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1.  Bradykinin B2 Receptors of Dendritic Cells, Acting as Sensors of Kinins Proteolytically Released by Trypanosoma cruzi, Are Critical for the Development of Protective Type-1 Responses  
PLoS Pathogens  2007;3(11):e185.
Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R−/− mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R−/− heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-γ-producing CD4+ and CD8+ T cells in the spleen of B2R−/− and wild-type mice. However, production of IFN-γ by effector T cells isolated from B2R−/− heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-γ-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R−/− mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R−/− mice was linked to upregulated secretion of IL-17 and TNF-α by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R−/− mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R−/− mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.
Author Summary
Antibodies and IFN-γ-producing effector T cells are essential for the immune control of infection by Trypanosoma cruzi, the intracellular protozoa that causes human Chagas disease. Despite the potency of anti-parasite immunity, the parasites are not cleared from their intracellular niches. Instead, a low grade chronic infection prevails, provoking severe immunopathology in the myocardium. Although it is well established that innate sentinel cells sense T. cruzi through receptors for microbial structures, such as Toll-like receptors, it remained unclear whether endogenous inflammatory signals also contribute to the development of adaptive immunity. The present study was motivated by awareness that T. cruzi trypomastigotes (extracellular infective forms) are equipped with proteases that liberate the pro-inflammatory bradykinin peptide from an internal segment of kininogens. Here we demonstrate that splenic dendritic cells (DCs), the antigen-presenting cells that coordinate the adaptive branch of immunity in lymphoid tissues, are potently activated via G-protein-coupled bradykinin B2 receptors (B2R). Analysis of the outcome of infection in B2R-knockout mice revealed that the mutant mice developed a typical susceptible phenotype, owing to impaired development of IFN-γ-producing effector T cells. Notably, the immune dysfunction of B2R-knockout mice was corrected upon cell transfer of wild-type DCs, thus linking development of protective T cells to DCs' sensing of endogenous danger signals (kinins) released by trypomastigotes.
PMCID: PMC2098834  PMID: 18052532
2.  Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes. 
Infection and Immunity  1996;64(6):1961-1967.
Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.
PMCID: PMC174023  PMID: 8675294
3.  NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection 
•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91phox−/− or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with •NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.
Author Summary
When pathogens enter their hosts, they are fought by several resistance strategies, including capture by phagocytes and the production of pathogen-toxic molecules. Nitric oxide, a free radical, has been extensively studied as one of these toxic molecules that successfully mediates intracellular parasite killing, including Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. On the other hand, reactive oxygen species also mediate resistance to several pathogens, mainly bacterial. In this study, we addressed the role of reactive oxygen species in the resistance to T. cruzi using gene-deficient mice, a species which phagocytes lack the ability to produce (phox−/− mice). We found that phagocyte-derived reactive oxygen species are not critical to mediate resistance to parasite in the knock-out animals. However, phox−/− mice presented higher mortality and lower blood pressure due to infection with T. cruzi than non-deficient mice. The blood pressure was restored to normal by an inhibitor of nitric oxide synthesis by phagocytes. We hypothesize that superoxide (one of the oxygen reactive species) controls blood pressure during infection with T. cruzi, by reacting with nitric oxide and preventing its action on blood vessels.
PMCID: PMC3279332  PMID: 22348160
4.  IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils 
PLoS Pathogens  2012;8(4):e1002658.
Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.
Author Summary
IL-17 family is comprised for six members (IL-17A to F) that have been reported to play protective effects in bacterial and fungal infections and contradictory roles in parasite infections. Using mice deficient in IL-17RA, the common receptor subunit for many IL-17 family members, we determined that these cytokines are required for host protection against the parasite Trypanosoma cruzi. In absence of IL-17 signaling, mice developed an aggravated infection with similar levels of parasite in blood but increased inflammation and tissue damage of vital organs such as liver. We evaluated the mechanisms underlying this increased susceptibility and determined that the absence of IL-17RA caused a reduced arrival of neutrophils to organs such as spleen and liver. Neutrophils are phagocytic cells with abilities to directly destroy pathogens and also to regulate the inflammatory response. Indeed, we determined that neutrophils from T. cruzi infected mice are poisoned to secrete the regulatory cytokine IL-10. Finally, by experiments of depletion and adoptive transfer of neutrophils we determined that, during T. cruzi infection, IL-17RA is required for the recruitment of neutrophils that destroy the parasite and that also regulate inflammatory responses and collateral tissue damage by secreting IL-10.
PMCID: PMC3343119  PMID: 22577359
5.  IFN-γ Plays a Unique Role in Protection against Low Virulent Trypanosoma cruzi Strain 
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-γ would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-α, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-γ we found that the latter is crucial for controlling infection by G strain amastigotes.
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-γ 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.
Author Summary
Trypanosoma cruzi, an obligate intracellular protozoan, is the etiological agent of Chagas disease that represents an important public health burden in Latin America. The infection with this parasite can lead to severe complications in cardiac and gastrointestinal tissue depending on the strain of parasite and host genetics. Currently, six genetic groups (T. cruzi I to VI) have been identified in this highly genetic and diverse parasite.The majority of published data concerning host immune response has been obtained from studying T. cruzi II to VI-infected mice, and the genetic differences between T. cruzi II to VI and T. cruzi I strains are large. Here we aimed to understand how amastigotes from T. cruzi I G strain are highly infective in vitro and do not contribute for a patent parasitemia in vivo. Our results showed that amastigotes from G strain are highly susceptible to IFN-γ treatment in vitro and secretion by immune cells in vivo. This information may represent important findings to design novel immune strategies to control pathology that may be caused by different strains in the same host.
PMCID: PMC3317909  PMID: 22509418
6.  Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: role of NK cells. 
Infection and Immunity  1996;64(1):128-134.
Gamma interferon (IFN-gamma) plays an important role in experimental Trypanosoma cruzi infections, presumably by controlling the early replication of parasites in host macrophages. In this work, we show that NK cells represent an important cell type responsible for the production of most of the IFN-gamma in the early stage of T. cruzi infection and that the in vivo treatment of mice with anti-NK1.1 monoclonal antibody made resistant animals susceptible to the infection. Through in vitro experiments, we demonstrate that normal splenocytes from euthymic or athymic nude mice cultivated for 48 h with live T. cruzi trypomastigotes produced elevated levels of IFN-gamma. In addition, NK-depleted splenocytes show a drastic reduction of IFN-gamma production in response to live T. cruzi trypomastigotes. We also demonstrated that IFN-gamma production is dependent on a factor secreted by adherent cells. Supernatants of spleen cells from athymic nude mice are able to induce IFN-gamma production by normal splenocytes when cultured with trypomastigotes. The addition of anti-interleukin-10 to these cultures resulted in a marked increase in IFN-gamma production. On the other hand, the absence of NK cells led to an increased secretion of interleukin-10 upon in vitro stimulation with T. cruzi. Taken together, these results suggest that NK cells are the major source of IFN-gamma that could be involved in limiting the replication of T. cruzi in host macrophages during the early acute phase of the infection.
PMCID: PMC173737  PMID: 8557330
7.  The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi 
PLoS Pathogens  2012;8(7):e1002799.
Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1−/− mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1−/− mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.
Author Summary
Chagas' disease caused by the intracellular protozoan Trypanosoma cruzi is the most important parasitic infection in Latin America affecting several million persons. Nonetheless, there is no therapy or vaccine available. Thus, more efforts are needed to identify new therapeutic targets. Here, we report that Slamf1, which controls phagosomal/lysosomal fusion and phagosomal NADPH-oxidase activity, is required for T.cruzi replication in macrophages and dendritic cells, but not in other cells, which do not express the receptor. In the absence of Slamf1 we detect reduced number of parasites in the heart compared to infected wt mice. This explains why T. cruzi-infected Slamf1 deficient mice do not succumb to myocarditis induced by a lethal challenge with T. cruzi in contrast to BALB/c mice. Perhaps more importantly, we demonstrate that parasite replication in phagocytes is of far greater importance for the pathogenesis of the cardiomyopathy than replication in other cells. Moreover, we found much lower IFN-γ production in the heart of Slamf1 deficient mice than in the heart of BALB/c mice. We corroborated those results using an alternative approach, blocking Slamf1 function in vivo by treating mice with anti-Slamf1 antibodies. Consequently, Slamf1 is an attractive novel therapeutic target for modulating T. cruzi infection.
PMCID: PMC3395606  PMID: 22807679
8.  Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages. 
Infection and Immunity  1995;63(12):4862-4867.
Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing.
PMCID: PMC173696  PMID: 7591147
9.  Immunization with Hexon Modified Adenoviral Vectors Integrated with gp83 Epitope Provides Protection against Trypanosoma cruzi Infection 
Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.
Methodology/Principal Findings
The “antigen capsid-incorporation” strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.
This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease.
Author Summary
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and has been detrimental to millions of people in Latin America since the early 1900s, and now this disease is having a global impact. Decades later, there is still no vaccine for this disease. Scientists have used the adenovirus vector in gene therapy as well as vaccine therapy for many diseases. In this study, adenovirus is used as the host vector for a potential vaccine against Chagas disease in a non-traditional way. Instead of the antigen being expressed as a transgene product, the antigen is incorporated on the capsid of the adenovirus to evoke an enhanced humoral immune response against T. cruzi. This study highlights the modified adenovirus' ability to elicit a strong anti-T. cruzi humoral response as well as providing protection against Trypanosoma cruzi infection. These findings illustrate the true potential of generating an effective T. cruzi vaccine by means of unique gene therapy-based techniques.
PMCID: PMC4140675  PMID: 25144771
10.  NLRP3 Controls Trypanosoma cruzi Infection through a Caspase-1-Dependent IL-1R-Independent NO Production 
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.
Author Summary
Inflammasomes are cytosolic innate receptors that are emerging as central effectors in the control of infections and inflammatory pathologies. NLRP3 is the most studied member of inflammasomes with established role in the control of bacterial and viral infections. This manuscript describes original studies on the involvement of NLRP3 inflammasome in the control of Trypanosoma cruzi, the etiological agent of Chagas disease, a chronic infectious illness that affects millions of people in the world. T. cruzi activates NLRP3 inflammasome by a mechanism involving cathepsin B. NLRP3−/− and caspase1−/− mice display high parasitemia during acute phase of T. cruzi infection, which could be explained by a severe defect in the production of nitric oxide (NO) and in the impairment of their macrophages to control intracellular parasites. Interestingly, inhibition of caspase-1, but not the neutralization of IL-1β and IL-18, the best-studied caspase-1 substrates, abrogated NO production by WT and MyD88−/− macrophages and rendered them as susceptible to T. cruzi infection as NLRP3−/− macrophages. Together, our results indicate a caspase-1-dependent and IL-1β and IL-18-independent pathway for NO production as a new effector mechanism played by NLRP3 to control T. cruzi infection.
PMCID: PMC3789781  PMID: 24098823
11.  The Endogenous Balance of Soluble Tumor Necrosis Factor Receptors and Tumor Necrosis Factor Modulates Cachexia and Mortality in Mice Acutely Infected with Trypanosoma cruzi 
Infection and Immunity  1999;67(11):5579-5586.
To better understand the role of tumor necrosis factor (TNF) during Trypanosoma cruzi infection in BALB/c mice, we have investigated the kinetics of circulating tumor necrosis factor (TNF), soluble TNF receptor 1 (sTNR1), and sTNFR2 levels, as well as the interactions between such factors, in relation to parasitemia, cachexia, and mortality of acutely infected animals. Our data show that the parasitemic phase of T. cruzi infection in mice is associated with high levels of circulating TNF and sTNFR2, resulting in the formation of cytokine-receptor complexes and some degree of neutralization of TNF bioactivity. Although sTNR2 levels always exceeded TNF levels, low sTNFR/TNF circulating ratios were associated with cachexia in all infected mice, whereas the lowest ratios were observed in dying animals harboring the highest parasitemia. We also studied the modulation of sTNFR/TNF ratios induced by anti-TNF antibodies administered to infected animals and their consequences on the outcome of the infection. The injection of anti-TNF monoclonal antibody (MAb) TN3 into infected mice resulted in a paradoxical overproduction of TNF (associated with a higher parasitemia), lowered the sTNFR/TNF circulating ratios, and considerably worsened cachexia and mortality of animals. Another anti-TNF MAb (1F3F3) decreased the in vivo availability of TNF as well as parasite levels and reduced cachexia. Altogether, such results highlight that, besides playing a beneficial role early in infection, TNF also triggers harmful effects in the parasitemic phase, which are limited by the in vivo simultaneous endogenous production of soluble receptors.
PMCID: PMC96929  PMID: 10531203
12.  Trypanosoma cruzi Infection in Tumor Necrosis Factor Receptor p55-Deficient Mice 
Infection and Immunity  1998;66(6):2960-2968.
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.
PMCID: PMC108295  PMID: 9596773
13.  Tumor Necrosis Factor Alpha-Mediated Toxic Shock in Trypanosoma cruzi-Infected Interleukin 10-Deficient Mice 
Infection and Immunity  2000;68(7):4075-4083.
Using interleukin-10 (IL-10)-deficient (IL-10−/−) mice, previous studies revealed a pathological immune response after infection with Trypanosoma cruzi that is associated with CD4+ T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals. T. cruzi-infected IL-10−/− mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-γ), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-α). Despite this early resistance, IL-10−/− mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-α overproduction. Indeed, high production of systemic TNF-α significantly correlated with mortality, and moribund mice died with critically high TNF-α concentrations in the blood. Consequent treatment with anti-TNF-α antiserum attenuated pathological changes in T. cruzi-infected IL-10−/− mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-α concentrations and the time of death. Since elevated serum IL-12 and IFN-γ concentrations were not affected by the administration of antiserum, these studies suggest that TNF-α is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-α-mediated toxic shock.
PMCID: PMC101698  PMID: 10858224
14.  Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection 
Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.
PMCID: PMC2119081  PMID: 1730915
15.  Effects of Interleukin-4 Deprivation and Treatment on Resistance to Trypanosoma cruzi 
Infection and Immunity  2000;68(4):1975-1979.
Trypanosoma cruzi (Y strain)-infected interleukin-4−/− (IL-4−/−) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-γ) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4−/− mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-γ synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-γ synthesis levels; in contrast, IL-10 neutralization increased IFN-γ production and addition of rIL-4 and/or rIL-10 diminished IFN-γ synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruzi infection but that IL-4 can, in association with IL-10, modulate IFN-γ production and resistance.
PMCID: PMC97375  PMID: 10722591
16.  Prophylactic Efficacy of TcVac2 against Trypanosoma cruzi in Mice 
Chagas disease is a major health problem in Latin America, and an emerging infectious disease in the US. Previously, we have screened the Trypanosoma cruzi sequence database by a computational/bioinformatics approach, and identified antigens that exhibited the characteristics of vaccine candidates.
We investigated the protective efficacy of a multi-component DNA-prime/protein-boost vaccine (TcVac2) constituted of the selected candidates and cytokine (IL-12 and GM-CSF) expression plasmids in a murine model. C57BL/6 mice were immunized with antigen-encoding plasmids plus cytokine adjuvants, followed by recombinant proteins; and two-weeks later, challenged with T. cruzi trypomastigotes. ELISA and flow cytometry were employed to measure humoral (antibody isotypes) and cellular (lymphocyte proliferation, CD4+ and CD8+ T cell phenotype and cytokines) responses. Myocardial pathology was evaluated by H&E and Masson's trichrome staining.
Principal Findings
TcVac2 induced a strong antigen-specific antibody response (IgG2b>IgG1) and a moderate level of lymphocyte proliferation in mice. Upon challenge infection, TcVac2-vaccinated mice expanded the IgG2b/IgG1 antibodies and elicited a substantial CD8+ T cell response associated with type 1 cytokines (IFN-γ and TNF-α) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8+ T cells and IFN-γ/TNF-α cytokines subsided, and IL-4/IL-10 cytokines became dominant in vaccinated mice. The tissue parasitism, inflammation, and fibrosis in heart and skeletal muscle of TcVac2-vaccinated chronic mice were undetectable by histological techniques. In comparison, mice injected with vector or cytokines only responded to T. cruzi by elicitation of a mixed (type 1/type 2) antibody, T cell and cytokine response, and exhibited persistent parasite burden and immunopathology in the myocardium.
TcVac2-induced activation of type 1 antibody and lymphocyte responses provided resistance to acute T. cruzi infection, and consequently, prevented the evolution of chronic immunopathology associated with parasite persistence in chagasic hearts.
Author Summary
Trypanosoma cruzi, a parasitic protozoan, is the etiologic agent of Chagas disease. Chagas disease is the most common cause of congestive heart failure related deaths among young adults in the endemic areas of South and Central America and Mexico. Vaccine development against Chagas disease has been dramatically limited because of extensive debate on the mechanisms involved in this pathology. It is now accepted that the presence of parasites in cardiac tissue is necessary to initiate and maintain the inflammatory responses and that therapeutic treatments or vaccines aimed at eliminating T. cruzi would limit or prevent the progression of chronic chagasic cardiomyopathy. In the present study, we have tested the protective efficacy of a multi-component heterologous DNA-prime/protein-boost vaccine TcVac2 in a murine model of T. cruzi infection. Immunization of mice with TcVac2 induced potent antibody, CD8+ T cell and cytokine responses that provided protection from acute parasitemia and chronic parasite persistence and immunopathology in chagasic mice in comparison to unvaccinated mice.
PMCID: PMC2919396  PMID: 20706586
17.  Interleukin-12 Enhances In Vivo Parasiticidal Effect of Benznidazole during Acute Experimental Infection with a Naturally Drug-Resistant Strain of Trypanosoma cruzi 
Antimicrobial Agents and Chemotherapy  1998;42(10):2549-2556.
The roles of gamma interferon (IFN-γ) and interleukin-12 (IL-12) in mediating and/or enhancing the in vivo trypanosomicidal activity of the nitroheterocyclic derivative benznidazole (Bz) were evaluated during early stages of experimental Chagas’ disease. Our results show that treatment of Trypanosoma cruzi-infected mice with anti-cytokine monoclonal antibodies (MAbs) had no apparent effect when the optimal dose of Bz (100 mg/kg of body weight) was used. In contrast, treatment with anti-IL-12 or anti-IFN-γ MAbs enhanced the parasitemia and accelerated the mortality of mice treated with a suboptimal dose of Bz (25 mg/kg). Simultaneous treatment with a suboptimal dose of Bz and recombinant IL-12 (rIL-12) enhanced the efficacy of drug treatment in terms of parasitemia and mouse survival. Interestingly, we found that drug-resistant T. cruzi strains were found to be poor inducers of IL-12 both in vitro and in vivo compared to strains of T. cruzi which are susceptible or partially resistant to Bz treatment. These results suggest that early activation of the cellular compartment of the immune system by IL-12 may favor in vivo Bz activity against T. cruzi. In order to test this hypothesis mice infected with the drug-resistant Colombiana strain of T. cruzi were treated with 100 mg of Bz per kg plus different concentrations of rIL-12. By using the results of PCR and serological and parasitological methods as the criteria of a cure, our results indicate that a higher percentage of mice treated with Bz combined with rIL-12 than mice treated with Bz alone are cured.
PMCID: PMC105888  PMID: 9756754
18.  Polyfunctional T Cell Responses in Children in Early Stages of Chronic Trypanosoma cruzi Infection Contrast with Monofunctional Responses of Long-term Infected Adults 
Adults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells.
Methodology/Principal Findings
To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4+ T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4+TNF-α+-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4+ T cells was evident in T. cruzi-infected children.
Our observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.
Author Summary
Chagas disease is a neglected tropical disease affecting approximately 10 million people in the world. As a consequence of migration flows, the disease has also become established in non-endemic countries. Previous studies have demonstrated that Trypanosoma cruzi-specific T cells inversely correlates with the severity of cardiac disease in the chronic phase of the infection, suggesting that the immune system becomes exhausted overtime. To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were measured in T. cruzi-infected children – who are presumed to have shorter-term infections - and compared with long-term T. cruzi-infected adults. The activation status of total T cells in T. cruzi-infected children was also evaluated. T. cruzi-infected children exhibit a more robust, and more highly functional parasite specific T cell responses compared to T. cruzi-infected adults. In spite of a more functional immune profile, T. cruzi-infected children have a heightened state of immune activation. These observations are compatible with the initial hypothesis that T cell responses specific for T. cruzi become exhausted overtime. The impairment in T cell responses might contribute to disease progression in long-term infected subjects.
PMCID: PMC3861186  PMID: 24349591
19.  Protective Role of Acetylsalicylic Acid in Experimental Trypanosoma cruzi Infection: Evidence of a 15-epi-Lipoxin A4-Mediated Effect 
Chagas' disease, produced by Trypanosoma cruzi, affects more than 8 million people, producing approximately 10,000 deaths each year in Latin America. Migration of people from endemic regions to developed countries has expanded the risk of infection, transforming this disease into a globally emerging problem. PGE2 and other eicosanoids contribute to cardiac functional deficits after infection with T. cruzi. Thus, the inhibition of host cyclooxygenase (COX) enzyme emerges as a potential therapeutic target. In vivo studies about the effect of acetylsalicylic acid (ASA) upon T. cruzi infection are controversial, and always report the effect of ASA at a single dose. Therefore, we aimed to analyze the effect of ASA at different doses in an in vivo model of infection and correlate it with the production of arachidonic acid metabolites. ASA decreased mortality, parasitemia, and heart damage in T. cruzi (Dm28c) infected mice, at the low doses of 25 and 50 mg/Kg. However, this effect disappeared when the high ASA doses of 75 and 100 mg/Kg were used. We explored whether this observation was related to the metabolic shift toward the production of 5-lipoxygenase derivatives, and although we did not observe an increase in LTB4 production in infected RAW cells and mice infected, we did find an increase in 15-epi-LXA4 (an ASA-triggered lipoxin). We also found high levels of 15-epi-LXA4 in T. cruzi infected mice treated with the low doses of ASA, while the high ASA doses decreased 15-epi-LXA4 levels. Importantly, 15-epi-LXA4 prevented parasitemia, mortality, and cardiac changes in vivo and restored the protective role in the treatment with a high dose of ASA. This is the first report showing the production of ASA-triggered lipoxins in T. cruzi infected mice, which demonstrates the role of this lipid as an anti-inflammatory molecule in the acute phase of the disease.
Author Summary
Chagas' disease is an infection produced by the parasite Trypanosoma cruzi. This pathology is endemic in Latin America and has become a public health issue in some non-endemic countries like the USA, Spain and Australia. There is no curative treatment against Chagas' disease. NSAIDs, like aspirin, have been assayed as drugs with therapeutic potential in Chagas' disease, but the studies about this issue show contradictory results; also, the mechanism involved in aspirin effect is not yet clear. In this study, we explore a broad range of doses the protective role of aspirin. We found that aspirin has a therapeutic effect at low doses, an effect that disappears when doses are increased. This phenomenon correlates with the presence of 15-epi-LXA4, a molecule known as an “aspirin-triggered lipoxin,” which increases at low doses of aspirin, and decreases when aspirin dose is increased. 15-epi-LXA4 has been related with the anti-inflammatory effect of aspirin; in this setting, we found that 15-epi-LXA4 is able to decrease the cardiac inflammation and others parameters related with Chagas' disease. Finally, we present the first study that shows that the protective effect of aspirin on Chagas' disease could be mediated by the synthesis of 15-epi-LXA4.
PMCID: PMC3630130  PMID: 23638194
20.  IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis 
Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi.
Methodology/Principal Findings
First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4+, CD8+ and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-γ and TNF-α and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, RORγt and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice.
These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.
Author Summary
Chagas disease is caused by the intracellular parasite Trypanosoma cruzi. This infection has been considered one of the most neglected diseases and affects several million people in the Central and South America. Around 30% of the infected patients develop digestive and cardiac forms of the disease. Most patients are diagnosed during the chronic phase, when the treatment is not effective. Here, we showed by the first time that IL-17 is produced during experimental T. cruzi infection and that it plays a significant role in host defense, modulating parasite-induced myocarditis. Applying this analysis to humans could be of great value in unraveling the elements involved in the pathogenesis of chagasic cardiopathy and could be used in the development of alternative therapies to reduce morbidity during the chronic phase of the disease, as well as clinical markers of disease progression. The understanding of these aspects of disease may be helpful in reducing the disability-adjusted life years (DALYs) and costs to the public health service in developing countries.
PMCID: PMC2821906  PMID: 20169058
21.  Cruzipain Induces Both Mucosal and Systemic Protection against Trypanosoma cruzi in Mice  
Infection and Immunity  2002;70(9):5065-5074.
Cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi, is expressed by all developmental forms and strains of the parasite and stimulates potent humoral and cellular immune responses during infection in both humans and mice. This information suggested that cruzipain could be used to develop an effective T. cruzi vaccine. To study whether cruzipain-specific T cells could inhibit T. cruzi intracellular replication, we generated cruzipain-reactive CD4+ Th1 cell lines. These T cells produced large amounts of gamma interferon when cocultured with infected macrophages, resulting in NO production and decreased intracellular parasite replication. To study the protective effects in vivo of cruzipain-specific Th1 responses against systemic T. cruzi challenges, we immunized mice with recombinant cruzipain plus interleukin 12 (IL-12) and a neutralizing anti-IL-4 MAb. These immunized mice developed potent cruzipain-specific memory Th1 cell responses and were significantly protected against normally lethal systemic T. cruzi challenges. Although cruzipain-specific Th1 responses were associated with T. cruzi protective immunity in vitro and in vivo, adoptive transfer of cruzipain-specific Th1 cells alone did not protect BALB/c histocompatible mice, indicating that additional immune mechanisms are important for cruzipain-specific immunity. To study whether cruzipain could induce mucosal immune responses relevant for vaccine development, we prepared recombinant attenuated Salmonella enterica serovar Typhimurium vaccines expressing cruzipain. BALB/c mice immunized with salmonella expressing cruzipain were significantly protected against T. cruzi mucosal infection. Overall, these data indicate that cruzipain is an important T. cruzi vaccine candidate and that protective T. cruzi vaccines will need to induce more than CD4+ Th1 cells alone.
PMCID: PMC128245  PMID: 12183554
22.  Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection 
The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 104 T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.
PMCID: PMC4147224  PMID: 25170304
mMGL; Trypanosoma cruzi; Proinflammatory cytokines; C-Type lectin receptor; Macrophages receptors.
23.  Impaired Innate Immunity in Tlr4−/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice 
PLoS Pathogens  2010;6(4):e1000870.
The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/− or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.
Author Summary
Innate and acquired immune responses are triggered during infection with T. cruzi, the etiologic agent of Chagas' disease, and are critical for host survival. Parasite burden is usually controlled by the time the adaptive response becomes operational. Nevertheless, T. cruzi manages to subsist within intracellular niches and establishes a chronic infection, leading to the development of cardiomyopathy in approximately one-third of infected individuals. Recently, Toll-like receptors (TLRs) have been shown to recognize T. cruzi molecules and mice lacking MyD88, the key adaptor for most TLRs, are extremely susceptible to infection. Although TLRs are known to link innate and adaptive responses, their role in the establishment of crucial effector mechanisms mediated by CD8+ T cells during T. cruzi infection has not yet been determined. We analyzed the induction of IFN-γ and cytotoxic activity in vivo in TLR2-, TLR4-, TLR9- or MyD88-deficient mice during infection, and found intact responses compared to WT mice. We also demonstrated that TLR4 is required for optimal production of inflammatory cytokines and nitric oxide and, consequently, for a better control of parasitemia levels. Understanding how TLR activation leads to resistance to infection might contribute to the development of better strategies to improve immune responses against this pathogen.
PMCID: PMC2861687  PMID: 20442858
24.  Granulocyte-macrophage colony-stimulating factor: involvement in control of Trypanosoma cruzi infection in mice. 
Infection and Immunity  1996;64(8):3429-3434.
Several cytokines play crucial roles in Trypanosoma cruzi infection in mice, but the involvement of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) is poorly documented. This report shows that T. cruzi infection of mice triggered an early and sharp increase in plasma GM-CSF during the ascending phase of parasitemia. The plasma GM-CSF concentration remained stable at the peak of parasitemia and subsequently increased in those mice that survived to the acute phase. GM-CSF level increased again sharply, while parasitemia was rapidly decreasing. Finally, GM-CSF was undetectable, soon after the disappearance of circulating parasites. Injection of T. cruzi-infected mice with neutralizing anti-GM-CSF monoclonal antibodies induced the early appearance of parasitemia and aggravated cumulative mortality. In contrast, recombinant mouse GM-CSF (rmGM-CSF) caused sharp decreases in both parasitemia and cumulative mortality in T. cruzi-infected mice. Peritoneal macrophages from rmGM-CSF-treated and infected or uninfected mice were less infected ex vivo than those from control mice. Taken together these data demonstrate the protective action of endogenous GM-CSF in T. cruzi infection. Neutralization of endogenous GM-CSF aggravates infection, while exogenous rmGM-CSF decreases both parasitemia and host mortality.
PMCID: PMC174243  PMID: 8757888
25.  BAFF Mediates Splenic B Cell Response and Antibody Production in Experimental Chagas Disease 
B cells and antibodies are involved not only in controlling the spread of blood circulating Trypanosoma cruzi, but also in the autoreactive manifestations observed in Chagas disease. Acute infection results in polyclonal B cell activation associated with hypergammaglobulinemia, delayed specific humoral immunity and high levels of non-parasite specific antibodies. Since TNF superfamily B lymphocyte Stimulator (BAFF) mediates polyclonal B cell response in vitro triggered by T. cruzi antigens, and BAFF-Tg mice show similar signs to T. cruzi infected mice, we hypothesized that BAFF can mediate polyclonal B cell response in experimental Chagas disease.
Methodology/Principal Findings
BAFF is produced early and persists throughout the infection. To analyze BAFF role in experimental Chagas disease, Balb/c infected mice were injected with BR3:Fc, a soluble receptor of BAFF, to block BAFF activity. By BAFF blockade we observed that this cytokine mediates the mature B cell response and the production of non-parasite specific IgM and IgG. BAFF also influences the development of antinuclear IgG and parasite-specific IgM response, not affecting T. cruzi-specific IgG and parasitemia. Interestingly, BAFF inhibition favors the parasitism in heart.
Our results demonstrate, for the first time, an active role for BAFF in shaping the mature B cell repertoire in a parasite infection.
Author Summary
Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Central and South America. It affects 20 million people and about 100 million people are at risk of infection in endemic areas. Some cases have been identified in non-endemic countries as a consequence of blood transfusion and organ transplantation. Chagas disease presents three stages of infection. The acute phase appears one to two weeks after infection and includes fever, swelling around the bite site, enlarged lymph glands and spleen, and fatigue. This stage is characterized by circulating parasites and many immunological disturbances including a massive B cell response. In general, the acute episode self-resolves in about 2 months and is followed by a clinically silent indeterminate phase characterized by absence of circulating parasites. In about one-third of the cases, the indeterminate phase evolves into a chronic phase with clinically defined cardiac or digestive disturbances. Current knowledge suggests that the persistence of parasites coupled with an unbalanced immune response sustain inflammatory response in the chronic stage. We believe that an effective treatment for chronic Chagas disease should combine antiparasitic drugs with immunomodulators aimed at reducing inflammation and autoreactive response. Our findings enlighten a new role of BAFF-BAFF-R signaling in parasite infection that partially controls polyclonal B cell response but not parasitespecific class-switched primary effectors B cells.
PMCID: PMC2864296  PMID: 20454564

Results 1-25 (1111500)