Trypanosoma cruzi Infection in Tumor Necrosis Factor Receptor p55-Deficient Mice

Tumor necrosis factor receptor p55 (TNFRp55) mediates host resistance to several pathogens by allowing microbicidal activities of phagocytes. In the studies reported here, TNFRp55^−/− mice infected with the intracellular parasite Trypanosoma cruzi showed clearly higher parasitemia and cumulative mortality than wild-type (WT) controls did. However, gamma interferon (IFN-γ)-activated macrophages from TNFRp55^−/− mice produced control levels of nitric oxide and killed the parasite efficiently in vitro. Trypanocidal mechanisms of nonphagocytic cells (myocardial fibroblasts) from both TNFRp55^−/− and WT mice were also activated by IFN-γ in a dose-dependent way. However, IFN-γ-activated TNFRp55^−/− nonphagocytes showed less effective killing of T. cruzi than WT control nonphagocytes, even when interleukin 1β (IL-1β) was added as a costimulator. In vivo, T. cruzi-infected TNFRp55^−/− mice and WT mice released similar levels of NO and showed similar levels of IFN-γ mRNA and inducible nitric oxide synthase mRNA in their tissues. Instead, increased susceptibility to T. cruzi of TNFRp55^−/− mice was associated with reduced levels of parasite-specific immunoglobulin G (IgG) (but not IgM) antibodies during infection, which is probably linked to abnormal B-cell differentiation in secondary lymphoid tissues of the mutant mice. Surprisingly, T. cruzi-infected TNFRp55^−/− mice showed increased inflammatory and necrotic lesions in several tissues, especially in skeletal muscles, indicating that TNFRp55 plays an important role in controlling the inflammatory process. Accordingly, levels of Mn²⁺ superoxide dismutase mRNA, a TNF-induced enzyme which protects the cell from the toxic effects of superoxide, were lower in mutant than in WT infected mice.     

Polyclonal Lymphocyte Responses to Murine Trypanosoma cruzi Infection

Intraperitoneal infection of young adult C57BL/6 males with 10(5) blood or cloned culture forms of Trypanosoma cruzi (CL strain) induced the appearance in spleen, blood, and lymph nodes of cytotoxic effector cells detectable in a lectin-dependent 51Cr-release assay. The effector cells were conventional cytotoxic T lymphocytes (CTL), since they were Thy 1+ and Lyt 2+, and the lysis of tumour target cells was strictly dependent on the presence of lectin. CTL activity is already detectable in spleen 2 days after infection, reaches a peak at 2 weeks, and returns to normal levels during the chronic phase (1 month onwards). Increased levels of CTL activity were also detected in lymph nodes with similar kinetics, even in animals that were splenectomized prior to infection. In contrast to spleen, significant levels of CTL activity persisted in lymph nodes in the chronic phases. This functional variable correlates with the appearance of high numbers of large Lyt 2+ lymphocytes in the same organs (50 to 100-fold higher than in control, uninfected mice). Very similar responses are detected in a T. cruzi sensitive mouse strain (C3H/HeJ). It appears, therefore, that T. cruzi infection results in a large polyclonal activation of Lyt 2+ lymphocytes, some of which differentiate to effector, cytolytic functions.     

Polyclonal Lymphocyte Responses to Murine Trypanosoma cruzi Infection

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.     

Interleukin-17-Mediated Control of Parasitemia in Experimental Trypanosoma congolense Infection in Mice

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.African trypanosomes are extracellular protozoan parasites that cause fatal disease in humans and domestic livestock in sub-Saharan Africa. The disease is endemic in 36 countries, and millions of people are at risk of suffering from human African trypanosomiasis. Trypanosomiasis in animals is caused by Trypanosoma congolense, Trypanosoma brucei brucei, and Trypanosoma vivax, but T. congolense is the most important cause of disease for livestock (29). It is estimated that the disease costs $1.3 billion to livestock producers and consumers every year (17).African trypanosomes have developed very sophisticated mechanisms to evade the host''s immune defenses (39, 40). The indigenous African and exotic European breeds of cattle are relatively resistant and susceptible, respectively, to African trypanosomiasis (28). In the laboratory, BALB/c mice are highly susceptible to experimental T. congolense infections, whereas C57BL/6 mice are relatively resistant, as measured by levels of parasitemia and survival time. When infected intraperitoneally (i.p.) with 10³ T. congolense, BALB/c mice have a mean survival time of 8.5 ± 0.5 days, whereas C57BL/6 mice survive for more than 100 days (40). Infected BALB/c mice die of systemic inflammatory response syndrome (SIRS), which is associated with high production of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, and gamma interferon (IFN-γ) (12, 37, 39, 40, 43, 45). Interestingly, blockade of IL-10 signaling either by anti-IL-10R monoclonal antibody (MAb) treatment (35, 36, 38) or knocking out the IL-10 gene (7) induces early death in the infected relatively resistant C57BL/6 mice. Thus, IL-10 is crucial for controlling proinflammatory cytokine storm that results lethal SIRS.Recently, T helper 17 (Th17) cells have been identified as a new T-helper subset (3, 10, 16, 33). A hallmark of Th17 cells is the production of IL-17A (also called IL-17), a proinflammatory cytokine. IL-17 and Th17 cells play important pathological role in several immune-mediated diseases, including inflammatory bowel disease, experimental autoimmune encephalopathy, and collagen-induced arthritis (5, 13, 30). IL-17A promotes inflammation by inducing the production of various proinflammatory cytokines (IL-6, TNF-α, and IL-1β) (3, 10, 16, 33). However, the protective function of Th17 and their effector cytokines in various infectious diseases has also been reported. For example, Mycobacterium tuberculosis (14), Pneumocystis carinii (34), Candida albicans (9), and Klebsiella pneumoniae (2) can trigger a strong Th17 response that mediates protective effects. These observations indicate that Th17 cells and their effector cytokines have both pathological and protective roles during inflammation and infections, respectively.There is as yet no report on the role of IL-17 and Th17 cells in resistance to African trypanosomes. Because T. congolense-infected BALB/c mice have high serum levels of IL-6, and their macrophages elaborate high amounts of IL-6 after in vitro infection (12), a cytokine that favors Th17 differentiation and IL-17 production (3, 16), we hypothesized that IL-17 and/or Th17 cells play important roles in resistance to T. congolense infection in mice by contributing to excessive inflammatory response. However, the data presented here suggest that IL-17 may be playing some protective role, particularly in controlling early parasitemia in mice infected with T. congolense.     

Suppression of Humoral Responses During Trypanosoma cruzi Infections in Mice

C57BL/6 mice exhibit low parasitemias and often survive Trypanosoma cruzi infections, whereas C3H(He) mice die during the acute phase with relatively high parasitemias. The present study showed that both strains of mice develop nonspecific immunosuppression to challenge with sheep erythrocytes during the course of infection. Several major differences in immunosuppression-related phenomena between the two strains of mice were determined, yet there is no apparent relationship between immunosuppression and resistance to T. cruzi. Both the number of direct plaque-forming cells and the titer of 2-mercaptoethanol-sensitive agglutinating antibody were significantly lower on day 11 for C57BL/6 mice and day 9 for C3H(He) mice. The number of indirect plaque-forming cells and the titer of mercaptoethanol-resistant agglutinating antibody were reduced by day 36 of infection in C57BL/6 mice and 13 days postinfection in C3H(He) mice. In both strains the degree of humoral response suppression of mice increased concomitant with the period of infection, but was not correlated with the changes in spleen cell numbers. Preliminary experiments designed to explore the mechanism underlying the induction and maintenance of immunosuppression in this hostparasite model disclosed the presence of suppressor substance in the serum of T. cruzi-infected mice. The passive transfer of serum from infected mice to syngeneic recipients elicited a state of immunosuppression to sheep erythrocytes, but did not diminish anti-erythrocyte activity in allogeneic recipients. The induction of immunosuppression in normal mice was further found to be dependent on the interval between serum transfer and challenge with antigen. No quantitative differences existed between the magnitude of suppressed humoral responses in mice infected for varying lengths of time and recipients of serum collected from similarly infected mice.     

Most B Cells in Acute Trypanosoma cruzi Infection Lack Parasite Specificity

The specificity of B lymphocytes activated in the acute phase of murine Trypanosoma cruzi infection was analysed in a panel of immunoglobulin-secreting hybridomas derived by fusion of lymph node cells 7 days after intraperitoneal parasite inoculation. The immunoglobulin isotype distribution of the hybrids reflected the total plaque-forming cell (PFC) response in the animal at this point, with a predominance of IgG2a, IgM, and IgG2b. Screening of the hybridoma antibodies on parasite antigens by three independent methods (western blot, ELISA, and immunofluorescence) revealed only one (out of a total of 51) that bound a parasite molecule with an apparent molecular mass of 180 kDa. In contrast, antibodies of both IgM and IgG classes were found to react with a panel of autologous antigens. These results establish that most B cells activated by T. cruzi infection are not specific for parasite antigens and therefore indicate the relevance of analysing the totality of host responses to infection.     

T-Cell Responses during Trypanosoma brucei Infections in Mice Deficient in Inducible Nitric Oxide Synthase

We have investigated the possibility that nitric oxide (NO) synthesis may affect the course of a trypanosome infection via T-cell responses using mice deficient in inducible NO synthase (iNOS). Parasitemia levels increased at the same rate in both iNOS-deficient homozygous and control heterozygous mice, and peak parasitemia values were the same in both groups. However, the heterozygous mice maintained higher parasitemia levels after the peak of an infection than the homozygous mice due to a decrease in the rate of clearance of parasites. In iNOS-deficient mice there was an increase in the numbers of total CD4(+) cells and activated (interleukin-2 receptor-expressing) CD4(+) cells in infected mice compared with the numbers in uninfected mice. Spleen cells from infected iNOS-deficient mice displayed increased proliferative responses and gamma interferon secretion when stimulated in vitro than those of control mice. These data suggest that NO production depresses T-helper 1-like responses generated during Trypanosoma brucei infections, thus promoting the survival of the parasite.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli Infection by Duplex PCR Assay Based on Telomeric Sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Trypanosoma cruzi Infection Impairs the Endocytosis of Zymosan A by Cardiomyocytes.

OBJECTIVE: We have previously reported that mannose receptors participate and are regulated during Trypanosoma cruzi cardiomyocyte (CM) infection. Our present aim is to characterize the endocytosis of mannosylated ligands like zymosan A (Zy) in uninfected and T. cruzi-infected CM. METHODS: CM infected or not by T. cruzi were incubated with Zy for different periods of time and their internalization was analyzed at light microscopy level. Fluorescent approaches were performed by treating Zy with concanavalin-A-TRITC and washing it exhaustively prior to incubation with CM. The cultures were further stained with phalloidin-FITC and DAPI for actin and DNA visualization, respectively. RESULTS: CM internalized Zy particles in a time-dependent fashion. The ligand specificity was confirmed by the addition of mannan, which efficiently blocked the Zy endocytosis. Designed fluorescent approaches extended and confirmed the Zy internalization by striated cells. Infected cultures displayed impairment in Zy endocytosis, which seems to be directly related to host infection rates. CONCLUSIONS: Altogether, our results show the ability of CM to ingest large particles such as the mannosylated ligand Zy. During their infection with T. cruzi, there is a loss in Zy internalization possibly due to the negative modulation of mannose receptors.     

Detection of Cruzipain,the Major Cysteine Proteinase from Trypanosoma cruzi and its C-Terminal Extension in Biological Fluids During Experimental Infection in Mice

Monoclonal antibodies (MoAbs) specific for unique epitopes of the catalytic domain of cruzipain (Crz) were used to develop a two-site sandwich ELISA specific for native Crz. In addition, the authors developed a sandwich ELISA that allowed the detection of the protease C-terminal domain (CT) using a combination of a MoAb specific for the CT and rabbit anti-Crz IgGs. Both assays were sensitive with detection limits of 2 ng/ml and 0.7 ng/ml, respectively. The assays were assessed for applicability in detection of antigens in serum and urine from experimentally infected BALB/c mice. The antigens were already detectable in serum by the third week after infection, reached their peak by week four, and decreased during the chronic phase of the infection. Throughout the infection the relative amount of CT detected was several-fold higher than that of native Crz, and the data demonstrate that the cT exposes highly immunogenic epitopes that are absent in native Crz. Since these observations have a potential application in diagnosis, the authors analysed the degree of cross-reactivity with antigens from T. rangeliT. bruceiLeishmania mexicana and L. panamensis, and determined that the assays were highly specific. Measurable amounts of the CT were also recorded in urine samples.     

Detection of Cruzipain, the Major Cysteine Proteinase from Trypanosoma cruzi and its C-Terminal Extension in Biological Fluids During Experimental Infection in Mice

Monoclonal antibodies (MoAbs) specific for unique epitopes of the catalytic domain of cruzipain (Crz) were used to develop a two-site sandwich ELISA specific for native Crz. In addition, the authors developed a sandwich ELISA that allowed the detection of the protease C-terminal domain (CT) using a combination of a MoAb specific for the CT and rabbit anti-Crz IgGs. Both assays were sensitive with detection limits of 2 ng/ml and 0.7 ng/ml, respectively. The assays were assessed for applicability in detection of antigens in serum and urine from experimentally infected BALB/c mice. The antigens were already detectable in serum by the third week after infection, reached their peak by week four, and decreased during the chronic phase of the infection. Throughout the infection the relative amount of CT detected was several-fold higher than that of native Crz, and the data demonstrate that the cT exposes highly immunogenic epitopes that are absent in native Crz. Since these observations have a potential application in diagnosis, the authors analysed the degree of cross-reactivity with antigens from T.  rangeli T. brucei Leishmania mexicana and L. panamensis , and determined that the assays were highly specific. Measurable amounts of the CT were also recorded in urine samples.     

Protein geranylgeranyltransferase-I of Trypanosoma cruzi

Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common alpha subunit and a distinct beta subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the beta subunit of PGGT-I. Co-expression of this protein and the alpha subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [(3)H]geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are approximately 100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.     

Characterization of Trypanosoma cruzi hexokinase

Properties of hexokinase (EC 2.7.1.1) from Trypanosoma cruzi epimastigote forms (Tulahuen strain) were studied and compared with enzymes from other sources. The enzyme activity was 37 units g-1 of wet cells (1.2 units mg-1 protein). Hexokinase showed Km values for glucose and ATP of 0.09 and 0.4 mM, respectively. The enzyme reacted with other nucleotides too. N-Acetylglucosamine was a competitive inhibitor with respect to glucose (Ki = 0.3 mM). ADP inhibited the enzyme competitively with respect to ATP (Ki = 1.5 mM) and noncompetitively with respect to glucose (Ki = 7 mM). The enzyme was markedly inhibited by 5-thioglucose, its Ki value was 0.4 mM. Hexokinase activity was not affected by glucose 6-phosphate.     

Trypanosoma cruzi Causes Paralyzing Systemic Necrotizing Vasculitis Driven by Pathogen-Specific Type I Immunity in Mice

Infectious agents are often considered potential triggers of chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. Here we show that following control of acute infection of mice with the myotropic Colombiana strain of Trypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Lesions occurred in many but not all organs and tissues, with skeletal muscle arteries being the most severely affected, and were associated with myositis, atrophy, paresis/paralysis, and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80⁺ macrophages, and T. cruzi tetramer-specific CD8⁺ T lymphocytes capable of producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) but not interleukin-17 (IL-17). T. cruzi-specific IgG was detected in sera from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus, T. cruzi infection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific type I immune responses.