Positive CD3, Gr1 and F4/80 hepatic leukocytes expressing TLR2, TLR4 and TLR9.

<p>The TLR expression, after gating on CD3, Gr1 and F4/80, are showed in A, B and C respectively. Absolute numbers of double positive cells for CD3 (A), Gr1 (B) or F4/80 (C) and TLR2, TLR4 or TLR9 are shown. (*) Statistical significant increase comparing leukocytes from infected mice vs non-infected control mice in each mouse strain. Arrowhead indicates statistical difference in TLR9 expression at 21dpi comparing BALB/c vs B6. These assays were performed with six animals per group. Data are representative of one of three independent experiments. A p-value <0.05 was considered significant using Two-way ANOVA test.</p

Cytokines produced by hepatic leukocytes from infected and control BALB/c and B6 mice.

<p>A–H) Purified leukocytes were cultured for 72 hs, and IL6, TNFα, IL12, IL1β, TGFβ, IL10, IFNγ and IL4 were assayed in supernatants. Six animals per group were analyzed and data are representative of one of three independent experiments. Statistical significance evaluated by Two-way ANOVA-Bonferroni's post-hoc test is indicated with a p-value <0.05. (*) To compare infected vs control mice.</p

iNOS expression on hepatic tissue and nitrite production by liver leukocytes.

<p>Kinetics of gp91 and p47-phox NADPDH oxidase expression in liver tissues from infected and control BALB/c and B6 mice. A) Liver iNOS expression at14, 21 and 24 dpi, analyzed by Western blot. Non-infected control mice are indicated as “C”. The square indicates an iNOS significant increase in infected compared to control B6 mice. B) Nitrites were measured in supernatants of cultured hepatic leukocytes from infected (14 and 21dpi) and control mice. C) PCR products in agarose gel electrophoresis and western blot assays for GAPDH, actin, gp91 and p47-phox. The square indicates a significant increase of p47-phox subunit at 24dpi in B6 compared to BALB/c. D) Immunofluorescence merge for gp91 and p47-phox in control and infected liver from BALB/c and B6 mice, at 24dpi, are shown. Six animals/group were analyzed and data are representative of one of three independent experiments. A p-value <0.05 was considered significant using Two-way ANOVA test. (*) To compare infected vs control mice in each mouse strain. (**) To compare B6 vs BALB/c mice.</p

Hepatic inflammation and steatosis in WT and TLR4-/- mice with or without <i>T</i>. <i>cruzi</i> infection.

<p><b>(A)</b> Liver tissue from all groups was subjected to hematoxylin and eosin (H&E at 400x) and Sudan Black IV staining (A, Insert) The arrows represent inflammatory foci. Scale bar = 20 μm. (<b>B)</b> Hepatic triglyceride and cholesterol contents were determined and expressed as mg/g of liver tissue of WT groups. (<b>C)</b> The numbers of IHLs were similar in mice of both genotypes with infection but the degree of steatohepatitis was significantly higher in MFD vs. LFD WT mice (*p < 0.05). All results are shown at 24 weeks of treatment and were representative of at least three independent experiments. Data are shown as mean ± SEM of more than 4 mice per group.</p

Medium-fat diet leads to increased parasitemia and parasite load in the liver.

<p><b>(A)</b> Parasitemia was determined at different time post <i>T</i>. <i>cruzi</i> infection in I+LFD and I+MFD WT mice, n = 10 per group. <b>(B)</b> Quantitative assessment of the parasite load by q-PCR in the liver of infected groups. Parasite quantification in DNA samples of I+LFD and I+MFD groups was performed amplifying a TCZ <i>T</i>. <i>cruzi</i> satellite sequence at 24 weeks post treatment. The relative load of parasites/liver was normalized against the eEF2 housekeeping gene control. Two positive samples, two negative samples and non-template DNA were included in every q-PCR. All data are shown as mean ± SEM of four mice per group from one experiment representative of two performed. A p<0.05 was considered significant using two-tailed T test.</p

The outburst of systemic inflammatory cytokines is highly dependent on TLR4 signaling and increased by parasite infection.

<p>The cytokine production was quantified by ELISA in plasma collected from all groups of mice at 24 weeks of treatment with LFD, MFD, I+LFD or I+MFD. <b>(A)</b> WT CCL2: **p < 0.01 MFD vs. LFD and *p < 0.05 I+LFD vs. LFD; IL6: *p < 0.05 MFD vs. LFD, **p < 0.01 I+LFD vs. LFD, ***p < 0.001 MFD vs. I+MFD and **p < 0.01 I+LFD vs. I+MFD; IFNγ: *p < 0.05 MFD vs. LFD, **p < 0.01 I+LFD vs. LFD, **p < 0.01 MFD vs. I+MFD and *p < 0.05 I+LFD vs. I+MFD; IL17: **p < 0.01 MFD vs. LFD, **p < 0.01 I+LFD vs. LFD, ***p < 0.001 MFD vs. I+MFD and *p < 0.05 I+LFD vs. I+MFD. <b>(B)</b> TLR4-/- CCL2: *p < 0.05 I+LFD vs. LFD; IFNγ: *p < 0.05 MFD vs. I+MFD; IL17: **p < 0.01 I+LFD vs. LFD and ***p < 0.001 MFD vs. I+MFD. All data are shown as mean ± SEM of four to eight mice per group from one experiment representative of two performed. A p-value <0.05 was considered significant using Two-way ANOVA test.</p

The local T cell response is induced by MFD and exacerbated by <i>T</i>. <i>cruzi</i> infection.

<p>IHLs from different groups of mice obtained at 24 weeks were stained with anti-CD3, anti-CD4, anti-CD8, anti-TNFα, anti-IFNγ, anti-IL17, anti-IL10, anti-CCL2, anti-CCL3 and anti-CD107 Abs. <b>(A)</b> The absolute numbers of CD3⁺ and <b>(B)</b> the absolute numbers of CD3⁺CD4⁺ and CD3⁺CD8⁺ cells in liver are indicated. <b>(C)</b> Percentage of TNFα and IFNγ producing CD4⁺ T cells in liver of WT are shown by intracellular staining. Percentage of <b>(D)</b> IL17 and <b>(E)</b> IL10 producing CD4⁺ T cells in liver of WT are shown by intracellular staining. <b>(F)</b> Percentages of CCL2 and CCL3 producing CD3+ T cells in liver of WT are shown by intracellular staining. <b>(G)</b> Percentages of CD107a⁺CD8⁺ T cells. IHLs from all groups were cultured in the presence of PMA plus ionomycin and monensin for 5 h and stained with corresponding antibodies. Data are shown as mean ± SEM of more than 4 mice per group from one experiment representative of three performed.</p