CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

Antiegressin acts late in the intracellular growth cycle of Trypanosoma cruzi to inhibit parasite egress

Trypanosoma cruzi is the causative agent of Chagas disease, which is characterized by acute and chronic phases. During the former, parasitemia rises dramatically, then decreases significantly during the chronic phase. Immune mechanisms responsible for the parasitemia reduction have not been thoroughly elucidated. The goal of the present study was to further characterize the immune response during chronic infection. Previously, we described antiegressin, an antibody in sera from chronically infected mice. The in vitro presence of antiegressin inhibits parasite egress from infected host cells. Antiegressin appears by day 14 of an in vivo infection and is maintained through at least day 280 postinfection. The in vitro functional activity of antiegressin is initiated late in the 4-6 days intracellular growth cycle of T. cruzi; antiegressin may be added at day 4, inhibiting parasite release at day 5. Immunocytochemical staining using antineuraminidase demonstrates the presence of mature parasites inside host BALB/c fibroblasts grown in the presence of antiegressin. These results demonstrate the ability of antiegressin to inhibit emergence of developmentally mature trypomastigotes from infected host cells late in their intracellular growth cycle. We believe this antibody plays an important and novel role in achieving the low-parasitemia characteristic of chronic Chagas disease.     

Polyclonal B lymphocyte activation during Trypanosoma cruzi infection

Infection of A/J mice with Trypanosoma cruzi results in the polyclonal activation of B lymphocytes in vivo as assessed by the spontaneous plaque-forming cell (PFC) response to trinitrophenyl and to goat, equine, and sheep erythrocytes. The peak response to these antigens is found at 5 to 6 days of infection. Additionally, a polyclonal response to syngeneic erythrocytes can be detected in infected mice by using aged but not fresh indicator cells. Polyclonally stimulated PFC to human gamma-PFC found late in infection during a period of marked splenomegaly and parasitemia. This trypanosoma-induced polyclonal B cell activation may well be responsible for the abnormalities in immunoglobulin synthesis and secretion that have been reported to occur during human infection with T. cruzi.     

Bias due to methods of parasite detection when estimating prevalence of infection of Triatoma infestans by Trypanosoma cruzi

The study aimed to quantify the bias from parasite detection methods in the estimation of the prevalence of infection of Triatoma infestans by Trypanosoma cruzi, the agent of Chagas disease. Three common protocols that detect T. cruzi in a sample of 640 wild‐caught T. infestans were compared: (1) the microscopic observation of insect fecal droplets, (2) a PCR protocol targeting mini‐exon genes of T. cruzi (MeM‐PCR), and (3) a PCR protocol targeting a satellite repeated unit of the parasite. Agreement among protocols was computed using Krippendorff Kα. The sensitivity (Se) and specificity (Sp) of each protocol was estimated using latent class models. The PCR protocols were more sensitive (Se > 0.97) than microscopy (Se = 0.53) giving a prevalence of infection of 17–18%, twice as high as microscopy. Microscopy may not be as specific as PCR if Trypanosomatid‐like organisms make up a high proportion of the sample. For small T. infestans, microscopy is not efficient, giving a prevalence of 1.5% when PCR techniques gave 10.7%. The PCR techniques were in agreement (Kα = 0.94) but not with microscopy (Kα never significant with both PCR techniques). Among the PCR protocols, the MeM‐PCR was the most efficient (Se=1; Sp=1).     

Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth

IFN-gamma is known to be required for host control of intracellular Trypanosoma cruzi infection in mice, although the basis of its protective function is poorly understood. LRG-47 is an IFN-inducible p47GTPase that has been shown to regulate host resistance to intracellular pathogens. To investigate the possible role of LRG-47 in IFN-gamma-dependent control of T. cruzi infection, LRG-47 knockout (KO) and wild-type (WT) mice were infected with the Y strain of this parasite, and host responses were analyzed. When assayed on day 12 after parasite inoculation, LRG-47 KO mice, in contrast to IFN-gamma KO mice, controlled early parasitemia almost as effectively as WT animals. However, the infected LRG-47 KO mice displayed a rebound in parasite growth on day 15, and all succumbed to the infection by day 19. Additional analysis indicated that LRG-47-deficient mice exhibit unimpaired proinflammatory responses throughout the infection. Instead, reactivated disease in the KO animals was associated with severe splenic and thymic atrophy, anemia, and thrombocytopenia not observed in their WT counterparts. In addition, in vitro studies revealed that IFN-gamma-stimulated LRG-47 KO macrophages display defective intracellular killing of amastigotes despite normal expression of TNF and NO synthetase type 2 and that both NO synthetase type 2 and LRG-47 are required for optimum IFN-gamma-dependent restriction of parasite growth. Together, these data establish that LRG-47 can influence pathogen control by simultaneously regulating macrophage-microbicidal activity and hemopoietic function.     

Interleukin-6 is required for parasite specific response and host resistance to Trypanosoma cruzi.

Infection with Trypanosoma cruzi, the agent of Chagas' disease, results in elevated levels of interleukin-6 (IL-6) in serum and infected tissues. However, it remains unknown whether IL-6 plays a role in host defence against T. cruzi. To determine whether IL-6 underlies disease progression, we followed the time course of T. cruzi-infected mice bearing IL-6 +/+ and minus sign/minus sign genotypes, respectively. We found that IL-6 minus sign/minus sign mice were more susceptible to T. cruzi infection as they exhibited about 3-fold higher parasitaemia and died earlier than wild-type animals. Unlike what might be expected, T. cruzi-infected IL-6 minus sign/minus sign mice did not show at peak infection a decrease in the secretion of IFN-gamma, a Th1 cytokine crucial for controlling the parasite. Instead, they exhibited a much reduced splenocyte recall response to T. cruzi antigens. Our results suggest that IL-6 mediates anti-parasite protective responses against T. cruzi.     

Trypanosoma cruzi: T-cell-dependent mechanisms of resistance during chronic infection

Effector mechanisms of resistance exerted by T cells from BALB/c mice chronically infected with Trypanosoma cruzi, Tulahuén strain, were studied. Spleen cells from chronically infected mice (Chro-SC) prestimulated with heat-killed trypomastigotes (HKT) and/or IL-2 destroyed PHA-labeled p-815 mastocytoma cells, HKT-pulsed macrophages, and normal peritoneal macrophages. However, HKT-stimulated Chro-SC did not affect the infectivity of free bloodstream forms of the parasite. Upon HKT stimulation, Chro-SC or their culture supernatant activated peritoneal macrophages for the destruction of intracellular amastigotes. The effect was abolished after Thy 1.2+ cell depletion. The addition of Cyclosporin A (CyA), which blocks T-cell activation, during HKT-stimulation of Chro-SC, diminished their ability to activate the trypanocidal activity of macrophages. CyA also inhibited the production of both macrophage-activating factors and interferon-gamma by HKT-stimulated Chro-SC. CyA administration to recipients of nylon-wool nonadherent spleen cells from chronically infected mice inhibited their adoptively acquired resistance against T. cruzi, suggesting that the conferred resistance depended on the effect of specifically activated cells. When administered during the chronic stage of the infection, CyA abrogated the antigen-specific delayed type hypersensitivity response but increased the levels of anti-T. cruzi IgG antibodies. Neither parasitemia, tissular parasitism in myocardium or skeletal muscle, nor mortality were detected after CyA treatment, suggesting the presence of a CyA nonsensitive mechanism(s) in the control of T. cruzi during the chronic phase of the infection.     

Beta-interferon inhibits cell infection by Trypanosoma cruzi

Preparations containing alpha/beta-interferon produced by L-929 cells were found to inhibit the capacity of bloodstream forms of Trypanosoma cruzi to associate with and infect mouse peritoneal macrophages or rat heart myoblasts. Marked reductions in the number of parasites per cell as well as in the percentage of cells associated with the trypanosomes were systematically observed in cultures of these cells that contained interferon. The inhibitory effect was abrogated in the presence of specific antibodies against alpha/beta-interferon, and purified beta-interferon induced a similar inhibitory effect, indicating that the active principle in the preparation was indeed interferon. Pretreatment of the parasites with alpha/beta-interferon reduced their infectivity for untreated host cells, whereas pretreatment of either type of host cell had no consequence on the interaction. The effect of interferon on the trypanosomes was reversible; the extent of the inhibitory effect was significantly reduced after 20 min, and was undetectable after 60 min when macrophages were used as host cells. Longer periods of time were required for the inhibitory effect to begin to subside (60 min) and to become undetectable or insignificant (120 min) when rat heart myoblasts were used. The results of additional studies performed with purified preparations of alpha- or beta-interferon revealed that only the latter was inhibitory of cell-parasite association. Because interferon is known to be produced shortly after T. cruzi infection and its administration has been shown to have a marked protective effect against this infection, our results suggest that the latter may involve inhibition of cell infection by interferon.     

Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity

Trypanosoma cruzi infection by oral route constitutes the most important mode of transmission in some geographical regions, as illustrated by reports on microepidemics and outbreaks of acute Chagas' disease acquired by ingestion of food contaminated with parasites from triatomine insects. In the mouse model, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium, a unique portal of entry for systemic infection. High efficiency of metacyclic forms in establishing infection by oral route is associated with expression of gp82, a stage-specific surface molecule that binds to gastric mucin and to epithelial cells. Gp82 promotes parasite entry by triggering the signaling cascades leading to intracellular Ca²⁺ mobilization. T. cruzi strains deficient in gp82 can effectively invade cells in vitro, by engaging the Ca²⁺ signal-inducing surface glycoprotein gp30. However, they are poorly infective in mice by oral route because gp30 has low affinity for gastric mucin. Metacyclic forms also express gp90, a stage-specific surface glycoprotein that binds to host cells and acts as a negative regulator of invasion. T. cruzi strains expressing gp90 at high levels, in addition to gp82 and gp30, are all poor cell invaders in vitro. Notwithstanding, their infectivity by oral route may vary because, unlike gp82 and gp30, which resist degradation by pepsin in the gastric milieu, the gp90 isoforms of different strains have varying susceptibility to peptic digestion. For instance, in a T. cruzi isolate, derived from an acute case of Chagas' disease acquired by oral route, gp90 is extensively degraded by gastric juice in the mouse stomach and this renders the parasite highly invasive towards target cells. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of the disease reported in outbreaks of oral infection.     

Trypanosoma cruzi: alpha-2-macroglobulin regulates host cell apoptosis induced by the parasite infection in vitro

A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form-A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment.     

Trypanosoma cruzi: early parasite proliferation and host resistance in inbred strains of mice

The extent of parasite proliferation following completion of the first cycle of intracellular replication was significantly higher in CD-1 nu/nu mice and in irradiated mice compared to other, including highly susceptible, mouse strains. A control of parasite proliferation thus occurs in normal mice as early as the first cycle of intracellular replication. The thymus dependency and radiation sensitivity of the early control of proliferation of Trypanosoma cruzi suggest that an immune response to the parasite is involved in the early control of proliferation. The BXH-2 recombinant inbred strain demonstrated an inability to control early proliferation and, 4-5 days after infection, had parasitemias several times higher than those observed in susceptible mouse strains. The BXH-2 strain appears to lack the early control mechanism. When the extent of proliferation of T. cruzi at completion of the first cycle of intracellular replication was compared in inbred strains of mice having varying levels of resistance to the parasite, the extent of proliferation correlated with host resistance, being lowest in the most resistant strains (C57BL/6, SJL) and highest in the most susceptible strains (C3H, A). It is suggested that the mechanism(s) controlling early parasite proliferation may be of primary importance as the basis for host resistance.     

Endogenous IFN-gamma is required for resistance to acute Trypanosoma cruzi infection in mice

In order to study the role of endogenous IFN-gamma in Trypanosoma cruzi infection in mice, a potent murine IFN-gamma-specific mAb was injected i.p. on days -1, 7, and 14, relative to infection. Irrespective of the parasite inocula (100 or 25,000), groups of antibody-treated mice had significantly greater cumulative mortality rates than did appropriate controls. In antibody-treated mice, mean survival times were also significantly shorter, and maximum mean parasitemia levels were significantly higher, than in controls. Moreover, the number of amastigote nests in tissues was higher than in control mice and attained a maximum at the same time as parasitemia. As evident from kinetic studies of neutralizing activity, injected mAb were rapidly consumed in infected, but not in noninfected, mice, which is suggestive of massive IFN-gamma production during the early parasitemic phase of the disease. Nevertheless, IFN-gamma remained undetectable in the sera of infected but untreated mice. Unexpectedly, however, a peak of IFN-like antiviral activity, characterizable as a mixture of IFN-gamma and IFN-beta, appeared in mAb-treated mice that survived to infection at a time when neutralizing activity of injected mAb had drastically decreased in the circulation. We hypothesize that this high level of artificially induced endogenous IFN-gamma, not neutralized by the amounts of injected mAb, was due to the more intense parasite multiplication occurring in mAb-treated mice, which in turn may have induced an increased amount of various cytokines. TNF-alpha was not found in the serum of our mice. The humoral immune response entered its exponential phase at a time point later than that when protection by endogenous IFN-gamma was evident. Treatment with IFN-gamma-specific antibody, as applied in our study, failed to affect the level of different Ig isotypes or of T. cruzi-specific antibodies. Our study clearly indicates that IFN-gamma is produced early in acute T. cruzi infection and exerts a protective effect that is probably independent from the humoral immune response.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.     

Trypanosoma cruzi: adhesion to the host cell and intracellular survival

Both invasion of the host cell by T. cruzi and its establishment into the mammalian host are critical steps. In this review, the adhesion step and the intracellular survival in non-professional phagocytes are particularly focused on, with special emphasis on the role of Gp85/trans-sialidase (Gp85/TS) superfamily. Excellent reviews have been published lately, some covering other aspects of T. cruzi-host interaction and will be cited instead of the original articles due to limited number of listed references.