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Infection and Immunity, August 2006, p. 4841-4848, Vol. 74, No. 8
0019-9567/06/$08.00+0     doi:10.1128/IAI.00026-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Streptococcus pneumoniae Infection Aggravates Experimental Autoimmune Encephalomyelitis via Toll-Like Receptor 2

Isabel Herrmann,¹ Markus Kellert,¹ Hauke Schmidt,² Alexander Mildner,² Uwe K. Hanisch,² Wolfgang Brück,² Marco Prinz,^2, and Roland Nau^1*,

Department of Neurology, Georg August University, D-37075 Göttingen, Germany,¹ Department of Neuropathology, Georg August University, D-37075 Göttingen, Germany²

Received 5 January 2006/ Returned for modification 9 March 2006/ Accepted 9 May 2006

	ABSTRACT

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The course of autoimmune inflammatory diseases of the central nervous system (CNS) can be influenced by infections. Here we assessed the disease-modulating effects of the most frequent respiratory pathogen Streptococcus pneumonia on the course of experimental autoimmune encephalomyelitis (EAE). Mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) peptide, challenged intraperitoneally with live S. pneumoniae type 3, and then treated with ceftriaxone. EAE was monitored by a clinical score for 35 days after immunization. EAE was unaltered in mice infected with S. pneumoniae 2 days before and 21 days after the first MOG_35-55 injection but was more severe in animals infected 7 days after the first MOG_35-55 injection. The antigen-driven systemic T-cell response was unaltered, and the intraspinal Th1 cytokine mRNA concentrations at the peak of disease were unchanged. The composition of CNS-infiltrating cells and subsequent tissue destruction were only slightly increased after S. pneumoniae infection. In contrast, the serum levels of tumor necrosis factor alpha and interleukin-6 and spinal interleukin-6 levels were elevated, and the expression of major histocompatibility complex class II molecules, CD80, and CD86 on splenic dendritic cells were enhanced early after infection. Serum cytokine concentrations were not elevated, and EAE was not aggravated by S. pneumoniae infection in Toll-like receptor 2 (TLR2)-deficient mice. In conclusion, infection with S. pneumoniae worsens EAE probably by elevation of proinflammatory cytokines and activation of dendritic cells in the systemic circulation via TLR2 and cross talk through the blood-brain barrier.

	INTRODUCTION

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Multiple sclerosis (MS) is a frequent inflammatory demyelinating disease of the central nervous system (CNS). Cell-mediated autoimmunity underlies its pathogenesis (21). Autoimmune diseases such as thyroiditis, diabetes type I (9, 10), and MS (1, 4) can be evoked or aggravated by infectious agents (11, 23, 26, 27). In MS patients in particular viral or virus-like infections (e.g., Mycoplasma pneumoniae and Chlamydia pneumoniae) of the respiratory tract can induce aggravation or cause relapse (4, 14, 42). Similar to the findings in humans, viral infections are important triggering factors for the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Examples are Semliki Forest virus or gammaherpesvirus infections, which can strongly evoke relapses (25, 48).

Thus far, only a few studies have addressed the question of whether bacterial infections can initiate relapses of MS or EAE. Enterotoxins from Staphylococcus aureus, which act as T-cell activating superantigens, can exacerbate or induce EAE (17, 30, 37, 38). Chlamydia pneumoniae injected intraperitoneally is able to induce or exacerbate EAE (7). The mode of action, how these pathogens probably influence the course of autoimmune diseases, became apparent after the discovery of Toll-like receptors (TLRs). TLRs recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity (28, 40). The quantity of TLR agonists released by bacteria is influenced by the mode of antibiotic treatment (3, 39, 45).

Here we report that EAE is aggravated by a mild infection with live Streptococcus pneumoniae, the most common pathogen of bacterial respiratory tract infections (41). Clinical aggravation of autoimmune encephalomyelitis was not accompanied by an augmentation of myelin oligodendrocyte glycoprotein (MOG)-specific autoreactive lymphocytes but by an increase of proinflammatory cytokines and the activation of dendritic cells in the systemic circulation. In TLR2^–/– mice the levels of tumor necrosis factor alpha (TNF-) and interleukin-6 (IL-6) were not increased after infection, and EAE was not worsened. Therefore, we postulate that exacerbation of EAE by S. pneumoniae infection is TLR2 dependent but T cell independent.

	MATERIALS AND METHODS

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Animals. Female C57BL/6 mice (age 6 to 8 weeks) were purchased from Charles River (Sulzfeld, Germany). TLR2^–/– mice (47) were bred in the Central Animal Care Facility of the University Hospital Göttingen, Germany. All animal experiments were approved by the District Government of Braunschweig, Lower Saxony. Mice were housed in pathogen-free conditions and received water and food ad libitum.

Induction of EAE and infection with S. pneumoniae. C57BL/6 mice were immunized subcutaneously (s.c.) in both flanks with 200 µg of myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) in 50 µl of phosphate-buffered saline (PBS) and 100 µl of incomplete Freund adjuvant (IFA; Sigma-Aldrich, Deisenhofen, Germany) containing 1 mg of desiccated M. tuberculosis (H37Ra; Difco Laboratories, Detroit, MI) on day 0 and 7. In addition, mice were injected intraperitoneally with 100 ng of Bordetella pertussis toxin (Sigma-Aldrich, Deisenhofen, Germany) in 300 µl of PBS on day 0 and 2 after the first immunization.

EAE mice were infected intraperitoneally with 2.5 x 10⁵ CFU of S. pneumoniae type 3 (39) in 0.5 ml of 0.9% NaCl either 2 days before immunization or 7 or 21 days after the first immunization. Control mice received an equal volume of saline. The infection time 2 days before the induction of EAE addressed the question whether an S. pneumoniae infection prior to EAE induction intensified the autoimmune process. The infection time 7 days after the first immunization was chosen to elucidate whether infection aggravated the course of EAE when it occurred within the early asymptomatic phase. By use of an infection time 21 days after the first immunization, we clarified whether infection could cause relapse beyond the peak of disease, when the clinical symptoms of EAE slowly resolved. Starting 12 h after infection, all mice received antibiotic treatment with ceftriaxone (Rocephin; Hoffmann-LaRoche, Grenzach-Wyhlen, Germany) at 100 mg/kg twice daily for 3 days. This short interval between S. pneumoniae application and antibiotic therapy was necessary to ensure a mild infection. At 12 h after infection blood was drawn from 11 EAE mice and plated on blood agar plates.

Clinical evaluation. Mice were weighed and starting from day 7 scored daily for clinical EAE signs according to the following scoring system: 0, no disease; 0.5, partial tail paralysis; 1, complete tail paralysis; 1.5, complete tail paralysis and discrete hind limb weakness; 2, complete tail paralysis and strong hind limb weakness; 2.5, unilateral hind limb paralysis; 3, complete hind limb paralysis; 3.5, complete hind limb paralysis and forelimb weakness; 4, tetraplegia; and 5, death of EAE (2). Mice were killed by cervical dislocation when the EAE score was >3.0 or the loss of weight was >20% of the maximum weight. One of thirty-four mice died of infection, and three of the thirty-three mice in the S. pneumoniae-infected group (see results in Fig. 1B) had to be killed because of an EAE score of 3.5.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Time dependence of the effect of S. pneumoniae infection on the course of autoimmune encephalomyelitis. MOG35-55 immunized mice received intraperitoneal injections of 2.5 x 105 CFU S. pneumoniae (; day –2, n = 12; day +7, n = 33; day +21, n = 12) or 0.9% NaCl (; day –2, n = 12; day +7, n = 32; day +21, n = 12) either prior (day –2, A) or after immunization (day 7, B; day 21, C). After 12 h, treatment with ceftriaxone was started twice daily for 3 days. Mice were weighed and scored for signs of EAE every day. Graphs show the mean clinical scores of the animals ± the SEM. When mice were infected 7 days after the first MOG35-55 immunization during the subclinical phase, the course of EAE was significantly aggravated (maximum disease score, P = 0.035; cumulative disease score, P = 0.046 [two-tailed unpaired t test]). S. pneumoniae infection 2 days before the first MOG35-55 immunization and during late EAE (day +21) did not influence the course of EAE.

Real-time PCR. Total RNA was extracted from spinal cords of EAE mice at the peak of disease using RNeasy Minikits (QIAGEN, Hilden, Germany). The samples were treated with DNase I (Roche, Mannheim, Germany), and 1 µg of RNA was transcribed into cDNA using oligo(dT) primers and the SuperScript II RT kit (Invitrogen, Carlsbad, CA). A total of 2.5 µl of cDNA was transferred into a 96-well Multiply PCR plate (Sarstedt, Germany), and 12.5 µl of ABsoluteQPCRSYBER Green master mix (ABgene, Surrey, United Kingdom) plus 19.6 µl of double-distilled H₂O was added. The following primer probe pairs were used: IL-10, sense (5'-GGTTGC CAA GCC TTA TCG GA-3') and antisense (5'-ACCTGC TCC ACT GCC TTG CT-3'); TNF-, sense (5'-CTT CTC AAA ATT CGA GTG ACA A-3') and antisense (5'-GAG TAG ACA AGG TAC AAC CC-3'); and GAPDH, sense (5'-TCCTGC ACC ACC AAC TGC TTA GCC-3') and antisense (5'-GTTCAG CTC TGG GAT GAC CTT GCC-3'). The primer pairs for IFN- and IL-2 were purchased from R&D Systems (Minneapolis, MN). After an initial Taq activation step at 95°C and 45 s at 63°C, amplicon accumulation was measured during the annealing phase. A total of 45 cycles were performed (iCycler, data analysis with iCycler analysis software version 2.3; Bio-Rad, Hercules, CA). The reaction efficiency for each primer was always at least 90%, based on the amplification efficiency in serial dilutions.

Flow cytometry. Mice were killed at the peak of disease, and spinal cord tissue was homogenized and passed through a 70-µm-pore-size nylon filter (Fisher Scientific, Schwerte, Germany). The homogenate was centrifuged, resuspended in 70% isotonic Percoll separation solution (Biochrom, Berlin, Germany), and overlaid with 37% isotonic Percoll. The gradient was centrifuged at 600 x g for 25 min at room temperature. Flow cytometry (FACSCalibur flow cytometer [CellQuest software], postacquisition analysis with WinMDI 2.8 software [Scripps Research Institute, La Jolla, CA]) was performed with the following antibodies: CD45-PerCP, CD11b-fluorescein isothiocyanate, and GR-1-phycoerythrin (Becton & Dickinson Bioscience, Heidelberg, Germany).

Fluorescence-activated cell sorting (FACS) analysis of CD11c⁺ cells was performed with samples from the spleens of immunized mice 13 h after infection with S. pneumoniae or intraperitoneal saline injection. Samples were prepared at 4°C in buffer solution (PBS containing 2% fetal calf serum and 0.2% NaN₃) and stained with fluorescein isothiocyanate (FITC)-labeled anti-CD80 or anti-major histocompatibility complex (MHC) class II and phycoerythrin-labeled anti-CD86 or anti-MHC class II (all BD Pharmingen). After lysis of erythrocytes with FACS lysis solution (Becton Dickinson, San Jose, CA) and washing, cell suspensions were analyzed on a FACSCalibur flow cytometer with CellQuest software. Viable cells were gated by the forward and side scatter of light.

Lymphocyte proliferation assay. Seven days after the first MOG_35-55 immunization (13 h after bacterial infection and 1 h after the first antibiotic dose) and 15 days after the first immunization, respectively, animals were killed, and draining axillary and inguinal lymph nodes were removed and homogenized (2). T cells were placed in a 96-well plate (5 x 10⁵/well) and cultured in triplicate in the presence of 50 µg of MOG_35-55 peptide/ml for 48 h in RPMI complete medium (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (PAA Lab GmbH, Pasching, Austria), 100 U of penicillin/ml, and 100 µg of streptomycin/ml (Biochrom, Berlin, Germany). The cells were harvested by using a Harvestor IH-110-96 (Inotech AG, Dottikon, Switzerland) after incubation for 15 h with 0.5 µCi of [³H]thymidine (Amersham Biosciences, Freiburg, Germany)/well. The thymidine incorporation was assessed by using a Microbeta 1450 BetaCounter (Perkin-Elmer, Boston, MA), and IFN- was measured in the supernatants by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Bergisch-Gladbach, Germany).

Cytokine ELISA. At 1 week after the initial MOG_35-55 immunization (13 h after bacterial infection, 1 h after the first antibiotic dose), the animals were decapitated and exsanguinated. Blood was allowed to clot and then centrifuged at 6,000 x g for 10 min. For the measurement of cytokine concentrations in the spinal cord, mice were transcardially perfused with PBS. Then the spinal cords were homogenized and centrifuged. The concentrations of TNF- and IL-6 in the sera and tissue supernatants were measured by commercially available ELISAs (Quantikine ELISA Systems; R&D Systems, Bergisch-Gladbach, Germany). The color reaction was read in a microplate reader (SLT; Spectra LabInstruments, Crailsheim, Germany) at an absorbance of 450 nm, with values obtained at 540 nm as a reference. Release was calculated as picograms of cytokine per milliliter.

Statistical analysis. Statistical evaluation and graphical presentation was performed by using GraphPad Prism 4.0 for the unpaired Student t test and GraphPad Instat 3.05 (GraphPad Software, San Diego, CA) for the Fisher exact test. Moreover, nonparametric repeated measures analysis of variance (ANOVA) was performed on the disease severity-versus-time curves of mice infected 7 days after the first immunization and the respective control animals. The data are expressed as means ± the standard error of the mean (SEM). Differences were considered statistically significant when the P value was <0.05.

	RESULTS

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Infection with S. pneumoniae increased the severity of autoimmune encephalomyelitis. S. pneumoniae infection was either performed 2 days before the first MOG immunization (Fig. 1A) or 7 (Fig. 1B) or 21 (Fig. 1C) days after the initial MOG_35-55 immunization. At 12 h after infection, animals were clinically asymptomatic. The blood contained (5.0 ± 2.4) x 10⁴ CFU of S. pneumoniae/ml. S. pneumoniae was not able to substantially modulate the course of autoimmune encephalomyelitis when mice were infected 2 days before or 21 days after the first immunization. In contrast, S. pneumoniae aggravated the disease course, when infection was performed 7 days after the initial MOG_35-55 immunization. The increased severity of EAE after S. pneumoniae infection was reflected by significant differences in the maximum disease score (infected versus uninfected mice, mean ± SEM: 2.7 ± 0.2 versus 2.0 ± 0.2; P = 0.035). Accordingly, the cumulative scores also differed among both groups (40.5 ± 3.5 in S. pneumoniae-infected mice and 29.5 ± 4.1 in saline-treated mice; P = 0.046). The disease severity-versus-time curves of mice infected 7 days after the first immunization and the respective control animals were significantly different (P = 0.048). Disease incidence was higher in the S. pneumoniae group (94%) compared to control animals (75%) (P = 0.044). All subsequent EAE experiments were then performed with S. pneumoniae infection at day 7 after the first MOG_35-55 immunization.

No substantial changes of the histopathology in the CNS after infection. In spinal cords examined histologically at the peak of disease (14 infected and 13 uninfected mice) (Fig. 2), the number of CD3-positive T cells was mildly elevated in S. pneumoniae-preinfected animals (160 ± 41 cells/mm² versus 96 ± 30 cells/mm² in controls) (P = 0.23, Fig. 2B). T-cell influx was accompanied by slightly more MAC3⁺ cells in mice after S. pneumoniae infection (101 ± 27 cells/mm²) than in the uninfected mice (82 ± 27 cells/mm²) (P = 0.63). The number of B220⁺ B cells was also subtly elevated in S. pneumoniae-infected mice (36 ± 9 cells/mm² versus 16 ± 5 cells/mm²) (P = 0.08). In the inflamed regions, APP-positive axonal structures indicating acute axonal damage were slightly enhanced in infected mice (57 ± 18 deposits/mm² compared to 37 ± 14 deposits/mm²; Fig. 2C) (P = 0.39), and the demyelinated area was slightly increased in mice challenged with S. pneumoniae (14% ± 4% compared to 10% ± 4%; Fig. 2D) (P = 0.44).

View larger version (91K): [in this window] [in a new window]   FIG. 2. Histopathological profile in EAE mice after S. pneumoniae infection. (A) Animals were killed at the peak of disease (10 days after the first clinical symptoms), and immunohistochemistry/histology was performed for demyelination (LFB), macrophages/microglia (MAC3), T cells (CD3), B cells (B220), and APP representing axonal damage (the scale bar in the top row is 500 µm; for all other rows it is 200 µm). In all cases, animals with high clinical scores are shown. (B to D) Quantification of mononuclear infiltrates (B), axonal damage (C) or demyelination (D) in S. pneumoniae-infected () and uninfected () mice. The data represent means ± the SEM from three spinal cord sections of 13 mice in the uninfected and 14 mice in the S. pneumoniae-treated group.

View larger version (44K): [in this window] [in a new window]   FIG. 3. Flow cytometric analysis of mononuclear infiltration in the CNS of EAE mice with or without prior S. pneumoniae infection. Spinal cords were taken at peak of disease (10 days after disease onset), and cells were stained with CD45, CD11b, and Gr-1. Infiltrating macrophages (CD11b+ CD45hi) could be distinguished from CD11b+ CD45lo endogenous microglia. Moreover, CD11b+ Gr1+ granulocytes were visualized. No substantial differences were seen when infected and uninfected mice were compared. The results for one of two representative experiments are shown.

View larger version (13K): [in this window] [in a new window]   FIG. 4. The induction of Th1 cytokines in the CNS during EAE was not influenced by S. pneumoniae. IFN-, IL-2, TNF-, and IL-10 mRNA levels were measured in the spinal cords of mice at peak of disease. The data are expressed as mean values per group ± the SEM (one of two representative experiments, n = 4 in each group).

View larger version (13K): [in this window] [in a new window]   FIG. 5. S. pneumoniae had no effect on T-cell priming and Th1 immune response. Recall responses of lymph node cells to MOG35-55 peptide were measured by evaluating [3H]thymidine uptake (A) and by IFN- ELISA (B). Mean values per group ± the SEM are shown (one of two representative experiments). No significant differences were noted, when infected and uninfected mice were compared. n.d., not detectable.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Induction of proinflammatory cytokines in the systemic circulation of mice after S. pneumoniae challenge. Sera of 10 mice per group for the IL-6 ELISA and 9 mice per group for the TNF- ELISA were collected 1 h after the start of antibiotic therapy (13 h after S. pneumoniae infection). TNF- and IL-6 were measured by ELISA. The graphs show the mean serum levels ± the SEM. Please note the strong differences of the cytokine concentrations in the sera of infected and uninfected animals. n.d., not detectable.

View larger version (8K): [in this window] [in a new window]   FIG. 7. Costimulatory molecules on CD11c+ dendritic cells were upregulated in mice infected during the subclinical phase of EAE. In three mice infected with S. pneumoniae (EAE SP3) and three control animals 7 days after the first MOG35-55 immunization (EAE NaCl), and in one mouse which did not receive MOG35-55 (isotype) the expression of MHC class II molecules and the costimulatory molecules CD80 and CD86 were studied in CD11c+ splenic dendritic cells 1 h after start of antibiotic therapy (13 h after S. pneumoniae infection) by flow cytometry. The mean fluorescence intensity (MFI) was determined (means ± the SEM). The expression of MHC class II, CD80, and CD86 were strongly increased in infected EAE mice.

View larger version (24K): [in this window] [in a new window]   FIG. 8. Disease modulation depends on the presence of TLR2. MOG35-55 immunized TLR2–/– mice received intraperitoneal injections of 2.5 x 105 CFU S. pneumoniae (, n = 14) or 0.9% NaCl (, n = 14) 7 days after the first immunization. The graph shows the mean clinical scores ± the SEM of the animals. No substantial differences in the course of EAE between both groups were noted.

	DISCUSSION

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In the present study, a mild S. pneumoniae infection during the preclinical phase of EAE led to a clear aggravation of clinical symptoms. In contrast, S. pneumoniae infection at earlier (2 days before the first immunization) or later (21 days after the first immunization) time points did not influence the course of EAE. It is tempting to speculate that humans with MS probably are not equally vulnerable to S. pneumoniae infections during the course of their disease, the susceptibility being greatest in the subclinical phase prior to the manifestation of an MS event. This finding speaks in favor of a dependence of the susceptibility to bacterial challenge on the time of infection. The present data clearly indicate that S. pneumoniae infections are capable of boosting the immune process of EAE. We therefore studied several mechanisms of potential relevance for this phenomenon.

Planimetry of the demyelinated areas, as well as of axonal damage, showed a tendency toward more pronounced tissue damage in infected animals; however, as a consequence of the high interindividual variation of demyelination and axonal injury within both groups, the differences failed to reach statistical significance. Accordingly, the intraspinal production of the key Th1 cytokines TNF-, IL-2, and IFN- was not substantially influenced by S. pneumoniae preincubation. These results make it unlikely that MOG-specific, encephalitogenic T lymphocytes and macrophages invading the brain after infection were responsible for disease aggravation. Indeed, when we examined the ability of S. pneumoniae to influence MOG-specific T-cell proliferation and IFN- production in vitro, no modulation was detectable. These data are in contrast to work by others who showed that systemic T-cell activation occurred after bacterial infection or administration of bacterial components in vivo (7, 20, 33, 46).

Teichoic and lipoteichoic acids and peptidoglycans are major constituents of the cell wall of S. pneumoniae. They are stimulants of the innate immune system and are ligands of TLR2 and Nod1/Nod2 (8, 40, 43, 49). Macrophages lack N-acetyl-muramyl L-alanine amidase required for the complete degradation of peptidoglycans (13). The persistence of peptidoglycan may be an important factor in various chronic autoimmune diseases, including MS (31). Cell wall components from gram-positive bacteria can substitute antigens from M. tuberculosis in EAE induction (46). TLR2 mRNA is upregulated during the course of EAE. Expression of the mRNA encoding TLR2 starts to increase 4 to 8 days after immunization with MOG and reaches its maximum at 3 weeks (50).

The action of pneumococcal cell wall products probably depends on a dendritic cell-mediated expression of costimulatory molecules and immunomodulatory cytokines. In the present study, dendritic cells from the systemic circulation were activated early after infection. Since in mice lacking TLR2 S. pneumoniae infection failed to increase the severity of EAE (Fig. 8), stimulation of TLR2 by pneumococcal components play a central role in the aggravation of EAE by this pathogen. TLR4, stimulated at sublytic concentrations by pneumolysin (8), does not appear as important as TLR2 for MS and EAE: the heterozygous Asp299Gly polymorphism of TLR4 did not influence the course of MS in 890 patients. The pro- and anti-inflammatory cytokine profile in these patients did not depend on the TLR4 genotype (15).

The immune system can signal the presence of microorganisms to the brain by several mechanisms (26). First, the circumventricular organs located at a strategic position in the midline ventricular system are not protected by the blood-brain barrier and therefore can function as communicating structures between brain and blood for microbial products (36). TLR2, TLR4, and TLR9 are expressed by microglial cells, astrocytes, and oligodendrocytes (24). Challenge by more than one TLR agonist synergistically activates microglial cells, suggesting that small concentrations of bacterial products acting on different TLR can stimulate microglial cells (8). Second, the vagus nerve can sense peripheral inflammation probably through cytokine receptors (16). Third, endothelial cells can be activated by circulating bacterial products and cytokines (44). The inflammatory mediators released by endothelial cells can directly interact with surrounding brain cells and also with adhering and invading blood cells (36). Fourthly, proinflammatory cytokines can also enter the brain tissue in regions without a tight blood-brain barrier. TNF- produced by the circulating pool of leukocytes plays an important role in the triggering of EAE events (18). Compared to viral infections, larger quantities of TNF- are produced during bacterial disease (19). IL-6, a Th-1 promoting cytokine, has been found to be upregulated in EAE in the preclinical phase (22), and IL-6-deficient mice are resistant to MOG-induced EAE, indicating that IL-6 also plays a crucial role in the course of this animal model (29). In the present study, TNF- and IL-6 concentrations in the blood of infected wild-type mice but not TLR2^–/– EAE mice were higher than in the respective uninfected controls.

In the CNS of MS patients, autoimmune T cells are found to be reactive for self-glycosphingolipids (self-GSL). Bacteria or their cell wall components can induce the release of IFN- by these autoimmunogenic T cells even in the absence of specific self-GSL (6). Although S. pneumoniae was not among the bacteria studied, this pathogen contains several compounds that should be able to activate self-GSL-specific T cells. Possibly, the aggravation of EAE by a mild S. pneumoniae infection observed in the present study was mediated by this mechanism.

In conclusion, even a mild infection with the gram-positive bacterium S. pneumoniae, the most abundant bacterial respiratory pathogen in humans, is able to aggravate autoimmune encephalomyelitis, when infection takes place in the asymptomatic preclinical phase. TLR2^–/– mice were protected against the S. pneumoniae-induced disease aggravation. The most likely mechanism in our model appears to be a sepsis-like symptom induced by the release of proinflammatory modulators by circulating immune cells and activation of dendritic cells in the presence of S. pneumoniae. In turn, this probably leads to a cross talk of activated leukocytes and microglial cells through the blood-brain barrier in the preclinical phase. The systemic inflammation was milder in infected TLR2^–/– mice, and there was no difference in the clinical course of EAE in these mice.

Our data stress the importance of early treatment of respiratory bacterial infections in MS patients. They encourage epidemiological studies on a possible link between MS onset or exacerbation and infections with S. pneumoniae. Whether the detrimental effect of bacterial infections on the course of EAE can be attenuated by antibiotic therapy tailored to minimize the release of proinflammatory bacterial products (3, 39, 45) remains to be studied.

	ACKNOWLEDGMENTS

 
This study was supported by grants from the Ely Lilly International Foundation (R.N., I.H., and W.B.), Fritz-Thyssen-Stiftung (grant 10.04.1.183 to M.P.), and Gemeinnützige Hertie-Stiftung (grant 1.01.1/05/002 to M.P.).

We thank Olga Dell for excellent technical assistance, Christine Crozier for critical reading of the manuscript, and Carola Werner for help in statistical analysis. We are grateful to Elke Pralle for help with the ELISAs.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Neurology, Georg-August-University, Robert-Koch-Str. 40, D-37075 Göttingen, Germany. Phone: 49-551-39-8455. Fax: 49-551-39-8405. E-mail: rnau{at}gwdg.de.

Editor: V. J. DiRita

M.P. and R.N. contributed equally to this study.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1.	Andersen, O., P. E. Lygner, T. Bergstrom, M. Andersson, and A. Vahlne. 1993. Viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study. J. Neurol. 240:417-422.[CrossRef][Medline]
2.	Becher, B., B. G. Durell, and R. J. Noelle. 2002. Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12. J. Clin. Investig. 110:493-497.[CrossRef][Medline]
3.	Brinkmann, K. C., A. J. Talati, R. E. Akbari, E. A. Meals, and B. K. English. 2005. Group B streptococci exposed to rifampin or clindamycin (versus ampicillin or cefotaxime) stimulate reduced production of inflammatory mediators by murine macrophages. Pediatr. Res. 57:419-423.[CrossRef][Medline]
4.	Buljevac, D., H. Z. Flach, W. C. Hop, D. Hijdra, J. D. Laman, H. F. Savelkoul, F. G. van der Meche, P. A. van Doorn, and R. Q. Hintzen. 2002. Prospective study on the relationship between infections and multiple sclerosis exacerbations. Brain 125:952-960.[Abstract/Free Full Text]
5.	De la Monte, S. M., A. H. Ropper, G. R. Dickersin, N. L. Harris, J. A. Ferry, and E. P. Richardson, Jr. 1986. Relapsing central and peripheral demyelinating diseases. Unusual pathologic features. Arch. Neurol. 43:626-629.[Abstract]
6.	De Libero, G., A. P. Moran, H. J. Gober, E. Rossy, A. Shamshiev, O. Chelnokova, Z. Mazorra, S. Vendetti, A. Sacchi, M. M. Prendergast, S. Sansano, A. Tonevitsky, R. Landmann, and L. Mori. 2005. Bacterial infections promote T-cell recognition of self-glycolipids. Immunity 22:763-772.[CrossRef][Medline]
7.	Du, C., S. Y. Yao, A. Ljunggren-Rose, and S. Sriram. 2002. Chlamydia pneumoniae infection of the central nervous system worsens experimental allergic encephalitis. J. Exp. Med. 196:1639-1644.[Abstract/Free Full Text]
8.	Ebert, S., J. Gerber, S. Bader, F. Mühlhauser, K. Brechtel, T. J. Mitchell, and R. Nau. 2005. Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination. J. Neuroimmunol. 159:87-96.[CrossRef][Medline]
9.	Forrest, J. M., M. A. Menser, and J. A. Burgess. 1971. High frequency of diabetes mellitus in young adults with congenital rubella. Lancet ii:332-334.
10.	Gianani, R., and N. Sarvetnick. 1996. Viruses, cytokines, antigens, and autoimmunity. Proc. Natl. Acad. Sci. USA 93:2257-2259.[Abstract/Free Full Text]
11.	Gilden, D. H. 2005. Infectious causes of multiple sclerosis. Lancet Neurol. 4:195-202.[Medline]
12.	Hamada, T., B. F. Driscoll, M. W. Kies, and E. C. Alvord, Jr. 1989. LPS augments adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat. Autoimmunity 2:275-284.[Medline]
13.	Hoijer, M. A., M. J. Melief, J. Calafat, D. Roos, R. W. van den Beemd, J. J. van Dongen, and M. P. Hazenberg. 1997. Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme. Blood 90:1246-1254.[Abstract/Free Full Text]
14.	Hunter, S. F., and D. A. Hafler. 2000. Ubiquitous pathogens: links between infection and autoimmunity in MS? Neurology 55:164-165.[Free Full Text]
15.	Kroner, A., F. Vogel, A. Kolb-Maurer, N. Kruse, K. V. Toyka, B. Hemmer, P. Rieckmann, and M. Maurer. 2005. Impact of the Asp299Gly polymorphism in the Toll-like receptor 4 (Tlr-4) gene on disease course of multiple sclerosis. J. Neuroimmunol. 165:161-165.[CrossRef][Medline]
16.	Maier, S. F., L. E. Goehler, M. Fleshner, and L. R. Watkins. 1998. The role of the vagus nerve in cytokine-to-brain communication. Ann. N. Y. Acad. Sci. 840:289-300.[Abstract/Free Full Text]
17.	Matsumoto, Y., and M. Fujiwara. 1993. Immunomodulation of experimental autoimmune encephalomyelitis by staphylococcal enterotoxin D. Cell. Immunol. 149:268-278.[CrossRef][Medline]
18.	Murphy, C. A., R. M. Hoek, M. T. Wiekowski, S. A. Lira, and J. D. Sedgwick. 2002. Interactions between hemopoietically derived TNF and central nervous system-resident glial chemokines underlie initiation of autoimmune inflammation in the brain. J. Immunol. 169:7054-7062.[Abstract/Free Full Text]
19.	Nadal, D., D. Leppert, K. Frei, P. Gallo, H. Lamche, and A. Fontana. 1989. Tumour necrosis factor-alpha in infectious meningitis. Arch. Dis. Child. 64:1274-1279.[Abstract]
20.	Nogai, A., V. Siffrin, K. Bonhagen, C. F. Pfueller, T. Hohnstein, R. Volkmer-Engert, W. Brück, C. Stadelmann, and T. Kamradt. 2005. Lipopolysaccharide injection induces relapses of experimental autoimmune encephalomyelitis in nontransgenic mice via bystander activation of autoreactive CD4+ cells. J. Immunol. 175:959-966.[Abstract/Free Full Text]
21.	Noseworthy, J. H., C. Lucchinetti, M. Rodriguez, and B. G. Weinshenker. 2000. Multiple sclerosis. N. Engl. J. Med. 343:938-952.[Free Full Text]
22.	Okuda, Y., S. Sakoda, and T. Yanagihara. 1998. The pattern of cytokine gene expression in lymphoid organs and peripheral blood mononuclear cells of mice with experimental allergic encephalomyelitis. J. Neuroimmunol. 87:147-155.[CrossRef][Medline]
23.	Olson, J. K., J. L. Croxford, and S. D. Miller. 2001. Virus-induced autoimmunity: potential role of viruses in initiation, perpetuation, and progression of T-cell-mediated autoimmune disease. Viral Immunol. 14:227-250.[CrossRef][Medline]
24.	Olson, J. K., and S. D. Miller. 2004. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J. Immunol. 173:3916-3924.[Abstract/Free Full Text]
25.	Peacock, J. W., S. F. Elsawa, C. C. Petty, W. F. Hickey, and K. L. Bost. 2003. Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68. Eur. J. Immunol. 33:1849-1858.[CrossRef][Medline]
26.	Perry, V. H., T. A. Newman, and C. Cunningham. 2003. The impact of systemic infection on the progression of neurodegenerative disease. Nat. Rev. Neurosci. 4:103-112.[CrossRef][Medline]
27.	Posnett, D. N., and D. Yarilin. 2005. Amplification of autoimmune disease by infection. Arthritis Res. Ther. 7:74-84.[Medline]
27a.	Prinz, M., F. Garbe, H. Schmidt, A. Mildner, J. Gutcher, K. Wolter, M. Piesche, R. Schroers, E. Weiss, C. J. Kirschning, C. D. Rochford, W. Brück, and B. Becher. 2006. Innate immunity mediated by TLR9 modulates pathogenicity in an animal model of multiple sclerosis. J. Clin. Investig. 116:456-464.[CrossRef][Medline]
28.	Prinz, M., M. Heikenwalder, P. Schwarz, K. Takeda, S. Akira, and A. Aguzzi. 2003. Prion pathogenesis in the absence of Toll-like receptor signalling. EMBO Rep. 4:195-199.[CrossRef][Medline]
29.	Samoilova, E. B., J. L. Horton, B. Hilliard, T. S. Liu, and Y. Chen. 1998. IL-6-deficient mice are resistant to experimental autoimmune encephalomyelitis: roles of IL-6 in the activation and differentiation of autoreactive T cells. J. Immunol. 161:6480-6486.[Abstract/Free Full Text]
30.	Schiffenbauer, J., H. M. Johnson, E. J. Butfiloski, L. Wegrzyn, and J. M. Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. USA 90:8543-8546.[Abstract/Free Full Text]
31.	Schrijver, I. A., M. van Meurs, M. J. Melief, A. C. Wim, D. Buljevac, R. Ravid, M. P. Hazenberg, and J. D. Laman. 2001. Bacterial peptidoglycan and immune reactivity in the central nervous system in multiple sclerosis. Brain 124:1544-1554.[Abstract/Free Full Text]
32.	Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J. Biol. Chem. 274:17406-17409.[Abstract/Free Full Text]
33.	Segal, B. M., J. T. Chang, and E. M. Shevach. 2000. CpG oligonucleotides are potent adjuvants for the activation of autoreactive encephalitogenic T cells in vivo. J. Immunol. 164:5683-5688.[Abstract/Free Full Text]
34.	Segal, B. M., D. M. Klinman, and E. M. Shevach. 1997. Microbial products induce autoimmune disease by an IL-12-dependent pathway. J. Immunol. 158:5087-5090.[Abstract]
35.	Shapira, L., S. Ayalon, and T. Brenner. 2002. Effects of Porphyromonas gingivalis on the central nervous system: activation of glial cells and exacerbation of experimental autoimmune encephalomyelitis. J. Periodontol. 73:511-516.[CrossRef][Medline]
36.	Sharshar, T., N. S. Hopkinson, D. Orlikowski, and D. Annane. 2005. Science review: the brain in sepsis—culprit and victim. Crit. Care. 9:37-44.[CrossRef][Medline]
37.	Soos, J. M., A. C. Hobeika, E. J. Butfiloski, J. Schiffenbauer, and H. M. Johnson. 1995. Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a non-V beta 8-specific superantigen. Proc. Natl. Acad. Sci. USA 92:6082-6086.[Abstract/Free Full Text]
38.	Soos, J. M., M. G. Mujtaba, J. Schiffenbauer, B. A. Torres, and H. M. Johnson. 2002. Intramolecular epitope spreading induced by staphylococcal enterotoxin superantigen reactivation of experimental allergic encephalomyelitis. J. Neuroimmunol. 123:30-34.[CrossRef][Medline]
39.	Stuertz, K., H. Schmidt, H. Eiffert, P. Schwartz, M. Mäder, and R. Nau. 1998. Differential release of lipoteichoic and teichoic acids from Streptococcus pneumoniae as a result of exposure to beta-lactam antibiotics, rifamycins, trovafloxacin, and quinupristin-dalfopristin. Antimicrob. Agents Chemother. 42:277-281.[Abstract/Free Full Text]
40.	Takeda, K., and S. Akira. 2005. Toll-like receptors in innate immunity. Int. Immunol. 17:1-14.[Abstract/Free Full Text]
41.	Tan, T. Q. 2002. Update on pneumococcal infections of the respiratory tract. Semin. Respir. Infect. 17:3-9.[CrossRef][Medline]
42.	Theil, D. J., I. Tsunoda, F. Rodriguez, J. L. Whitton, and R. S. Fujinami. 2001. Viruses can silently prime for and trigger central nervous system autoimmune disease. J. Neurovirol. 7:220-227.[CrossRef][Medline]
43.	Travassos, L. H., S. E. Girardin, D. J. Philpott, D. Blanot, M. A. Nahori, C. Werts, and I. G. Boneca. 2004. Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition. EMBO Rep. 5:1000-1006.[CrossRef][Medline]
44.	Tsao, N., H. P. Hsu, C. M. Wu, C. C. Liu, and H. Y. Lei. 2001. Tumour necrosis factor-alpha causes an increase in blood-brain barrier permeability during sepsis. J. Med. Microbiol. 50:812-821.[Abstract/Free Full Text]
45.	Van Langevelde, P., J. T. Van Dissel, E. Ravensbergen, B. J. Appelmelk, I. A. Schrijver, and P. H. Groeneveld. 1998. Antibiotic-induced release of lipoteichoic acid and peptidoglycan from Staphylococcus aureus: quantitative measurements and biological reactivities. Antimicrob. Agents Chemother. 42:3073-3078.[Abstract/Free Full Text]
46.	Visser, L., J. de Heer, L. A. Boven, D. van Riel, M. van Meurs, M. J. Melief, U. Zahringer, J. van Strijp, B. N. Lambrecht, E. E. Nieuwenhuis, and J. D. Laman. 2005. Proinflammatory bacterial peptidoglycan as a cofactor for the development of central nervous system autoimmune disease. J. Immunol. 174:808-816.[Abstract/Free Full Text]
47.	Werts, C., R. I. Tapping, J. C. Mathison, T. H. Chuang, V. Kravchenko, I. Saint Girons, D. A. Haake, P. J. Godowski, F. Hayashi, A. Ozinsky, D. M. Underhill, C. J. Kirschning, H. Wagner, A. Aderem, P. S. Tobias, and R. J. Ulevitch. 2001. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat. Immunol. 2:346-352.[CrossRef][Medline]
48.	Wu, L. X., M. J. Makela, M. Roytta, and A. Salmi. 1998. Effect of viral infection on experimental allergic encephalomyelitis in mice. J. Neuroimmunol. 18:139-153.
49.	Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and D. Golenbock. 1999. Cutting edge: recognition of gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163:1-5.[Abstract/Free Full Text]
50.	Zekki, H., D. L. Feinstein, and S. Rivest. 2002. The clinical course of experimental autoimmune encephalomyelitis is associated with a profound and sustained transcriptional activation of the genes encoding Toll-like receptor 2 and CD14 in the mouse CNS. Brain Pathol. 12:308-319.[Medline]

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