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

Expression of Toxoplasma gondii-Specific Heat Shock Protein 70 during In Vivo Conversion of Bradyzoites to Tachyzoites

Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain of Toxoplasma gondii. In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-γ) or anti-tumor necrosis factor alpha (TNF-α) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-γ or anti-TNF-α treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis

Heme oxygenase-1 activity is involved in the control of Toxoplasma gondii infection in the lung of BALB/c and C57BL/6 and in the small intestine of C57BL/6 mice

Abstract Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice

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

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

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

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

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure

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