During acute experimental infection with the reticulotropic Trypanosoma cruzi strain Tulahuen IL-22 is induced IL-23-dependently but is dispensable for protection.
ABSTRACT: Protective immunity against Trypanosoma cruzi, the causative agent of Chagas disease, depends on the activation of macrophages by IFN-γ and IL-17A. In contrast, IL-10 prevents immunopathology. IL-22 belongs to the IL-10 cytokine family and has pleiotropic effects during host defense and immunopathology, however its role in protection and pathology during T. cruzi infection has not been analyzed yet. Therefore, we examined the role of IL-22 in experimental Chagas disease using the reticulotropic Tulahuen strain of T. cruzi. During infection, IL-22 is secreted by CD4-positive cells in an IL-23-dependent fashion. Infected IL-22(-/-) mice exhibited an increased production of IFN-γ and TNF and displayed enhanced numbers of activated IFN-γ-producing T cells in their spleens. Additionally, the production of IL-10 was increased in IL-22(-/-) mice upon infection. Macrophage activation and by association the parasitemia was not affected in the absence of IL-22. Apart from a transient increase in the body weight loss, infected IL-22(-/-) mice did not show any signs for an altered immunopathology during the first fourteen days of infection. Taken together, although IL-22 is expressed, it seems to play a minor role in protection and pathology during the acute systemic infection with the reticulotropic Tulahuen strain of T. cruzi.
Project description: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.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-gamma and TNF-alpha) that resulted in control of acute parasite burden. During chronic phase, antibody response persisted, splenic activation of CD8(+) T cells and IFN-gamma/TNF-alpha 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.
Project description:Infection with Trypanosoma cruzi, the etiologic agent of Chagas disease is accompanied by an intense inflammatory reaction. Our laboratory group has identified adipose tissue as one of the major sites of inflammation during disease progression. Because adipose tissue is composed of many cell types, we were interested in investigating whether the adipocyte per se was a source of inflammatory mediators in this infection. Cultured adipocytes were infected with the Tulahuen strain of T. cruzi for 48-96 h. Immunoblot and quantitative PCR (qPCR) analyses demonstrated an increase in the expression of proinflammatory cytokines and chemokines, including interleukin (IL)-1 beta, interferon-gamma, tumor necrosis factor-alpha, CCL2, CCL5, and CXCL10 as well as an increase in the expression of Toll-like receptors-2 and 9 and activation of the notch pathway. Interestingly, caveolin-1 expression was reduced while cyclin D1 and extracellular signal-regulated kinase (ERK) expression was increased. The expression of PI3kinase and the activation of AKT (phosphorylated AKT) were increased suggesting that infection may induce components of the insulin/IGF-1 receptor cascade. There was an infection-associated decrease in adiponectin and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These data provide a mechanism for the increase in the inflammatory phenotype that occurs in T. cruzi-infected adipocytes. Overall, these data implicate the adipocyte as an important target of T. cruzi, and one which contributes significantly to the inflammatory response observed in Chagas disease.
Project description:Trypanosoma cruzi is the etiologic agent of Chagas' disease, a major health problem in Latin America and an emerging infectious disease in the United States. Previously, we screened a T. cruzi sequence database by a computational-bioinformatic approach and identified antigens that exhibited the characteristics of good vaccine candidates. In this study, we tested the vaccine efficacy of three of the putative candidate antigens against T. cruzi infection and disease in a mouse model. C57BL/6 mice vaccinated with T. cruzi G1 (TcG1)-, TcG2-, or TcG4-encoding plasmids and cytokine (interleukin-12 and granulocyte-macrophage colony-stimulating factor) expression plasmids elicited a strong Th1-type antibody response dominated by immunoglobulin G2b (IgG2b)/IgG1 isotypes. The dominant IgG2b/IgG1 antibody response was maintained after a challenge infection and was associated with 50 to 90% control of the acute-phase tissue parasite burden and an almost undetectable level of tissue parasites during the chronic phase, as determined by a sensitive T. cruzi 18S rRNA gene-specific real-time PCR approach. Splenocytes from vaccinated-and-infected mice, compared to unvaccinated-and-infected mice, exhibited decreased (approximately 50% lower) proliferation and gamma interferon (IFN-gamma) production when stimulated in vitro with T. cruzi antigens, thus suggesting that protection from challenge infection was not provided by an active T-cell response. Subsequently, the serum and cardiac levels of IFN-gamma and tumor necrosis factor alpha and infiltration of inflammatory infiltrate in the heart were decreased in vaccinated mice during the course of infection and chronic disease development. Taken together, these results demonstrate the identification of novel vaccine candidates that provided protection from T. cruzi-induced immunopathology in experimental mice.
Project description:The identification of anti-inflammatory mediators can reveal important targetable molecules capable of counterbalancing Trypanosoma cruzi-induced myocarditis. Composed of Ebi3 and IL-27p28 subunits, IL-27 is produced by myeloid cells and is able to suppress inflammation by inducing IL-10-producing Tr1 cells, thus emerging as a potential candidate to ameliorate cardiac inflammation induced by T. cruzi. Although IL-27 has been extensively characterized as a suppressive cytokine that prevents liver immunopathogenesis after T. cruzi infection, the mechanisms underlying its effects on T. cruzi-induced myocarditis remain largely unknown. Here, wild-type (WT) and Ebi3-deficient animals were intraperitoneally infected with trypomastigotes of T. cruzi Y strain and used to evaluate the potential anti-inflammatory properties of Ebi3 during T. cruzi infection. The survival rates of mice were daily recorded, the frequency of inflammatory cells was analyzed by flow cytometry and inflammatory mediators were measured by ELISA, real-time PCR and PCR array. We reported that T. cruzi-induced myocarditis was prevented by Ebi3. Stressors mainly recognized by TLR2 and TLR4 receptors on myeloid cells were essential to trigger IL-27p28 production. In addition, Ebi3 regulated IFN-?-mediated myocarditis by promoting an anti-inflammatory environment through IL-10, which was most likely produced by Tr1 cells rather than classical regulatory T cells (Tregs), in the heart tissue of T. cruzi-infected animals. Furthermore, in vivo IFN-? blockade ameliorated the host survival without compromising the parasite control in the bloodstream. In humans, IL-27p28 was correlated with cardiac protection during Chagas disease. Patients with mild clinical forms of the disease produced high levels of IL-27p28, whereas lower levels were found in those with severe forms. In addition, polymorphic sites at Ebi3 gene were associated with severe cardiomyopathy in patients with Chagas disease. Collectively, we describe a novel regulatory mechanism where Ebi3 dampens cardiac inflammation by modulating the overproduction of IFN-?, the bona fide culprit of Chagas disease cardiomyopathy.
Project description:CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with Trypanosoma cruzi, the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 (ccl3+/+) and CCL3-deficient (ccl3-/-) mice by infection with the Colombian Type I strain. In ccl3+/+ mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with ccl3+/+, chronically infected ccl3-/- mice presented reduced cardiac parasitism and inflammation due to CD8+ cells and macrophages. Leukocytosis was decreased in infected ccl3-/- mice, paralleling the accumulation of CD8+ T cells devoid of activated CCR5+ LFA-1+ cells in the spleen. Further, T. cruzi-infected ccl3-/-mice presented reduced frequency of interferon-gamma (IFN?)+ cells and numbers of parasite-specific IFN?-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFN? improved parasite control, in a milieu with reduced nitric oxide (NOx) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically T. cruzi-infected ccl3+/+ counterparts, ccl3-/- mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with ccl3+/+, infected ccl3-/- mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of ccl3+/+ NI controls, most of the CD8+ T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10+, while in infected mice these cells were mainly TNF+. Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected ccl3+/+ mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic T. cruzi infection CCL3 takes part in parasite persistence and contributes to form a CD8+ T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFN? and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.
Project description:BACKGROUND:Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8+ T cell responses in HLA-A0201+ individuals chronically infected by T. cruzi. METHODS AND FINDINGS:Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8+ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8+ T cell responses to promiscuous epitopes revealed that the CD8+ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-gamma alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-gamma/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection. CONCLUSIONS:This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.
Project description:Chagas disease is transmitted by the parasite, Trypanosoma cruzi. Acute infection is characterized by acute myocarditis, although it is largely asymptomatic. Initial cardiac insult could be a determinant to the posterior development of chronic Chagasic cardiomyopathy, usually after 10 years in only approximately 30% of chronically infected patients. Herein, we characterized the acute gene expression profiling in heart tissue of two strains of mice infected with T. cruzi (tulahuen strain) at 4 weeks and their respective controls. Gene sequence data are available at NCBI under GEO accession number: GSE63847. The output of the genes expression suggests differences in involvement of protein kinase B (AKT), NCAM1, HLA-DRA, and ubiquitin C genes networks. These gene activation differences may correlate with myocardial contractility during the acute infection.
Project description:The inflammatory cytokine interferon-gamma (IFN?) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFN? is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFN? 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 IFN? mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFN?+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFN? 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 IFN? 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 IFN? fuels astrocyte infection by T. cruzi and critically implicate IFN?-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.
Project description:Trypanosoma cruzi, the protozoan parasite that causes human Chagas' disease, induces a type I interferon (IFN) (IFN-?/?) response during acute experimental infection in mice and in isolated primary cell types. To examine the potential impact of the type I IFN response in shaping outcomes in experimental T. cruzi infection, groups of wild-type (WT) and type I IFN receptor-deficient (IFNAR(-/-)) 129sv/ev mice were infected with two different T. cruzi strains under lethal and sublethal conditions and several parameters were measured during the acute stage of infection. The results demonstrate that type I IFNs are not required for early host protection against T. cruzi. In contrast, under conditions of lethal T. cruzi challenge, WT mice succumbed to infection whereas IFNAR(-/-) mice were ultimately able to control parasite growth and survive. T. cruzi clearance in and survival of IFNAR(-/-) mice were accompanied by higher levels of IFN-? production by isolated splenocytes in response to parasite antigen. The suppression of IFN-? in splenocytes from WT mice was independent of IL-10 levels. While the impact of type I IFNs on the production of IFN-? and other cytokines/chemokines remains to be fully determined in the context of T. cruzi infection, our data suggest that, under conditions of high parasite burden, type I IFNs negatively impact IFN-? production, initiating a detrimental cycle that contributes to the ultimate failure to control infection. These findings are consistent with a growing theme in the microbial pathogenesis field in which type I IFNs can be detrimental to the host in a variety of nonviral pathogen infection models.
Project description:BACKGROUND:The severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-? in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS:PBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-?-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-?-producing cells (i.e. IFN-? producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-? nonproducers) parasite-specific T-cell responses. IFN-? producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-? producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-? production in IFN-? producers but not in IFN-? nonproducers. CONCLUSIONS/SIGNIFICANCE:Alterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-? production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.