Comparison of the proinflammatory response in TLR2- and TLR9-deficient mice during the acute phase of <i>T.cruzi</i> infection.

<p>Cytokine levels in spleen cell culture (A) and serum (B) from either control (NI) or infected (I) C57BL/6 WT, TLR2^−/−, TLR9^−/− mice evaluated seven days post-infection. The data represent the mean of two experiments. *p<0.05 and **p<0.01 indicate statistical significance when comparing cytokine level in serum or in splenocyte culture from knockout versus C57BL/6 WT infected mice.</p

Evaluation of cytokine production by splenic cells from <i>T.cruzi</i> infected mice in response to TLR agonists.

<p>Cytokine levels in spleen cell culture stimulated or not with LPS (1 µg/ml), Pam3Cys (1 µg/ml) or CpG DNA (1 µg/ml) from either control (non-infected) or infected C57BL/6 WT, TLR2^−/−, and TLR9^−/− mice seven days post-infection. Supernatants from spleen cell cultures from C57BL/6 WT (A), TLR2^−/− (B) and TLR9^−/− mice (C) were analyzed for IL-12/IL-23p40 or TNF-α after 48h. Data are representative of two independent experiments. *p<0.05 indicates statistical significance when comparing cytokine release by spleen cells stimulated or not with TLR agonist in a same group (infected or not infected mice).</p

Schematic representation of the complementary effect of TLR2 and TLR9 activation during <i>T.cruzi</i> infection.

<p>A) The early release of IFN-γ induces an increase of TLR9 expression in DC and primes cells to TLR9 response (1). The high levels of IL-12/IL-23p40 secreted by DCs down-regulate the TLR9 responses of monocytes/macrophages by modulating the TLR9 expression (2). On the other hand, TLR2 is used by macrophage population to produce TNF-α (3). B) In DCs, TLR2 regulates negatively TLR9-dependent IL-12/IL-23p40 production by modulating signaling pathway.</p

Evaluation of the capacity of F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high populations to produce TNF-α and IL-12/IL-23p40 during the acute phase of <i>T.cruzi</i> infection.

<p>Splenic cells were analyzed seven days post-infection. (A) Representative flow cytometry plots showing the exclusion of debris and non-interest population of interest (FSC-H X SSC-H) and the assortment of immune cells from non-infected or infected mice. Representative flow cytometry plots showing intracellular cytokine in the different cells from non-infected or infected mice. (B) Frequencies of IL-12/IL-23p40⁺ or TNF-α⁺ splenic cells (F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ or MHCII⁺CD11c^high) (mean ± SD of four mice) isolated from non-infected or infected mice. Data are representative of two independent experiments. **p<0.01 indicates statistical significance when comparing the percentage of the same cell population from infected versus non infected mice involved in TNF-α or IL-12/IL-23p40 production.</p

Comparison of the capacity of F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high populations to respond to TLR9 agonist during acute phase of <i>T.cruzi</i> infection.

<p>Intracellular TNF-α and IL-12/IL-23p40 were analyzed by flow cytometry in spleen from C57BL/6 mice seven days post-infection. Splenic cells were cultured in medium alone, with CpG DNA (1 µg/ml), LPS (1 µg/ml) or Pam3Cys (1 µg/ml). (A) Frequencies of splenic TNF-α⁺ or IL-12/IL-23p40⁺ cells (F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high) after stimulation with CpG DNA (1 µg/ml) (mean ± SD of four mice) or (B) frequencies of splenic TNF-α⁺ or IL-12/IL-23p40⁺ cells (F4/80⁺CD11b⁺ and F4/80^lowCD11b⁺) stimulated with LPS (1 µg/ml) or Pam3Cys (1 µg/ml) (mean ± SD of four mice) isolated from infected and non-infected mice. Data are representative of three independent experiments. *p<0.05 and **p<0.01 indicate statistical significance when comparing the percentage of the same cell population from infected or not infected mice cultured in the same conditions.</p

Adoptive transfer of WT macrophages in TLR9^−/− mice allow normal TLR response of F4/80⁺CD11b⁺ cells after <i>T.cruzi</i> infection.

<p>Representative flow cytometry plots showing (A) IL-12/IL-23p40⁺MHCII⁺CD11c^high and (B, C) IL-12/IL-23p40⁺F4/80⁺CD11b⁺ cells, stimulated or not with CpG DNA (1 µg/ml), from non-infected or infected C57BL/6 WT, TLR9^−/−, TLR2^−/− and TLR9^−/− or TLR2^−/− mice that received WT macrophages (Rec TLR9^−/− or Rec TLR2^−/− mice). (D) Frequencies of IL-12/IL-23p40⁺F4/80⁺CD11b⁺ cells stimulated with CpG DNA (mean ± SD of four mice) isolated from non-infected or infected C57BL/6 WT, TLR9^−/−, and Rec TLR9^−/− mice. **p<0.01 indicates statistical significance when compared the percentage IL-12/IL-23p40⁺F4/80⁺CD11b⁺ cells after stimulation with CpG DNA in infected C57BL/6 WT or Rec TLR9^−/− mice.</p

Role of TLR2 and TLR9 in TNF-α and IL-12/IL-23p40 production by F4/80⁺CD11b⁺, F4/80^lowCD11b⁺ and MHCII⁺CD11c^high populations from mice acutely infected with <i>T.cruzi</i>.

<p>Intracellular cytokine was analyzed by flow cytometry in spleen from C57BL/6 WT, TLR2^−/−, and TLR9^−/− mice seven days post-infection. Frequencies of splenic TNF-α⁺ (A) or IL-12/IL-23p40⁺ (B) cells (mean ± SD of four mice) isolated from infected and non-infected mice. Data are representative of two independent experiments. *p<0.05 and **p<0.01 indicate statistical significance when comparing the percentage of the same cell population from knockout versus C57BL/6 WT infected mice involved in TNF-α or IL-12/IL-23p40 production.</p

Knockdown of CD36-like receptors in hemocytes reduced phagocytosis and favored <i>Leishmania</i> proliferation in flies.

<p>(A) While control flies did not allow parasite proliferation, the RNAi lines had a significant increase in parasite burden, determined by two-way ANOVA, n = 10. (B) Hemocytes were harvested from adult flies 1 h post-infection for microscopic scoring of infection. The knockdown of CD36-like receptors reduced the number of intracellular parasites (**: <i>p</i> < 0.01 one-way ANOVA, n = 4). (C) Two out of three RNAi fly lines had a higher percentage of promastigotes at 8 days post-infection. One-way ANOVA, n = 10. Whiskers represent 10%-90% limits of the sample. Representative data from two independent assays.</p

An <i>in vivo</i> RNAi screen identified factors required for <i>Drosophila</i> resistance to <i>L</i>. <i>amazonensis</i> infection.

<p>Nine hemocyte-expressed factors were identified as being required to control <i>Leishmania</i> infection after screening a collection of 32 RNAi or mutant lines (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005669#ppat.1005669.t001" target="_blank">Table 1</a>). Three general phagocytic factors (Rac2, β’COP and ftz-f1, A-C), and six scavenger receptors (D-I) were identified. The receptor hits fell into three classes: two were Class C receptors (a group unique to some Diptera) (D, E), one Nimrod type (F), and three CD36-like receptors (SR-B family): CG10345, CG31741 and croquemort (G-J). Control flies for all RNAi assays included the <i>Hml(Δ)</i>-Gal4 driver but no UAS-RNAi (A-I), while heterozygous flies were used as controls for the <i>crq</i> mutant (J). At least 60 flies were used per sample, survival curve analysis was performed using a Log-rank (Mantel-Cox) test, indicated <i>p</i>-values were determined from the comparison of control and RNAi/mutant-infected flies.</p

Plasmatocytes from adult flies phagocytose and control <i>Leishmania</i> parasites.

<p>DsRed-expressing amastigotes were injected alone or with polystyrene beads in <i>Hml(Δ)</i>Gal4-eGFP flies. At indicated times, hemocytes were harvested from the hemolymph and analyzed by confocal microscopy. (A) In eGFP-expressing hemocytes from flies injected with polystyrene beads and amastigotes, the cells contained large amounts of intracellular beads (blue) and just one intracellular parasite (red) confirming the inhibitory effect of beads in phagocytosis. (B) 24 h post-injection, plasmatocytes from flies injected only with parasites contained several amastigote forms, while at 72 h after infection some amastigotes differentiated to the long and flagellated promastigote form and were observed inside plasmatocytes (arrow in (C)). (D) Phagocytosis blockage by injection of polystyrene beads increased the parasite burden of flies over time. The control group, flies not injected with beads, prevented parasite growth without completely clearing infection. Parasite burdens were assayed by limiting dilution of single flies, with a minimum of 12 animals per time point. Whiskers represent 10%-90% limits of the sample. Statistical significance was determined by two-way ANOVA.</p