Toll-like receptor-2 deficiency is associated with enhanced brain TNF gene expression during pneumococcal meningitis

Journal of Neuroimmunology 168 (2005) 21 – 33 www.elsevier.com/locate/jneuroim

Toll-like receptor-2 deficiency is associated with enhanced brain TNF gene expression during pneumococcal meningitis Maryse Letiembre a,1, Hakim Echchannaoui a,1, Fabrizia Ferracin a, Serge Rivest b, Regine Landmann a,* a

Division of Infectious Diseases, Department of Research, University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland b Universite´ Laval, Que´bec, Canada Received 10 February 2005; accepted 10 June 2005

Abstract TNF is a marker of disease activity in bacterial meningitis. To investigate TNF modulation by Toll-like receptor-2 (TLR2), we studied temporal and anatomical expression patterns of TLR2 and TNF in a pneumococcal meningitis model in wild type (wt) and TLR2 / mice. We show by in situ hybridization that transcripts of TLR2 and of the comolecules CD14, MD-2, TLR1/6 strongly increased and colocalized with TNF in CD45-positive infiltrating cells in the ventricles, corpus callosum and the meninges. TNF gene and protein expression was stronger in TLR2 / than wt brains and associated with increased IjB expression suggesting that TLR2 is controlling inflammation via TNF regulation. D 2005 Elsevier B.V. All rights reserved. Keywords: Toll-like receptor 2; TNF; Brain; Streptococcus pneumoniae; Meningitis

1. Introduction Bacterial meningitis is a severe and frequent disease of the central nervous system (Sigurdardottir et al., 1997). In adults, S. pneumoniae meningitis is the most prevalent and has the highest mortality rate. In the pathogenesis of this disease, penetration of bacteria across the blood – brain (BBB) or the blood – CSF barrier and the subsequent inflammation with brain edema and secondary ischemia are of prime importance. Infiltrating leukocytes release proinflammatory substances and reactive oxygen products which contribute to the bad prognosis of the disease. TNF is one of the inflammatory mediators detected in CSF of patients with bacterial meningitis (Nadal et al., 1989) and is a marker of disease severity (van Deuren et al., 1995). TNF Abbreviations: PMN, polymorphonuclear leukocytes; S. pneumoniae, Streptococcus pneumoniae; TLR, Toll-like receptor; Wt, wild type. * Corresponding author. Tel.: +41 61 265 23 25; fax: +41 61 265 23 50. E-mail address: [email protected] (R. Landmann). 1 Equal contribution. 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.06.016

is rapidly produced by infiltrating cells (Leist et al., 1988); in vitro studies show that microglia (Hanisch et al., 2001), endothelia (Freyer et al., 1999), dendritic cells (Fischer and Reichmann, 2001) and infiltrating macrophages (van der Flier et al., 1995), are also able to express and release TNF upon stimulation with bacterial components. TNF has an ambiguous function in meningitis. Host defense was found disturbed in TNF-deficient mice undergoing meningitis (Gerber et al., 2004). Conversely, TNF increased BBB permeability in a hematogenous meningitis model (Tsao et al., 2001), and contributed to hippocampal neuronal damage, as demonstrated by the beneficial effect of antiTNF in neonatal rat Streptococcus B meningitis (Bogdan et al., 1997). Finally, combined therapy with inhibitors of matrix metalloprotease and of TNF-a converting enzyme (TACE) strongly reduced disease severity and TNF levels in CSF (Leib et al., 2001). Similarly IL-10, which is accumulating in CSF in bacterial meningitis, and can be induced by TNF (Frei et al., 1993; van der Poll et al., 1994), has an ambiguous function in this disease. IL-10 deficient mice showed a

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stronger meningeal inflammation in streptococcal meningitis than wt mice ((Zwijnenburg et al., 2003) and own unpublished data). Conversely, systemic IL-10 pretreatment reduced inflammation in experimental pneumococcal meningitis (Koedel et al., 1996). This can be explained by the fact that in wt mice, IL-10 inhibits TNF production, and at the same time enhances in an autocrine manner its own production and the suppressor of cytokine signaling-3 (SOCS-3) (Donnelly et al., 1999), therefore acts as an anti-inflammatory molecule. Additionally, IL-10 in the CSF suppressed killing of intracellular bacteria (Frei et al., 1993). In conclusion, IL-10 is linked to TNF in an autocrine feedback loop; it is required, but high levels are harmful. TLRs have been recognized as host pattern recognition receptors with a broad specificity of ligands derived from pathogens and are candidate receptors for bacterial components involved in meningeal inflammation. TLR2 is used in the cellular activation by lipoteichoic acid (LTA) (Schwandner et al., 1999) and membrane lipoproteins (Brightbill et al., 1999) from Gram-positive bacteria. Specificity for activating compounds is broadened by the formation of heteromeric complexes between TLR2 and TLR1 or TLR6, respectively and by association with CD36 and/or CD14 (Bulut et al., 2001; Hajjar et al., 2001; Hoebe et al., 2005). TLR2 activation by Gram-positive cell wall components leads via NF-nB activation to TNF transcription and release (Schwandner et al., 1999). However TLR2 activation can also cause IL-10 induction e.g., after stimulation with schistosomal lysophosphatidylserine (Van der Kleij et al., 2002) or with Aspergillus fumigatus hyphae (Netea et al., 2003). Indeed the Yersinia virulence factor primarily induces IL-10 and weakly TNF via TLR2 (Sing et al., 2002b). Therefore it appears that, depending on the stimulus, TLR2 mediates either pro- or anti-inflammatory cytokines. Live bacteria use many receptors for infection and TLR2 is not required, but has a modulatory function. This is evident from our and other studies with Grampositive bacteria in wt and TLR2 / mice. In S. aureus sepsis and S. pneumoniae meningitis, TLR2 was found protective. In contrast, in oral Y. enterocolitica infection, TLR2 was found harmful (Sing et al., 2002a). This indicates that in infection the balance of pro- and anti-inflammatory signals determines outcome, and that different cell types involved may have different abilities to induce anti-versus pro-inflammation. Furthermore the effect of TLR2 activation can be beneficial or detrimental depending on the disease. In a previous study, we found TLR2-deficiency associated with an increase in disease severity and earlier mortality in meningitis. The aggravated clinical course resulted from an increased inflammatory response 24 h after infection. Indeed, TNF concentration was significantly higher in CSF of TLR2 / than of wt mice and TNF levels were significant related to BBB damage and to disease severity (Echchannaoui et al., 2002). The pathogenetic importance of TNF is underlined by the fact that

TACE inhibitor treatment prolonged survival in TLR2 / mice to the values observed in untreated wt mice (submitted). In this study, our aim was to investigate the role of TLR2 in TNF regulation during meningitis. We first identified the cells expressing TLR2 mRNA and determined the kinetics of TLR2 induction. We next specified TNF expressing cells during infection and found the signal enhanced in TLR2 / mice. After demonstrating TNF and TLR2 colocalization in infiltrating cells, we investigated whether the absence of TLR2 coincided with an altered signaling of the inflammatory response. We found the activation of the NF-nB dependent IjB gene stronger in infected brains of TLR2 / than of wt mice.

2. Materials and methods 2.1. Experimental meningitis models Six to eight week old C57BL/6 (wild type, wt) were bred at the Animal House of the University Hospital Basel. TLR2 / mice, which had been backcrossed for 10 generations on a C57BL/6 background were provided by William J. Rieflin (Tularik, South San Francisco, CA). All animals were kept under specific pathogen-free conditions in the Animal House of the Department of Research, University Hospital Basel according to the regulations of the Swiss veterinary law. Mice underwent experimental meningitis as previously described (Echchannaoui et al., 2002). Animals were anaesthetized via intraperitoneal (i.p.) injection of 100 mg/kg Ketamine (Ketalar*; WarnerLambert AG, Baar, Switzerland) and 20 mg/kg Xylazinum (Xylapan*; Graeub AG, Bern, Switzerland), and subsequently subarachnoidally inoculated into the left forebrain with either 0.9% NaCl, live S. pneumoniae (clinical isolate of serotype 3) or Listeria monocytogenes (strain EGD, provided by Dr. R. M. Zinkernagel, University Hospital, Zu¨rich, Switzerland) in a 25 Al volume. The infectious dose of bacteria was retrospectively confirmed by plating each inoculum. At the indicated time points, the animals were euthanized by i.p. injection of 100 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL). Mice were perfused into the left cardiac ventricle with either Ringer’s solution (Braun Medical AG, Emmenbru¨cke, Switzerland) for RNA analysis by Northern blot, RT-PCR or Western blot or with 4% paraformaldehyde (Sigma) in 0.1 M borax (Sigma) buffer for in situ hybridization. CSF was collected by puncture of the cisterna magna as previously described (Echchannaoui et al., 2002), and centrifuged at 800 g for 7 min (room temperature). The CSF cells were resuspended in FACS buffer for flow cytometry. After CSF collection, total brains or the brain regions hippocampus, brain stem, cortex and cerebellum were quickly removed and immersed in TRIzol reagent (Invitrogen, The Netherlands) for Northern analysis. For in situ hybridization, brains were fixed in 4%

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paraformaldehyde for 2 – 8 days and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde –borax buffer overnight at 4 -C. The frozen brains were mounted on a microtome (Reichert-Jung, Cambridge Instruments Company, Deerfield, IL) and cut into 20 Am coronal sections from the olfactory bulb to the end of the medulla. The slices were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer pH 7.3, 30% ethylene glycol, 20% glycerol) and stored at 20 -C. For Western blot the organs were frozen in liquid nitrogen and kept at 70 -C until analysis. For the leukocyte depletion treatment, cyclophosphamide (Sigma) was reconstituted with sterile PBS and injected i.p. (300 mg/kg in 0.2 ml) 48 h before S. pneumoniae inoculation. 2.2. RNA extraction and northern blot analysis Whole brain and cerebellum were immersed into TRIzol reagent before homogenization with a polytron and total RNA was extracted following the protocol given by the supplier. Total RNA from hippocampi, brain stem and cortex were extracted with the RNeasy kit (QIAGEN AG). 20 Ag RNA was fractionated on a 1% denaturing agarose – formaldehyde gel and transferred onto a nylon membrane. The amount of RNA applied onto the electrophoresis gel was visualised by ethidium bromide stained 28s and 18s ribosomal RNA. Full length mouse TLR2 cDNA was used as a probe after radioactive-labelling with 32P dCTP (500,000 cpm/ml) and denaturation for 10 min at 95 -C. Pre-hybridization and hybridization were performed at 60 -C (1 h and overnight, respectively) with Dextran-sulphate 2.5%, SSC 2, SDS 0.1%, EDTA 2 mM, Na2H2P2O7 0.1%, Denhards 10x, Salmon sperm DNA 100 Ag/ml. After hybridization, the membranes were washed 3 times 30 min with SSC 2, SDS 0.1%, Na2H2P2O7 0.1%, EDTA 2 mM at 60 -C; SSC 1, SDS 0.1%, EDTA 2 mM at 45 -C and SSC 0.4, SDS 0.1%, EDTA 2 mM at 45 -C, respectively. The blots were exposed to a phosphor-imager (Molecular Dynamics). Signals were quantified with the NIH Image software. 2.3. Semi-quantitative RT-PCR 1 Ag of total RNA isolated from the different tissues was reverse-transcribed into cDNA using random primers (Promega, Catalys, San Luis Obispo, USA). Standard PCR was performed for 27 cycles or 24 cycles to cover the linear range of the amplification kinetics for TLR2 or h-actin, respectively. PCR for amplification of TLR2 was performed using primers amplifying part of their extracellular domain (anti-sense TLR2 primer 5V-AACATCCAACACCTCCAGC-3V; sense TLR2 primer 5VCTCGTTCTCCCAGCATTTA-3V). Amplification of the house-keeping gene h-actin, served as a reference for the semi-quantification using NIH image 1.62 program.

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2.4. cRNA probes and in situ hybridization Plasmids were linearized and the sense and antisense riboprobes synthesized as described (Laflamme et al., 2001). Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in MgCl2 6 mM, Tris (pH 7.9) 40 mM, spermidine 2 mM, NaCl 10 mM, dithiothreitol 10 mM, ATP/GTP/CTP 0.2 mM, 100 ACi of a-35S-UTP (Dupont NEN, #NEG 039H), 20 U RNasin (Promega, Catalys, San Luis Obispo, USA) and 10 U of either T7, SP6 or T3 RNA polymerase for 60 min at 37 -C. Unincorporated nucleotides were removed using ammonium-acetate precipitation method; 100 Al of DNase solution (1 Al DNase, 5 Al of 5 mg/ml tRNA, 94 Al of 10 mM Tris/10 mM MgCl2) was added, and 10 min later, a phenol-chloroform extraction was performed. The cRNA was precipitated with 80 Al of 5 M ammonium acetate and 500 Al of 100% ethanol for 20 min on dry ice. The pellet was dried and resuspended in 50 Al of 10 mM Tris/1 mM EDTA. A concentration of 107 cpm/probe was mixed into 1 ml of hybridization solution (500 Al formamide, 60 Al 5 M NaCl, 10 Al 1 M Tris [pH 8.0], 2 Al 0.5 M EDTA [pH 8.0], 50 Al 20 Denhart’s solution, 200 Al 50% dextran sulfate, 50 Al 10 mg/ml tRNA, 10 Al 1 M DTT, [118 Al DEPC water- volume of probe used]). This solution was mixed and heated for 10 min at 65 -C before being spotted on slides. Hybridization histochemical localization of TLR2, TNF, CD14, MD-2 and TLR6 mRNA was carried out on every twelfth sections of the whole rostro-caudal extent of each brain using 35S-labeled cRNA probes as described previously (Laflamme et al., 2001). The sections were exposed at 4 -C to X-ray films (Biomax, Kodak, Rochester, NY) for 1 –3 days. The slides were thereafter defatted in xylene, dipped in NTB-2 nuclear emulsion (Kodak; diluted 1 : 1 with distilled water), exposed for 15 days (TLR2, MD-2, TLR6 transcripts) or 10 days (TNF, CD14, IjB, IL-10 transcripts). The slides were then developed in D19 developer (Kodak) for 3.5 min at 14– 15 -C, washed 15 s in water, and fixed in rapid fixer (Kodak) for 5 min. Tissues were thereafter rinsed in running distilled water for 1 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with distrene plasticizer xylene (DPX) mounting medium (Electron Microscopy Science, Washington, PA). TNF signal intensity was assessed by measuring the areas in Am2 of the signals from different wt (86 cells, 7 different slides) or TLR2 / (117 cells, 9 different slides) cells with the Analysis Program using a Zeiss microscope under bright-field illumination. An average of cell areas was made from each slide. 2.5. Immunohistochemistry and dual-labeling procedure Immunohistochemistry was combined with in situ hybridization to determine the cell types, which express TLR2 and TNF transcripts in the mouse brain after S.

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pneumoniae infection. Anti-ionized calcium binding adapter molecule 1 (Iba1, provided by Dr. Y. Imai, Nationale Institute of Neuroscience, Kodaira, Japan, 473 ng/ml) for microglial cells, GFAP (Pharmingen, 500 ng/ml) for astrocytes, CD45 (Pharmingen, 180 ng/ml) for leukocytes, Gr1 (Pharmingen, 200ng/ml) for granulocytes, F4/80 (ATCC HB-198, 1 Ag/ml), for macrophages and NeuN (Serotec, 300 ng/ml) for neurons were used as markers for the specified cells. Every twelfth brain section was processed by using the avidin– biotin amplification bridge method with peroxidase as a substrate as described previously (Laflamme et al., 2001) and mounted on polyl-lysine coated slides (20 sections/brain/slide). 2.6. Western blot analysis and TNF ELISA Lysates were obtained by homogenizing tissues or LPS (Escherichia coli 0127: B8, Difco Laboratory, Detroit, MI) stimulated murine macrophage cell line RAW 264.7 with lysis buffer (n-Octyl-h-d-Glucopyranoside 60 mM, Tris pH 8.0 10 mM, NaCl 150 mM, Triton X-100 1%, PMSF 1 mM) and incubated on ice for 30 min. After centrifugation, the supernatant was carefully transferred into a new tube and protein concentration was measured by the Bradford method. 10 Ag of lysate was loaded onto a 10% SDSacrylamide gel. After transfer onto nitrocellulose, the membrane was blocked for 3 h at RT with non-fat milk 1%, PBS 1 and Tween-20 0.1%. Rabbit anti-mouse TLR2 antibody directed against the intracellular domain (149902092, eBioscience, San Diego, USA), was applied overnight in milk 1%, Tween-20 0.1%, PBS 1 at 4 -C. After 3 washes, the membrane was incubated with the secondary antibody labelled with horseradish peroxidase for 2 h at RT. Detection of the antibody was done by enhanced chemiluminescence. For TNF protein measurement, brains were removed and homogenized with a Polytron homogenizer in 1 ml PBS containing the protease inhibitor PMSF 1 mM and incubated on ice for 30 min. After centrifugation, TNF was measured in the supernatant by ELISA (R and D Systems Europe, Abingdon, UK), according to the instructions of the manufacturers.

mM Hepes and 0.1% Saponin. Cells were washed in FACS buffer (PBS with 2% FBS and 10 mM sodium azide), incubated 15 min at room temperature (RT) with 10 Ag/ml Fc block (rat anti-mouse CD16/CD32, BD) to block unspecific binding, then for 30 min on ice with 10 Ag/ml of phycoerythrin (PE)-conjugated rat anti-mouse TLR2 (clone 6C2, rat IgG2b, eBioscience, San Diego, CA). Fluorescence was analyzed in a FACScan with the Cellquest software. 2.8. Statistical analysis Areas of TNF mRNA signal intensity and levels of TNF protein in brain homogenates from wt and TLR2 / cells were compared with the non-parametric analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

3. Results 3.1. TLR2 mRNA and protein expression To understand the regulatory function of TLR2 in meningitis, we first studied TLR2 mRNA expression in whole brain. In northern blots, TLR2 mRNA was detected 24 h after infection and the signal remained for at least 48 h (Fig. 1A). The more sensitive RT-PCR, performed on the same RNA, showed a low basal expression of TLR2 mRNA in NaCl-injected whole brains (Fig. 1B), and TLR2 signals were clearly enhanced 24 and 48 h after S. pneumoniae infection (Fig. 1A, B). TLR2 mRNA induction was not

2.7. Flow cytometry of CSF leukocytes CSF cells from S. pneumoniae infected mice were harvested and collected directly in RPMI 5% fetal bovine serum (FBS) containing Brefeldin A (Sigma) at 10 Ag/ml. Cells were washed and fixed with ice cold 4% paraformaldehyde (Sigma) for 5 min. The fixative was removed and cells were washed in wash solution composed by Hanks’ balanced salt solution (HBSS, Life Technologies GIBCO), 10 mM Hepes (Calbiochem) and 0.1% Saponin (Sigma). After washing, cells were incubated overnight at 4 -C with rat anti-mouse TNF (BD Pharmingen 554641) or rat IgG1 mAbs at 40 Ag/ml in wash solution containing HBSS, 10

Fig. 1. Northern blot analysis (A, C) and semi-quantitative RT-PCR (B, D) of TLR2 mRNA in whole brain 24 h after subarachnoidal injection of NaCl (<)- or 6, 24 or 48 h after-infection with S. pneumoniae (A, B) or L. monocytogenes (C, D). EtBr stain of 28s and 18s RNA (Northern) or hactin (RT-PCR) signal are used as loading controls. E) Western blotting of lysates from whole brain following subarachnoidal NaCl (<)-injection or 3 to 72 h after infection with S. pneumoniae. An anti-mouse TLR2 antibody recognizing an epitope in the intracellular part of TLR2 was used. The arrow indicates TLR2 proteins at MW of 92 kDa. LPS-stimulated RAW cells were used as positive control.

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modulated by the infection dose used, since different S. pneumoniae inocula yielded similar results (data not shown). Semi-quantitative RT-PCR and Northern blot analysis of TLR2 was also performed on hippocampus, cortex, brain stem and cerebellum, obtained from brains of NaCl- or S. pneumoniae-injected animals. A weak constitutive TLR2 mRNA expression was detected in all tested brain areas. Furthermore TLR2 mRNA could already be detected 6 h post-infection in hippocampus and cortex and was found after 24 and 48 h in all tested regions (data not shown) A similar TLR2 mRNA induction was also found in mouse brains 24 and 48 h after infection with Listeria monocytogenes (Fig. 1C, D). In summary, these results show a TLR2 induction which was not restricted to one specific brain region or Gram-positive pathogen. As we found TLR2 mRNA increased in the mouse whole brain during bacterial meningitis, we tested whether TLR2 protein was also upregulated. With an antibody against its intracellular domain, TLR2 protein could be detected already 24 to 72 h after infection with S. pneumoniae with a strong signal at 48 and 72 h but not in NaCl-injected brains (Fig. 1E). Similar results were obtained after L. monocytogenes infection (data not shown). The LPS-stimulated murine macrophage cell line RAW 264.7 served as a positive control (Fig. 1E). The TLR2 upregulation and localization in brain was further documented by in situ hybridization. In wt NaClinjected mice no or a very faint signal was found (Fig. 2A). A weak expression of TLR2 mRNA appeared in the corpus callosum in the brain of six hours after infection (data not shown). It increased 12 h after infection (Fig. 2B) and was maximal after 24 h (Fig. 2C). At this time point, the message was particularly strong in the entire corpus callosum (Fig. 2E), in the median eminence (Fig. 2C), around the ependymal cells (Fig. 2F) and in the meninges (Fig. 2G). Numerous TLR2-expressing cells were detected in a scattered manner across the whole brain parenchyma (Fig. 2H). This distribution explains that TLR2 upregulation

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was found in all brain regions by northern blot analysis. At 48 h after infection, TLR2 signals were still detected but to a lesser extent (Fig. 2D). In summary TLR2 mRNA was induced in selective areas, where infiltrating cells are found, and scattered in cortex. 3.2. CD14, MD-2 and TLR6 mRNA expression TLR2 associates with CD14 and with TLR1 or TLR6 for signaling, and CD14 functions as receptor for the same microbial ligands as TLR2 (Muta and Takeshige, 2001; Schroder et al., 2000; Schwandner et al., 1999). Therefore, we investigated whether mRNA regulation of these comolecules during S. pneumoniae meningitis was similar to the one of TLR2 (Bulut et al., 2001; Hajjar et al., 2001). Finally, we investigated MD-2 as it was shown that TLR2 cotransfected with MD2 enhanced TLR2 expression (Dziarski et al., 2001) and as MD-2 was found to physically associate with TLR2 (Matsumura et al., 2000). Twenty four hours after infection CD14 (Fig. 3C, D), and MD-2 (Fig. 3G, H) mRNA were strongly upregulated in brains from wt and TLR2 / infected mice. All signals were stronger in infected TLR2 / than wt brains. The TLR6 signal was weak in wt and strong in TLR2 / mice (Fig. 3E, F). None of the control mice injected with NaCl showed a signal for any of the transcripts tested (Fig. 3A, B data for CD14). Since the whole sequence of TLR6 was used for the in situ hybridization cRNA probe and as there is a high homology between TLR1 and TLR6, the obtained signal indicates the presence of either TLR1 or TLR6. The localization of the co-molecule transcripts was identical to the one of the TLR2 transcript; all the signals were found in the corpus callosum, around the ependymal cells and in the meninges (data not shown). As for TLR2-expressing cells, transcripts for CD14 were detected in a scattered manner across the whole brain parenchyma and the signal was weaker after 48 than after 24 h (data not shown). In summary, mRNA of TLR2 co-

Fig. 2. Coronal sections showing the hybridization signal for TLR2 mRNA in brains from wt and TLR2 / animals 24 h after NaCl-injection (A) or 12 (B), 24 (C) and 48 h (D) after infection with S. pneumoniae. The darkfield photomicrographs of nuclear emulsion-dipped sections made 24 h after infection depict a localized positive signal in the corpus callosum marked with dotted square in C (E), in the parenchyma marked with a solid line square in C (F), in the meninges (G) and in the parenchyma (H). In situ hybridization was performed with radioactive cRNA mouse TLR2 probe. One representative result out of three similar experiments is shown. Magnifications are 25 (E); 400 (F, H) and 200 (G).

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also augmented. Indeed TNF concentration was significantly higher in brain lysates of TLR2 / than wt mice ( p < 0.001, Fig. 4G). 3.4. Cellular distribution of TNF and TLR2 transcripts To investigate whether infiltrating leukocytes were the cellular source of TNF and TLR2 transcripts, we analyzed both signals in infected mice rendered leukopenic by

Fig. 3. Coronal sections showing the hybridization signal for CD14 (C, D), TLR1/6 (E, F) and MD-2 (G, H) mRNA in brains from wt and TLR2 / animals after 24 h NaCl-injection (A, B) or S. pneumoniae infection (C – H). Representative pictures of brains hybridized with each of the three transcripts are shown for NaCl-injected mice (A, B). In situ hybridization was performed with radioactive mouse CD14, mouse TLR6 or mouse MD-2 probes. One representative result out of three similar experiments is shown.

molecules was induced during pneumococcal meningitis at the same localization as TLR2 itself, with a stronger signal in TLR2 / than in wt mice. 3.3. TNF mRNA and protein expression In order to understand the contribution of TLR2 to TNF regulation, we studied TNF mRNA expression during meningitis in wt and TLR2 / mice by in situ hybridization. As shown in Fig. 4, TNF mRNA was not detectable in NaCl-injected brains (Fig. 4A, B), but a clear signal could be detected 24 h after S. pneumoniae infection in both mouse strains (Fig. 4C, D). The signal was stronger in infected TLR2 / (Fig. 4D) than in wt mice (Fig. 4C). Surprisingly, it was found in the entire corpus callosum, around ependymal cells, and in the meninges (data not shown). In the parenchyma, less cells were TNF-positive (Fig. 4E, F) compared to the TLR2positive cells (Fig. 2C, H). In view of the increased TNF mRNA in TLR2 / compared to wt brains, we tested whether TNF protein was

Fig. 4. Coronal sections showing the hybridization signal for TNF mRNA in brains from wt (A, C, E) and TLR2 / (B, D, F) mice 24 h after NaClinjection (A, B) or S. pneumoniae infection (C – F). The darkfield photomicrographs of nuclear emulsion-dipped sections depict a localized positive signal in the parenchyma marked with squares in C and D (E, F). In situ hybridization was performed with radioactive cRNA mouse TNF probe. One representative result out of three similar experiments is shown. Magnification is 400 (E, F). TNF protein levels in brain homogenates (G) from wt (n = 6) and TLR2 / mice (n = 6) 24 h after S. pneumoniae infection. Mean values T SD (*p < 0.001, ANOVA).

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cyclophosphamide. As shown in Fig. 5, a TNF signal could not be detected in wt (Fig. 5D) or TLR2 / (Fig. 5E) infected leukopenic mice. Similarly, no TLR2 upregulation could be detected in wt infected leukopenic animals (Fig. 5F). These results indicate that TLR2 and TNF were originating in infiltrating cells. To further relate TNF expression to TLR2, we investigated the cellular distribution of TLR2 and TNF mRNA in brains 24 h after infection by immunohistochemistry combined with in situ hybridization. Table 1 summarizes the percentage of transcript-positive cells, which were also expressing cell-specific markers and Fig. 6 illustrates these results. In both mouse strains, TNF mRNA was expressed by the same cell populations; transcripts mostly colocalized (86%) with CD45-positive leukocytes, however interestingly, only a small fraction of all CD45-positive cells (< 1%) expressed TNF (Table 1, Fig. 6A, B); only 15 –22% of TNF mRNA colocalized with F4/80-positive macrophages (Table 1, Fig. 6D, E) and 13– 18% with Gr1-positive granulocytes (Table 1, Fig. 6G, H). This indicates that not all TNFpositive CD45-positive leukocytes could be classified as monocytes or granulocytes with these markers. It is unlikely that other leukocytes such as lymphocytes expressed TNF mRNA, because differential analysis of CSF cells and HE stains of infected brains did not show lymphocyte infiltration (Cauwels et al., 1999). We excluded that the markers F4/80 and Gr1 were lost during the in situ hybridization procedure following immunohistochemistry, since double immunofluorescent stains of infected brain with Gr1/CD45 and F4/80/CD45 similarly showed that among all CD45positive cells only a minority carried the subpopulation markers (30% were Gr1-positive and 5% were F4/80positive), while 90% of Gr1- and F4/80-positive cells were also CD45-positive (data not shown). TNF mRNA colocalized very rarely with Iba1-positive microglia (< 5%, Fig. 6J, K) or GFAP-positive astrocytes (< 5%, Fig. 6M, N) and was never found in neurons (NeuN staining, Table 1). The

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Table 1 Percentage of transcript (TNF or TLR2)-positive cells, which co-express the specified cellular marker (CD45, F4/80, Gr1, Iba1, GFAP, NeuN) in S. pneumoniae infected wt and TLR2 / animals TNF mRNA

CD45 F4/80 Gr1 Iba1 GFAP NeuN

TLR2 mRNA /

wt

TLR2

88.0 T 3.5 15.0 T 7.0 13.2 T 6.2 3.0 T 1.7 4.2 T 1.0 –

84.3 T 4.0 22.0 T 13.9 18.0 T 2.6 5.5 T 6.4 1.0 T 0.5 –

wt 95.0 T 0.5 43.3 T 12.2 12.3 T 6.4 5.0 T 2.0 24.0 T 4.2 3.0 T 0.5

mRNA signals were assessed in 100 cells and the colocalization with the different phenotypic markers was expressed as % using a Zeiss microscope under bright-field illumination as described in Materials and methods. Values are mean and SD from 20 sections/slide and from slides of 2 to 3 mice. The percentage of TNF mRNA-positive cells, which co-express the different cellular markers, was similar in TLR2 / and wt mice ( p > 0.05).

absence of TNF from microglia was confirmed by Iba1/TNF mRNA double staining in cyclophosphamide-treated mice. Strongly Iba1-positive/TNF-negative cells with typical microglial morphology remained unaltered, whereas TNFpositive cells, which weakly stained Iba1-positive monocytes, disappeared after leukocyte depletion (data not shown). The number of TNF-positive cells was higher in the corpus callosum and the average signal intensity of TNF mRNA-positive cells was stronger in brains from TLR2 / than from wt mice (Fig. 7). Similar to TNF, more than 90% of TLR2 transcripts were confined to CD45-positive leukocytes (Table 1, Fig. 6C). Half of the TLR2 transcripts colocalized with F4/80-positive macrophages (Table 1, Fig. 6F) and 15% with Gr1-positive granulocytes (Table 1, Fig. 6I). 24% coexpressed the astrocyte marker GFAP (Table 1, Fig. 6O); a small fraction was labelling Iba1-positive microglia (Table 1, Fig. 6L) and NeuN-positive neurons. In the parenchyma TLR2 mRNApositive cells were more abundant compared to TNF mRNA-

Fig. 5. Coronal sections of wt and TLR2 / brains showing the hybridization signal for TNF mRNA (A, B, D, E) or TLR2 (C, F) transcripts in immunocompetent (A – C) and leukocyte-depleted (D – F) animals 24 h after S. pneumoniae infection. In situ hybridization was performed with radioactive mouse TNF or TLR2 probes. One representative result out of three similar experiments is shown.

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Fig. 6. Immunohistochemistry for different cellular markers was performed combined with in situ hybridization with radioactive cRNA for TNF (A, D, G, J, M for wt animals; B, E, H, K, N for TLR2 / animals) or TLR2 (C, F, I, L, O) probes. Co-localization of TNF or TLR2 with leukocytes (CD45), macrophages (F4/80), granulocytes (Gr1), microglial cells (Iba1) and astrocytes (GFAP) are shown.

positive cells (Fig. 2 compared to Fig. 4). In summary, dual stainings confirmed the results of the cyclophosphamide experiment, which showed that TNF and TLR2 transcripts were originating in infiltrating leukocytes. 3.5. Colocalization of TNF and TLR2 proteins in infiltrating cells of CSF The immunohistochemical demonstration of TLR2 and TNF transcripts did not prove the TLR2 and TNF colocalization within the same cell. Therefore we performed

double immunostaining of intracellular TNF and membrane TLR2 in CSF cells from mice 24 h after infection. Infiltrating cells were mainly PMN (90%), as identified by cytospin staining and flow cytometry using the granulocyte specific marker anti-Gr1 (data not shown). In wt mice, all infiltrating PMN showed intracellular TNF staining and expression of TLR2 within the same cell (Fig. 8C). In TLR2 / mice, all PMN contained also intracellular TNF with similar fluorescence intensity to wt cells (Fig. 8D). This result indicates that TLR2 and TNF colocalized within the same cell in CSF leukocytes, yet that intracellular

M. Letiembre et al. / Journal of Neuroimmunology 168 (2005) 21 – 33

Fig. 7. Semi-quantitative analysis of TNF signal areas (Am2) in wt and TLR2 / brains 24 h after S. pneumoniae infection. From each slide (n = 7 in wt and n = 9 in TLR2 / brains), 50 TNF dot areas were measured with the Analysis Program using the Zeiss microscope, the average from each slide was made and drawn. Mean values T SD ( p < 0.001, ANOVA).

TNF protein did not accumulate more in TLR2 wt cells.

/

than in

3.6. IL-10 and IjB mRNA expression The anti-inflammatory cytokine IL-10 was found induced via TLR2 with pathogenic antigens such as Yersinia V antigen (Sing et al., 2002b). More recently,

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IL-10 has been reported to be upregulated in brain tissue during intranasal murine pneumococcal meningitis and IL10 deficiency was associated with increased brain cytokine concentrations (Zwijnenburg et al., 2003). Therefore we investigated, whether the lack of TLR2 and increased TNF mRNA were accompanied by reduced IL-10. By RT-PCR, IL-10 mRNA was weakly upregulated after 24 h of infection in wt mice but not in infected TLR2 / mice, in which we detected a slight IL-10 upregulation only after 48 h (data not shown). By in situ hybridization, no IL-10 mRNA was detected in NaCl-injected mouse brains (data not shown), but a signal appeared 24 h after S. pneumoniae infection in wt (Fig. 9A) as well as in TLR2 / (Fig. 9B) brains in the same localization as TLR2 and TNF; it was weaker in the majority of the TLR2 / brains. The signal was maintained after 48 h in wt mice (Fig. 9C) while it was weakly detectable in TLR2 / mice (Fig. 9D). IL-10 protein could not be detected 24 h after infection in CSF of both wt and TLR2 / mice (data not shown). Therefore reduced IL-10 was possibly contributing to the increased TNF in TLR2 / mice. To investigate the pathway of TLR2-regulated TNF activation, we studied induction of a NF-nB responsive gene. The activation of NF-nB causes the upregulation of IjB transcription, which serves to regulate the NF-kB signal (Hoffmann et al., 2002; Iwai et al., 2005). Therefore we choose to analyse InB transcription. InB which was not

Fig. 8. Double staining of intracellular TNF and membrane TLR2 (C, D) or staining with isotype control (A, B) antibodies in CSF PMN of wt (left) and TLR2 / (right) mice 24 h after infection with S. pneumoniae. One out of 3 similar experiments is shown.

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M. Letiembre et al. / Journal of Neuroimmunology 168 (2005) 21 – 33

Fig. 9. Coronal sections showing the hybridization signal for IL-10 (A – D) or InB (E – F) mRNA in brains from wt (A, C, E, G) and TLR2 / (B, D, F, H) animals after 24 h NaCl-injection (E, F) or S. pneumoniae infection (A – D, G, H). The signal was localized in the corpus callosum and in the parenchyma. In situ hybridization was performed with radioactive cRNA mouse IL-10 or InB probes. One representative result out of three similar experiments is shown.

expressed in non-infected brains; it was induced more strongly in TLR2 / than in wt mice (Fig. 9). It was found at the same localization as TLR2 in corpus callosum, the median eminence, in the meninges and scattered in the cortex. In addition a signal was detected in the hippocampus. Thus InB was an indicator of stronger NF-nB dependent gene activation in TLR2-deficient brains.

4. Discussion The present study was prompted by the fact that TLR2 / mice showed higher TNF levels and earlier mortality during S. pneumoniae meningitis than wt mice (Echchannaoui et al., 2002). Furthermore, there are no in vivo studies on the mechanism of interaction between TLR2 and TNF gene regulation in brain during bacterial meningitis. Our findings show first, the temporal and

spatial distribution of TLR2 mRNA during meningitis, second, the induction of TNF gene expression in the same infiltrating cells as TLR2 and third, the dysregulation of TNF mRNA in the absence of TLR2 with a weakened IL-10 and an increase in NF-nB-dependent InB gene activation. The first finding is that the TLR2 gene, which showed a weak constitutive expression, was upregulated in whole brain and in different brain regions after pneumococcal or Listeria infection as assessed by Northern blot or RT-PCR. We demonstrated by leukocyte depletion experiments with cyclophosphamide, that the gene was activated in infiltrating leukocytes. Immunohistochemistry combined with in situ hybridization permitted the exact localization of this upregulation in CD45-positive infiltrating leukocytes; half of TLR2-expressing cells could be classified as F4/80positive macrophages and 12% as Gr1-positive granulocytes. The TLR2-positive cells were in the ventricles, in the meninges and sparsely scattered in the parenchyma surrounding the ventricles. We could exclude microglial cells and neurons as sources of TLR2 transcripts. We found, however TLR2 mRNA in a small fraction of GFAP-positive astrocytes. Since this signal was abolished in cyclophosphamide-pretreated mice, either GFAP labelled a population of immigrating cells or cylophosphamide prevented the infection-induced TLR2 expression in these cells. In a previous report (Laflamme et al., 2001) Gram-positive cell wall components such as PGN and LTA failed to modulate the TLR2 transcript, while here and in a report from Koedel (Koedel et al., 2003), live Grampositive bacteria enhanced TLR2 message. This indicates that live bacteria contain other TLR2-stimulatory molecules than LTA. Our results added live bacteria to the broad range of in vitro stimuli including LPS, PGN, TNF, IL1-a, IL-6 or GM-CSF, which were found in previous studies to enhance TLR2 gene expression in different cells including macrophages. This effect required NFnB activation and was transcriptionally regulated (Liu et al., 2001; Matsuguchi et al., 2000; Musikacharoen et al., 2001). Since in our model, TLR2 mRNA upregulation occurred early with a maximum after 24 h and simultaneously to that of TNF mRNA, it is unlikely that TNF contributed to TLR2 induction. The analysis of the TLR2 protein was more difficult, likely because of the lack of suitable reagents and its very low expression. Although, induction of the TLR2 protein was documented in whole brain by Western blot following infection with S. pneumoniae, the exact cellular distribution of the translated protein could not be determined by immunohistochemistry. A strong transcriptional activation of the comolecules CD14, TLR1/6 and MD2 was found in the same localization as TLR2. While the induction of TLR1/6 and MD2 was weak and limited to the ventricles, CD14 mRNA was also found spread in cells around the ventricles and the in situ hybridization picture was superposable to the one of TLR2.

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Interestingly the induction of comolecules was stronger in TLR2 / mice; it is not known how and whether these molecules participate in the S. pneumoniae recognition, but this observation suggests that they were affected by the absence of TLR2. The second finding is that TNF mRNA was essentially induced in the same area as TLR2, mainly in the corpus callosum around the ventricles, along the ependyma and in the meninges, in infiltrating cells as demonstrated with the leukocyte depletion experiment. Nearly all TNF-positive cells carried the CD45 antigen, but only 30% to 40% were classifiable as macrophages or granulocytes. Since we did not find lymphocytes in brain and an artefactual phenotype loss due to the in situ hybridization procedure could be excluded, we postulate that the brain-infiltrating phagocytes had lost these antigens in vivo with the adherence in the subventricular space. This particular localization of the TNF signal was not found, when intraperitoneal administration of LPS was performed, suggesting that cells responsible for the inflammatory response are different in these two models (Laflamme et al., 2001). In contrast, such a special localization had already been detected for MIP-1a and MIP-2 expression in the brain following infection with Listeria monocytogenes (Seebach et al., 1995). In this report, the authors showed that PMN and macrophages were the source of MIP-1a and MIP-2 transcripts, since they co-localized with the chemokine mRNA. Neonatal rat meningitis model, intracisternal injection of group B streptococcus led to an early induction of TNF mRNA along the ventricles and the ependyma (Kim et al., 2004); in that study the signal was not attributed to a given cell population. The third finding is that in the absence of TLR2, infection led to an enhanced TNF response. It supports a previous description, where in a candidiasis model TLR2 mainly induced anti-inflammatory signals (Netea et al., 2004). In situ hybridization showed that TNF gene expression was stronger in brains from TLR2 / than from wt mice after infection. Similar TNF mRNA accumulation in brains of infected wt and TLR2 / mice was found in an intracisternal pneumococcal meningitis when mRNA was analyzed by quantitative RT-PCR (Koedel et al., 2003). Importantly, we showed that increased TNF transcription lead also to increased TNF protein in brain lysates of TLR2 / mice, which extended our previous finding of higher TNF levels in CSF of these mice (Echchannaoui et al., 2002). Since we showed co-expression of TNF and TLR2 in the same cells, augmented TNF in TLR2-deficiency probably reflects altered signaling for TNF. Our data suggest the hypothesis that TLR2, when activated by infection with bacteria may dampen rather than increase the TNF response. They indicate also that live S. pneumoniae did not require TLR2 for signaling, that it must exist another receptor, which permits the recognition of bacteria and signals the inflammatory response. This receptor can be another already

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described TLR or an unknown receptor, which appears modulated by TLR2, since its activity was enhanced in TLR2 / mice. The enhanced signalling appeared NF-nBdependant, since InB, which is an NF-nB dependent gene, was more strongly induced in TLR2 / brains. Further studies will identify upstream molecules, which were leading to increased NF-nB activation in TLR2 / mice. Possible candidates are IRAK-M, SOCS, ST2 or SIGIRR, which are not only induced by TLR2, but also by other TLRs or IL-1, and which control TLR2 signalling (Kobayashi et al., 2002; Trajkovic et al., 2004; Wald et al., 2003; Yoshimura et al., 2003). Since it is known that IL-10 can also be induced by TLR2 activation (Sing et al., 2002b) and suppresses TNF transcription (Donnelly et al., 1999), a delay of IL-10 induction in TLR2 / as compared to wt mice could explain an excess TNF in these animals. In our study, IL-10 mRNA was weakly upregulated, and this effect was less in TLR2 / than wt mice, therefore it can be concluded that weak IL-10 may have contributed to enhanced change of TNF in TLR2 / mice. In addition, the TLR2-dependency of IL-10 production in infection has been shown in a C. albicans infection model (Netea et al., 2004). Furthermore, differences in infiltrating leukocyte numbers or viability, and post-transcriptional changes in TNF production could explain the enhanced TNF in TLR2 / mice. In our earlier studies, infiltrating leukocytes number in the CSF were similar 24 h after infection in wt and TLR2 / strains (Echchannaoui et al., 2002), excluding increased leukocyte numbers at the origin of enhanced TNF gene expression in TLR2 / mice. Additionally all cells were viable, thus excluding that TNF in TLR2 / mice resulted from a lack of TLR2-mediated apoptosis (Aliprantis et al., 1999). We demonstrated here by in situ hybridization that the increased TNF mRNA was due to an excess of gene activation in each TLR2 / infected CD45-positive leukocyte in the tissue. FACS analysis of intracellular TNF in CSF cells did not reveal an increased intracellular fluorescence in TLR2 / as compared to wt leukocytes. This indicates that the increased TNF protein measured in TLR2 / CSF was due to increased transcription without accumulation of intracellular TNF. In conclusion, we show expression and upregulation of TLR2 in mouse brain during experimental Gram-positive meningitis and document that TLR2 regulates TNF mRNA via NF-nB in S. pneumoniae-infected brain. We also demonstrate that excessive TNF in TLR2 / mice was derived from infiltrating cells. This indicates that TLR2 contributes to host defence in Gram-positive meningitis.

Acknowledgements We thank Zarko Rajacic for his technical help and Dr. Gennaro De Libero for stimulating discussions of the manuscript.

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