Cutting Edge: Natural DNA Repetitive Extragenic Sequences from Gram-Negative Pathogens Strongly Stimulate TLR9

Abstract

Clinical and epidemiologic studies have suggested a connection between infectious agents and autoimmune diseases (1-3). The innate immune response to microbial DNA could be one of the connections between infectious agents and diseases such as chronic inflammatory intestinal diseases (4) or systemic lupus erythematosus (5-7). Furthermore, bacterial DNA could be playing a key role in severe conditions like Gram-negative septic shock (8), in which a dysregulated innate immune response mediated by TLRs has been confirmed (9 -11). Immunostimulatory effects of bacterial DNA on mammalian cells are exerted via the intracellular TLR9 (12). TLR9 distinguishes bacterial DNA from self-DNA detecting special patterns in DNA fragments (12). Studies with oligodeoxynucleotides (ODNs) have allowed the determination of features favoring the immunostimulatory ability of DNA sequences (13): DNA fragments with lengths of ϳ25 nt; the presence of unmethylated CpG motifs following the pattern "not C, C, G, not G" separated by AT-rich stretches of ϳ2-4 nt; and palindromicity.These features have been determined analyzing the immunostimulatory potential of synthetic ODNs, but which kinds of natural bacterial sequences have immunostimulatory potential is unknown. This could be critical to the understanding of how bacteria activate immune responses.In this study, we searched for bacterial DNA sequence candidates as natural TLR9 ligands. Our results prove that bacterial repetitive extragenic palindromic (REPs) sequences are natural TLR9 ligands that allow the innate immune system to distinguish bacterial DNA. Materials and Methods Bioinformatics analysisTo detect REP sequences we used a BLAST-based strategy specially designed to detect repetitive extragenic and palindromic sequences. We developed Cϩϩ programs to assist in the analysis of REPs in the entire genomes. The genomes analyzed were: Escherichia coli K12 (GenBank U00096.2; RefSeq NC_000913.2), Salmonella enterica subsp. enterica serovar typhi (GenBank AL513382.1; RefSeq NC_003198.1), Pseudomonas aeruginosa (PAO1 GenBank AE004091.2; RefSeq NC_002516.2), and Neisseria meningitidis serogroup B strain MC58 (GenBankAE002098.2; RefSeq NC_003112.2). For multiple alignment of the identified REPs in each genome, we used the program Multalign (14) available at http://prodes.toulouse.inra.fr/ multalin/multalin.html.REPs to be experimentally tested were selected based on the fulfillment of the consensus REPs of every species, on the abundance of identical or very similar copies in the genome and on the presence of CpG motifs. AnimalsTLR9 knockout mice (B6.129P2-Tlr9 tm1Aki ) were a gift from S. Akira (Re-

Activation of Toll-like receptor 9 and production of epitope specific antibody by liposome-encapsulated CpG-DNA

“…To enhance the immunostimulatory activity of PO-ODN, several liposomes were applied as a vehicle for delivery. When PO-ODN was encapsulated in DOTAP, the uptake of PO-ODN and the production of IFN-α were enhanced in hPBMCs (18,19). Previously, we screened a natural immunostimulatory PO-ODN from bacterial chromosomal DNA, but the PO-ODN was much less potent in inducing HLA-DRA and hBD-2 promoter activation in RPMI 8226 cells than in mice (13,14,20).…”

Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate tlr-dependent signal pathway and supress apoptosis

“…Плазмида p(СатIII) обогащена АТ-парами (область 1q12 1-й хромосомы человека), которые не содержат ни лигандов, ни супрессоров TLR9, однако в составе вектора pBR322 присутствуют CpG-богатые участки ДНК. В качестве положительного контроля использовали ДНК Е. coli в концентрации 50 нг/мл, которая является известным лигандом, взаимодействующим с рецепторами TLR9 [16].…”

The production and immunostimulatory activity of double-stranded CpG-DNA

“…PO-ODN activates the innate immune system in mice, but triggers relatively low activity in human cells (2). However, it has been reported that activation of the innate immune response in human cells was induced with liposome-encapsulated PO-ODN (DOTAP and lipofectin) (26,27). In testing whether encapsulated dsCpG-DNA has immunostimulatory activity, we found that IL-8 promoter activation, HLA-DRA expression, and IL-8 expression were significantly enhanced in a CG-sequence-independent manner (Figs.…”