Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.

Streptococcus pneumoniae is the leading causative pathogen in community-acquired pneumonia and a major cause of morbidity and mortality in humans (1, 2, 3). Pneumococci account for up to 36% of adult community-acquired pneumonia in the United States. An estimated 570,000 cases of pneumococcal pneumonia occur annually in the United States, including 175,000 hospitalized cases (4). The rise in antibiotic resistance of this pathogen urges further efforts to understand the host response mechanisms involved in pneumococcal pneumonia (5, 6).

The first line of defense against invading bacteria is provided by the innate immune system, which recognizes pathogen-associated molecular patterns (PAMPs),3 conserved microbial patterns shared by large groups of pathogens, but not found in higher eukaryotes (7, 8, 9). Over the last few years, it has become evident that both the recognition and the subsequent response to pathogens is mainly transferred by members of the Toll-like receptor (TLR) family (for review, see Refs. 8 , 10 , and 11)). Ten TLRs have been described so far, among which TLR2 and TLR4 are the best-investigated family members. TLR4, the LPS receptor, is important for the recognition of Gram-negative bacteria, whereas TLR2 has been designated the major receptor for Gram-positive bacteria by virtue of its capacity to recognize major cell wall constituents of Gram-positive microorganisms, such as peptidoglycan (PGN), lipoteichoic acid (LTA), and lipoproteins (12, 13, 14, 15, 16). TLR2, together with TLR1 and TLR6, have been demonstrated to migrate to phagosomes within phagocytic cells, where they might sample the contents and signal the presence of an invader (17, 18). TLR2 has to cooperate and heterodimerize with other TLRs like TLR6 or TLR1 to activate TNF-α production in macrophages (18). Moreover, evidence exists that TLR2 is directly involved in bacterial killing by monocytes and macrophages (19).

The role of TLRs in the innate recognition of S. pneumoniae has been the subject of several recent investigations. In a model of a Chinese hamster ovary (CHO) reporter fibroblast cell line, heat-killed S. pneumoniae were found to partially stimulate CHO cells in the absence of TLR expression (13). The responsiveness of CHO cells markedly increased after TLR2 expression, suggesting that S. pneumoniae stimulates both a TLR2-dependent and a TLR2-independent pathway (13). In the human embryonic kidney cell line 293, transfection with either TLR2 or TLR4 resulted in cellular activation by S. pneumoniae (20). However, peritoneal macrophages harvested from mice lacking functional TLR2 and/or TLR4 responded normally to S. pneumoniae, suggesting that, for activation of these cells, neither TLR2 nor TLR4 is required (20). Two investigations examined the role of TLR2 in host defense against S. pneumoniae in vivo, both making use of a meningitis model in which pneumococci were injected directly into the CNS (20, 21). Both studies reported an increased disease severity in TLR2-deficient (TLR2−/−) mice together with a moderately elevated bacterial outgrowth in the CNS when compared with normal wild-type (wt) mice. Very recently, Malley et al. (22) reported an important role for TLR4 in the innate immune response to S. pneumoniae in the nasopharynx (22). Indeed, C3H/HeJ mice, which display a mutant nonfunctional TLR4, were found to be more susceptible to invasive disease after colonization of the nasopharynx with pneumococci.

Knowledge of the role of TLR2 in host defense against respiratory tract infections is highly limited. Therefore, in the present study, we sought to determine the role of TLR2 in the innate immune response to pneumococcal pneumonia.

Pathogen-free 10- to 12-wk-old male C57BL/6 wt mice were purchased from Harlan Sprague-Dawley (Horst, The Netherlands). TLR2−/− mice were generated as described previously (12), backcrossed to C57BL/6 background six times, and bred in the animal facility of the Academic Medical Center in Amsterdam, The Netherlands. Age- and sex-matched mice were used in all experiments. The Animal Care and Use Committee of the University of Amsterdam approved all experiments.

Pneumonia was induced as described previously (23, 24, 25). Briefly, S. pneumoniae serotype 3 obtained from American Type Culture Collection (ATCC 6303; Manassas, VA) were grown for 6 h to midlogarithmic phase at 37°C using Todd-Hewitt broth (Difco, Detroit, MI), harvested by centrifugation at 1500 × g for 15 min, and washed twice in sterile isotonic saline. Bacteria were then resuspended in sterile isotonic saline at a concentration of 2 × 103–5 × 105 CFU/50 μl, as determined by plating serial 10-fold dilutions on sheep blood agar plates. Mice were lightly anesthetized by inhalation of isoflurane (Upjohn, Ede, The Netherlands), and 50 μl (containing 2 × 103–5 × 105 CFU, depending on the experiment) was inoculated intranasally (i.n.).

Total RNA was extracted from lungs of wt mice 24 h after inoculation with S. pneumoniae or saline using chloroform extraction and isopropanol precipitation. After treatment with RQ1 RNase-free DNase (Promega, Madison, WI), total RNA was reverse transcribed using oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, U.K.) according to the supplier’s recommendations. cDNA samples were standardized based on the content of β-actin cDNA as housekeeping gene. β-Actin cDNA was evaluated by performance of a β-actin PCR on multiple dilutions of each cDNA sample. The amount of amplified product was estimated by densitometry of ethidium bromide-stained 1.2% agarose gels using a charge-coupled device camera and Imagemaster VDS software (Pharmacia, Uppsala, Sweden). Primers for TLR2 were 5′-TCT GGG CAG TCT TGA ACA TTT-3′ (sense primer) and 5′-AGA GTC AGG TGA TGG ATG TCG-3′ (antisense primer), yielding a 321-bp fragment; primers for murine β-actin were 5′-TAA AAC GCA GCT CAG TAA CAG TCG G-3′ (sense primer) and 5′-TGC AAT CCT GTG GCA TCC ATG AAA C-3′ (antisense primer). Using appropriate dilutions of the cDNA, PCR was performed as described previously (26). Levels of TLR2 mRNA expression were evaluated by densitometric image analysis, and relative TLR2 mRNA levels were calculated by comparison of band intensities of the TLR2 RT-PCR products with standard curves prepared by PCR amplifications on dilution series of a highly concentrated murine lung cDNA.

AM were harvested from TLR2−/− and wt mice by bronchoalveolar lavage (n = 8 per strain). The trachea was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter (Abott, Sligo, Ireland). Bronchoalveolar lavage was performed by instilling two 0.5-ml aliquots of sterile saline. Total cell numbers were counted from each sample using a hemocytometer. Cells of two mice were pooled, washed, and resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM l-glutamine, penicillin, streptomycin, and 10% FCS in a final concentration of 1 × 105cells/ml. Cells were then cultured in 96-well microtiter plates (Greiner, Alphen a/d Rijn, The Netherlands) for 2 h and washed with RPMI 1640 to remove nonadherent cells. Adherent monolayer cells were stimulated with LPS (from Escherichia coli O55:B5; 1 μg/ml; Sigma-Aldrich, St. Louis, MO), LTA (from Staphylococcus aureus; 10 μg/ml; kind gift of Dr. T. Hartung (University of Konstanz, Konstanz, Germany) (27)), heat-killed S. pneumoniae (5 × 107 CFU/ml; ATCC 6303; American Type Culture Collection), or RPMI 1640 for 16 h. Supernatants were collected and stored at −70°C until assayed for TNF-α and KC.

Six or 48 h after infection, mice were anesthetized with Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Roche, Meidrecht, The Netherlands) and sacrificed by bleeding out the vena cava inferior. Blood was collected in EDTA-containing tubes. Whole lungs were harvested and homogenized at 4°C in 4 vol of sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, OK). CFUs were determined from serial dilutions of lung homogenates and blood, plated on blood agar plates, and incubated at 37°C for 16 h before colonies were counted.

For cytokine measurements, lung homogenates were diluted 1/2 in lysis buffer containing 300 mM NaCl, 30 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 1% Triton X-100, and pepstatin A, leupeptin, and aprotinin (all 20 ng/ml; pH 7.4), and incubated at 4°C for 30 min. Homogenates were centrifuged at 1500 × g at 4°C for 15 min, and supernatants were stored at −20°C until assays were performed.

Lungs for histology were harvested at 6, 48, or 72 h after infection, fixed in 4% formalin, and embedded in paraffin. Sections (4 μm) were stained with H&E and analyzed by a pathologist who was blinded for groups. To score lung inflammation and damage, the entire lung surface was analyzed with respect to the following parameters: interstitial inflammation, intra-alveolar inflammation, edema, endothelialitis, bronchitis, pleuritis, and thrombi formation. Each parameter was graded on a scale from 0 to 3 as follows: 0, absent; 1, mild; 2, moderate; and 3, severe. The total lung inflammation score was expressed as the sum of the scores for each parameter, the maximum being 21. Granulocyte staining was done as described previously (23, 28). Briefly, slides were deparaffinized, and endogenous peroxidase activity was quenched by a solution of methanol/0.03% H2O2 (Merck, Darmstadt, Germany). After digestion with a solution of pepsine 0.25% (Sigma-Aldrich) in 0.01 M HCl, the sections were incubated in 10% normal goat serum (DAKO, Glostrup, Denmark) and then exposed to FITC-labeled anti-mouse Ly-6G mAb (BD PharMingen, San Diego, CA). Slides were incubated with a rabbit anti-FITC Ab (DAKO) followed by a further incubation with a biotinylated swine anti-rabbit Ab (DAKO), rinsed again, incubated in a streptavidin-ABC (avidin/biotin complex) solution (DAKO), and developed using 1% H2O2 and 3,3′-diaminobenzidine-tetrahydrochloride (Sigma-Aldrich) in Tris-HCl. The sections were mounted in glycerin gelatin without counterstaining and analyzed. All Abs were used in concentrations recommended by the manufacturers.

Lungs from wt and TLR2−/− mice (n = 8 per group) were harvested 48 h after induction of pneumonia. Pulmonary lung suspensions were obtained by crushing lungs through a 40-μm cell strainer (BD PharMingen); cells were washed and resuspended in PBS, and cytospins were prepared. Endogenous peroxidase activity was quenched by a solution of 0.3% H2O2 in PBS/0.1% NaN3 (Merck). After a blocking step with 10% normal goat serum (DAKO), slides were incubated with rabbit-anti-mouse TNF-α Abs (Biosource, Camarillo, CA) in the presence of 1% saponin (Sigma-Aldrich) followed by exposure to streptavidin goat anti-rabbit Ab (ImmunoLogic, Duiven, The Netherlands), and developed using 1% H2O2 and 3,3′-diaminobenzidine-tetrahydrochloride (Sigma-Aldrich) in Tris-HCl. Abs were used in concentrations recommended by the manufacturers.

Cytokines and chemokines (TNF-α, IL-1β, IL-6, IL-10, KC, and macrophage-inflammatory protein (MIP)-2) were measured using specific ELISAs (R&D Systems, Minneapolis, MN) according to the manufacturers’ instructions. The detection limits were 31 pg/ml for TNF-α and IL-10, 8 pg/ml for IL-1β, 16 pg/ml for IL-6, 12 pg/ml for KC, and 94 pg/ml for MIP-2.

Differences between groups were analyzed using Mann-Whitney U test. For survival analyses, Kaplan-Meier analysis followed by log rank test was performed. Results of the in vitro stimulation were calculated with one-way ANOVA followed by Bonferroni’s multiple comparison tests. Values are expressed as mean ± SEM. A value of p ≤ 0.05 was considered statistically significant.

The pulmonary TLR2 expression was determined in lungs from healthy control mice (receiving saline) and animals challenged with S. pneumoniae to obtain constitutive and infection-induced TLR2 mRNA levels. As depicted in Fig. 1, TLR2 mRNA was constitutively expressed in healthy lung tissue and markedly enhanced following pulmonary infection with pneumococci.

FIGURE 1.

Lung TLR2 mRNA expression after infection with S. pneumoniae. Pulmonary TLR2 mRNA expression was determined by specific RT-PCR amplification of lung cDNA samples of wt mice inoculated i.n. with saline (control; C) or S. pneumoniae (pneumonia; P). S. pneumoniae induced an increased expression of TLR2 compared with saline-treated controls. Results shown are representative of three independent RT-PCR of lung cDNA samples; cDNA was standardized for β-actin content. Semiquantitative data were generated by densitometric evaluation of RT-PCR products, which were compared with a standard curve obtained by amplification of a serial dilution of highly concentrated cDNA.

FIGURE 1.

Lung TLR2 mRNA expression after infection with S. pneumoniae. Pulmonary TLR2 mRNA expression was determined by specific RT-PCR amplification of lung cDNA samples of wt mice inoculated i.n. with saline (control; C) or S. pneumoniae (pneumonia; P). S. pneumoniae induced an increased expression of TLR2 compared with saline-treated controls. Results shown are representative of three independent RT-PCR of lung cDNA samples; cDNA was standardized for β-actin content. Semiquantitative data were generated by densitometric evaluation of RT-PCR products, which were compared with a standard curve obtained by amplification of a serial dilution of highly concentrated cDNA.

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To obtain a first insight into the function of TLR2 in the pulmonary host response to S. pneumoniae, we determined the responsiveness of AM to heat-killed pneumococci and, as controls, LPS (signaling in a TLR2-independent way) and LTA (signaling in a TLR2-dependent way) (12, 15, 27). Freshly isolated AM from TLR2−/− mice failed to release TNF-α or KC upon stimulation with heat-killed S. pneumoniae, whereas wt AM released high amounts of TNF-α and KC (p < 0.05; Fig. 2). As expected, LPS stimulated TLR2−/− and wt AM to release similar amounts of TNF-α and KC, whereas LTA induced TNF-α and KC secretion in cultures of wt but not of TLR2−/− AM (Fig. 2). Hence, the responsiveness of AM to S. pneumoniae depended on the presence of TLR2.

FIGURE 2.

TLR2 is required for responsiveness of AM to S. pneumoniae. Freshly isolated AM of wt and TLR2−/− mice (n = 8 per group: cells from two mice were pooled yielding four samples per group) were incubated with RPMI 1640 (control), LPS (1 μg/ml), LTA (10 μg/ml), or heat-killed S. pneumoniae (equivalent of 5 × 107 CFU/ml) for 16 h before TNF-α and KC production was measured (∗, p < 0.05, TLR2−/− vs wt).

FIGURE 2.

TLR2 is required for responsiveness of AM to S. pneumoniae. Freshly isolated AM of wt and TLR2−/− mice (n = 8 per group: cells from two mice were pooled yielding four samples per group) were incubated with RPMI 1640 (control), LPS (1 μg/ml), LTA (10 μg/ml), or heat-killed S. pneumoniae (equivalent of 5 × 107 CFU/ml) for 16 h before TNF-α and KC production was measured (∗, p < 0.05, TLR2−/− vs wt).

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Having established that TLR2 mediates S. pneumoniae induced TNF-α and KC release by AM in vitro, we next determined whether the pulmonary cytokine/chemokine response to respiratory tract infection with pneumococci was altered in the absence of TLR2 in vivo. For this, the concentrations of TNF-α, IL-1β, IL-6, IL-10, KC, and MIP-2 were assessed in lung homogenates obtained from TLR2−/− and wt mice 48 h after infection with a low (104 CFU), intermediate (approximate LD60; 105 CFU), or high (lethal; 5 × 105 CFU) S. pneumoniae dose. We chose to measure these mediators 48 h postinfection, because we previously established that this time point is suitable for this purpose in this model (23, 24, 29). Mediator levels did not differ between TLR2−/− and wt mice (data not shown), except for KC and MIP-2 concentrations after infection with 105S. pneumoniae CFU, which were lower in TLR2−/− mice (1.6 ± 0.5 and 8.3 ± 2.1 ng/ml, respectively) than in wt mice (3.1 ± 0.5 and 17.9 ± 4.7 ng/ml, respectively; both p ≤ 0.05 for the difference between groups). Because the in vivo finding of unaltered TNF-α concentrations in the lungs of wt and TLR2−/− mice contrasted with in vitro studies using AM (Fig. 2), we decided to investigate the cellular source of TNF-α in vivo and performed immunohistochemical stainings on lung cell suspensions obtained 48 h after induction of pneumonia. Pulmonary macrophages proved to be the main cellular source of TNF-α in both wt and TLR2−/− mice in vivo (Fig. 3). To further evaluate the role of TLR2 in lung inflammation induced by S. pneumoniae in vivo, lung histology slides, obtained 48 h after infection with either 104 or 105 CFU S. pneumoniae, were scored as described in Materials and Methods. At both inoculum doses, TLR2−/− mice displayed significantly less inflammation, edema, and pleuritis, when compared with wt mice (Fig. 4, Table I). In accordance, granulocyte staining of lung slides from TLR2−/− mice revealed less granulocytes than lung tissue obtained from wt mice (Fig. 4, insets).

FIGURE 3.

TNF-α is produced by macrophages in vivo. Representative immunohistochemical stainings for TNF-α on lung cell suspensions of wt (A) and TLR2−/− (B) mice 48 h after infection with 5 × 103 CFU S. pneumoniae (n = 7 per group) showing macrophages with similar staining intensities. Epithelial cells and granulocytes did not display positive staining.

FIGURE 3.

TNF-α is produced by macrophages in vivo. Representative immunohistochemical stainings for TNF-α on lung cell suspensions of wt (A) and TLR2−/− (B) mice 48 h after infection with 5 × 103 CFU S. pneumoniae (n = 7 per group) showing macrophages with similar staining intensities. Epithelial cells and granulocytes did not display positive staining.

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FIGURE 4.

Reduced lung inflammation in TLR2−/− mice 48 h after infection. Representative lung histology of wt (A and C) and TLR2−/− (B and D) mice 48 h after infection with 104 CFU (A and B) or 105 CFU (C and D) S. pneumoniae showing significantly more inflammation, pleuritis, edema, bronchitis, and endothelialitis in wt mice compared with TLR2−/− animals. The insets are representative pictures of immunostaining for granulocytes, confirming the dense granulocytic infiltration in wt mice. The histological lung sections are representative for at least five mice per group. H&E staining, Magnification, ×4. Inset (Ly-6 staining), Magnification, ×20.

FIGURE 4.

Reduced lung inflammation in TLR2−/− mice 48 h after infection. Representative lung histology of wt (A and C) and TLR2−/− (B and D) mice 48 h after infection with 104 CFU (A and B) or 105 CFU (C and D) S. pneumoniae showing significantly more inflammation, pleuritis, edema, bronchitis, and endothelialitis in wt mice compared with TLR2−/− animals. The insets are representative pictures of immunostaining for granulocytes, confirming the dense granulocytic infiltration in wt mice. The histological lung sections are representative for at least five mice per group. H&E staining, Magnification, ×4. Inset (Ly-6 staining), Magnification, ×20.

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Table I.

Less severe pulmonary inflammation in TLR2−/− compared with wt micea

S. pneumoniae
104 CFU105 CFU
wtTLR2−/−wtTLR2−/−
Inflammation score 10.9 ± 1.6 6.4 ± 1.6* 17.0 ± 0.5 9.6 ± 2.8* 
S. pneumoniae
104 CFU105 CFU
wtTLR2−/−wtTLR2−/−
Inflammation score 10.9 ± 1.6 6.4 ± 1.6* 17.0 ± 0.5 9.6 ± 2.8* 
a

Inflammation scores (as described in Materials and Methods) in wt and TLR2−/− mice 48 h after infection with 105 or 104 CFU S. pneumoniae, respectively. Data are presented as mean ± SEM of n = 7–8 mice per strain. ∗, p < 0.05 vs wt mice.

The clearance of bacteria from the respiratory tract during pneumococcal pneumonia strongly depends on the efficacy to mount a local inflammatory response (30, 31). To evaluate whether the lack of TLR2 interferes with bacterial clearance, we determined bacterial loads in lungs 48 h after infection with 5 × 103, 104, 105, or 5 × 105 CFU S. pneumoniae. At all four bacterial doses, TLR2−/− mice tended to have less S. pneumoniae CFU in their lungs than wt mice, although the differences between the two mouse strains did not reach significance (Fig. 5). Likewise, the proportion of bacteremic mice tended to be lower in TLR2−/− mice, irrespective of the size of inoculum (data not shown). Moreover, survival did not differ between TLR2−/− and wt mice after infection with a high (105 CFU) or low (5 × 103 CFU) bacterial inoculum (Fig. 6), whereas both mouse strains completely cleared pneumococci from their lungs after infection with a bacterial dose of 2 × 103 CFU after 72 h. Hence, these data suggest that TLR2 does not contribute to local antibacterial defense or containment of the infection during pneumococcal pneumonia.

FIGURE 5.

TLR2−/− mice display an unaltered bacterial clearance. wt and TLR2−/− mice were inoculated with 5 × 105, 105, 104, or 5 × 103 CFU S. pneumoniae, and bacterial outgrowth in lungs was obtained 48 h later. Data represent mean ± SEM of n = 7–8 mice per strain, 48 h after infection.

FIGURE 5.

TLR2−/− mice display an unaltered bacterial clearance. wt and TLR2−/− mice were inoculated with 5 × 105, 105, 104, or 5 × 103 CFU S. pneumoniae, and bacterial outgrowth in lungs was obtained 48 h later. Data represent mean ± SEM of n = 7–8 mice per strain, 48 h after infection.

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FIGURE 6.

Unaltered survival in TLR2−/− mice. wt and TLR2−/− mice (n = 8–9) were i.n. inoculated with 2 × 105 (A) or 5 × 103 (B) S. pneumoniae, and survival was monitored.

FIGURE 6.

Unaltered survival in TLR2−/− mice. wt and TLR2−/− mice (n = 8–9) were i.n. inoculated with 2 × 105 (A) or 5 × 103 (B) S. pneumoniae, and survival was monitored.

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Postulating that TLR2 might play a more important role in the early initiation phase of the host response to pneumococcal pneumonia, we infected mice with 105 CFU S. pneumoniae and sacrificed them 6 h later. IL-1β, IL-6, and KC levels were significantly lower in TLR2−/− than in wt mice at this early time point, whereas TNF-α, IL-10, and MIP-2 levels did not differ between the two mouse strains (Table II). The extent of lung inflammation was found to be slightly lower in TLR2−/− mice at this time point (data not shown). No difference in bacterial loads in lungs of TLR2−/− and wt mice was observed (2.4 ± 1.5 × 104 CFU/ml and 5.4 ± 1.9 × 104 CFU/ml, respectively; NS), and blood cultures were all negative.

Table II.

Reduced lung concentrations of IL-1β, IL-6, and KC in TLR2−/− mice 6 h after infection with S. pneumoniaea

pg/mlwtTLR2−/−
TNF-α 2631 ± 297 2374 ± 173 
IL-1β 1787 ± 424 549 ± 88* 
IL-6 153 ± 36 78 ± 4* 
IL-10 462 ± 57 464 ± 35 
KC 3132 ± 504 1880 ± 87* 
MIP-2 4328 ± 632 4421 ± 417 
pg/mlwtTLR2−/−
TNF-α 2631 ± 297 2374 ± 173 
IL-1β 1787 ± 424 549 ± 88* 
IL-6 153 ± 36 78 ± 4* 
IL-10 462 ± 57 464 ± 35 
KC 3132 ± 504 1880 ± 87* 
MIP-2 4328 ± 632 4421 ± 417 
a

Cytokines and chemokines measured in lung homogenates 6 h after infection with 105 CFU S. pneumoniae. Data are means ± SEM; n = 9 per strain. ∗, p < 0.05 vs wt mice.

S. pneumoniae is the most frequently isolated pathogen in community-acquired pneumonia and responsible for an estimated 10 million deaths annually, making pneumococcal pneumonia a major health threat worldwide (32, 33). Previous observations have pointed to TLR2 as the key pattern recognition receptor in the immune response to Gram-positive bacteria (12, 13, 14, 34). In this report, we sought to obtain insight into the contribution of TLR2 in Gram-positive pneumonia and investigated the requirement for TLR2 during both in vitro and in vivo infection with S. pneumoniae. We used AM in in vitro studies and showed that TLR2 is required for responsiveness to S. pneumoniae. However, although TLR2 played a role in the induction of an inflammatory response in the lung during pneumococcal pneumonia, TLR2−/− mice displayed an uncompromised antibacterial defense against pneumococci in the respiratory tract using infectious inocula ranging from nonlethal to lethal. Together, these data indicate that TLR2 is not indispensable in the innate immune response to S. pneumoniae pneumonia.

There is abundant evidence from in vitro observations that TLR2 is the predominant receptor signaling the presence of cell wall components of Gram-positive bacteria, such as PGN or LTA (13, 14, 15, 35, 36). However, data that confirm these observations in vivo are still scarce. Both LTA and PGN alone are capable of inducing acute lung inflammation in vivo, indicating that these PAMPs likely contribute to the local inflammatory response during Gram-positive pneumonia (28). In this report, we demonstrated that AM require TLR2 to mount an inflammatory response to LTA. Similarly, TLR2−/− AM did not respond to S. pneumoniae in vitro. Considering that AM are major players in the induction and regulation of lung inflammation during respiratory tract infections (30), we expected TLR2−/− mice to mount a reduced inflammatory response to pneumococcal pneumonia, and as a consequence thereof an impaired bacterial clearance. We indeed observed less inflammation in lungs of TLR2−/− mice, albeit the differences with wt mice were modest. Importantly, antibacterial defense was indistinguishable in TLR2−/− and wt mice. Given the multitude of factors involved in the innate immune response against pneumococcal pneumonia, such as natural Abs and complement, these mediators are likely candidates to compensate for the lack of TLR2 (37). This might also explain why in vivo TLR2−/− macrophages stained positive for TNF-α during pneumonia, whereas incubation of isolated TLR2−/− macrophages with S. pneumoniae in vitro did not result in TNF-α production. Thus, these results suggest that TLR2 plays a modest role in the immune response to S. pneumoniae in the respiratory tract, which does not influence the overall antibacterial defense during pneumococcal pneumonia.

Two recent reports investigated the role of TLR2 in pneumococcal meningitis in vivo and demonstrated an aggravated course in mice lacking TLR2 using a clinical severity score (20, 21). One study provided survival data showing a 100% fatality rate in both TLR2−/− and wt mice (21). In both investigations, in which S. pneumoniae was injected intracisternally, differences between TLR2−/− and wt mice in bacterial clearance were modest at best. Indeed, in the study by Echchannaoui et al. (21), bacterial counts in blood and cerebrospinal fluid were similar in both mouse strains. Using luciferase-tagged S. pneumoniae, these investigators reported higher fluorescence intensity in brains of TLR2−/− mice than in wt brains. In the study by Koedel et al. (20), TLR2−/− mice displayed more pneumococci in their cerebellum and blood, but not in their spleen. The recruitment of neutrophils to the CNS and blood cytokine concentrations were unaltered (20, 21). In contrast to this CNS infection model, we tried to reproduce the natural route of pulmonary infection and infected mice i.n. with S. pneumoniae. By using this approach, we found no major difference between wt and TLR2−/− mice in their pulmonary host response to pneumococci. In S. aureus sepsis, Takeuchi et al. (34) reported higher susceptibility and impaired bacterial clearance in TLR2−/− mice when high bacterial doses were administered, whereas no clear difference between wt and TLR2−/− animals could be found when mice were treated with lower bacterial doses. We investigated the role of TLR2 in host defense against S. pneumoniae using high and low infectious doses and did not observe differences between TLR2−/− and wt mice. Together, the observations of moderately impaired host defense in pneumococcal meningitis (20, 21) and our results of an unaltered host defense of TLR2−/− mice in pneumococcal pneumonia, point toward the simultaneous involvement of different recognition receptors in the innate immune response to S. pneumoniae. TLR2 is apparently one participating receptor, but in vivo findings thus far could not reveal an unequivocal role for TLR2 as the prime receptor in this Gram-positive infection. Moreover, the absence of myeloid differentiation factor 88 rendered mice more susceptible to S. aureus sepsis than the lack of TLR2 alone, suggesting the involvement of additional myeloid differentiation factor 88-dependent mechanisms (34).

The immune response to S. pneumoniae continues to be a point of interest. In vitro experiments using CHO cells could not demonstrate an equally strong dependency on TLR2 as it was found for S. aureus, because a partial, although less pronounced, immune response to whole S. pneumoniae could be observed in the absence of TLR2 (13). Evidence is increasing that the innate immune response to pathogens does not solely depend on the composition of the outer cell wall of bacteria but also involves factors produced and released by bacteria. Moreover, that these bacterial factors can also signal in a TLR-dependent way has been shown for group B streptococci and Yersinia (38, 39, 40). Of great interest, a recent report demonstrated that pneumolysin, known as a major toxic component of S. pneumoniae, signals via TLR4 (22). Pneumolysin gets released upon lysis of pneumococci during multiplication of bacteria and has many biological activities like induction of cytokines and cytotoxicity (41, 42, 43). Malley et al. (22) used a model of i.n. inoculation in unanesthetized mice, in which the infecting organism is confined to the upper respiratory tract, not progressing to the lung. These investigators monitored nasopharyngeal colonization using luminescent pneumococci and found a much higher bacterial nasopharyngeal burden (as determined by photon emission) in TLR4 mutant than in wt mice. Although Malley et al. (22) focused on the role of TLR4 during nasopharyngeal colonization with S. pneumoniae, our laboratory recently studied the role of this receptor in the pneumonia model also used in the current investigation (44). In S. pneumoniae pneumonia, TLR4 appeared to play a modest protective role, as indicated by slightly higher bacterial loads in lungs of TLR4 mutant mice than in wt mice (44). Interestingly, in the model by Malley et al. (22), TLR4 mutant mice demonstrated an increased mortality when infected with wt but not with pneumolysin-deficient pneumococci, indicating that the interaction between pneumolysin and TLR4 during colonization of the nasopharynx is important for protection against invasive pneumococcal disease. This report also showed that the host response to pneumolysin was synergistically potentiated by costimulation with TLR2-dependent cell wall components of S. pneumoniae. The lack of costimulatory signals in the absence of TLR2 likely contributed to the diminished pulmonary inflammation we observed in TLR2−/− mice. Of note, the immune response to S. pneumoniae in the lungs likely includes other receptors and/or pathways. Thus, the roles of, for example, TLR6 and TLR1, which can form heterodimers with TLR2 for optimal detection of some Gram-positive PAMPs (18, 45), and TLR9, which recognizes bacterial DNA (46), in S. pneumoniae pneumonia remain to be established.

In conclusion, we show in this study that TLR2 does not importantly contribute to host defense in pneumococcal pneumonia. By ruling out the absolute requirement for TLR2 in host defense against S. pneumoniae in vivo, our data contribute valuable information to the paradigm of complex interactions between multiple pattern recognition receptors in host defense against a single pathogen in vivo.

We thank Ingvild Kop and Joost Daalhuisen for expert technical assistance, and Nike Claessen for the Ly-6G immunohistochemical stainings.

1

This work was supported by grants from the Fonds zur Foerderung der wissenschaftlichen Forschung in Oesterreich (to S.K.), Mr. Willem Bakhuys Roozeboom Foundation (to C.W.W.), and The Netherlands Research Organization (to S.F.).

3

Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; PGN, peptidoglycan; LTA, lipoteichoic acid; CHO, Chinese hamster ovary; wt, wild type; i.n., intranasal(ly); AM, alveolar macrophage; MIP, macrophage-inflammatory protein.

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