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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

L. major and vertebrate DNA differ in their DNAse sensitivity.DNA degradation was analysed by electrophoresis on a 0.7% agarose gel, stained by EtBr. Increasing amounts of DNAse I (left), DNase II (middle) or cytoplasmic extract from C57BL/6 BMDCs (right) was added to a same amount of full-length genomic L. major or vertebrate DNA (1 µg). L stands for the Kb ladder (λDNA-Hind III). The data represent one representative of three experiments.
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pntd-0003308-g004: L. major and vertebrate DNA differ in their DNAse sensitivity.DNA degradation was analysed by electrophoresis on a 0.7% agarose gel, stained by EtBr. Increasing amounts of DNAse I (left), DNase II (middle) or cytoplasmic extract from C57BL/6 BMDCs (right) was added to a same amount of full-length genomic L. major or vertebrate DNA (1 µg). L stands for the Kb ladder (λDNA-Hind III). The data represent one representative of three experiments.

Mentions: Degradation of exogenous DNA by DNAses may be a limit to TLR9 activation. Since the DNase content is much higher in phagocytic cells, such as DCs, than in other cells, we compared the relative sensitivity of L. major and mouse genomic DNA to increasing concentrations of both DNase I or II (Figure 4). DNase I and II nucleases are usually involved in the digestion of DNA that originated outside the nucleus [36]. We observed that the complete degradation of L. major DNA requires ten times more purified DNase I or II or 2 times more cytoplasmic extract than for vertebrate DNA (Figure 4). Thus, L. major DNA is intrinsically more resistant to DNase than vertebrate DNA suggesting that the parasitic DNA could persist longer in the cells.


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)

L. major and vertebrate DNA differ in their DNAse sensitivity.DNA degradation was analysed by electrophoresis on a 0.7% agarose gel, stained by EtBr. Increasing amounts of DNAse I (left), DNase II (middle) or cytoplasmic extract from C57BL/6 BMDCs (right) was added to a same amount of full-length genomic L. major or vertebrate DNA (1 µg). L stands for the Kb ladder (λDNA-Hind III). The data represent one representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003308-g004: L. major and vertebrate DNA differ in their DNAse sensitivity.DNA degradation was analysed by electrophoresis on a 0.7% agarose gel, stained by EtBr. Increasing amounts of DNAse I (left), DNase II (middle) or cytoplasmic extract from C57BL/6 BMDCs (right) was added to a same amount of full-length genomic L. major or vertebrate DNA (1 µg). L stands for the Kb ladder (λDNA-Hind III). The data represent one representative of three experiments.
Mentions: Degradation of exogenous DNA by DNAses may be a limit to TLR9 activation. Since the DNase content is much higher in phagocytic cells, such as DCs, than in other cells, we compared the relative sensitivity of L. major and mouse genomic DNA to increasing concentrations of both DNase I or II (Figure 4). DNase I and II nucleases are usually involved in the digestion of DNA that originated outside the nucleus [36]. We observed that the complete degradation of L. major DNA requires ten times more purified DNase I or II or 2 times more cytoplasmic extract than for vertebrate DNA (Figure 4). Thus, L. major DNA is intrinsically more resistant to DNase than vertebrate DNA suggesting that the parasitic DNA could persist longer in the cells.

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