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The Journal of Immunology, 2007, 179, 31 -35
Copyright © 2007 by The American Association of Immunologists, Inc.
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Cutting Edge: Natural DNA Repetitive Extragenic Sequences from Gram-Negative Pathogens Strongly Stimulate TLR91

Mattias Magnusson2,*, Raquel Tobes2,{dagger}, Jaime Sancho{ddagger} and Eduardo Pareja3,{dagger}

* Department of Rheumatology and Inflammation Research, Göteborg University, Göteborg, Sweden; {dagger} Era7 Information Technologies Societal Limitada, Business Innovation Center, Granada Centro Europeo de Empresas e Innovación, Parque Tecnológico de Ciencias de la Salud-Armilla Granada, Armilla, Spain; and {ddagger} Instituto de Parasitologia y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Armilla, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-{alpha} production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9tm1Aki knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Clinical and epidemiologic studies have suggested a connection between infectious agents and autoimmune diseases (1, 2, 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, 6, 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, 10, 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 ~25 nt; the presence of unmethylated CpG motifs following the pattern "not C, C, G, not G" separated by AT-rich stretches of ~2–4 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
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Bioinformatics analysis

To detect REP sequences we used a BLAST-based strategy specially designed to detect repetitive extragenic and palindromic sequences. We developed C++ 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.

Animals

TLR9 knockout mice (B6.129P2-Tlr9tm1Aki) were a gift from S. Akira (Research Institute for Microbial Diseases, Department of Host Defense, Osaka University, Osaka, Japan). C57BL/6 mice were purchased from B&K. 129 mice were purchased from the Institut National de la Recherche Agronomique. All mice were housed in the animal facility of the Department of Rheumatology and Inflammation Research, Göteborg University (Göteborg, Sweden). Mice were kept under standard conditions of temperature and light and fed laboratory chow and water ad libitum. This study was approved by the Ethical Committee of Göteborg University (ethical no. 313-2004) and the requirements of the National Board for Laboratory Animals were followed.

Reagents

The studied REP sequences (Table I) were selected from the REP sequences found in E. coli, S. enterica subsp. Enterica serovar typhi, P. aeruginosa, and N. meningitidis (see above and supplemental material in Fig. 1, a and b).5 Phosphodiester ODNs representing the selected ODNs were synthesized in the Instituto de Parasitología y Biomedicina López Neyra core facilities and HPLC purified. The C-methylated modified ODNs were purchased lyophilized from MWG-Biotech. ODNs were suspended in sterile, endotoxin-free distilled water and stored at –20°C until use. They were handled under aseptic conditions to prevent both microbial and endotoxin contamination. For methylated ODNs, 5-methylcytidine was used in the ODN synthesis. To form dsDNA of the REPs, equimolar amounts of sense and antisense ODNs were mixed and incubated for 30 min at room temperature. This procedure has been verified to yield double-stranded molecules by comparing the migration rate in 20% nondenaturing PAGE of equal amounts of hybridized and nonhybridized molecules (6). The dsODNs were diluted in Iscove’s incomplete (without FCS) medium (50 µM 2-ME, 4 mM L-glutamine, and 20 µg/ml gentamicin) supplemented or not with lipofectin, which was prepared according to the manufacturer’s instruction’s (Invitrogen Life Technologies). The mixture was incubated for 15 min at room temperature and then added to cell cultures (see below). UV-inactivated HSV was a gift from Kristina Eriksson (Göteborg University) and used at a final concentration of 4 x 105 PFU/ml cell culture. dsRNA was prepared from Rota virus as described previously (15) or purchased as synthetic poly(I):poly(C) (Sigma-Aldrich) and used at a final concentration of 50 µg/ml cell culture.


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  		Table I. REP and control sequences used in this studya

 
Cell stimulation

Murine splenocytes were isolated aseptically and passed through nylon mesh. Erythrocytes were depleted by hypotonic lysis and the remaining splenocytes were resuspended in Iscove’s complete medium (20% FCS (10% is the final concentration in cell cultures), 50 µM 2-ME, 4 mM L-glutamine, and 20 µg/ml gentamicin). Cultures were established by mixing splenocytes with the various inducers (see above) in a total volume of 0.2 ml. The splenocytes were cultured in flat-bottom 96-well plates (Nunc) at 4 x 106 cells/ml. The cultures were incubated at 37°C and in 5% CO2/95% humidity for 24 h. All cultures were conducted in triplicates.

Immunoassay

The supernatants from cell cultures were analyzed for the presence of IFN-{alpha} as described previously (16).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Using a Bioinformatics search for bacterial DNA sequence candidates as natural TLR9 ligands, we selected a special kind of repetitive bacterial DNA sequence named REP. The term "REP sequences" encompasses repetitive and palindromic sequences with a length between 21 and 65 bases detected in the extragenic space of some bacterial genomes constituting >0.5% of the total extragenic space (17). These sequences are present in many Gram-negative bacteria (17, 18), playing important roles in DNA physiology and genomic plasticity (19). REPs perfectly fulfill the criteria to be strong immunostimulatory ODNs because they have an appropriate length, are rich in unmethylated CpG motifs, and are palindromic. REPs palindromicity allows one to envisage possible stem-loop secondary structures that they could adopt (20). DNA secondary or tertiary structures could endow REPs with higher stability and DNase resistance. Furthermore, REPs have two additional advantageous features for being a target of immune recognition of bacteria: abundance and conservation. In this study, we selected a set of important Gram-negative human pathogens having REPs in their genomes to test their in vitro immunostimulatory activity. The selected bacteria were E. coli, S. enterica typhi, N. meningitidis, and P. aeruginosa. There are many copies of a REP sequence in a genome. Thus, we found 454 E. coli REPs in E. coli K12 genome, 228 Salmonella REPs in S. enterica subsp. Enterica serovar typhi genome, 226 P. aeruginosa REPs in P. aeruginosa PAO1 genome, and 753 N. meningitidis REPs in N. meningitidis serogroup B strain MC58. Alignments and sequences are displayed in supplemental material Fig. 1, a and b. The abundance of REPs and the stable stem-loop secondary structures that they probably adopt could facilitate their detection by TLR9. DNA tertiary structures, stable even under denaturing conditions, have been suggested as responsible for the high IFN-{alpha} stimulatory ability of A-type CpG ODNs (21). REPs are conserved within every species or even within a group of related species (as is the case for E. coli and Salmonella) and the involvement of REPs in important bacterial functions precludes their genetic loss for immune escape (19). In any case, all REPs from different species share the palindromicity and richness in unmethylated CpG motifs common in stimulatory ODNs.

We decided to use dsODNs with a natural phosphodiester backbone to mimic natural REPs. Thus, double-stranded phosphodiester oligonucleotides with the sequence of representative REPs from each bacteria (see Materials and Methods) were synthesized (Table I) and were tested for their ability to activate production of the proinflammatory cytokine IFN-{alpha}. All REP sequences tested induced production of IFN-{alpha} when they were preincubated with lipofectin that facilitates cellular uptake. In the absence of cationic lipid, the REPs induced little or no IFN-{alpha} production (Fig. 1, A and B). To activate TLR9 and thereby the production of IFN-{alpha}, CpG DNA must localize to the endosomal compartment and induce endosomal maturation (22). During infection, engulfed bacteria may be directed to late endosomal compartments in dendritic cells (23), where REPs may interact with TLR9. The requirement of lipofection for REPs to induce IFN-{alpha} production strongly suggests that naked REP sequences are not efficiently internalized in vitro and, therefore, they do not reach the endosomal compartment, whereas REPs complexed with cationic lipids were endowed with such ability. Indeed, complexation of, otherwise non-interferogenic, phosphodiester ODNs with cationic lipids resulting in both translocation of DNA to endosomes and release of IFN-{alpha} has been reported for several cell types (24). In contrast, the activity of the A class stimulatory CpG ODN2216, used in our study as a positive control, was little enhanced by pretreatment with lipofectin (data not shown). ODN2216 contains flanking nuclease-resistant phosphorothioate guanosine nucleotides, which are known to form nanoparticles via G tetrads (21). These G tetrads may both facilitate interaction with scavenger receptors for cell entry and subcellular localization (25), as well as endosomal maturation, crucial for the IFN-{alpha} production in response to A class CpG ODNs (26). Moreover, preventing secondary structure formation of ODN2216 abrogates its interferogenic activity in the absence of cationic lipids and addition of poly(Gs) to a self-complementary CpG ODN abrogated dependence on cationic lipids for IFN-{alpha}-inducing activity (27).


Figure 1
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  		FIGURE 1. REP sequences induce IFN-{alpha} production mediated by TLR9 in vitro. A and B, Induction of IFN-{alpha} production by REP sequences from E. coli and S. enterica typhi (A) and from P. aeruginosa and N. meningitidis (B). Splenocytes (4 x 106/ml) from 129 mice were stimulated with REP sequences (1.16 µM) with ({blacksquare}) or without (Figure 1) 5 µg/ml lipofectin. Medium (med) was used as negative control and UV-inactivated HSV as positive control. The strong IFN-{alpha} stimulatory capability of REPs depends on the presence of lipofectin ({blacksquare}). The p values comparing the IFN-{alpha} production induced by REPs from E. coli, S. enterica typhi, P. aeruginosa, and N. meningitidis in the presence of lipofectin vs the IFN-{alpha} level induced by medium plus lipofectin were <0.05. C and D, Capability of REP sequences to activate production of IFN-{alpha} in splenocytes from wild-type (wt) C57BL6 mice compared with TLR9 knockout mice (B6.129P2-Tlr9tm1Aki provided by S. Akira). The bar graphs show a strong stimulation of IFN-{alpha} production by E. coli, S. typhi, P. aeruginosa, and N. meningitidis REPs in wild-type mice compared with the lack of stimulation in TLR9 knockout mice (p < 0.0001). Likewise, UV-inactivated HSV and CpG ODN2216 (3 µg/ml equivalent to 0.47 µM) induce IFN-{alpha} in a TLR9-dependent manner, whereas dsRNA (50 µg/ml)-induced production of IFN-{alpha} is TLR9 independent. ODN2216 non-CpG (0.47 µM) was used as a negative control to CpG- ODN. E, Loss of the ability of N. meningitidis REP sequences devoid of CpG motifs or with methylated CpG motifs to induce production of IFN-{alpha} in comparison to the production of IFN-{alpha} of N. meningitidis REPs at 1.16 µM (p < 0.001) tested in splenocytes from 129 strain mice. Cell cultures were in triplicates and the levels of IFN-{alpha} (U/ml; mean ± SD) in supernatants were determined by an immunoassay.

 
In the presence of lipofectin, the REP sequences induced levels of IFN-{alpha} comparable to that of UV-inactivated HSV or A-type CpG ODN2216 (21, 28), indicating that they could be strong natural immune activators (Fig. 1). The IFN-{alpha}-inducing ability of all REP sequences tested was totally dependent on TLR9-signaling because splenocytes from TLR9 knockout mice did not produce IFN-{alpha} when stimulated with REP sequences (Fig. 1, C and D). To confirm that the activating capacity is indeed CpG dependent, the CpG motifs in one of the REP sequences (from N. meningitidis) were turned into either non-CpG sequences or C-methylated (Table I). Both of these modifications totally abolished the IFN-{alpha}-inducing capacity of the N. meningitidis REP sequence (Fig. 1E).

The single-stranded sense and antisense ODNs representing the REPs from N. meningitidis and P. aeruginosa were also tested for their interferogenic activity. Single-stranded REPs also induced high levels of IFN-{alpha} in the presence of lipofectin in a TLR9- and CpG-dependent manner (data not shown).

These experiments prove that the abundant natural bacterial DNA sequences named REPs are able to produce innate immune system stimulation. The absence of any stimulatory effect in TLR9 knockout mice proves that the immunostimulatory effect of REPs is mediated by TLR9 receptors. In addition, the fact that REPs with methylated or deleted CpG motifs had no stimulatory effect further corroborates the dependence on unmethylated CpG motifs for the immunostimulatory effects of natural bacterial DNA. Furthermore, the interferogenic activity of natural bacterial REPs heavily relies on the presence of unmethylated CpG dinucleotides, in contrast to other experimentally used ODNs that in some instances retain immunostimulatory activity in the absence of CpG (6, 24).

TLR9 recognizes (CpG) dinucleotides in specific sequence contexts (CpG motifs). Experimental studies using synthetic ODNs suggest that sequence composition, secondary and tertiary structures, and the sugar, base, or backbone modifications within this CpG sequence context also determine the immune profile of ODNs. Based on these factors, four classes of CpG ODNs (A, B, C, and S) have been defined (29). REPs offer novel interferogenic natural sequences with a motif different than the motifs defining the four classes of CpG ODNs. This REPs motif could be described as a GC-rich palindrome with CpG motifs and a CTA sequence at 3'.

Taking into account that Gram-negative bacteria, and especially the four bacterial species whose REPs have been studied, are involved in human septic shock (30, 31), it is probable that REPs play a role in their pathogenesis. REPs could be a potential novel target to be blocked, with the aim of ameliorating the unbalanced immune response that takes place in this life-threatening condition.

Synthetic ODNs as TLR9 agonists are being applied in cancer vaccination and treatment, infectious diseases, asthma, and other allergic diseases (32, 33, 34). On the other hand, blocking TLR9 has shown beneficial effects on systemic lupus erythematosus and rheumatoid arthritis prevention (32). The finding of REPs as natural TLR9 stimulatory ligands supplies new knowledge that can be useful for the development of new therapeutic applications. Thus, REP sequences could be used as templates for designing new ODNs to be applied in the fields in which synthetic ODNs have proved their usefulness. In addition, REPs could be novel targets to control TLR9-mediated immune responses in processes with an unbalanced innate immune response such as Gram-negative sepsis (11) or chronic intestinal inflammation (35). Signaling of TLRs through the adaptor proteins MyD88 and/or TRIF, which are both involved in the production of type I IFN, is fundamental during endotoxic shock or polymicrobial sepsis (11). Accordingly, it has been shown that MyD88 deficiency results in beneficial effects for the outcome of septic peritonitis because it reduces systemic hyperinflammation (11). Recent data implicate signaling byTLRs and the common adaptor molecule MyD88 in the pathogenesis of inflammatory bowel disease (36). REPs from microflora could be exerting a continuous stimulatory effect mediated by TLR9 and could be involved in inflammatory bowel disease pathogenesis. Altered response to infection is an important component in many autoimmune diseases. The ability of REP sequences to stimulate TLR9 could be playing a role in some of them and the control of this response could provide novel therapeutic alternatives.


   Acknowledgments
 
We thank Emma Skoogh and Antonio Mérida for technical assistance. We thank S. Akira for providing B6.129P2-Tlr9tm1Aki mice.


   Disclosures
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 Abstract
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 Materials and Methods
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 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Ministerio de Educación y Ciencia (Grant SAF2005-06056-C02-01) and by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (Grant P05-CVI-00908) to J.S. Back

2 M.M. and R.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Eduardo Pareja, Immunology Unit, Era7 Information Technologies Societal Limitada, Business Innovation Center, Granada Centro Europeo de Empresas e Innovación, Parque Tecnológico de Ciencias de la Salud, Armilla, Granada 18100, Spain. E-mail address: epareja{at}era7.com Back

4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; REP, repetitive extragenic palindromic. Back

5 The online version of this article has supplemental material. Back

Received for publication February 23, 2007. Accepted for publication April 30, 2007.


   References
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 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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