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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

Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.L. major 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. (A) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (B) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA (left) then blotted and revealed with anti-HMGB1 antibody (right). (C) Sonicated Trypanosomatidae 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. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.
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pntd-0003308-g009: Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.L. major 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. (A) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (B) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA (left) then blotted and revealed with anti-HMGB1 antibody (right). (C) Sonicated Trypanosomatidae 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. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.

Mentions: As shown in Figure 9A, HMGB1 complexed with DNA had a different electrophoretic mobility than the HMGB1 alone (lane H), that did not migrate into the gel. Importantly, HMGB1 formed complexes with sonicated L. major DNA even for the lowest ratios of HMGB1/DNA, whereas it barely interacted with vertebrate DNA at the highest ratios. In HMGB1/L. major DNA lanes 3–7 (left part Figure 9A), little free HMGB1 is found, except at the highest molar ratios in lanes 6 (25∶1) and 7 (50∶1). In contrast, in HMGB1/vertebrate DNA lanes 3–7 (right part Figure 9A) a larger amount of free HMGB1 is observed in lanes 5 to 7. Quantification showed an increasing amount of bound HMGB1 on L. major DNA (from lane 3 to 7) while very low amount of HMGB1 was complexed with vertebrate DNA in the same conditions (Figure S7A). This result indicates that HMGB1 binding differs between -vertebrate and L.major DNA.


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)

Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.L. major 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. (A) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (B) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA (left) then blotted and revealed with anti-HMGB1 antibody (right). (C) Sonicated Trypanosomatidae 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. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003308-g009: Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.L. major 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. (A) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (B) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA (left) then blotted and revealed with anti-HMGB1 antibody (right). (C) Sonicated Trypanosomatidae 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. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.
Mentions: As shown in Figure 9A, HMGB1 complexed with DNA had a different electrophoretic mobility than the HMGB1 alone (lane H), that did not migrate into the gel. Importantly, HMGB1 formed complexes with sonicated L. major DNA even for the lowest ratios of HMGB1/DNA, whereas it barely interacted with vertebrate DNA at the highest ratios. In HMGB1/L. major DNA lanes 3–7 (left part Figure 9A), little free HMGB1 is found, except at the highest molar ratios in lanes 6 (25∶1) and 7 (50∶1). In contrast, in HMGB1/vertebrate DNA lanes 3–7 (right part Figure 9A) a larger amount of free HMGB1 is observed in lanes 5 to 7. Quantification showed an increasing amount of bound HMGB1 on L. major DNA (from lane 3 to 7) while very low amount of HMGB1 was complexed with vertebrate DNA in the same conditions (Figure S7A). This result indicates that HMGB1 binding differs between -vertebrate and L.major DNA.

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