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Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8 + T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim +/- , Bim -/- mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim -/- mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim +/- mice. At the peak of parasitemia, peritoneal macrophages of Bim -/- mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim -/- splenocytes. Moreover, an impaired anti-T. cruzi CD8 + T-cell response was found in Bim -/- mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim -/- mice and place Bim as an important protein in the control of T. cruzi infections., (© 2021. The Author(s).)

Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite and the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people worldwide. Although the role of TLR and Nod1 in the control of T. cruzi infection is well-established, the involvement of inflammasomes remains to be elucidated. Herein, we demonstrate for the first time that T. cruzi infection induces IL-1β production in an NLRP3- and caspase-1-dependent manner. Cathepsin B appears to be required for NLRP3 activation in response to infection with T. cruzi, as pharmacological inhibition of cathepsin B abrogates IL-1β secretion. NLRP3(-/-) and caspase1(-/-) mice exhibited high numbers of T. cruzi parasites, with a magnitude of peak parasitemia comparable to MyD88(-/-) and iNOS(-/-) mice (which are susceptible models for T. cruzi infection), indicating the involvement of NLRP3 inflammasome in the control of the acute phase of T. cruzi infection. Although the inflammatory cytokines IL-6 and IFN-γ were found in spleen cells from NLRP3(-/-) and caspase1(-/-) mice infected with T. cruzi, these mice exhibited severe defects in nitric oxide (NO) production and an impairment in macrophage-mediated parasite killing. Interestingly, neutralization of IL-1β and IL-18, and IL-1R genetic deficiency demonstrate that these cytokines have a minor effect on NO secretion and the capacity of macrophages to control T. cruzi infection. In contrast, inhibition of caspase-1 with z-YVAD-fmk abrogated NO production by WT and MyD88(-/-) macrophages and rendered them as susceptible to T. cruzi infection as NLRP3(-/-) and caspase-1(-/-) macrophages. Taken together, our results demonstrate a role for the NLRP3 inflammasome in the control of T. cruzi infection and identify NLRP3-mediated, caspase-1-dependent and IL-1R-independent NO production as a novel effector mechanism for these innate receptors.

Chagas disease is a Trypanosoma cruzi-induced zoonosis that has no natural cure. Local damage induced by the parasite and the immune response causes chronic heart and digestive lesions. Efforts to develop a therapeutic vaccine that boosts the immune response to completely clear the parasite are needed because there is no effective treatment for chronically infected patients. In an attempt to modify the host-parasite equilibrium to increase parasite destruction, we analyzed cardiopathy and the immune response in chronically infected mice that were challenged with live homologous parasites. Challenge with a single dose of parasite increased CD4(+) and CD8(+) T cell populations, gamma interferon (IFN-γ) production, and serum-specific IgG levels. However, subpatent parasitemias and cardiac tissue were not affected. Because of the short duration of the immune boost after a single challenge, we next evaluated the impact of four parasite doses, administered 3 weeks apart. At 1 to 2 months after the last dose, the numbers of CD4(+) T cells and IFN-γ-producing CD4(+) memory cells and the CD4(+) T cell proliferative response to T. cruzi antigen were increased in the spleen. The frequency of IFN-γ-producing CD8(+) memory cells in the blood was also increased. However, the sustained challenge did not favor TH1 development; rather, it induced an increase in serum-specific IgG1 levels and mixed TH1/TH2 cytokine production. Moreover, there were no significant changes in cardiac lesions and subpatent parasitemias. In conclusion, we believe that this study may help in elucidating the necessary elements for a successful therapeutic vaccine which may reduce cardiomyopathy in chronically infected human patients.

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ) subsets, namely small peritoneal MØ (SPM) and large peritoneal MØ (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for β-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5x10(6) trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+)CD8(+), CD69(+)CD4(+) and CD25(+)CD122(+)CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Background/aim: Chagas' disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process., Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis., Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p < 0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p < 0.01) and NOS (544%, p < 0.001). Moreover, the intensity of nitrotyrosine (16%, p < 0.01), NOS (38%, p < 0.05) and S100beta (21%, p < 0.001) immunoreactivities were also augmented. No IFN-gamma-labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled., Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100beta may contribute to NOS activation in the absence of IL-12., (Copyright 2009 S. Karger AG, Basel.)

The physiopathology of Chagas' disease has been largely defined in murine infections with virulent strains which partially represent parasite diversity. This report reviews our studies with Sylvio X10/4 parasites, a Trypanosoma cruzi clone that induces no acute phase but in C3H/He mice leads to chronic myocarditis resembling the human disease.

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas' disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite-specific IgG serum levels in immune-deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN-gamma was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN-gamma or IL-12p40, a molecule shared by IL-12 and IL-23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL-12p40-KO mice produced IgG2a and IgG1 and IFN-gamma-KO mice produced only IgG1. The IFN-gamma-protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS-KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4(+) or CD8(+) T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28-KO mice were unable to produce anti-T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28-mediated signalling are essential for the non-sterile control of Sylvio X10/4 parasites.

Protective immunity against lethal infection is developed when BALB/c or C57BL/6 mice are immunized with plasmids containing genes from the protozoan parasite Trypanosoma cruzi. However, genetic vaccination of the highly susceptible mouse strain A/Sn promoted limited survival after challenge. This observation questioned whether this type of vaccination would be appropriate for highly susceptible individuals. Here, we compared the protective efficacy and the immune response after individual or combined genetic vaccination of A/Sn mice with genes encoding trans-sialidase (TS) or the amastigote surface protein-2 (ASP-2). After challenge, a significant proportion of A/Sn mice immunized with either the asp-2 gene or simultaneously with asp-2 and ts genes, survived infection. In contrast, the vast majority of mice immunized with the ts gene or the vector alone died. Parasitological and histological studies performed in the surviving mice revealed that these mice harbored parasites; however, minimal inflammatory responses were seen in heart and striated muscle. We used this model to search for an in vitro correlation for protection. We found that protective immunity correlated with a higher secretion of interferon- by spleen cells on in vitro restimulation with ASP-2 and the presence of ASP-2-specific CD8 cells. Depletion of either CD4 or CD8 or both T-cell subpopulations prior to the challenge rendered the mice susceptible to infection demonstrating the critical contribution of both cell types in protective immunity. Our results reinforce the prophylactic potential of genetic vaccination with asp-2 and ts genes by describing protective immunity against lethal T. cruzi infection and chronic tissue pathology in a highly susceptible mouse strain.

Challenge of 1-yr Trypanosoma cruzi chronically infected mice with trypomastigotes results in a consistent reduction of parasite dissemination that correlates with spleen activation and increase in the anti-T. cruzi effector immune mechanisms. That is, parasite challenge results not only in elimination of the inoculum but also in a drastic decrease in basal subpatent parasitemia levels as revealed by transferring blood samples to immunosuppressed mice. Parasite elimination correlated with (1) a brief and intense burst in the ability of spleen cells to produce interferon-gamma, (2) an increase in total IgG2a-producing spleen cells, (3) higher parasite-specific IgG2a serum levels, and (4) an accumulation of non-B, non-T class II+ cells in the spleen. Furthermore, challenged, chronically infected mice had increased numbers of B, CD4+, and CD8+ large spleen cells. Besides reinforcing the activation of protective Th1 effector mechanisms, challenge with T. cruzi also induced Th2 effector molecules, such as interleukin (IL)-10 and IL-4, and IL-4-dependent IgG1. Our results are the first evidence that the immune system of T. cruzi chronically infected mice can be optimized in its ability to restrict parasite dissemination, opening the possibility that therapeutic vaccination could be used to reduce the parasite load and pathology of patients with chronic Chagas' disease.

Chagas' disease is a chronic infection caused by Trypanosoma cruzi and represents an important public health burden in Latin America. Frequently the disease evolves undetectable for decades, while in a significant fraction of the affected individuals it culminates in death by heart failure. Here, we describe a novel murine model of the chronic infection with T. cruzi using a stable clone isolated from a human patient (Sylvio X10/4). The infection in the C3H/HePAS mouse strain progresses chronically and is mainly characterized by intense cardiac inflammatory lesions that recapitulate the chronic cardiac pathology observed in the human disease. Moderate striated muscle lesions are also present in C3H/HePAS mice. Viable parasites are detected and recovered from the chronic heart lesions of C3H/HePAS mice, supporting the current notion that development of heart pathology in Chagas' disease is related to parasite persistence in the inflamed tissue. By contrast, in infected A/J mice, chronic inflammatory lesions are targeted to the liver and the skeletal muscle, while pathology and parasites are undetectable in the heart. The phenotypic analysis of F(1) (A/J x C3H/HePAS) and F(2) (A/J x C3H/HePAS) mice suggests that the genetic predisposition to develop the inflammatory lesions caused by T. cruzi (Sylvio X10/4 clone) is heterogeneous because the heart and liver pathology segregate in the F(2) generation. These findings raise the hypothesis that the pathology heterogeneity observed in humans with Chagas' disease (absence and presence of cardiac or digestive chronic lesions) may be attributable to host genetic factors.

To obtain low and high parasite loads in the acute phase of Chagas' disease, A/J mice were infected with 10(3) or 10(5) Trypanosoma cruzi trypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8(+) and B220(-) CD5(-) cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220(+)), CD4(+), and CD8(+) large lymphocytes; (iii) a shift of CD4(+) cells towards a CD45RBLow phenotype; (iv) increased frequencies of both CD45RBLow and CD45RBHigh large CD4(+) cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas' disease pathology at the late chronic phase of the disease.

We have recently described that Trypanosoma cruzi parasites of the reticulotropic Y strain increase their resistance to antibody-induced clearance during their interaction with the vertebrate host immune system. In the present study, we observed that trypomastigotes of the myotropic CL strain isolated from normal host also display an increased resistance to immune clearance when compared to parasites obtained from immunosuppressed donors. Through fluorescence-activated cell sorting analysis, we have observed that the high expression of membrane-bound IgM antibodies on Y and CL trypomastigotes correlates with their enhanced resistance to Ig-induced clearance. Trypomastigotes from normal mice were essentially refractory to the in vitro binding of immunoglobulins, showing that their membrane structures were completely covered by IgM antibodies. These findings suggest that this isotype does not efficiently mediate immune clearance. Moreover, membrane-bound IgM antibodies limited the amount of IgG attached to the parasite and, as a consequence, impaired efficient immune clearance. Through this mechanism, trypomastigotes of T. cruzi could increase their persistence in the bloodstream thus favoring parasite transmission to its hematophagous host vector in the early acute phase of the disease.

To investigate functional changes in Trypanosoma cruzi parasites induced during their interaction with the vertebrate host, we compared the blood clearance profiles of blood forms isolated from infected normal mice (Reg-Tc) or from infected mice immunodepressed after treatment with cyclophosphamide (Cy-Tc). Parasite blood numbers were measured at various time intervals in animals injected intravenously (i.v.) with 1-2 x 10(6) T. cruzi of either isolate. In the absence of added immune sera (spontaneous clearance), Reg-Tc and Cy-Tc were cleared from blood at similar rates. However, when acute immune mouse serum (Ac-IMS) was injected i.v. 2 min after inoculation of parasites, a significant proportion of Cy-Tc only was cleared from the blood an hour later, whereas Reg-Tc were not, their clearance profile being identical to that observed in mice injected with normal mouse serum. Cy-Tc susceptibility to Ac-IMS was not the result of a toxic effect of cyclophosphamide over T. cruzi as parasites recovered from animals immunodepressed by irradiation before infection were cleared similarly by acute serum. Contrary to Ac-IMS, chronic immune mouse serum induced similar rates of disappearance of Reg-Tc and Cy-Tc from blood. Our results suggest the occurrence of T. cruzi selection or modification during the acute phase, which leads to an increased parasite resistance to the clearance properties of acute-phase antibodies.