Effects of Toll-like receptor 2 agonist Pam 3 CysSK 4 on inflammation and brain damage in experimental pneumococcal meningitis Johann Sellner a , Denis Grandgirard b , Christian Gianinazzi b , Regine M. Landmann c , Stephen L. Leib b, ⁎ a Department of Neurology, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str.22, D-81675 München, Germany b Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland c Division of Infectious Diseases, Department of Research, University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland abstract article info Article history: Received 11 February 2008 Received in revised form 14 October 2008 Accepted 14 October 2008 Available online xxxx Keywords: Pneumococcal meningitis Pam 3 CysSK 4 Toll-like receptor2 Inflammation Apoptosis Hippocampal injury TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam 3 CysSK 4 was applied intracisternally (0.5 μg in 10 μl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam 3 CysSK 4 on apoptosis was investigated by intracisternal application of 0.5 μg in 10 μl saline and 40 μg in 20 μl saline. Pam 3 CysSK 4 neither induced apoptosis in sham- operated mice nor aggravated apoptosis in acute infection. However, Pam 3 CysSK 4 induced pleocytosis, TNF-α and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam 3 CysSK 4 at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Bacterial invasion of the meninges induces a rapid inflammatory response, which is mediated by the innate immune system. Recogni- tion relies, in the case of Gram-positive pathogens, on the binding of bacterial lipoproteins to TLR2. Activation of TLR2 was shown to be critical for sensing and activating the initial immune response with regulation of bacterial burden and inflammatory reaction in pneumo- coccal infections e.g. pneumococcal meningitis (Koedel et al., 2003). Apoptotic neuronal death within the subgranular zone of the hippocampus is a characteristic feature of pneumococcal meningitis in humans and corresponding animal models, and is considered as a putative morphological correlate of neuropsychological sequelae in the survivors. Components of the host inflammatory response including excitatory amino acids, cytokines and metalloproteinases, but also pneumococcal products, e.g. pneumolysin and hydrogen peroxide, may contribute to the occurrence of this entity of hippocampal damage (Klein et al., 2006). An involvement of TLR2 in the pathogenesis of hippocampal injury was previously suggested on the basis of the observation that apoptosis was found after intrathecal administration of the TLR2 agonist Pam 3 CysSK 4 and of pneumococci in C57BL/6 but not in TLR2 -/- mice (Hoffmann et al., 2007). The synthetic lipopeptide Pam 3 CysSK 4 (N-palmitoyl-2-(2,3-bis (palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine) has been characterized as a specific TLR2 receptor agonist acting through TLR2/1 heterodimeric receptors (Jin et al., 2007). Here, we evaluated the effect of Pam 3 CysSK 4 on mediators of a neuro-inflammatory host response and on the development and extent of hippocampal apoptosis in experimental pneumococcal meningitis in infant rats. 2. Materials and methods 2.1. Infecting organism A clinical isolate of Streptococcus pneumoniae (serogroup 3) was grown on blood agar plates, cultured overnight in 10 ml of brain heart infusion medium, diluted in fresh medium, and grown for 6 h to logarithmic phase. The suspension was centrifuged for 10 min (5000 × g), and re-suspended in sterile saline to the desired density. The accuracy of the inoculum size was confirmed by quantitative cultures. 2.2. Model of meningitis and Pam 3 CysSK 4 treatment The animal studies were approved by the Animal Care and Experimentation Committee of the Canton of Bern, Switzerland, and NIH guidelines were followed for the performance of animal experi- ments. Nursing Wistar rats (Charles River, Germany) were infected (n = 41) on postnatal day 11 by direct intracisternal injection with 10 μl of saline containing log 10 7.13±0.38 colony forming units (CFU)/ml of S. pneumoniae as described previously (Sellner and Leib, 2006). Saline was given to sham-infected controls (n = 29). Animals were randomized Journal of Neuroimmunology xxx (2008) xxx–xxx ⁎ Corresponding author. Institute of Infectious Diseases, University of Bern, Friedbühlstrasse 51, 3010 Bern, Switzerland. Tel.: +41 31 632 4949; fax: +41 31 632 3550. E-mail address: stephen.leib@ifik.unibe.ch (S.L. Leib). JNI-474722; No of Pages 4 0165-5728/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.10.004 Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim ARTICLE IN PRESS Please cite this article as: Sellner, J., et al., Effects of Toll-like receptor 2 agonist Pam 3 CysSK 4 on inflammation and brain damage in experimental pneumococcal meningitis, J. Neuroimmunol. (2008), doi:10.1016/j.jneuroim.2008.10.004