11 research outputs found
Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.
<p>The table shows the S/I ratio that corresponds to (number of stimulatory motif/number of inhibitory motif) calculated for each motif and genome. The number of motifs observed in each genome is reported as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>, A and B corresponding to the stimulatory motifs and C and D to the inhibitory ones. The S/I ratios are significantly higher in <i>trypanosomatidae</i> DNA than in mouse DNA, according to Wilcoxon test (p<0.5).</p><p>Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.</p
The representation of stimulatory and inhibitory motifs in <i>L. major</i> genome is shared by other <i>Trypanosomatidae</i> genomes.
<p>The data represent the ratio of observed/expected number rO/E for each motif [stimulatory (RRCGYY and HRWCGTTN) or inhibitory (WKKVGGGG and CCNDDNNGGG)] from the analysis of <i>Trypanosomatidae</i> complete genomes (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>). The dotted line represent the ratio of observed/expected rO/E sequences which is 1, when no selection pressure is exerted on the genome in a neutral environment.</p
<i>L. major</i> and vertebrate DNA differ in their DNAse sensitivity.
<p>DNA degradation was analysed by electrophoresis on a 0.7% agarose gel, stained by EtBr. Increasing amounts of DNAse I <b><i>(left)</i></b>, DNase II <b><i>(middle)</i></b> or cytoplasmic extract from C57BL/6 BMDCs <b><i>(right)</i></b> was added to a same amount of full-length genomic <i>L. major</i> or vertebrate DNA (1 µg). L stands for the Kb ladder (λDNA-Hind III). The data represent one representative of three experiments.</p
Competition with vertebrate DNA prevents the immunostimulatory activity of <i>L. major</i> DNA.
<p>C57BL/6 BMDCs were stimulated <i>in vitro</i> for 6 h with 10 µg of <i>L. major</i> DNA (<b>A–B</b>) or <i>T. cruzi</i> DNA (<b>C</b>), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (<b>A and C</b>) The data represent the mean and SEM of three independent experiments. Significant differences are indicated (*, p<0.05; **, p<0.01; ***, p<0.001). (<b>B</b>) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to <i>L. major</i> DNA. Percentage (%) of inhibition = [100-{cytokines production by <i>L. major</i> with vertebrate DNA/cytokines production by <i>L. major</i> DNA alone}]×100. The results represent the mean and SEM of three independent experiments.</p
Stimulation of human plasmacytoid dendritic cells by <i>L. major</i> and vertebrate DNA.
<p>Human plasmacytoid cell line (Gen2.2) was stimulated by <i>L. major</i>, mouse and human DNA alone <b><i>(left)</i></b> or complexed with DOTAP <b><i>(right)</i></b>. In the indicated lanes, cells were treated with chloroquine (20 µM) before <i>L. major</i> stimulation. Expression of the indicated cytokines was determined by real time RT-PCR. The mRNA expression levels were normalized to the expression of the HPRT gene and calculated as the n-fold difference with the expression in unstimulated cells. The results represent the mean and SEM of three independent experiments (*p<0,05, **p<0,01).</p
Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.
<p><i>L. major</i> or vertebrate sonicated DNA (250 ng) were incubated with increasing amounts of HMGB1. Lane 1 shows the migration of DNA alone and lane H the HMGB1 alone. In lane 2 to 7, DNA was complexed to HMGB1, respectively at a molar ratio of 2.5, 5, 7.5, 15, 25 and 50. (<b>A</b>) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (<b>B</b>) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA <b><i>(left)</i></b> then blotted and revealed with anti-HMGB1 antibody <b><i>(right)</i></b>. (<b>C</b>) Sonicated <i>Trypanosomatidae</i> DNA alone (250 ng) (lane 1) or complexed with HMGB1 at a molar ratio of 25 and 50 (lanes 6 and 7) were analysed by electrophoresis with the procedure described in <b>B</b>. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.</p
The contribution of HMGB1 to TLR9 activation.
<p>C57BL/6 BMDCs were stimulated with <i>L. major</i> or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (<b>A</b>) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by <i>L. major</i> or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (<b>B</b>) IL-6 and TNFα production was measured in supernatants by ELISA. (<b>C</b>) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), <i>L. major</i> or vertebrate DNA (20 µg). <b><i>(left)</i></b> Analysis was performed by Western Blot at 30 and 60 min post-induction. <b><i>(right)</i></b> Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (<b>D</b>) C57BL/6 BMDCs were stimulated with <i>L. major</i> DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition = [100-{cytokines production by <i>L. major</i> DNA with glycyrrhizin/cytokines production by <i>L. major</i> DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).</p
Selection for inhibitory and against stimulatory TLR9 motifs in vertebrate genome.
<p>(<b>A</b>) Poly G<sub>8</sub> (GGGG)<sub>2</sub>, stimulatory GACGTT and inhibitory TTAGGG motifs were quantified in <i>L. major</i> and mouse genomes. Stimulatory GTCGTT motif was quantified in <i>L. major</i> and human genomes. The datas are represented as copy number per 10<sup>6</sup> bases pair ( = copy number/genome size (bp)*10<sup>6</sup>). (<b>B</b>) Stimulatory (RRCGYY and HRWCGTTN) and inhibitory (WKKVGGGG and CCNDDNNGGG) motifs were quantified in each <i>L. major</i>, mouse or human chromosome. They are represented as the ratio of observed/expected sequences rO/E, as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>. For each chromosome, the ratio is represented by a single symbol. Significant differences between <i>L. major</i> and vertebrate chromosomes are indicated (***, p<0.001). The dotted line represent the ratio of observed/expected rO/E sequences which is 1, when no selection pressure is exerted on the genome in a neutral environment.</p
TLR9-dependent activation of BMDCs specific to <i>Trypanosomatidae</i> DNA.
<p>C57BL/6 or TLR9<sup>-/-</sup> BMDCs were stimulated <i>in vitro</i> either with <i>Trypanosomatidae</i> or vertebrate DNA for 6 hours (<b>A–C</b>) IL-6 and TNFα production was measured by ELISA in the supernatants of stimulated BMDCs for 6 h (<b>A</b>) with <i>Trypanosomatidae</i> DNA alone or complexed with DOTAP, (<b>B</b>) with different concentrations of <i>L. major</i> or vertebrate (mouse or pig) DNA alone or complexed with DOTAP, (<b>C</b>) with CpG 1826 (0,25 µg/ml) and LPS (100 ng/ml) as controls, (<b>D</b>) with <i>L. major</i> or mouse DNA sonicated in 200 base pair (bp) fragments. The data represent the mean and SEM of three independent experiments. Significant differences were found between C57BL/6 and TLR9<sup>-/-</sup> in A, B, C (*, p<0.05) and between <i>L. major</i> and vertebrate DNA (mouse or pig) in C and D (*, p<0.05; **, p<0.01; ***, p<0.001).</p
TLR9 Activation Is Triggered by the Excess of Stimulatory versus Inhibitory Motifs Present in Trypanosomatidae DNA
International audienceDNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived-or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA