Limits...
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

Comparative analysis of mouse and L. major DNA cell uptake.Analysis by confocal microscopy (A, B) and flow cytometry (C) of BMDCs stimulated 1 h by 10 µg of full-length DNA or sonicated DNA (200 bp) from L. major or mouse labelled with propidium iodide (PI) (red). (A, B) Nuclei are stained with DAPI (blue). Quantification was performed by counting 250–300 BMDCs per condition per experiment. (C) Internalized DNA was detected in FL3 channel in BMDCs labelled with an anti-CD11c APC (FL4 channel). 1) Dot plots represent BMDCs alone (a) or incubated with L. major DNA (b), vertebrate DNA (c) or both DNAs (d). The percentage of BMDCs PI+CD11c+ (respectively in FL3 and FL4 channels) is indicated for each dot plot. 2) Filled and open histograms represent unstimulated or stimulated BMDCs with PI stained DNA. L. major DNA (L.m), vertebrate DNA (V) and L. major (10 µg) with vertebrate DNA (10 µg) (L.m+V) are represented respectively with a thin, solid and a dashed line. 3) Percentage of BMDCs with exogenous DNA. In the experience of uptake competition, only L. major DNA was stained with PI. The data represent three independent experiments: one representative for A C1 and C2 and the mean and SEM in B and C3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230925&req=5

pntd-0003308-g003: Comparative analysis of mouse and L. major DNA cell uptake.Analysis by confocal microscopy (A, B) and flow cytometry (C) of BMDCs stimulated 1 h by 10 µg of full-length DNA or sonicated DNA (200 bp) from L. major or mouse labelled with propidium iodide (PI) (red). (A, B) Nuclei are stained with DAPI (blue). Quantification was performed by counting 250–300 BMDCs per condition per experiment. (C) Internalized DNA was detected in FL3 channel in BMDCs labelled with an anti-CD11c APC (FL4 channel). 1) Dot plots represent BMDCs alone (a) or incubated with L. major DNA (b), vertebrate DNA (c) or both DNAs (d). The percentage of BMDCs PI+CD11c+ (respectively in FL3 and FL4 channels) is indicated for each dot plot. 2) Filled and open histograms represent unstimulated or stimulated BMDCs with PI stained DNA. L. major DNA (L.m), vertebrate DNA (V) and L. major (10 µg) with vertebrate DNA (10 µg) (L.m+V) are represented respectively with a thin, solid and a dashed line. 3) Percentage of BMDCs with exogenous DNA. In the experience of uptake competition, only L. major DNA was stained with PI. The data represent three independent experiments: one representative for A C1 and C2 and the mean and SEM in B and C3.

Mentions: Differential DNA uptake could account for the difference for TLR9 signaling between L. major and vertebrate DNAs. To compare their uptake in BMDCs, both purified DNAs were labeled with propidium iodide (PI) and added to the cells. To avoid PI diffusion, the process of DNA uptake was analysed after one-hour incubation with the cells by confocal microscopy (Figure 3A and 3B) or flow cytometry (Figure 3C). We detected 6–10% of BMDCs containing exogenous full-length DNA with both techniques. Surprisingly we found the same proportion of DNA-containing BMDCs when exogenous sonicated DNA was used (Figure 3A, 3B and 3C). Thus, the uptake of L. major and vertebrate DNA is not significantly different. We also investigated whether L. major and vertebrate DNAs could compete for cellular uptake and tracked their internalization in BMDCs by flow cytometry. No difference in L. major DNA uptake was observed in presence of vertebrate DNA (Figure 3).


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)

Comparative analysis of mouse and L. major DNA cell uptake.Analysis by confocal microscopy (A, B) and flow cytometry (C) of BMDCs stimulated 1 h by 10 µg of full-length DNA or sonicated DNA (200 bp) from L. major or mouse labelled with propidium iodide (PI) (red). (A, B) Nuclei are stained with DAPI (blue). Quantification was performed by counting 250–300 BMDCs per condition per experiment. (C) Internalized DNA was detected in FL3 channel in BMDCs labelled with an anti-CD11c APC (FL4 channel). 1) Dot plots represent BMDCs alone (a) or incubated with L. major DNA (b), vertebrate DNA (c) or both DNAs (d). The percentage of BMDCs PI+CD11c+ (respectively in FL3 and FL4 channels) is indicated for each dot plot. 2) Filled and open histograms represent unstimulated or stimulated BMDCs with PI stained DNA. L. major DNA (L.m), vertebrate DNA (V) and L. major (10 µg) with vertebrate DNA (10 µg) (L.m+V) are represented respectively with a thin, solid and a dashed line. 3) Percentage of BMDCs with exogenous DNA. In the experience of uptake competition, only L. major DNA was stained with PI. The data represent three independent experiments: one representative for A C1 and C2 and the mean and SEM in B and C3.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003308-g003: Comparative analysis of mouse and L. major DNA cell uptake.Analysis by confocal microscopy (A, B) and flow cytometry (C) of BMDCs stimulated 1 h by 10 µg of full-length DNA or sonicated DNA (200 bp) from L. major or mouse labelled with propidium iodide (PI) (red). (A, B) Nuclei are stained with DAPI (blue). Quantification was performed by counting 250–300 BMDCs per condition per experiment. (C) Internalized DNA was detected in FL3 channel in BMDCs labelled with an anti-CD11c APC (FL4 channel). 1) Dot plots represent BMDCs alone (a) or incubated with L. major DNA (b), vertebrate DNA (c) or both DNAs (d). The percentage of BMDCs PI+CD11c+ (respectively in FL3 and FL4 channels) is indicated for each dot plot. 2) Filled and open histograms represent unstimulated or stimulated BMDCs with PI stained DNA. L. major DNA (L.m), vertebrate DNA (V) and L. major (10 µg) with vertebrate DNA (10 µg) (L.m+V) are represented respectively with a thin, solid and a dashed line. 3) Percentage of BMDCs with exogenous DNA. In the experience of uptake competition, only L. major DNA was stained with PI. The data represent three independent experiments: one representative for A C1 and C2 and the mean and SEM in B and C3.
Mentions: Differential DNA uptake could account for the difference for TLR9 signaling between L. major and vertebrate DNAs. To compare their uptake in BMDCs, both purified DNAs were labeled with propidium iodide (PI) and added to the cells. To avoid PI diffusion, the process of DNA uptake was analysed after one-hour incubation with the cells by confocal microscopy (Figure 3A and 3B) or flow cytometry (Figure 3C). We detected 6–10% of BMDCs containing exogenous full-length DNA with both techniques. Surprisingly we found the same proportion of DNA-containing BMDCs when exogenous sonicated DNA was used (Figure 3A, 3B and 3C). Thus, the uptake of L. major and vertebrate DNA is not significantly different. We also investigated whether L. major and vertebrate DNAs could compete for cellular uptake and tracked their internalization in BMDCs by flow cytometry. No difference in L. major DNA uptake was observed in presence of vertebrate DNA (Figure 3).

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