CatB^-/- and WT BMDCs respond similarly to TLR-agonists or <i>L</i>. <i>major</i>.

<p>BMDCs from WT and CatB^-/- mice were stimulated with LPS (100ng/ml) and CpG (250ng/ml) or with live or killed <i>L</i>. <i>major</i> promastigotes (1:5 MOI) for 24h to determine secreted cytokines by ELISA (<b>A</b> and <b>B</b>) or for 6h to assess mRNA by RT-PCR (<b>C</b> and <b>D</b>). <b>(A)</b> Concentration of IL-6 and TNF in supernatants of BMDCs from WT (filled bars) and CatB^-/- (empty bars) mice stimulated with LPS or CpG or <b>(B)</b> with live or killed <i>L</i>. <i>major</i>. <b>(C)</b> Level of expression of IL-12p40, IL-12p35, IL-6, IFNβ, IL-10 and TNF transcripts from BMDCs of WT (filled bars) and CatB^-/- (empty bars) mice stimulate with LPS or CpG or <b>(D)</b> with live or killed <i>L</i>. <i>major</i>. The mRNA expression levels were normalized to the expression of the HPRT gene and calculated as the n-fold difference with the level of expression in unstimulated cells. Data depicted represent the mean and SEM of three independent experiments. No statistical differences were found.</p

Adaptive immune cell dynamics in responses to <i>L</i>. <i>major</i> infection of WT and CatB^-/- mice.

<p>WT and CatB^-/- were subcutaneously inoculated with 3x10⁶ stationary phase promastigotes of <i>L</i>. <i>major</i> in the footpads and experimental read-outs were measured weekly as indicated. <b>(A)</b> Gating strategy used for analysis. Cell numbers in draining lymphnodes of <i>L</i>. <i>major</i> -infected WT (filled bars) and CatB^-/- (empty bars) mice <b>(B)</b>. Percentages of CD19+ B cells <b>(C)</b>, CD3+ T cells <b>(D)</b>, CD4+ T cells <b>(E)</b> and CD8+ T cells <b>(F)</b> gated on the lymphocyte gate and foxp3+CD25+ Tregs <b>(G)</b> gated on the CD4+ gate from lymph nodes of <i>L</i>. <i>major</i> -infected WT (filled bars) and CatB^-/- (empty bars) mice. Data depicted represent the mean and SEM of at least 3 independent experiments with n ≥ 4 mice/group/time point. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001</p

CatB^-/- T cells secrete more IFNγ but less IL-2 and have impaired proliferation as compared to WT T cells.

<p><i>In vitro</i> CD3 T cells purified from spleens of naïve WT and CatB^-/- mice were labelled with CFSE or not and stimulated overnight with PMA or with anti-CD3. Supernatants were harvested for cytokine detection at 24, 48 and 72h and CFSE dilution was assessed by FACS at 72h. Concentration of IFNγ <b>(A)</b> and IL-2 <b>(B)</b> in supernatants of PMA stimulated CD3 T cells from WT (filled bars) and CatB^-/- (empty bars) mice. <b>(C)</b> Percentage of CD4 cells that have undergone 1, 2 or 3 divisions based on CFSE labelling at 72h after stimulation with plate-bound anti-CD3 of purified T cells from WT (filled bars) and CatB^-/- (empty bars) mice. <i>In vivo</i> purified CD3 T cells were transferred into recipient RAG2^-/-γc^-/- mice and BrdU assay was performed on day 21 after transfer. <b>(D)</b> Percentage of BrdU+ CD4 and CD8 cells identified among CD45+ cells in spleens of RAG2^-/-γc^-/- mice reconstituted with purified CD3 T cells from WT (filled bars) and CatB^-/- (empty bars) mice at day 21 after transfer. Figures depict representative data pooled from 3 independent repeats with n ≥ 5 mice/group * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001</p

Innate immune cytokine responses of <i>L</i>. <i>major</i> infected WT and CatB^-/- mice.

<p>WT and CatB^-/- were subcutaneously inoculated with 3x10⁶ stationary phase promastigotes of <i>L</i>. <i>major</i> in the footpads and experimental read-outs were measured weekly as indicated. Levels of TNF <b>(A)</b> and IL-1β <b>(B)</b> were measured in footpads of <i>L</i>. <i>major</i> -infected WT (filled bars) and CatB^-/- (empty bars) mice by specific ELISA. Level of expression of TNF <b>(C)</b> and IL-6 <b>(D)</b> transcripts in footpads or TNF <b>(E)</b> and IL-6 <b>(F)</b> transcripts in lymph nodes of <i>L</i>. <i>major</i> -infected WT (filled bars) and CatB^-/- (empty bars) mice were measured by RT-PCR. All PCR data values are normalized to the expression of the HPRT gene. Data depicted represent the mean and SEM of at least 3 independent experiments with n ≥ 4 mice/group/time point. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001</p

Similar antigen presentation capacity of BMDCs from WT and CatB^-/- mice.

<p>WT and CatB^-/- were infected subcutaneously with 3x10⁶ stationary phase promastigotes of <i>L</i>. <i>major</i> in the footpads. On days 21 and 28, lymph nodes were harvested and whole cells or purified CD4 cells from these were co-cultured overnight with WT or CatB^-/- BMDCs that were non-stimulated as control or activated by PMA, killed or live <i>L</i>. <i>major</i>. Supernatants were harvested for cytokine determination by specific ELISA and unadherent cells were harvested for intracellular cytokine staining by FACS. <b>(A-B)</b> IFNγ levels secreted by lymph node (dLNs) cells and (<b>C-D)</b> percentage of IFNγ+ cells gated on CD4+ cells in these dLNs from <i>L</i>. <i>major</i>-infected WT <b>(A, C)</b> or CatB^-/- <b>(B, D)</b> mice after co-culture with WT (filled bars) or CatB^-/- (empty bars) BMDCs. <b>(E-F)</b> IFNγ levels secreted by CD4+ cells and <b>(G-H)</b> percentage of IFNγ+ cells gated on CD4+ cells from purified CD4+ cells of <i>L</i>. <i>major</i>-infected WT <b>(E, G)</b> or CatB^-/- <b>(F, H)</b> mice that were co-cultured with WT (filled bars) or CatB^-/- (empty bars) BMDCs. Data depicted represent the mean and SEM of at least 3 independent experiments with n ≥ 4 donor mice/group.</p

CatB^-/- mice resolve L. major subcutaneous infection faster than WT.

<p>WT, TLR9^-/-, AEP^-/-, CatL^-/-, CatS^-/- and CatB^-/- were subcutaneously inoculated with 3x10⁶ stationary phase promastigotes of <i>L</i>. <i>major</i> in the footpads and experimental read-outs were measured weekly as indicated. (<b>A)</b> Mean footpad thickness (±SEM) of infected mice (WT–filled circle; TLR9^-/-- empty circle). (<b>B)</b> Mean footpad thickness (±SEM) of infected mice (WT–filled circle; AEP^-/-- empty circle). (<b>C)</b> Mean footpad thickness (±SEM) of infected mice (WT–filled circle; CatL^-/-- empty circle). (<b>D)</b> Mean footpad thickness (±SEM) of infected mice (WT–filled circle; CatS^-/-- empty circle) (<b>E)</b> Mean footpad thickness (±SEM) of infected mice (WT–filled circle; CatB^-/-- empty circle). <b>(F)</b> Logarithm base 10 of limit dilution for parasite burden from footpads of infected mice (WT—filled bars; CatB^-/-- empty bars), middle line represents mean value per group. Figures depict representative data pooled from at least 3 independent repeats with n ≥ 8 mice/group/time point. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001</p

The representation of stimulatory and inhibitory motifs in <i>L. major</i> genome is shared by other <i>Trypanosomatidae</i> genomes.

<p>The data represent the ratio of observed/expected number rO/E for each motif [stimulatory (RRCGYY and HRWCGTTN) or inhibitory (WKKVGGGG and CCNDDNNGGG)] from the analysis of <i>Trypanosomatidae</i> complete genomes (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>). The dotted line represent the ratio of observed/expected rO/E sequences which is 1, when no selection pressure is exerted on the genome in a neutral environment.</p

<i>L. major</i> and vertebrate DNA differ in their DNAse sensitivity.

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

Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.

<p>The table shows the S/I ratio that corresponds to (number of stimulatory motif/number of inhibitory motif) calculated for each motif and genome. The number of motifs observed in each genome is reported as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003308#pntd-0003308-t001" target="_blank">Table 1</a>, A and B corresponding to the stimulatory motifs and C and D to the inhibitory ones. The S/I ratios are significantly higher in <i>trypanosomatidae</i> DNA than in mouse DNA, according to Wilcoxon test (p<0.5).</p><p>Ratio between stimulatory and inhibitory motifs in <i>trypanosomatidae</i> and vertebrate genomes.</p

Competition with vertebrate DNA prevents the immunostimulatory activity of <i>L. major</i> DNA.

<p>C57BL/6 BMDCs were stimulated <i>in vitro</i> for 6 h with 10 µg of <i>L. major</i> DNA (<b>A–B</b>) or <i>T. cruzi</i> DNA (<b>C</b>), alone or with 10 µg of vertebrate DNA. Cytokines production was measured by ELISA in the supernatants of cultures. (<b>A and C</b>) The data represent the mean and SEM of three independent experiments. Significant differences are indicated (*, p<0.05; **, p<0.01; ***, p<0.001). (<b>B</b>) The percentage of inhibition of BMDCs activation obtained by adding increasing amount of vertebrate DNA (mouse, pig or sheep) to <i>L. major</i> DNA. Percentage (%) of inhibition  =  [100-{cytokines production by <i>L. major</i> with vertebrate DNA/cytokines production by <i>L. major</i> DNA alone}]×100. The results represent the mean and SEM of three independent experiments.</p