IFN-gamma Plays a Unique Role in Protection against Low Virulent Trypanosoma cruzi Strain

Background: T. cruzi strains have been divided into six discrete typing units (DTUs) according to their genetic background. These groups are designated T. cruzi I to VI. In this context, amastigotes from G strain (T. cruzi I) are highly infective in vitro and show no parasitemia in vivo. Here we aimed to understand why amastigotes from G strain are highly infective in vitro and do not contribute for a patent in vivo infection. Methodology/Principal Findings: Our in vitro studies demonstrated the first evidence that IFN-gamma would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-alpha, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-gamma we found that the latter is crucial for controlling infection by G strain amastigotes. Conclusions/Significance: Our results showed that amastigotes from G strain are highly infective in vitro but did not contribute for a patent infection in vivo due to its susceptibility to IFN-gamma production by host immune cells. These data are useful to understand the mechanisms underlying the contrasting behavior of different T. cruzi groups for in vitro and in vivo infection.CAPES [3038.005295/2011-40]CAPESFAPEMIGFAPEMIG [APQ-00621-11]CNPqCNPqFAPESPFAPESP [10-50959-4

Monitoring presence of activated NK cells in bloodstream post amastigote intraperitoneal inoculation.

<p>Flow cytometry was performed with mononuclear cells prepared from mice left without any inoculation (a, b, c, d and e), and mice that were given intraperitoneal G strain amastiogotes at day-8 post-inoculation (a′, b′, c′, d′ and e′) at day-25 (b″, c″, d″ and e″). Note the higher percentage of activated NK cells at day-8 post-inoculation (p<0.01). Gates: L – lymphocytes and LGL – large granular lymphocytes (NK cells).</p

Inflamatory and IFN-γ treated naive macrophages impaired cell-cycling trypomastigotes differentiate from amastigotes.

<p>Amastigotes did not multiply in inflammatory peritoneal macrophages in an <i>ex-vivo</i> assay (<b>A</b>). Treatment with recombinant IFN-γ controlled in a dose dependent manner trypomastigotes release from bone marrow derived naive macrophages (<b>B</b>) (p<0.001).</p

G strain parasitemia is only detected after dexmethasone treatment.

<p>G strain amastigote progeny is generated in Hela and MEF cells <i>in vitro</i> but no trypomastigotes are released at a detectable level in the bloodstream in C57BL/6 mice except if the latter are given dexmethasone. Amastigotes from G strain invasion (<b>A</b>) and multiplication (<b>B</b>) in HeLa and MEF cells. Parasitemia was not observed in wild type C57BL/6 and BALB/c mice (<b>C</b>). C57BL/6 mice that were given 100,000 amastigotes intraperitoneally at day 0 and that were given dexamethasone from day 10 onward displayed parasitemia from days 24 post amastigote inoculation (<b>D</b>). *p<0.001.</p

Deletion in IL12p40 and IFN-γ induced bloodstream parasitemia.

<p>Though with distinct profiles, in mice genetically deleted from either IL12p40, IFN-γ, the G strain trypomastigote progeny was detected in the bloodstream, while in mice deleted from either IL-18 or TNF-α, no trypomastigote progeny was detected. Wild type and IL-12p40 (a, e), IL-18 (b, f), TNF-α (c, g) and IFN-γ (d, h) knockout mice were given intraperitoneally 100,000 G strain amastigotes. Parasitemia values were monitored in mouse blood at 7, 12, 19 and 26 days post-inoculation; survival was checked every day until 30 post-inoculation . (n = 5 mice per group). It was observed parasitemia peak and mortality only for IL-12p40 and IFN-γ KO mice. *p<0.01; ***p<0.001.</p

In iNOS and gp91 KO mice no trypomastigote progeny was detected.

<p>Wild type, iNOS (a, c) and gp91 KO (b, d) mice were given intraperitoneally 100 000 G strain amastigotes. Parasitemia values were monitored in mouse blood at 7, 12, 19 and 26 days post-inoculation ; survival was checked every day until 30 post-inoculation . (n = 5 mice per group).</p

Whatever gene (CD4, CD8, Nod2 and Myd88) deletion, trypomastigotes were never detected in mice bloodstream.

<p>Wild type and CD4 (a, e), CD8 (b, f) Nod2 (c, g) and Myd88 (d, h) knockout mice mice were given intraperitoneally 100,000 G strain amastigotes. Parasitemia values were monitored in mouse blood at 7, 12, 19 and 26 days post-inoculation; survival was checked every day until 30 post-inoculation . (n = 5 mice per group).</p