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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

The contribution of HMGB1 to TLR9 activation.C57BL/6 BMDCs were stimulated with L. major or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (A) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by L. major or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (B) IL-6 and TNFα production was measured in supernatants by ELISA. (C) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), L. major or vertebrate DNA (20 µg). (left) Analysis was performed by Western Blot at 30 and 60 min post-induction. (right) Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (D) C57BL/6 BMDCs were stimulated with L. major DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition  =  [100-{cytokines production by L. major DNA with glycyrrhizin/cytokines production by L. major DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).
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pntd-0003308-g008: The contribution of HMGB1 to TLR9 activation.C57BL/6 BMDCs were stimulated with L. major or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (A) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by L. major or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (B) IL-6 and TNFα production was measured in supernatants by ELISA. (C) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), L. major or vertebrate DNA (20 µg). (left) Analysis was performed by Western Blot at 30 and 60 min post-induction. (right) Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (D) C57BL/6 BMDCs were stimulated with L. major DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition  =  [100-{cytokines production by L. major DNA with glycyrrhizin/cytokines production by L. major DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).

Mentions: To determine whether HMGB1 could modify the immunostimulatory properties of DNA, BMDCs were stimulated either with L. major or vertebrate DNA alone, as well as with pre-formed HMGB1-DNA complexes. Stimulation with L. major DNA-HMGB1 complexes doubled cytokine mRNA expression and secretion compared with DNA alone (Figure 8A and 8B), whereas HMGB1-vertebrate DNA complex or HMGB1 alone did not. Similarly, no cellular stimulation was observed with sheep, pig or mouse DNA complexed with SLPI or LL37 peptides under these conditions (Figure S6). Therefore, enhanced DCs activation by SLPI, LL37, HMGB1 is only observed in the presence of parasitic 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)

The contribution of HMGB1 to TLR9 activation.C57BL/6 BMDCs were stimulated with L. major or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (A) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by L. major or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (B) IL-6 and TNFα production was measured in supernatants by ELISA. (C) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), L. major or vertebrate DNA (20 µg). (left) Analysis was performed by Western Blot at 30 and 60 min post-induction. (right) Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (D) C57BL/6 BMDCs were stimulated with L. major DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition  =  [100-{cytokines production by L. major DNA with glycyrrhizin/cytokines production by L. major DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230925&req=5

pntd-0003308-g008: The contribution of HMGB1 to TLR9 activation.C57BL/6 BMDCs were stimulated with L. major or vertebrate DNA (20 µg) complexed with rHMGB1 (1 µg) or alone (as control and reference) for 6 h. (A) Cytokines mRNA were detected by real-time PCR. The data are expressed as the n-fold increase with the expression in stimulated BMDCs by L. major or vertebrate DNA alone. The mRNA expression levels were normalized to the expression of the HPRT gene. nd: not detectable. (B) IL-6 and TNFα production was measured in supernatants by ELISA. (C) Time-course analysis of HMGB1 in the cytoplasm and nucleus of BMDCs not stimulated (NS) or stimulated by CpG 1826 (1 µg), L. major or vertebrate DNA (20 µg). (left) Analysis was performed by Western Blot at 30 and 60 min post-induction. (right) Quantification of HMGB1 in cytoplasmic extracts was normalized to that of actin and expressed as the n-fold difference with the unstimulated BMDCs. The mean and SEM of six independent experiments, each including both 30 min and 60 min extracts, is represented by the histogram. (D) C57BL/6 BMDCs were stimulated with L. major DNA alone (20 µg) or with glycyrrhizin (10 or 20 µg/ml) for 6 h. Percentage (%) of inhibition  =  [100-{cytokines production by L. major DNA with glycyrrhizin/cytokines production by L. major DNA alone}]×100. The data represent three independent experiments: one representative for C and the mean and SEM in A, B, D (*p<0,05, **p<0,01).
Mentions: To determine whether HMGB1 could modify the immunostimulatory properties of DNA, BMDCs were stimulated either with L. major or vertebrate DNA alone, as well as with pre-formed HMGB1-DNA complexes. Stimulation with L. major DNA-HMGB1 complexes doubled cytokine mRNA expression and secretion compared with DNA alone (Figure 8A and 8B), whereas HMGB1-vertebrate DNA complex or HMGB1 alone did not. Similarly, no cellular stimulation was observed with sheep, pig or mouse DNA complexed with SLPI or LL37 peptides under these conditions (Figure S6). Therefore, enhanced DCs activation by SLPI, LL37, HMGB1 is only observed in the presence of parasitic 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