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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

Stimulation of human plasmacytoid dendritic cells by L. major and vertebrate DNA.Human plasmacytoid cell line (Gen2.2) was stimulated by L. major, mouse and human DNA alone (left) or complexed with DOTAP (right). In the indicated lanes, cells were treated with chloroquine (20 µM) before L. major 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).
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pntd-0003308-g002: Stimulation of human plasmacytoid dendritic cells by L. major and vertebrate DNA.Human plasmacytoid cell line (Gen2.2) was stimulated by L. major, mouse and human DNA alone (left) or complexed with DOTAP (right). In the indicated lanes, cells were treated with chloroquine (20 µM) before L. major 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).

Mentions: To address whether human TLR9 could also discriminate between L. major and vertebrate DNA, we investigated TLR9 signaling in human plasmacytoid DCs (pDCs). Because of their low frequency in human blood, we used a human pDCs cell line GEN2.2 CD123+ HLA-DR+, derived from leukemic pDCs [32], which were activated by TLR9 and TLR7 agonists (CpG and Cl-097) (Figure S3). Only L. major DNA induced the increase of IFNα2 and IFNβ in pDCs (Figure 2). No comparable activation was observed with the same quantity of vertebrate DNA, even in the presence of DOTAP. The activation was impaired by chloroquine treatment, which inhibits the endosomal acidification necessary for TLR9 activation. Thus, in bone marrow derived DCs from mouse and in a human plasmacytoid cell line that have been only investigated, we showed that L. major DNA induced TLR9 signaling at least 10 times more efficiently than vertebrate 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)

Stimulation of human plasmacytoid dendritic cells by L. major and vertebrate DNA.Human plasmacytoid cell line (Gen2.2) was stimulated by L. major, mouse and human DNA alone (left) or complexed with DOTAP (right). In the indicated lanes, cells were treated with chloroquine (20 µM) before L. major 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).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003308-g002: Stimulation of human plasmacytoid dendritic cells by L. major and vertebrate DNA.Human plasmacytoid cell line (Gen2.2) was stimulated by L. major, mouse and human DNA alone (left) or complexed with DOTAP (right). In the indicated lanes, cells were treated with chloroquine (20 µM) before L. major 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).
Mentions: To address whether human TLR9 could also discriminate between L. major and vertebrate DNA, we investigated TLR9 signaling in human plasmacytoid DCs (pDCs). Because of their low frequency in human blood, we used a human pDCs cell line GEN2.2 CD123+ HLA-DR+, derived from leukemic pDCs [32], which were activated by TLR9 and TLR7 agonists (CpG and Cl-097) (Figure S3). Only L. major DNA induced the increase of IFNα2 and IFNβ in pDCs (Figure 2). No comparable activation was observed with the same quantity of vertebrate DNA, even in the presence of DOTAP. The activation was impaired by chloroquine treatment, which inhibits the endosomal acidification necessary for TLR9 activation. Thus, in bone marrow derived DCs from mouse and in a human plasmacytoid cell line that have been only investigated, we showed that L. major DNA induced TLR9 signaling at least 10 times more efficiently than vertebrate 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