streptomycin and 10% heat-inactivated FBS.

Stimulation assays
Prior to simulation assays, human THP-1 monocytes were differentiated by pre-treatment with IFN-{gamma} (500 U ml–1) for 48 h as previously described (23). Differentiated THP-1 monocytes were then washed and re-suspended in fresh medium at 106 cells ml–1, and 0.5 ml of this suspension was distributed in 24-well plates (FalconTM, Becton Dickinson, Bedford, MA, USA). For stimulation assays with TLR2–/–, MyD88–/– and WT mouse macrophages, cell cultures were re-suspended in fresh medium at 106 cells ml–1 and 0.5 ml of this suspension distributed in 24-well plates. Streptococcus suis strain 31533 or the mutant strain B218, diluted to 109 CFU ml–1 in culture medium, was then added to the culture plates. In selected experiments, cells were stimulated with purified S. suis cell wall (150 µg ml–1) or purified CPS (100 µg ml–1). These concentrations were chosen based on our previous results (20, 22, 24). Cells stimulated with highly purified LPS (50 ng ml–1) and MALP-2 (10 ng ml–1) served as positive controls. Cells cultured in medium alone served as controls for spontaneous cytokine release and basal level of mRNA expression. Cell culture plates were incubated at 37°C with 5% CO2. At different time intervals (see Results), culture supernatants were harvested from individual wells, aliquoted and frozen at –20°C until cytokine analysis. The cell pellet was then treated with 1 ml of Trizol reagent (Invitrogen, Burlington, Ontario, Canada) to extract RNA as instructed by the manufacturer. The final RNA pellet was re-suspended in 20 µl of DEPC-treated water, and RNA concentration was measured using the Ribogreen RNA quantitation kit (Invitrogen). RNA was stored at –80°C for future use.

Reverse transcriptase–PCR assay for TLR2, TLR4 and CD14
Reverse transcription of the mRNA samples to cDNA was performed with Superscript II (Invitrogen) and random primers (Roche, Laval, Quebec, Canada). Total mRNA (2 µg) was mixed with 200 ng of random primers, 10 mM deoxynucleoside triphosphate mixture (dNTP) (Amersham Biosciences, Baie d'Urfe, Quebec, Canada) and DEPC-treated water up to 20 µl, then heated at 65°C for 5 min and cooled at 4°C in a T-Gradient thermocycler Whatman (Biometra GmbH, Göttingen, Germany). Then, 5x first-strand buffer (GIBCO), 0.1 M dithiothreitol and RNase Guard ribonuclease inhibitor (Amersham Biosciences) were added and the reaction sample was incubated for 10 min at 25°C, followed by 2 min at 42°C. Superscript II reverse transcriptase (RT) 400 U l–1 (Invitrogen) was added and the resulting mixture was incubated for 50 min at 42°C, 15 min at 70°C and held at 4°C. Samples were conserved at –20°C until used. Primers (Invitrogen) used for PCR were of the following sequences: TLR2, forward 5'-ATGAAAATGATGTGGGCCTG-3' and reverse 5'-TTACCCAAAATCCTTCCCGC-3'; TLR4, forward 5'-CTGCAATGGATCAAGGACCA-3' and reverse 5'-TCCCACTCCAGGTAAGTGTT-3', and CD14, forward 5'-AGGACTTGCACTTTCCAGCTTG-3' and reverse 5'-TCCCGTCCAGTGTCAGGTTATC-3'. Human ß-actin served as housekeeping gene and was amplified using the following primers: forward 5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' and reverse 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'. The PCR mixture contained 2 µl of the cDNA, 2.5 µl 10x PCR buffer (Roche), 10 mM dNTP (DNA polymerization mixture, Amersham Biosciences), 1 µl Taq DNA polymerase (Roche) and 0.4 µl of each gene primer in a total volume of 25 µl. Amplification was performed in a T-Gradient thermocycler Whatman (Biometra, Kirkland, QC, Canada) as follows: TLR2 and CD14, 32 cycles of denaturation at 95°C for 40 s, annealing at 55°C for 40 s and elongation at 72°C for 1 min and TLR4, 29 cycles of denaturation at 95°C for 40 s, annealing at 55°C for 40 s and elongation at 72°C for 1 min. Amplified samples were visualized on 1.8% agarose gels stained with ethidium bromide and photographed under UV. The intensity of bands was quantified by densitometry with an AlphaImager 2000 Multimage camera (Alpha Innotech Corp., San Leandro, CA, USA) and software (AlphaEase 3.2). To compare the relative mRNA expression levels from each of the samples, the values are presented as the ratio of the band intensities of the cytokine RT–PCR product over the housekeeping RT–PCR product run simultaneously in the same tube. Absence of competition between specific gene and housekeeping primers was confirmed before selection of the primers. The conditions and amount of cycles for each of the mRNA assessed by RT–PCR were carefully selected at the linear phase.

ELISA for cytokines
The concentration of tumor necrosis factor-{alpha} (TNF-{alpha}), IL-1ß, IL-6, IL-8 and MCP-1 produced in vitro by human monocytes, and of IL-6 and MCP-1 by mouse macrophages, were measured by ELISA using pair-matched mAbs and cytokine standards from R&D Systems (Minneapolis, MN, USA), according to the manufacturer's recommendations. Dilutions of supernatants were added in duplicate wells to each ELISA plate. Analyses were performed at least four times for each individual monocyte stimulation assay.

TLR2 and TLR4 blockade
In selected experiments, anti-human TLR2 or anti-human TLR4 blocking mAbs were used to assess whether cytokine production induced by S. suis or its purified components follows a TLR2- or a TLR4-dependent pathway. Differentiated THP-1 cells were cultured in the presence of mouse anti-human TLR2 (TL2.1; 15 µg ml–1), anti-human TLR4 (HTA125; 15 µg ml–1) or Ig isotype-matched (IgG2a) control mAbs. mAbs were obtained from e-Bioscience (San Diego, CA, USA) and added 1 h prior to the bacterial stimuli to ensure complete blocking. After 8 h of stimulation, culture supernatants were harvested and analyzed for cytokine production as described above.

Endotoxin contamination
All solutions and bacterial preparations used in these experiments were tested for the absence of endotoxin using a Limulus amebocyte lysate gel-clot test (Pyrotell STV, Cape Cod, MA, USA) with a sensitivity limit of 0.03 EU ml–1. In some experiments, absence of endotoxin contamination during cell stimulation was controlled by parallel assays with polymyxin B at 10 µg ml–1 (data not shown).

Statistical analysis
Each cell stimulation test was performed at least in triplicate. Semi-quantitative values for RT–PCR are expressed as the mean sample mRNA/housekeeping ratios from three independent experiments, whereas quantitative cytokine values obtained by ELISA are expressed as means ± SEs of pg ml–1 values. Each sample was analyzed in triplicate in the ELISA test. Differences were analyzed for significance by using the Student's unpaired t-test (two-tailed P value). A P value < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Streptococcus suis modulates the expression levels of TLR2 and CD14, but not TLR4, mRNA
It has been reported that THP-1 monocytes express TLR2 and TLR4 as measured by RT–PCR (49). However, the transcriptional regulation of these two receptors following stimulation with whole S. suis has not been previously described. To analyze whether S. suis modulates the mRNA expression levels of these pattern recognition receptors, THP-1 human monocytes were stimulated with encapsulated S. suis strain 31533 or its isogenic non-encapsulated mutant B218. MALP-2 (a lipopeptide originally isolated from Mycoplasma fermentans) and LPS were used as positive controls for the induction of mRNA expression of TLR2 and CD14/TLR4, respectively (50, 51). As shown in Fig. 1, non-stimulated THP-1 cells expressed a relatively constant basal level of TLR2, TLR4 and CD14 mRNA throughout the incubation period. In contrast, 30 min after stimulation, both the encapsulated strain and its non-encapsulated mutant up-regulated the expression of TLR2 mRNA to similar levels as those induced by the TLR2 ligand, MALP-2 (Fig. 1A). This up-regulation was followed by a decrease in mRNA expression between 4 and 8 h, a pattern which was more pronounced with the non-encapsulated mutant B218. A second peak of up-regulation was observed at 18 h after stimulation of monocytes with the encapsulated strain 31533 as well as with MALP-2, but was delayed to 24 h after incubation with mutant B218 (Fig. 1A). The kinetics of CD14 mRNA expression differed from those observed for TLR2 mRNA (Fig. 1B). Indeed, no differences in CD14 mRNA expression levels were observed during the first 8 h of incubation. Expression levels of CD14 mRNA started to increase at 18 h and continued to increase until at least 24 h after stimulation of monocytes with both S. suis strains, as well as with LPS-positive control (Fig. 1B). As reported by Tamai et al. (52), LPS also enhanced the expression of TLR4 in THP-1 cells in a time-dependent manner. This increase in TLR4 mRNA levels was observed at 4–8 h of incubation and continued to increase up to at least 24 h after LPS treatment (Fig. 1C). However, no changes in the expression levels of TLR4 mRNA were obtained after stimulation of cells with encapsulated or non-encapsulated S. suis strains compared with cells incubated with medium alone. Taken together, these data suggest that THP-1 cells respond to stimulation with S. suis as indicated by changes in the expression levels of TLR2 and CD14, which may indicate a possible interaction between S. suis and these pattern recognition receptors.


Figure 1
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  		Fig. 1. Streptococcus suis modulates the expression of TLR2 and CD14 mRNA, but not TLR4 mRNA. THP-1 cells (5 x 105 cells ml–1) were incubated with medium alone, heat-killed S. suis strain 31533 or the non-encapsulated mutant strain B218 (109 CFU ml–1). LPS (50 ng ml–1) and MALP-2 (10 ng ml–1) were used as positive controls for TLR4 and TLR2, respectively. At different time intervals, total RNA was extracted and TLR2, CD14 and TLR4 mRNA expression was analyzed by RT–PCR. Results are representative of three independent experiments performed in duplicate. It should be noted that relative mRNA expression levels from each of the samples were calculated as the ratio of the band intensities of the RT–PCR product over the housekeeping RT–PCR product run simultaneously in the same tube. The ß-actin bands displayed in the micrographs are thus representative of one sample.

 
Purified components of S. suis also modulate the expression of TLR2 and CD14 mRNA
To further characterize the mechanism by which S. suis is recognized by phagocytic cells, we analyzed the capacity of purified components from the cell surface of S. suis to modulate the expression of TLR2 and CD14 mRNA. Human THP-1 monocytes were stimulated with whole bacteria, purified CPS or bacterial cell wall. Based on the results obtained in kinetic studies of TLR2 and CD14 expression (Fig. 1), mRNA levels were analyzed at 30 min, 8 and 24 h for TLR2 and 24 h for CD14 analysis. As illustrated in Fig. 1, an early up-regulation of TLR2 mRNA followed by a decrease at 8 h to reach a higher level of transcription at 24 h occurred when cells were stimulated with either whole encapsulated bacteria or the non-encapsulated mutant strain (Fig. 2A). Similar trends were obtained when THP-1 cells were stimulated with purified CPS or bacterial cell wall. However, these purified components showed a lower capacity to induce the expression of TLR2 transcripts within <30 min of incubation compared with whole bacteria. In addition, purified cell wall and, to a lesser extent, CPS induced a late up-regulation of CD14 expression in a similar manner as observed with whole bacteria (Fig. 2B). Thus, purified components of the bacterial surface may be responsible, at least in part, for the modulation of TLR2 and CD14 mRNA expression observed with whole bacteria.


Figure 2
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  		Fig. 2. Purified components of Streptococcus suis modulate the expression of TLR2 and CD14 mRNA in the same manner as whole bacteria. (A) Comparative study of TLR2 mRNA expression of THP-1 cells (5 x 105 cells ml–1) alone or stimulated with heat-killed S. suis strain 31533, S. suis non-encapsulated mutant B218 (109 CFU ml–1), purified CPS (100 µg ml–1), purified cell wall (150 µg ml–1) or MALP-2 (10 ng ml–1, used as positive control) for 30 min, 8 and 24 h or (B) for 24 h. Results are representative of two independent experiments performed in duplicate. It should be noted that relative mRNA expression levels from each of the samples were calculated as the ratio of the band intensities of the RT–PCR product over the housekeeping RT–PCR product run simultaneously in the same tube. The ß-actin bands displayed in the micrographs are thus representative of one sample.

 
Differences in cytokine production between the encapsulated strain and its non-encapsulated mutant
To verify the capacity of S. suis to induce an inflammatory response that may parallel the results on the modulation of TLR2 and CD14 expression, we analyzed the level of cytokine and chemokine production after cell stimulation with whole bacteria. Incubation of THP-1 monocytes with encapsulated S. suis resulted in a time-dependent production of cytokines. TNF-{alpha} production was highest at 8 h after stimulation, whereas production of the four other cytokines tested gradually increased up to 24 h after stimulation (Fig. 3). Similar kinetics of cytokine production were observed with the isogenic non-encapsulated mutant B218. However, depending on the presence or absence of CPS, three different patterns of cytokine production were observed: (i) B218 mutant induced significantly higher levels of TNF-{alpha} and IL-1ß than the encapsulated strain (P < 0.05) (Fig. 3A and B); (ii) although levels of IL-6 and IL-8 induced by mutant B218 were higher than those induced by the encapsulated strain at 8 h of incubation, both strains induced similar peak production levels of these cytokines following 18–24 h of cell stimulation (P > 0.05) (Fig. 3C and D) and (iii) the non-encapsulated strain B218 induced lower levels of MCP-1 production than the encapsulated strain (P < 0.05) (Fig. 3E). Thus, both encapsulated and non-encapsulated S. suis induced the release of all five cytokines and the quantitative differences observed are related to the presence or absence of CPS.


Figure 3
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  		Fig. 3. Differences in cytokine production between the WT strain and its non-encapsulated mutant. Time course production of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), IL-8 (D) and MCP-1 (E) by THP-1 cells stimulated with heat-killed Streptococcus suis 31533 strain or S. suis non-encapsulated B218 mutant (109 CFU ml–1). Culture supernatant were harvested at different time intervals and analyzed in triplicate for cytokine production by ELISA. Data are expressed as mean pg ml–1 ± SE from three independent experiments. *P < 0.05 compared with cells stimulated with the WT strain 31533.

 
Purified cell wall induces the release of cytokines and chemokines whereas purified CPS only stimulates MCP-1 production
Since different patterns of cytokine production were observed after stimulation of human monocytes with encapsulated or non-encapsulated S. suis strains, we wanted to analyze whether purified S. suis cell wall and CPS induce differential release of pro-inflammatory cytokines and chemokines. As shown in Fig. 4, panels (A–E), only purified cell wall of S. suis was able to induce the release of significant levels of IL-1ß, IL-6, IL-8, MCP-1 and, to a lesser extent, TNF-{alpha}. Although, in general, the cell wall induced lower levels of cytokine production than those observed after activation with whole bacteria (Fig. 3), this purified component demonstrated a stimulating activity similar to or higher than that of MALP-2 or LPS (Fig. 4). In contrast, when THP-1 cells were stimulated with purified CPS, only significant MCP-1 production was detected (P < 0.001 versus control medium), whereas levels of other cytokines were very low (Fig. 4; P > 0.05). Thus, although both purified CPS and purified cell wall modulate the relative expression of TLR2 and CD14, only purified cell wall induced a significant release of all five pro-inflammatory cytokines.


Figure 4
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  		Fig. 4. Purified components of Streptococcus suis differently induce the release of pro-inflammatory cytokines. Comparative study of TNF-{alpha} (A), IL-1ß (B), IL-6 (C), IL-8 (D) and MCP-1 (E) production by THP-1 cells stimulated with the positive controls, LPS (50 ng ml–1) and MALP-2 (10 ng ml–1), or with the S. suis purified components, cell wall (150 µg ml–1) or CPS (100 µg ml–1). Data are expressed as mean pg ml–1 ± SE from three independent experiments. *P < 0.05 compared with non-stimulated cells (cells alone).

 
Blockade of TLR2, but not TLR4, inhibits the production of pro-inflammatory cytokines by THP-1 cells stimulated with S. suis
We have previously demonstrated that blocking membrane-bound CD14 receptors on the surface of THP-1 cells with two different mAbs (clones MY4 and RMO52/IOM2) partially inhibited TNF-{alpha}, IL-1ß and IL-6 release induced by S. suis, whereas MCP-1 was only slightly reduced and the production of IL-8 was independent of CD14 (23). To assess the role of TLR2 and/or TLR4 as part of the receptor complex used by S. suis to trigger cytokine production by human monocytes, THP-1 cells were pre-treated with mAbs against human TLR2 (clone TL2.1) or human TLR4 (clone HTA125) or with isotype controls before stimulation with either encapsulated S. suis strain 31533 or the non-encapsulated mutant B218. As previously reported, these mAbs selectively block the release of cytokine production by THP-1 cells, depending on the stimuli used (53, 54). As expected, pre-treatment of THP-1 cells with anti-TLR4 mAb significantly inhibited the production of all five cytokines tested in response to LPS (P < 0.01) (Fig. 5F–J). However, no significant effect was observed when cells pre-treated with anti-TLR4 mAb or with an isotype control were stimulated with encapsulated S. suis, non-encapsulated mutant or MALP-2 (P > 0.05). On the other hand, pre-treatment of cells with anti-TLR2 mAb inhibited WT S. suis-induced TNF-{alpha}, IL-1ß, IL-6, IL-8 and MCP-1 production by 82, 83, 87, 75 and 65%, respectively (P > 0.001) (Fig. 5A–E). Non-encapsulated mutant-induced TNF-{alpha}, IL-1ß, IL-6 and IL-8 production was inhibited by 65, 71, 67 and 50%, respectively (P < 0.001), while MCP-1 production induced by this mutant strain was not significantly altered by pre-treatment of monocytes with anti-TLR2 mAb (21% of inhibition, P > 0.05). The specificity of TL2.1 mAb was clearly demonstrated by the lack of inhibition of LPS-induced cytokine production and by the significant inhibition of MALP-2 stimulatory activity (Fig. 5A–E). These data indicate that blockade of TLR2 resulted in more efficient inhibition of TNF-{alpha}, IL-1ß and IL-6 than of IL-8 and MCP-1 release induced by both WT and non-encapsulated S. suis strains. On the other hand, the inflammatory activity induced by the non-encapsulated mutant, especially MCP-1 production, was generally less affected by treatment of cells with TL2.1 mAb than that induced by the WT strain.


Figure 5
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  		Fig. 5. Effect of anti-TLR mAbs on cytokine production induced by Streptococcus suis. THP-1 cells were treated for 1 h with medium only (control), with 15 µg ml–1 of anti-TLR2 mAb TLR2.1 or anti-TLR4 mAb HTA125 or an equivalent concentration of IgG2a isotype control. Cells were then stimulated for 8 h with heat-killed S. suis 31533, non-encapsulated mutant strain S. suis B218 (109 CFU ml–1), cell wall (150 µg ml–1) or CPS (100 µg ml–1). To evaluate the efficacy of the anti-TLR mAbs, THP-1 cells were also stimulated with LPS (50 ng ml–1) or MALP-2 (10 ng ml–1). Induction of TNF-{alpha} (A and F), IL-1ß (B and G), IL-6 (C and H), IL-8 (D and I) and MCP-1 (E and J) were evaluated by ELISA. Data are expressed as mean pg ml–1 ± SE from three independent experiments. *P < 0.05 compared with untreated cells or isotype control-treated cells.

 
We also evaluated the effect of TLR2 blockade on cytokine production induced by the purified bacterial cell wall as well as MCP-1 production induced by CPS. As shown in Fig. 5, panels (A–E), pre-treatment of cells with TL2.1 mAb significantly inhibited cell wall-induced TNF-{alpha}, IL-1ß, IL-6, IL-8 and MCP-1 production by 87, 89, 87, 71 and 72%, respectively (P < 0.01). Surprisingly, the CPS-induced production of MCP-1, the only cytokine produced in significant amounts by human monocytes activated with this bacterial component, was shown to be independent of TLR2 (2830 ± 650 versus 2400 ± 130; P > 0.05). It should be noted that the blockade of TLR4 by HTA125 mAb had no effect on the pro-inflammatory activity of either the cell wall or CPS (Fig. 5F–J). These data confirm that the mechanisms underlying S. suis stimulation of human monocytes involve mainly a TLR2- rather than a TLR4-dependent pathway and suggest that activation of TLR2-mediated cytokine induction may involve interaction of this receptor with bacterial cell wall components.

MyD88 is the major downstream mediator of TLR-dependent S. suis-induced cytokine production
To confirm the role of TLR2 in S. suis-induced cytokine production and to determine whether MyD88 is involved as an adaptor molecule in downstream signaling events induced by S. suis interaction with TLRs, we compared the capacity of bone marrow-derived macrophages from WT, TLR2–/– or MyD88–/– mice to respond to S. suis stimulation. Macrophages were incubated in the presence of either WT or non-encapsulated S. suis or with purified components of the bacterial surface, that is, CPS and the cell wall. Supernatants were harvested at 12 and 24 h after stimulation and analyzed for MCP-1 and IL-6 release. These incubation times were shown to be optimal for the production of these cytokines (data not shown).

Encapsulated S. suis induced production of both IL-6 and MCP-1 by WT macrophages (Fig. 6A and B). This activity was significantly impaired in TLR2–/– macrophages (P < 0.01). The non-encapsulated mutant induced similar levels of IL-6 and MCP-1 production by WT macrophages compared with the parent strain. However, whereas B218 mutant-induced IL-6 was significantly reduced in TLR2–/– macrophages (P < 0.01), MCP-1 production was unchanged (Fig. 6A and B). These results paralleled those observed after mAb-mediated blocking of TLR2 on human monocytes. It should be noted that cytokine release induced by MALP-2 was significantly inhibited in TLR2–/– macrophages (P < 0.001), whereas cytokine levels induced by LPS were similar to those observed in WT cells (P > 0.05).


Figure 6
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  		Fig. 6. Bone marrow-derived macrophages from TLR2–/– and MyD88–/– mice exhibit a defective response in IL-6 and, to a lesser extent, MCP-1 production in response to stimulation with Streptococcus suis. Macrophages (5 x 105 cells ml–1) from WT, TLR2–/– and MyD88–/– mice were incubated with heat-killed S. suis strain 31533, heat-killed S. suis non-encapsulated mutant strain B218 (109 CFU ml–1), cell wall (150 µg ml–1) or CPS (100 µg ml–1). LPS (50 ng ml–1) and MALP-2 (10 ng ml–1) were used as positive controls. Concentrations of IL-6 (A and C) and MCP-1 (B and D) in 24 and 12 h culture supernatants, respectively, were analyzed by ELISA. Data are expressed as mean pg ml–1 ± SE from three independent experiments. *P < 0.05 compared with WT macrophages.

 
In contrast to whole bacteria, purified S. suis cell wall induced low levels of IL-6 release and relatively high levels of MCP-1 production by WT macrophages (Fig. 6A and B). In cell wall-stimulated TLR2–/– macrophages, IL-6 production was completely impaired (P < 0.01) and MCP-1 release was reduced by 41% compared with WT cells (P < 0.001). It should be noted that, in agreement with results obtained with antibody treatment, MCP-1 production induced by purified CPS was not significantly affected in TLR2–/– macrophages (P > 0.05).

Since MyD88 is an adaptor protein that plays a critical role in signaling for most TLRs, MyD88–/– macrophages were compared with WT cells to determine its relative contribution to S. suis-mediated cell activation. As expected, the stimulatory activity of LPS and MALP-2 was significantly abrogated in MyD88–/– macrophages (P < 0.001). In addition, as shown in Fig. 6 (C and D), the stimulatory activities of WT or non-encapsulated S. suis as well as that of the purified cell wall were either completely suppressed (IL-6) or markedly inhibited (MCP-1) in MyD88–/– macrophages (P < 0.001). However, in accordance with the results described above, MCP-1 production induced by purified CPS was not significantly affected in MyD88–/– macrophages compared with WT cells (P > 0.05).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Despite advances in anti-microbial therapy, infections with encapsulated bacteria that are of importance to humans and/or veterinary medicine, such as Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, GBS, Haemophilus parasuis and S. suis, continue to cause serious clinical and economic problems. Although S. suis is a pathogen of increasing importance in the swine industry and is also an emerging human infectious disease in Asia (3), our understanding of the mechanisms involved in its pathogenesis is still limited. Previous studies showed that S. suis induces the release of pro-inflammatory cytokines and chemokines, as well as the expression of adhesion molecules, by different types of host cells (1). However, the mechanisms by which S. suis is recognized by target cells remain unknown. In the present study, we demonstrated for the first time that encapsulated S. suis is able to interact with TLR2, probably in association with CD14 (23), and to induce the expression of several cytokines and chemokines. Furthermore, S. suis was shown to modulate the transcriptional levels of these receptors. Indeed, whereas S. suis does not seem to modulate the level of TLR4 mRNA expression, recognition of encapsulated S. suis by monocytes was associated with changes in both TLR2 and CD14 mRNA expression. TLR2 transcription was up-regulated as early as 30 min after induction. Although the mRNA expression pattern of TLR2 may not precisely reflect its protein level, an early increase of TLR2 production at the protein level after treatment of THP-1 cells with bacterial lipoprotein has been previously observed. This early up-regulation was followed by a decrease in the protein level of TLR2 as soon as 2 h after stimulation (55). Our results suggest similar mechanisms given that S. suis-induced up-regulation of TLR2 expression was followed by a decrease in relative mRNA levels. This down-modulation of TLR2 mimics that reported for the immune receptors of LPS-mediated activation, which is, at least in part, associated with immunotolerance (56, 57). This negative feedback observed during in vitro activation of monocytes by S. suis was followed by a second up-regulation of TLR2 as well as a later peak of CD14 mRNA expression, which may correspond to a second phase of activation. One hypothesis to explain the late up-regulation of CD14 mRNA expression is that the requirement for the THP-1 cell line to be pre-differentiated with IFN-{gamma} 24 h before induction leads to, among other effects, increased expression of CD14 during the differentiation process (23, 52). On the other hand, dysregulation of CD14 expression was not observed during LPS or bacterial lipoprotein tolerization (55, 56, 58, 59). Thus, although these results do not demonstrate a direct binding of S. suis to TLR2 and CD14, they strengthen the hypothesis that S. suis is recognized by host cells via TLR2 in combination with CD14.

Indeed, our previous studies demonstrated that under the same experimental conditions, blocking CD14 on THP-1 cells markedly inhibited encapsulated S. suis-induced TNF-{alpha}, IL-1ß and IL-6 release but had no significant effect on MCP-1 or IL-8 production (23). In the present study, we further demonstrated that a strong inhibition of encapsulated S. suis-induced TNF-{alpha}, IL-1ß and IL-6 (>80%), a partial inhibition of IL-8 and, to a lesser extent, MCP-1 production are obtained after treatment of monocytes with a mAb against TLR2 but not TLR4. These observations suggest that TLR2 is the main receptor responsible for the recognition of encapsulated S. suis, and CD14 might also contribute to recognition of this bacterium, as previously reported for other Gram-positive bacteria (31, 33, 34). Although CD14 was reported to act as co-receptor for TLR2 activation, Mambula et al. (60) reported species-specific disparities concerning the relative contribution of CD14 in TLR2-dependent activation of cells by Aspergillus fumigatus. Thus, the specific contribution of CD14 in recognition of S. suis warranties further investigation. Interestingly, the chemokines IL-8 and MCP-1 appear to show different patterns of activation, since both were only partially suppressed even after combined anti-TLR2 and anti-CD14 antibody treatment (Segura, M.; unpublished observations). Thus, TLR2-independent pathways might also contribute to the activation of chemokine production by encapsulated S. suis. For example, the participation of an amplification loop in the inflammatory cascade induced by cytokines produced early during cell activation cannot be ruled out and might reduce the effect of antibody treatment. Indeed, we previously reported that blockade of TNF-{alpha} and IL-1ß significantly reduced IL-8 and MCP-1 production induced by encapsulated S. suis (23).

An important role for TLR2 was confirmed using TLR2–/– mouse macrophages. We demonstrated strong inhibition of IL-6 (~80%), but partial inhibition of MCP-1 release after activation of TLR2–/– mouse macrophages with encapsulated S. suis, paralleling the results obtained with anti-TLR2 antibody. However, IL-6 production was completely abrogated and MCP-1 release highly impaired in MyD88–/– mouse macrophages. Thus, although TLR2 is the principal receptor involved in the recognition of encapsulated S. suis leading to pro-inflammatory cytokine release, a second pathway involving other TLRs may play a minor role and might also contribute directly or indirectly to the activation of the inflammatory cascade, namely chemokine production as mentioned above. In addition, since a profound, albeit not complete, inhibition of MCP-1 release was observed with MyD88–/– mouse macrophages, a third pathway which is MyD88 and, by extension, TLR independent might also contribute to MCP-1 production. Henneke et al. (33) showed that MyD88–/– but not TLR2–/– mouse macrophages had decreased responses to whole GBS compared with WT cells. In addition, MyD88 deficiency has been associated with a more profound inability to either control GBS infection or produce cytokine responses than TLR2 deficiency in a mouse model (61). Similarly, WT and TLR2–/– mouse spleen cells secrete equivalent amounts of cytokines after stimulation with different doses of S. pneumoniae, whereas MyD88–/– spleen cells fail to respond to this bacterial stimulation (62). However, our data suggest a greater role for TLR2 in S. suis recognition compared with GBS or S. pneumoniae, given that significant impairment of cytokine production with TLR2–/– macrophages or TLR2 blockade was observed at a comparable dose of S. suis bacteria.

To better understand the differential modulation of cytokine production by S. suis, we compared the inflammatory activity of encapsulated bacteria with an isogenic non-encapsulated mutant strain, as well as with the activities of purified bacterial cell wall and CPS. Treatment of human monocytes with the non-encapsulated mutant resulted in strong modulation of TLR2 and CD14, but not TLR4, mRNA expression with a similar pattern and kinetics as its parental strain. In addition, the non-encapsulated mutant induced comparable or even higher amounts of TNF-{alpha}, IL-1ß, IL-6 and IL-8. Similarly, purified bacterial cell wall exhibited high modulatory and pro-inflammatory activities. The cytokine-inducing activities of the cell wall were drastically impaired by TLR2 blockade or in TLR2- and MyD88-deficient macrophages, whereas its chemokine-inducing activities were only partially affected, confirming the role of alternative pathways for chemokine induction, as observed for the WT S. suis strain. To a lesser extent, a similar pattern of inhibition of TNF-{alpha}, IL-1ß, IL-6 and IL-8 release induced by the non-encapsulated mutant was observed following TLR2 blockade. The relative reduction in inhibition produced by the antibody treatment could be related to either a higher level of exposure of cell wall components in the non-encapsulated mutant resulting in a stronger inflammatory response (20, 24) or to the exposure of uncovered antigens which do not interact with TLR2. Interestingly, IL-6 production induced by the mutant strain was completely abrogated in MyD88-deficient macrophages, as observed for the purified cell wall. Taken together, these results strongly suggest a role for S. suis cell wall components as the predominant mediators of cytokine production via their interaction mainly with TLR2 as well as with presently unidentified TLRs in a MyD88-dependent manner.

Although the role of CPS in facilitating bacterial survival within the host is well established, the mechanisms underlying protective immunity to encapsulated pathogenic bacteria have yet to be fully elucidated. Indeed, the concept of CPS-mediated modulation of bacterial interactions with TLRs or a direct interaction of CPS with TLRs is still controversial. Neisseria meningitidis engages a TLR4- and TLR2-dependent pro-inflammatory response irrespective of the presence of a capsule (63), whereas encapsulated Salmonella enterica serotype Typhi shows impaired TLR5- and TLR4/MD2/CD14-dependent IL-8 responses (39). In the present study, we observed that although the presence of the S. suis capsule partially interferes with the interaction between cell wall components and TLRs, it is required for optimal MCP-1 production by human monocytes and, to a lesser extent, by mouse macrophages. Accordingly, MCP-1 was the only cytokine produced by these cells upon activation with purified CPS. We previously reported reduced MCP-1 production by human brain microvascular endothelial cells, human THP-1 cells and porcine whole-blood leukocytes activated with two distinct non-encapsulated mutants or naturally-occurring less encapsulated strains (20, 24). Furthermore, purified CPS was shown to strongly activate MCP-1 mRNA expression by porcine whole-blood leukocytes (24). Taken together, these findings support a contributory role for CPS in the activation of MCP-1 secretion by several types of host cells. However, in contrast to CPS from type II S. pneumoniae, which was reported to induce secretory and cellular macrophage responses in part through TLR2–CD14 pathways (38, 64), we demonstrated here that S. suis CPS type 2 induces MCP-1 production through a TLR2- and MyD88-independent pathway. This activity might explain the above postulated third pathway for MCP-1 production by the encapsulated strain, as indicated by the lack of complete abrogation of MCP-1 production induced by intact bacteria in TLR2–/– and MyD88–/– macrophages. Surprisingly, the remaining MCP-1 activity of the non-encapsulated strain, thought to be mediated by uncovered cell wall antigens, was not inhibited by TLR2 blockade or impaired in TLR2–/– macrophages but was markedly, although not completely, suppressed in MyD88–/– cells. This observation, together with results obtained with purified bacterial cell wall, suggests that multiple antigens of either CPS or cell wall origin, as well as multiple receptor pathways, are involved in the unique MCP-1 modulatory activity of encapsulated S. suis.

In addition, CPS was shown to induce late activation of TLR2 and CD14 mRNA expression. Since CPS does not seem to interact with TLRs, this up-regulation of receptor expression might be an indirect consequence of MCP-1 downstream activation of NF-{kappa}B and AP-1 transcription factors (65), which are known to be involved in tlr2 and cd14 promoter regulation, respectively (66–68). MCP-1 is an important regulator of leukocyte recruitment to sites of inflammation and has previously been reported to destabilize the integrity of the BBB. Indeed, Song et al. (69) showed that MCP-1 can alter tight junction-associated proteins by promoting vasoactive effects which, in turn, are linked to changes in endothelial junction integrity. Encapsulation has been suggested to be down-regulated during colonization of epithelial cells (lungs, nasopharynx), and once the bacteria are in the bloodstream, up-regulation of capsule-mediated production protects them from the immune system while allowing interaction with cells of the BBB (8, 16, 24, 70). Thus, the capacity of well-encapsulated S. suis to induce the release of MCP-1 by different pathways may be an important advantage to bacterial dissemination and thus may constitute one of the mechanisms used by S. suis to cross the BBB, gain access to the CNS and subsequently cause disease. Nevertheless, it should be noted that although encapsulated S. suis induced lower levels of TNF and IL-1 than the non-encapsulated mutant strain, these levels were still remarkably high and thus might also contribute to BBB breakdown.

In conclusion, this work is a first step in understanding the mechanisms used by the host to recognize the presence of S. suis, as well as those used by this encapsulated bacterium to overcome or subvert the immune response to its own benefit. Streptococcus suis is mainly recognized via TLR2, likely in combination with CD14, and this interaction results in the release of pro-inflammatory mediators. The presence of CPS plays an important role by interfering with these interactions and by means of its own immunomodulatory activities. These in vitro observations were recently confirmed by in vivo studies which indicate a strong TLR2, CD14 and MCP-1, but not TLR4, activation in the brains of mice experimentally infected with S. suis serotype 2 (71). Nevertheless, presently undefined mechanisms and receptors, including other TLRs, may mediate the recognition of this encapsulated pathogen by macrophages and participate in the activation of the immune response as well as in mechanisms of pathogenesis of the CNS infection.


   Acknowledgements
 
This work was supported by the Natural Sciences and Engineering Research Council of Canada grant no. 0680154280 and by the Centre de Recherche en Infectiologie Porcine (FQRNT).


   Abbreviations
 
BBB, blood–brain barrier
CFU, colony-forming unit
CNS, central nervous system
CPS, capsular polysaccharide
dNTP, deoxynucleoside triphosphate mixture
FBS, fetal bovine serum
GBS, Group B Streptococcus
LTA, lipoteichoic acid
MALP-2, Mycoplasma fermentans 2-kDa macrophage-activating lipopeptide
MCP-1, monocyte chemoattractant protein-1
MyD88, myeloid differentiation factor 88
NF-{kappa}B, nuclear factor-kappa B
PGN, peptidoglycan
RT, reverse transcriptase
THB, Todd–Hewitt broth
TLR, Toll-like receptor
TNF-{alpha}, tumor necrosis factor-{alpha}
WT, wild type

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: E. Vivier

Received 9 November 2006, accepted 12 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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