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TLR9 activation is triggered by the excess of stimulatory versus inhibitory motifs present in Trypanosomatidae DNA.

Khan ME, Borde C, Rocha EP, Mériaux V, Maréchal V, Escoll P, Goyard S, Cavaillon JM, Manoury B, Doyen N - PLoS Negl Trop Dis (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département Infection et Epidémiologie, Unité Cytokines & Inflammation, Paris, France.

ABSTRACT
DNA 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.

No MeSH data available.


Related in: MedlinePlus

Competition with vertebrate DNA prevents the immunostimulatory activity of L. major DNA.C57BL/6 BMDCs were stimulated in vitro for 6 h with 10 µg of L. major DNA (A–B) or T. cruzi DNA (C), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (A and C) 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) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to L. major DNA. Percentage (%) of inhibition  =  [100-{cytokines production by L. major with vertebrate DNA/cytokines production by L. major DNA alone}]×100. The results represent the mean and SEM of three independent experiments.
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pntd-0003308-g005: Competition with vertebrate DNA prevents the immunostimulatory activity of L. major DNA.C57BL/6 BMDCs were stimulated in vitro for 6 h with 10 µg of L. major DNA (A–B) or T. cruzi DNA (C), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (A and C) 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) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to L. major DNA. Percentage (%) of inhibition  =  [100-{cytokines production by L. major with vertebrate DNA/cytokines production by L. major DNA alone}]×100. The results represent the mean and SEM of three independent experiments.

Mentions: Given the different properties of L. major and vertebrate DNA regarding TLR9 activation and DNase sensitivity, we wondered whether these DNA might be in competition for TLR9 activation. The addition of different vertebrate DNA (mouse or pig) to L. major DNA inhibited the activation of BMDCs induced by L. major DNA alone. Indeed, the production of IL-6 and TNFα by BMDCs is significantly reduced (Figure 5A). This inhibition increases in relation to the concentration of vertebrate DNA (Figure 5B). The percentage of inhibition reached approximatively 30 to 50% depending on the species with an identical amount of vertebrate and L. major DNA and reaches up to 70% to 85% in the presence of a two fold excess vertebrate DNA. It should be noted a slightly higher inhibition with pig versus mouse DNA (10 µg) characterized by a lower production of IL-6 (Figure 5A) but not statistically different with 20 µg of DNA (Figure 5B). Neither toxic effect nor inhibition of LPS activation was observed with high concentration of DNA (20 or 40 µg) (Figure 1B and Figure S4), indicating that this inhibition was specific of the TLR9 activation pathway. We also noticed that sonicated vertebrate DNA and inhibitory oligonucleotide inhibited the activation induced by L. major DNA (Figure S4). Since sonicated vertebrate DNA did not cause cellular activation (Figure 1D) while exhibiting an inhibitory capacity (Figure S4) we could conclude that full-length DNA and degraded vertebrate DNA have the same properties. Importantly this would suggest that the higher sensitivity of vertebrate DNA to cellular DNAses should not interfere with its inhibitory capacity.


TLR9 activation is triggered by the excess of stimulatory versus inhibitory motifs present in Trypanosomatidae DNA.

Khan ME, Borde C, Rocha EP, Mériaux V, Maréchal V, Escoll P, Goyard S, Cavaillon JM, Manoury B, Doyen N - PLoS Negl Trop Dis (2014)

Competition with vertebrate DNA prevents the immunostimulatory activity of L. major DNA.C57BL/6 BMDCs were stimulated in vitro for 6 h with 10 µg of L. major DNA (A–B) or T. cruzi DNA (C), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (A and C) 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) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to L. major DNA. Percentage (%) of inhibition  =  [100-{cytokines production by L. major with vertebrate DNA/cytokines production by L. major DNA alone}]×100. The results represent the mean and SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230925&req=5

pntd-0003308-g005: Competition with vertebrate DNA prevents the immunostimulatory activity of L. major DNA.C57BL/6 BMDCs were stimulated in vitro for 6 h with 10 µg of L. major DNA (A–B) or T. cruzi DNA (C), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (A and C) 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) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to L. major DNA. Percentage (%) of inhibition  =  [100-{cytokines production by L. major with vertebrate DNA/cytokines production by L. major DNA alone}]×100. The results represent the mean and SEM of three independent experiments.
Mentions: Given the different properties of L. major and vertebrate DNA regarding TLR9 activation and DNase sensitivity, we wondered whether these DNA might be in competition for TLR9 activation. The addition of different vertebrate DNA (mouse or pig) to L. major DNA inhibited the activation of BMDCs induced by L. major DNA alone. Indeed, the production of IL-6 and TNFα by BMDCs is significantly reduced (Figure 5A). This inhibition increases in relation to the concentration of vertebrate DNA (Figure 5B). The percentage of inhibition reached approximatively 30 to 50% depending on the species with an identical amount of vertebrate and L. major DNA and reaches up to 70% to 85% in the presence of a two fold excess vertebrate DNA. It should be noted a slightly higher inhibition with pig versus mouse DNA (10 µg) characterized by a lower production of IL-6 (Figure 5A) but not statistically different with 20 µg of DNA (Figure 5B). Neither toxic effect nor inhibition of LPS activation was observed with high concentration of DNA (20 or 40 µg) (Figure 1B and Figure S4), indicating that this inhibition was specific of the TLR9 activation pathway. We also noticed that sonicated vertebrate DNA and inhibitory oligonucleotide inhibited the activation induced by L. major DNA (Figure S4). Since sonicated vertebrate DNA did not cause cellular activation (Figure 1D) while exhibiting an inhibitory capacity (Figure S4) we could conclude that full-length DNA and degraded vertebrate DNA have the same properties. Importantly this would suggest that the higher sensitivity of vertebrate DNA to cellular DNAses should not interfere with its inhibitory capacity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département Infection et Epidémiologie, Unité Cytokines & Inflammation, Paris, France.

ABSTRACT
DNA 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.

No MeSH data available.


Related in: MedlinePlus