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

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

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

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

Additional file 2: Figure S2. of Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B

Representative FACS density plots showing the gate strategy for the identification of iNOS within F4/80+ gated on live CD45+ leucocytes in the trigeminal ganglia (a) and spleen (b) from a single HSV1-infected WT mouse. A minimum of 100,000 events was acquired for analysis. (PPTX 2700 kb

Additional file 1: Figure S1. of Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B

Representative FACS density plots showing the gate strategy for the identification of IL-1β within CD11c+MHCIIhigh gated on live CD45+ leucocytes in the trigeminal ganglia (a) and spleen (b) from a single HSV1-infected WT mouse. A minimum of 100,000 events was acquired for analysis. (PPTX 386 kb