Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart

Figures

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  Figure 1. Trypanosoma cruzi were grouped in high, medium, and low virulent strains according to parasitemia and survival in mice. Parasitemia (A) and survival (B) in Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed high (Colombian + Y), medium (PL1.10.14/TcIII + CL/TcVI), and low (AM64/TcIV + 3253/TcV) virulence. The data are representative of two independent experiments (n = 10, ten animals were infected with each strain).

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  Figure 2. Intensity of myocarditis in the acute phase in mice is related to Trypanosoma cruzi strain virulence. Representative histological sections of cardiac tissue from Swiss mice, not infected (A) and infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (strains: AM64 + 3253) (n = 10) (B), medium (PL1.10.14 + CL) (n = 10) (C) and high (Colombian + Y) (n = 10) (D) virulence and euthanized 15 days after infection. Quantification of inflammatory mononuclear cells (E) and amastigote nests (F) in the heart tissue of mice. Hematoxylin and eosin staining was used. ×400 magnification. Arrow indicates parasites. The results are expressed as the means ± standard errors. *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 3. Virulence of strains is correlated with TLR2, TLR4, TLR5, and TLR9 inhibition in heart from mice. The mRNA expression levels of TLR1 (A), TLR2 (B), TLR3 (C), TLR4 (D), TLR5 (E), TLR6 (F), TLR7 (G), TLR8 (H), and TLR9 (I) were determined by real-time PCR in heart of Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (AM64 + 3253) (n = 10), medium (PL1.10.14 + CL) (n = 10) and high (Colombian + Y) (n = 10) virulence. The expression levels were normalized to the expression level of GAPDH. The results are expressed as the means ± standard errors. *p < 0.05; **p < 0.01; ***p < 0.001. UC, uninfected control mice (n = 10).

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  Figure 4. Virulence of strains is correlated with TRIF, Myd88, IL-6, IL-12 inhibition in the heart from mice.The mRNA expression levels of TRIF (A), Myd88 (B), IL-10 (C), IL-6 (D), IL-12p35 (E), IL-12p40 (F), and IFN-γ (G) were determined by real-time PCR in the heart from Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (AM64 + 3253) (n = 10), medium (PL1.10.14 + CL) (n = 10), and high (Colombian + Y) (n = 10) virulence. The expression levels were normalized to the expression level of GAPDH. The results are expressed as the means ± standard errors. *p < 0.05; **p < 0.01; ***p < 0.001. UC, uninfected control mice (n = 10).

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  Figure 5. Virulence of strains is correlated with NLRP-3, caspase-1, IL-1β, TNF-α, and iNOS increase in heart from mice. The mRNA expression levels of NLRP3 (A), ASC (B), caspase-1 (C), NOD1 (D), NOD2 (E), RIP2 (F), IL-1β (G), TNF-α (H), IL-18 (I), and iNOS (J) were determined by real-time PCR in the heart from Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of Trypanosoma cruzi strains that showed low (AM64 + 3253) (n = 10), medium (PL1.10.14 + CL) (n = 10), and high (Colombian + Y) (n = 10) virulence. The expression levels were normalized to the expression level of GAPDH. The results are expressed as the means ± standard errors. *p < 0.05; **p < 0.01; ***p < 0.001. UC, uninfected control mice (n = 10).

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  Figure 6. Low TLR2, TLR4, TLR5, TLR7, TLR9, TRIF, IL-6, IL-10, IL-12p35, IL-12p40, IFN-γ and high TNF-α, IL-1β, iNOS mRNA expression in cardiac tissue are correlated with high Trypanosoma cruzi virulence. The mRNA expression levels of TLR2 (A), TLR4 (B), TLR5 (C), TLR7 (D), TLR9 (E), TRIF (F), IL-6 (G), IL-10 (H), IL-12p35 (I), IL-12p40 (J), IFN-γ (K), TNF-α (L), IL-1β (M), and iNOS (N) were determined by real-time PCR in cardiac tissue from Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (AM64 + 3253) (n=10), medium (PL1.10.14 + CL) (n=10), and high (Colombian + Y) (n = 10) virulence. The expression levels were normalized to the expression level of GAPDH. The results are expressed as the means ± standard errors and Spearman test was used. *p < 0.05; ***p < 0.001.

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  Figure 7. Low TLR4, TLR5, TRIF, IL-6, IL-10, IL-12p35 and high TNF-α, IL-1β, iNOS mRNA expression in cardiac tissue are correlated with high Trypanosoma cruzi virulence. The mRNA expression levels of TLR4 (A), TLR5 (B), TRIF (C), IL-6 (D), IL-10 (E), IL-12p35 (F), TNF-α (G), IL-1β (H) and iNOS (I) were determined by real-time PCR in cardiac tissue from Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (AM64 + 3253) (n = 10), medium (PL1.10.14 + CL) (n = 10), and high (Colombian + Y) (n = 10) virulence. The expression levels were normalized to the expression level of GAPDH. The results are expressed as the means ± standard errors and Spearman test was used. *p < 0.05; ***p < 0.001.

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  Figure 8. IL-1β and TNF-α in sera from mice infected with Trypanosoma cruzi are correlated with strain virulence (parasitemia, survival, and myocarditis). IL-1β (A), TNF-α (B), IL-12 (C) and IFN-γ (D) were quantified in the sera (15 days after infection) by ELISA from Swiss mice infected by the intraperitoneal route with 1 × 104 blood trypomastigote forms of T. cruzi strains that showed low (AM64 + 3253) (n = 10), medium (PL1.10.14 + CL) (n = 10), and high (Colombian + Y) (n = 10) virulence. The results are expressed as the means ± standard errors. *p < 0.05; **p < 0.01; ***p < 0.001. UC, uninfected control mice (n = 10).