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

Abstract

Resistance or susceptibility to T. cruzi infection is dependent on the host immunological profile. Innate immune receptors, such as Toll-like receptors (TLRs/TLR2, TLR4, TLR7, and TLR9) and Nod-like receptors (NLRs/NOD1 and NLRP3 inflammasome) are involved with the resistance against acute experimental T. cruzi infection. Here, we evaluated the impact of T. cruzi virulence on the expression of innate immune receptors and its products in mice. For that, we used six T. cruzi strains/isolates that showed low (AM64/TcIV and 3253/Tc-V), medium (PL1.10.14/TcIII and CL/TcVI), or high (Colombian/Tc-I and Y/TcII) virulence and pathogenicity to the vertebrate host and belonging to the six discrete typing units (DTUs)-TcI to TcVI. Parasitemia, mortality, and myocarditis were evaluated and correlated to the expression of TLRs, NLRs, adapter molecules, cytokines, and iNOS in myocardium by real time PCR. Cytokines (IL-1β, IL-12, TNF-α, and IFN-γ) were quantified in sera 15 days after infection. Our data indicate that high virulent strains of T. cruzi, which generate high parasitemia, severe myocarditis, and 100% mortality in infected mice, inhibit the expression of TLR2, TLR4, TLR9, TRIF, and Myd88 transcripts, leading to a low IL-12 production, when compared to medium and low virulent T. cruzi strains. On the other hand, the high virulent T. cruzi strains induce the upregulation of NLRP3, caspase-1, IL-1β, TNF-α, and iNOS mRNA in heart muscle, compared to low and medium virulent strains, which may contribute to myocarditis and death. Moreover, high virulent strains induce higher levels of IL-1β and TNF-α in sera compared to less virulent parasites. Altogether the data indicate that differential TLR and NLR expression in heart muscle is correlated with virulence and pathogenicity of T cruzi strains. A better knowledge of the immunological mechanisms involved in resistance to T. cruzi infection is important to understand the natural history of Chagas disease, can lead to identification of immunological markers and/or to serve as a basis for alternative therapies.

Keywords: NLRP3 inflammasome; Nod-like receptor; Toll-like receptor; Trypanosoma cruzi; innate immune receptors; mice; virulence.

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).

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.

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).

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).

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).

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.

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.

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).