Chagasic cardiomyopathy is marked by a unique signature of activated CD4+ T cells.

Chagas disease is a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagas disease cardiomyopathy (CCC) is the most prominent clinical form and can lead to heart failure, thromboembolism, and sudden death. While previous reports have supported a role for CD4+ T lymphocytes in the pathogenesis of CCC a comprehensive analysis of these cells during different clinical forms is lacking. Here, we used high-dimensional flow cytometry to assess the diversity of circulating CD4+ T cells in patients with distinct clinical forms. We found increased frequencies of CD4+CD69+ T cells in patients compared to controls. CD39+ regulatory T cells, represented by mesocluster 6 were reduced in mild CCC patients compared to controls. Cytotoxic CD4+ T cells co-expressing granzyme B and perforin were expanded in patients with Chagas disease and were higher in patients with mild CCC compared to controls. Furthermore, patients with mild CCC displayed higher frequencies of multifunctional effector memory CD4+ T cells. Our results demonstrate an expansion in activated CD4+ T cells and a decrease in a functional subset of regulatory T cells associated with the onset of Chagas cardiomyopathy, suggesting their role in the establishment of cardiac lesions and as potential biomarkers for disease aggravation.

Behavioral alterations in long-term Toxoplasma gondii infection of C57BL/6 mice are associated with neuroinflammation and disruption of the blood brain barrier.

The Apicomplexa protozoan Toxoplasma gondii is a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition, T. gondii infection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type II T. gondii strain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic T. gondii infection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronic T. gondii infection.

Circulating inflammatory mediators as biomarkers of ocular toxoplasmosis in acute and in chronic infection.

Toxoplasmosis is highly endemic worldwide. In Brazil, depending on the geographical region and socioeconomic status, 40-70% of individuals become seropositive at some point in their lives. A significant proportion of Toxoplasma gondii-chronically infected individuals who are otherwise immunocompetent develop recurrent ocular lesions. The inflammatory/immune mechanisms involved in development of ocular lesion are still unknown and, despite previous investigation, there are no reliable immune biomarkers to predict/follow disease outcome. To better understand the impact of the immune response on parasite control and immunopathology of ocular toxoplasmosis, and to provide insights on putative biomarkers for disease monitoring, we assessed the production of a large panel of circulating immune mediators in a longitudinal study of patients with postnatally acquired toxoplasmosis stratified by the presence of ocular involvement, both at the early acute stage and 6 months later during chronic infection, correlating them with presence of ocular involvement. We found that T. gondii-infected patients, especially during the acute stage of the disease, display high levels of chemokines, cytokines, and growth factors involved in the activation, proliferation, and migration of inflammatory cells to injured tissues. In particular, major increases were found in the IFN-induced chemokines CXCL9 and CXCL10 in T. gondii-infected patients regardless of disease stage or clinical manifestations. Moreover, a specific subgroup of circulating cytokines and chemokines including GM-CSF, CCL25, CCL11, CXCL12, CXCL13, and CCL2 was identified as potential biomarkers that accurately distinguish different stages of infection and predict the occurrence of ocular toxoplasmosis. In addition to serving as predictors of disease development, these host inflammatory molecules may offer promise as candidate targets for therapeutic intervention.

Increased frequencies of circulating CCR5+ memory T cells are correlated to chronic chagasic cardiomyopathy progression.

The infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease, a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, culminating in heart failure and high rates of sudden death. CCC pathogenesis is influenced by both host and parasite factors and is proposed to be mostly immune-driven. Chemokines are crucial players in orchestrating immune cell recruitment to infected tissues and inflammation. Herein, we investigated inflammatory chemokine receptor expression on circulating T cells in patients stratified by CCC severity. Compared to asymptomatic individuals, we found increased percentages of effector CD4+ T cells and central memory CD4+ and CD8+ T cells expressing CCR5 in patients with structural cardiopathy, but normal global ventricular function and no symptoms of chronic heart failure. Even naïve T cells expressed CCR5 in these patients. In contrast, reduced frequencies of CD4+ and CD8+ effector T cells expressing CXCR3 were observed in patients presenting with severe heart disease. Patients with increased left ventricular diameter, heart enlargement, and insufficiency had higher frequencies of CCR5+ effector and effector memory CD8+ T cells. Moreover, the percentage of effector CCR5+ CD8+ T cells was increased in patients with a reduced ejection fraction. Our results show that high expression CCR5 and low expression of CXCR3 on circulating T cells are associated with worse prognosis, possibly reflecting immune-mediated cardiac remodeling of CCC.

Low-Level Parasite Persistence Drives Vasculitis and Myositis in Skeletal Muscle of Mice Chronically Infected with Trypanosoma cruzi.

In chronic Trypanosoma cruzi infection, the cause of Chagas disease, life-threatening inflammatory diseases develop over time in the heart, esophagus, and colon of some patients. C57BL/6 mice infected with the myotropic Colombiana strain of T. cruzi model many of the immunological and parasitological features of human infection but succumb to chronic paralyzing myositis and skeletal muscle vasculitis, not cardiomyopathy or gastrointestinal disease. Here we show that T cell depletion in the chronic phase of this model increased tissue parasitism to acute-phase levels and induced neutrophilic skeletal muscle inflammation. Conversely, after daily treatment with the trypanocide benznidazole for 8 weeks during the chronic phase, viable parasites were no longer detectable, myositis completely resolved, vasculitis was ∼80% reduced, fibrosis was reduced, and myofiber morphology normalized. After the drug was discontinued, parasitism rebounded, and immunopathology recurred. The parasite load was statistically strongly correlated with the severity of inflammation. Thus, both T cell immunity and trypanocidal pharmacotherapy suppress to very low levels, but do not cure, T. cruzi infection, which is necessary and possibly sufficient to induce crippling chronic skeletal muscle myositis and vasculitis in the model.

Down Modulation of Host Immune Response by Amino Acid Repeats Present in a Trypanosoma cruzi Ribosomal Antigen.

Several antigens from Trypanosoma cruzi, the causative agent of Chagas disease (CD), contain amino acid repeats identified as targets of the host immune response. Ribosomal proteins containing an Ala, Lys, Pro-rich repeat domain are among the T. cruzi antigens that are strongly recognized by antibodies from CD patients. Here we investigated the role of amino acid repeats present in the T. cruzi ribosomal protein L7a, by immunizing mice with recombinant versions of the full-length protein (TcRpL7a), as well as with truncated versions containing only the repetitive (TcRpL7aRep) or the non-repetitive domains (TcRpL7aΔRep). Mice immunized with full-length TcRpL7a produced high levels of IgG antibodies against the complete protein as well as against the repeat domain, whereas mice immunized with TcRpL7aΔRep or TcRpL7aRep produced very low levels or did not produce IgG antibodies against this antigen. Also in contrast to mice immunized with the full-length TcRpL7a, which produced high levels of IFN-γ, only low levels of IFN-γ or no IFN-γ were detected in cultures of splenocytes derived from mice immunized with truncated versions of the protein. After challenging with trypomastigotes, mice immunized with the TcRpL7a were partially protected against the infection whereas immunization with TcRpL7aΔRep did not alter parasitemia levels compared to controls. Strikingly, mice immunized with TcRpL7aRep displayed an exacerbated parasitemia compared to the other groups and 100% mortality after infection. Analyses of antibody production in mice that were immunized with TcRpL7aRep prior to infection showed a reduced humoral response to parasite antigens as well as against an heterologous antigen. In vitro proliferation assays with mice splenocytes incubated with different mitogens in the presence of TcRpL7aRep resulted in a drastic inhibition of B-cell proliferation and antibody production. Taken together, these results indicate that the repeat domain of TcRpL7a acts as an immunosuppressive factor that down regulates the host B-cell response against parasite antigens favoring parasite multiplication in the mammalian host.

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.

Interferon-gamma promotes infection of astrocytes by Trypanosoma cruzi.

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.

Genetic deletion of chemokine receptor Ccr7 exacerbates atherogenesis in ApoE-deficient mice.

AIMS: Recent evidence suggests that both Ccr7 and its ligands, Ccl19 and Ccl21, are present in mouse and human atherosclerotic plaques; however, the role of Ccr7 in atherogenesis is still controversial. Here, we addressed this question by using the classic apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: Ccr7(-/-)ApoE(-/-) double knockout mice and Ccr7(+/+)ApoE(-/-) littermates were generated and maintained on a high-fat Western diet for 8 weeks to induce atherosclerosis. Ccr7(-/-)ApoE(-/-) mice showed an ~80% increase in atherosclerotic lesion size in the whole aorta and a two-fold increase in the aortic root compared with Ccr7(+/+)ApoE(-/-) mice. Ccr7(-/-)ApoE(-/-) mice had increased T cells in the blood, bone marrow, and spleen, as well as in atherosclerotic lesions. Competitive repopulation experiments revealed that T cells from Ccr7(-/-)ApoE(-/-) mice migrated poorly into lymph nodes but better into mouse aortas compared with T cells from Ccr7(+/+)ApoE(-/-) mice. Transplantation of the bone marrow from Ccr7(-/-)ApoE(-/-) mice into lethally irradiated Ccr7(+/+)ApoE(-/-) mice resulted in ~60% more atherosclerotic lesions compared with Ccr7(+/+)ApoE(-/-) donor bone marrow, suggesting that exacerbation was mediated by a Ccr7(+) bone marrow-derived cell(s). Furthermore, in Ccr7(-/-)ApoE(-/-) mice the serum level of IL-12 was markedly increased, whereas the level of transforming growth factor beta (TGF-ß) was significantly decreased, suggesting an imbalance of T cell responses in these mice. CONCLUSION: Our data suggest that genetic deletion of Ccr7 exacerbates atherosclerosis by increasing T cell accumulation in atherosclerotic lesions.

Mannose-binding lectin regulates host resistance and pathology during experimental infection with Trypanosoma cruzi.

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas' disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas' disease. In this study we employed MBL(-/-) mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas' disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL(-/-) mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL(-/-) mice. Importantly, MBL(-/-) mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.