Coinfection with Different Trypanosoma cruzi Strains Interferes with the Host Immune Response to Infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3+ and CD4+ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice

Flow cytometry analysis of splenic CD4⁺ T and CD8⁺ T cells from BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and flow cytometry analysis of splenic CD4⁺ T and CD8⁺ T cells were performed at 7, 14 and 21 days p.i. Symbols as follow: (A) TNF-α⁺/CD4⁺; (B) TNF-α⁺/CD8⁺; (C) IL-10⁺/CD4⁺; (D) IL-10⁺/CD8⁺; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mice groups, respectively.</p

Representative flow cytometry charts illustrating the cytokine synthesis by spleen MAC-3⁺ and CD4⁺ T cells from BALB/c mice infected with JG and/or CL Brener.

<p>Results are presented in density plot format. The analyses were performed by quadrant statistics expressed as the percentage of cytokine⁺ cells within gated MAC-3⁺ at 14 days p.i., and CD4⁺ cells at 14 and 21 days p.i. in splenocytes cultures from naïve, JG single infected, CL Brener single infected and JG and CL Brener coinfected mice. Cytokine flow cytometry charts demonstrate the enhanced percentage of cytokine⁺ cells in all infected mice. Outstanding levels of TNF-α-producing CD4⁺ T cells were contra balanced by high frequency of IL-10-producing MAC-3⁺ and CD4⁺ T cells in coinfected mice.</p

Hemoglobin level, hematocrit and red blood cell count in JG and/or CL Brener infected mice at 21 days p.i.

*<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection); or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 proportion via intraperitoneal route, and haemoglobin level, hematocrit determination and red blood cell count were assessed at 7, 14 and 21 days p.i. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments).</p>*<p>and ^†represent <i>P</i><0.05 compared with naïve and JG mice groups, respectively.</p

Genetic characterization of nuclear and mitochondrial markers of the JG and CL Brener <i>T. cruzi</i> strains.

a<p>Major lineages nomenclature for <i>T. cruzi</i> strains in accordance with Zigales <i>et al.</i> (2009).</p>b<p><i>Alu</i>I restriction fragment length polymorphism (RFLP) of the <i>T. cruzi</i> cytochrome oxidase subunit II (COII) gene (Freitas <i>et al.</i>, 2006).</p>c<p><i>rDNA 24Sα</i> group of the <i>T. cruzi</i> as defined by Souto <i>et al.</i> (1996).</p

Flow cytometry analysis of splenic MAC-3⁺ and NK cells from BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and flow cytometry analysis of splenic MAC-3⁺ and NK cells was performed at 7, 14 and 21 days p.i. Symbols as follows: (A) TNF-α⁺/MAC-3⁺; (B) TNF-α⁺/NK; (C) IL-10⁺/MAC-3⁺; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mouse groups, respectively.</p

Heart histopathology, morphometric analysis and parasitism in BALB/c mice infected with JG and/or CL Brener.

<p>HE-stained representative myocardial sections from mice infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route were collected at 21 days p.i. (A–D) and were histopathologically analyzed in original magnifications of 10× or 40× (details). Total number of parasite nests (E) and nucleus area quantification (cellularity) were assessed using computer-aided morphometry (F), Symbols as follows: A or white bar: Naïve mice; B or light gray bar: JG infected mice; C or black bar: CL Brener infected mice; and D or dark gray bar: JG and CL Brener infected mice. ^aRepresents number of parasite nests counted in three semi-consecutive sections. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mice groups, respectively.</p

Serum cytokine levels in BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and serum cytokine levels were assessed at 7, 14 and 21 days p.i. Symbols as follows: (A) TNF-α; (B) CCL2; (C) IL-6; (D) IFN-γ; (E) IL-10; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 compared with naïve, JG and CL Brener mouse groups, respectively.</p

Serum TNF-α, CCL2 or IFN-γ to serum IL-10 ratios in BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and TNF-α/, CCL2 or IFN-γ to serum IL-10 ratios were assessed at 7, 14 and 21 days p.i. Symbols as follows: (A) TNF-α/IL-10; (B) CCL2/IL-10; (C) IFN-γ/IL-10; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mouse groups, respectively.</p