Toll-like receptor 9 (TLR-9) recognizes unmethylated CpG dinucleotides which are abundant in prokaryotic DNA and yet are rare in eukaryotic DNA. Little is known about the significance of TLR-9 in terms of recognition of different bacterial DNA species. In this study HEK293 cells stably transfected with human TLR-9 were used to analyze the immunostimulatory properties of 15 bacterial DNA preparations. In addition, bacterial genome data were analyzed for the frequency of unmethylated cytosine-guanosine ([CG]) dinucleotides. We observed that DNA samples of different bacteria showed considerable differences in their potential to stimulate TLR-9. This correlated with the frequency of [CG] dinucleotides. Based upon data from our experiments the estimate of immunostimulatory bacterial DNA concentrations translated to as high as 10 9 bacteria/ml. Application of the transfection reagent DOTAP resulted in a more efficient delivery of DNA into the cell, and this went along with increased TLR-9 activation. The data indicate that bacterial DNA preparations from different species differ in their capacity to activate TLR-9, which is dependent on the individual [CG] content. Moreover, increased intracellular delivery results in a marked enhancement of immunostimulation.Innate immunity recognizes conserved microbial structures that are referred to as pathogen-associated molecular patterns (15). This is achieved by use of pattern recognition receptors, of which Toll-like receptors have been identified to play a crucial role. Prototypical substances constituting microbial patterns are components of the prokaryotic cell wall, e.g., lipopolysaccharide or lipoteichoic acid. However, it has been recognized early that also DNA possesses immunostimulatory potential when Tokunaga et al. identified DNA-containing fractions of mycobacteria to mediate immune modulation (23,25). Later it was shown that this is due to the relative abundance of unmethylated cytosine-guanosine ([CG]) dinucleotides (11). Indeed, vertebrate DNA shows a suppression of [CG] dinucleotide frequency and, moreover, is characterized by an increased rate of C-5 methylation of cytosine residues (24). TLR-9 has been identified as the DNA-recognizing receptor (7).It was noted early that stimulation by bacterial DNA could be mimicked by the use of [CG]-containing synthetic oligodesoxynucleotides (CpG-ODNs) (11). Since CpG-ODNs turned out to possess a considerable potential for immune modulation and since these compounds were easy to synthesize, subsequent work in the TLR-9 field has mainly focused on the use synthetic ODNs. It is intriguing that, despite the fact that basic principles in bacterial DNA/TLR-9 recognition have been known for years, information concerning the meaning of TLR-9 for infections or for the recognition of bacteria is still extremely limited.Differences in the C/G composition (implying differences in [CG] content) are typical for different genera of bacteria. Since TLR-9 recognizes [CG]-containing DNA motifs, it can be speculated that DNA from differen...

Filarial parasites have to trespass many barriers to successfully settle within their mammalian host, which is equipped with mechanical borders and complex weaponry of an evolved immune system. However, little is known about mechanisms of early local events in filarial infections. In this study, bone marrow-derived dendritic cells not only upregulated activation markers CD40 and CD80 upon in vitro stimulation with filarial extracts, but also secreted CCL17, a chemokine known to be produced upon microbial challenge. Mice deficient for CCL17 had an up to 4-fold higher worm burden compared with controls by day 10 of infection with the murine filaria Litomosoides sigmodontis. Also, numbers of mast cells (MCs) invading the skin and degranulation were significantly increased, which was associated with enhanced vascular permeability and larval establishment. This phenotype was reverted by inhibition of MC degranulation with disodium cromoglycate or by blockade of histamine. In addition, we showed that CCL17-mediated vascular permeability was dependent on the presence of Wolbachia endosymbionts and TLR2. Our findings reveal that CCL17 controls filarial larval entry by limiting MC-dependent vascular permeability.