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

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

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

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

Immunostimulatory and adjuvant activity of TLR9 agonists derived from <i>T. cruzi</i> genome.

<p>(<b>A</b>) PBMCs derived from healthy donors were stimulated with human B-class-like CpG ODNs derived from the <i>T. cruzi</i> genome with four different concentrations (3.0, 1.0, 0.3, and 0.1 µM) and the levels of IFN-α measured in the cell culture supernatants 24 h later. The CpG ODN 2007 was used as positive controls for human B-class ODNs. PBMC experiments were performed in three different donors, yielding similar results. (<b>B</b>) Proinflammatory activity of mouse B class-like CpG motifs was evaluated in inflammatory macrophages from WT (C57BL/6), <i>TLR4</i>^−/− and <i>TLR9</i>^−/− mice. ODNs were tested at different concentrations (1.5, 0.3 and 0.06 µM) and LPS, as well as CpG ODN 7909 were used as positive controls for TLR4 and TLR9 activation, respectively. IL-12 (p40) was measured in the macrophage culture supernatants 24 h after cellular stimulation. (<b>C</b>) C57BL/6 mice received three immunization doses with alum alone, OVA plus alum or OVA plus alum associated with either CpG ODNs B344, B287, B128 or 7909 (positive control). The levels of OVA-specific total IgG, IgG1 and IgG2c were assessed by ELISA. (<b>D</b>) Amount of IFN-γ secreted by splenocytes after stimulation with OVA derived CD4⁺ T or CD8⁺ T cell epitopes was evaluated in culture supernatants 72 hours post-stimulation. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.</p

Sequences of synthesized CpG oligonucleotide.

*<p>CpG ODNs used for immunization protocolsz.</p

<i>T. cruzi</i> derived GIPLs are TLR4 agonists and promote high levels of antigen-specific IgG2c antibodies as well as IFN-γ production by CD4⁺ T cells.

<p>(<b>A</b>) CHO cells control (TLR2⁻/TLR4⁻) or expressing TLR2 (TLR2⁺) or TLR4 (TLR4⁺) were either left untreated (solid gray) or exposed to 100 µg/ml of GIPLs from <i>Trypanosoma cruzi</i> Tulahuen (GTH), Y strain (GY) (black line). MALP-2 (10 ηg/ml) and LPS (200 ηg/ml) were used as positive controls for activation of TLR2⁺ or TLR4⁺, respectively. (<b>B</b>) TLR4⁺ cells were activated with different preparations of GIPLs in the presence of polymyxin B (PB). LPS was used as control. (<b>C</b>) OVA specific immune responses induced by immunization with TLR2 or TLR4 agonists associated with OVA absorbed in alum. Mice were immunized with three doses on days 0, 14 and 28. The production of total IgG, IgG1 and IgG2c were assessed by ELISA using the sera from immunized mice, at day 9 after the second boost. (<b>D</b>) To assess the levels of IFN-γ production by T lymphocytes from vaccinated mice, splenocytes were collected 21 days after the third immunization dose and stimulated with either CD4⁺ T or CD8⁺ T cell epitopes from OVA. The results are representative of two independent experiments yielding similar results. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.</p