Genetic Ablation and Pharmacological Blockade of Bradykinin B1 Receptor Unveiled a Detrimental Role for the Kinin System in Chagas Disease Cardiomyopathy.

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.

Treatment with benznidazole and pentoxifylline regulates microRNA transcriptomic profile in a murine model of Chagas chronic cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is one of the leading causes of morbidity and mortality due to cardiovascular disorders in endemic areas of Chagas disease (CD), a neglected tropical illness caused by the protozoan parasite Trypanosoma cruzi. CCC is characterized by parasite persistence and inflammatory response in the heart tissue, which occur parallel to microRNA (miRNA) alterations. Here, we investigated the miRNA transcriptome profiling in the cardiac tissue of chronically T. cruzi-infected mice treated with a suboptimal dose of benznidazole (Bz), the immunomodulator pentoxifylline alone (PTX), or the combination of both (Bz+PTX), following the CCC onset. At 150 days post-infection, Bz, PTX, and Bz+PTX treatment regimens improved electrocardiographic alterations, reducing the percentage of mice afflicted by sinus arrhythmia and second-degree atrioventricular block (AVB2) when compared with the vehicle-treated animals. miRNA Transcriptome profiling revealed considerable changes in the differential expression of miRNAs in the Bz and Bz+PTX treatment groups compared with the control (infected, vehicle-treated) group. The latter showed pathways related to organismal abnormalities, cellular development, skeletal muscle development, cardiac enlargement, and fibrosis, likely associated with CCC. Bz-Treated mice exhibited 68 differentially expressed miRNAs related to signaling pathways like cell cycle, cell death and survival, tissue morphology, and connective tissue function. Finally, the Bz+PTX-treated group revealed 58 differentially expressed miRNAs associated with key signaling pathways related to cellular growth and proliferation, tissue development, cardiac fibrosis, damage, and necrosis/cell death. The T. cruzi-induced upregulation of miR-146b-5p, previously shown in acutely infected mice and in vitro T. cruzi-infected cardiomyocytes, was reversed upon Bz and Bz+PTX treatment regimens when further experimentally validated. Our results further our understanding of molecular pathways related to CCC progression and evaluation of treatment response. Moreover, the differentially expressed miRNAs may serve as drug targets, associated molecular therapy, or biomarkers of treatment outcomes.

Pro-Inflammatory Profile of Children Exposed to Maternal Chikungunya Virus Infection during the Intrauterine Period: A One-Year Follow-Up Study.

Chikungunya virus (CHIKV) vertical transmission occurs due to maternal viremia in the prepartum. Clinical presentation in neonates can be varied; however, the consequences of intrauterine exposure on the immune response are unclear. Thus, we aimed to analyze inflammatory alterations in children exposed to maternal CHIKV infection. This is a cross-sectional study that included children exposed to maternal CHIKV infection (confirmed by RT-qPCR and/or IgM). Circulant immune mediators were analyzed by a multiplex assay. RESULTS: We included 33 children, with a mean age of 3 ± 2.9 months-old, and 19 (57.6%) were male. Only one child presented neurological alterations. CHIKV-exposed infants showed elevated levels of MIP-1α, MIP-1ß, and CCL-2 (p < 0.05). Pro-inflammatory cytokines such as TNFα, IL-6, and IL-7 (p < 0.0001) were also increased. In addition, lower levels of PDGF-BB and GM-CSF were observed in the same group (p < 0.0001). Principal component (PC) analysis highlighted a distinction in the inflammatory profile between groups, where PC explained 56.6% of the alterations. Our findings suggest that maternal exposure to CHIKV can affect the circulating levels of pro-inflammatory cytokines during the infants' first year of life. The long-term clinical consequences of these findings should be investigated.

Treatment With Suboptimal Dose of Benznidazole Mitigates Immune Response Molecular Pathways in Mice With Chronic Chagas Cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe form of Chagas disease, a neglected tropical illness caused by the protozoan Trypanosoma cruzi, and the main cause of morbimortality from cardiovascular problems in endemic areas. Although efforts have been made to understand the signaling pathways and molecular mechanisms underlying CCC, the immunological signaling pathways regulated by the etiological treatment with benznidazole (Bz) has not been reported. In experimental CCC, Bz combined with the hemorheological and immunoregulatory agent pentoxifylline (PTX) has beneficial effects on CCC. To explore the molecular mechanisms of Bz or Bz+PTX therapeutic strategies, C57BL/6 mice chronically infected with the T. cruzi Colombian strain (discrete typing unit TcI) and showing electrocardiographic abnormalities were submitted to suboptimal dose of Bz or Bz+PTX from 120 to 150 days postinfection. Electrocardiographic alterations, such as prolonged corrected QT interval and heart parasite load, were beneficially impacted by Bz and Bz+PTX. RT-qPCR TaqMan array was used to evaluate the expression of 92 genes related to the immune response in RNA extracted from heart tissues. In comparison with non-infected mice, 30 genes were upregulated, and 31 were downregulated in infected mice. Particularly, infection upregulated the cytokines IFN-γ, IL-12b, and IL-2 (126-, 44-, and 18-fold change, respectively) and the T-cell chemoattractants CCL3 and CCL5 (23- and 16-fold change, respectively). Bz therapy restored the expression of genes related to inflammatory response, cellular development, growth, and proliferation, and tissue development pathways, most probably linked to the cardiac remodeling processes inherent to CCC, thus mitigating the Th1-driven response found in vehicle-treated infected mice. The combined Bz+PTX therapy revealed pathways related to the modulation of cell death and survival, and organismal survival, supporting that this strategy may mitigate the progression of CCC. Altogether, our results contribute to the better understanding of the molecular mechanisms of the immune response in the heart tissue in chronic Chagas disease and reinforce that parasite persistence and dysregulated immune response underpin CCC severity. Therefore, Bz and Bz+PTX chemotherapies emerge as tools to interfere in these pathways aiming to improve CCC prognosis.

CCL3/Macrophage Inflammatory Protein-1α Is Dually Involved in Parasite Persistence and Induction of a TNF- and IFNγ-Enriched Inflammatory Milieu in Trypanosoma cruzi-Induced Chronic Cardiomyopathy.

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 (IFNγ)+ cells and numbers of parasite-specific IFNγ-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ 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 IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

Single nucleotide polymorphisms of cytokine-related genes and association with clinical outcome in a Chagas disease case-control study from Brazil.

BACKGROUND: The severity of chronic chagasic cardiomyopathy (CCC), the most frequent clinical outcome of Chagas disease (CD), has been associated with cytokine-enriched heart tissue inflammation, and high serum levels of transforming growth factor (TGFß), interferon-gamma (IFNγ), and tumour necrosis factor (TNF). Conversely, increased interleukin (IL)-10 serum concentrations have been associated with asymptomatic CD. Cytokines and cytokine-related gene polymorphisms may control cytokine expression and have been proposed to contribute to CCC outcomes. OBJECTIVES: We evaluated the association of 13 cytokine-related genes (TGFB: rs8179181, rs8105161, rs1800469; IL10: rs1800890, rs1800871, rs1800896; IFNG: rs2430561; TNF: rs1800629; BAT1: rs3853601; LTA: rs909253, rs2239704; TNFR1: rs767455; TNFR2: rs1061624) with risk and progression of CCC. FINDINGS: Four hundred and six seropositive patients from CD endemic areas in the state of Pernambuco, north-eastern Brazil, were classified as non-cardiopathic (A, 110) or cardiopathic (mild, B1, 163; severe, C, 133). We found no evidence of TGFB, IL10, TNF, or TNFR1/2 gene polymorphisms associated with CCC risk or progression. Only BAT1 rs3853601 -22G carriers (B1 vs. C: OR = 0.5; p-value = 0.03) and IFNG rs2430561 +874AT (A vs. C: OR = 0.7; p-value = 0.03; A vs. B1+C: OR = 0.8; p-value = 0.02) showed a significant association with protection from cardiopathy in a logistic regression analysis with adjustment for gender and ethnicity; however, the association disappeared after performing adjustment for multiple testing. A systematic review of TNF rs1800629 -308G>A publications included five studies for meta-analysis (534 CCC and 472 asymptomatic patients) and showed no consensus in pooled odds ratio (OR) estimates for A allele or A carriers (OR = 1.4 and 1.5; p-values = 0.14 and 0.15, respectively). In CD patients, TNF serum levels were increased, but not affected by the TNF rs1800629 -308A allele. MAIN CONCLUSIONS: Our data suggest no significant contribution of the analysed gene variants of cytokine-related molecules to development/severity of Chagas' heart disease, reinforcing the idea that parasite/host interplay is critical to CD outcomes.

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5-/- mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Trypanosoma cruzi activates mouse cardiac fibroblasts in vitro leading to fibroblast-myofibroblast transition and increase in expression of extracellular matrix proteins.

BACKGROUND: Cardiac fibrosis is a consequence of chronic chagasic cardiomyopathy (CCC). In other cardiovascular diseases, the protagonist role of fibroblasts in cardiac fibrosis is well established. However, the role of cardiac fibroblasts (CFs) in fibrosis during the CCC is not clear. Here, our aim was to investigate the effect of Trypanosoma cruzi, the etiological agent of Chagas disease on CFs activation. METHODS: Cardiac fibroblasts were purified from primary cultures of mouse embryo cardiac cells. After two passages, cells were infected with T. cruzi (Y strain) and analyzed at different times for determination of infectivity, activation and production of extracellular matrix components (fibronectin, laminin and collagen IV) by immunofluorescence and western blot. RESULTS: At second passage, cultures were enriched in CFs (95% of fibroblasts and 5% of cardiomyocytes), as revealed by presence of alpha-smooth muscle actin (α-SMA) and discoidin domain receptor 2 (DDR2) and absence of sarcomeric tropomyosin (ST) protein expression. Trypanosoma cruzi infection induced fibroblast-myofibroblast transition, with increased expression of α-SMA after 6 and 24 h post-infection (hpi). Fibronectin was increased at 6, 24 and 48 hpi, laminin was increased at 6 and 24 hpi and collagen IV was increased at 6 hpi. CONCLUSIONS: Our results showed that T. cruzi activates CFs, inducing activation and exacerbates ECM production. Furthermore, our data raise the possibility of the involvement of CFs in heart fibrosis during Chagas disease.

Single nucleotide polymorphisms of cytokine-related genes and association with clinical outcome in a Chagas disease case-control study from Brazil

BACKGROUND The severity of chronic chagasic cardiomyopathy (CCC), the most frequent clinical outcome of Chagas disease (CD), has been associated with cytokine-enriched heart tissue inflammation, and high serum levels of transforming growth factor (TGFβ), interferon-gamma (IFNγ), and tumour necrosis factor (TNF). Conversely, increased interleukin (IL)-10 serum concentrations have been associated with asymptomatic CD. Cytokines and cytokine-related gene polymorphisms may control cytokine expression and have been proposed to contribute to CCC outcomes. OBJECTIVES We evaluated the association of 13 cytokine-related genes (TGFB: rs8179181, rs8105161, rs1800469; IL10: rs1800890, rs1800871, rs1800896; IFNG: rs2430561; TNF: rs1800629; BAT1: rs3853601; LTA: rs909253, rs2239704; TNFR1: rs767455; TNFR2: rs1061624) with risk and progression of CCC. FINDINGS Four hundred and six seropositive patients from CD endemic areas in the state of Pernambuco, north-eastern Brazil, were classified as non-cardiopathic (A, 110) or cardiopathic (mild, B1, 163; severe, C, 133). We found no evidence of TGFB, IL10, TNF, or TNFR1/2 gene polymorphisms associated with CCC risk or progression. Only BAT1 rs3853601 −22G carriers (B1 vs. C: OR = 0.5; p-value = 0.03) and IFNG rs2430561 +874AT (A vs. C: OR = 0.7; p-value = 0.03; A vs. B1+C: OR = 0.8; p-value = 0.02) showed a significant association with protection from cardiopathy in a logistic regression analysis with adjustment for gender and ethnicity; however, the association disappeared after performing adjustment for multiple testing. A systematic review of TNF rs1800629 −308G>A publications included five studies for meta-analysis (534 CCC and 472 asymptomatic patients) and showed no consensus in pooled odds ratio (OR) estimates for A allele or A carriers (OR = 1.4 and 1.5; p-values = 0.14 and 0.15, respectively). In CD patients, TNF serum levels were increased, but not affected by the TNF rs1800629 −308A allele. MAIN CONCLUSIONS Our data suggest no significant contribution of the analysed gene variants of cytokine-related molecules to development/severity of Chagas' heart disease, reinforcing the idea that parasite/host interplay is critical to CD outcomes.

Integration of miRNA and gene expression profiles suggest a role for miRNAs in the pathobiological processes of acute Trypanosoma cruzi infection.

Chagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America. Its acute phase is associated with high parasitism, myocarditis and profound myocardial gene expression changes. A chronic phase ensues where 30% develop severe heart lesions. Mouse models of T. cruzi infection have been used to study heart damage in Chagas disease. The aim of this study was to provide an interactome between miRNAs and their targetome in Chagas heart disease by integrating gene and microRNA expression profiling data from hearts of T. cruzi infected mice. Gene expression profiling revealed enrichment in biological processes and pathways associated with immune response and metabolism. Pathways, functional and upstream regulator analysis of the intersections between predicted targets of differentially expressed microRNAs and differentially expressed mRNAs revealed enrichment in biological processes and pathways such as IFNγ, TNFα, NF-kB signaling signatures, CTL-mediated apoptosis, mitochondrial dysfunction, and Nrf2-modulated antioxidative responses. We also observed enrichment in other key heart disease-related processes like myocarditis, fibrosis, hypertrophy and arrhythmia. Our correlation study suggests that miRNAs may be implicated in the pathophysiological processes taking place the hearts of acutely T. cruzi-infected mice.

Priming astrocytes with TNF enhances their susceptibility to Trypanosoma cruzi infection and creates a self-sustaining inflammatory milieu.

BACKGROUND: In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. METHODS: Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. RESULTS: rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain. CONCLUSIONS: Our data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.

MicroRNA Transcriptome Profiling in Heart of Trypanosoma cruzi-Infected Mice: Parasitological and Cardiological Outcomes.

Chagas disease is caused by the parasite Trypanosoma cruzi, and it begins with a short acute phase characterized by high parasitemia followed by a life-long chronic phase with scarce parasitism. Cardiac involvement is the most prominent manifestation, as 30% of infected subjects will develop abnormal ventricular repolarization with myocarditis, fibrosis and cardiomyocyte hypertrophy by undefined mechanisms. Nevertheless, follow-up studies in chagasic patients, as well as studies with murine models, suggest that the intensity of clinical symptoms and pathophysiological events that occur during the acute phase of disease are associated with the severity of cardiac disease observed during the chronic phase. In the present study we investigated the role of microRNAs (miRNAs) in the disease progression in response to T. cruzi infection, as alterations in miRNA levels are known to be associated with many cardiovascular disorders. We screened 641 rodent miRNAs in heart samples of mice during an acute infection with the Colombiana T.cruzi strain and identified multiple miRNAs significantly altered upon infection. Seventeen miRNAs were found significantly deregulated in all three analyzed time points post infection. Among these, six miRNAs had their expression correlated with clinical parameters relevant to the disease, such as parasitemia and maximal heart rate-corrected QT (QTc) interval. Computational analyses identified that the gene targets for these six miRNAs were involved in networks and signaling pathways related to increased ventricular depolarization and repolarization times, important factors for QTc interval prolongation. The data presented here will guide further studies about the contribution of microRNAs to Chagas heart disease pathogenesis.

Pentoxifylline reverses chronic experimental Chagasic cardiomyopathy in association with repositioning of abnormal CD8+ T-cell response.

BACKGROUND: Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8+ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8+ T-cell abnormalities and cardiac alterations using a model of experimental Chagas' heart disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice chronically infected by the Colombian Trypanosoma cruzi strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8+ cells induced by T. cruzi infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8+ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin+ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction. CONCLUSIONS/SIGNIFICANCE: PTX therapy ameliorates critical aspects of CCC and repositioned CD8+ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas' heart disease and to improve prognosis.

A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.

Tumor necrosis factor is a therapeutic target for immunological unbalance and cardiac abnormalities in chronic experimental Chagas' heart disease.

BACKGROUND: Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure. AIM OF THE STUDY: To challenge the hypothesis that TNF plays a key role in Trypanosoma cruzi-induced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas' heart disease. RESULTS: Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFNγ levels; diminished the frequency of IL-17A(+) but increased IL-10(+) CD4(+) T-cells; reduced TNF(+) but augmented IL-10(+) Ly6C(+) and F4/80(+) cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFNγ-producing VNHRFTLV-specific CD8(+) T-cells in spleen and reduced the number of perforin(+) cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue. CONCLUSIONS: Our data support that TNF is a crucial player in the pathogenesis of Chagas' heart disease fueling immunological unbalance which contributes to cardiac abnormalities.

Levels of circulating anti-muscarinic and anti-adrenergic antibodies and their effect on cardiac arrhythmias and dysautonomia in murine models of Chagas disease.

SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as ß 1 and ß 2 adrenergic (anti-ß 1-AR and anti-ß 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-ß 1-AR Ab are detected only in the chronic phase, while anti-ß 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60 dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.

Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease.

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas' heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas' heart disease.

Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas’ heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas’ heart disease.

Trypanosoma cruzi-induced depressive-like behavior is independent of meningoencephalitis but responsive to parasiticide and TNF-targeted therapeutic interventions.

Inflammatory cytokines and microbe-borne immunostimulators have emerged as triggers of depressive behavior. Behavioral alterations affect patients chronically infected by the parasite Trypanosoma cruzi. We have previously shown that C3H/He mice present acute phase-restricted meningoencephalitis with persistent central nervous system (CNS) parasitism, whereas C57BL/6 mice are resistant to T. cruzi-induced CNS inflammation. In the present study, we investigated whether depression is a long-term consequence of acute CNS inflammation and a contribution of the parasite strain that infects the host. C3H/He and C57BL/6 mice were infected with the Colombian (type I) and Y (type II) T. cruzi strains. Forced-swim and tail-suspension tests were used to assess depressive-like behavior. Independent of the mouse lineage, the Colombian-infected mice showed significant increases in immobility times during the acute and chronic phases of infection. Therefore, T. cruzi-induced depression is independent of active or prior CNS inflammation. Furthermore, chronic depressive-like behavior was triggered only by the type I Colombian T. cruzi strain. Acute and chronic T. cruzi infection increased indoleamine 2,3-dioxygenase (IDO) expression in the CNS. Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine abrogated the T. cruzi-induced depressive-like behavior. Moreover, treatment with the parasiticide drug benznidazole abrogated depression. Chronic T. cruzi infection of C57BL/6 mice increased tumor necrosis factor (TNF) expression systemically but not in the CNS. Importantly, TNF modulators (anti-TNF and pentoxifylline) reduced immobility. Therefore, direct or indirect parasite-induced immune dysregulation may contribute to chronic depressive disorder in T. cruzi infection, which opens a new therapeutic pathway to be explored.

Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury.

BACKGROUND: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/-)) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. METHODOLOGY: Rhesus monkeys and C57BL/6 and Nos2(-/-) mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+) cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue. RESULTS: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+) cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/-) mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. CONCLUSION: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas' heart disease.