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1.
Microb Pathog ; : 104884, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33864876

RESUMO

Dogs are a reservoir for Chagas disease, caused by Trypanosoma cruzi (T. cruzi), and other companion vector-borne diseases, including ehrlichiosis (Ehrlichia canis and Ehrlichia ewingii), anaplasmosis (Anaplasma phagocytophilum and Anaplasma platys), dirofilariasis (Dirofilaria immitis) and Lyme disease (Borrelia burgdorferi). This study has two key objectives: 1) to determine seroreactivity against T. cruzi in dogs from the town of Colón, in Portoviejo city, in the central coast of Ecuador; and 2) to establish the coinfection frequency of other companion vector-borne diseases in dogs positive for T. cruzi. Antibodies against T. cruzi were detected using two enzyme-linked immunosorbent assays. Diagnostic consensus between ELISA tests was established using the Cohen's Kappa coefficient. Other haemoparasitic diseases were detected using the IDEXX SNAP® 4Dx® kit in dogs previously diagnosed as T. cruzi-seropositive. From 84 dogs sampled, 57.14% (48/84) tested positive for T. cruzi. Co-infection analysis of 25 dogs positive for T. cruzi revealed antibodies also against Ehrlichia spp. (48%), Anaplasma spp. (28%), and Dirofilaria immitis (12%). These results provide a novel perspective regarding the status of these pathogens which co-infect dogs in Colón. Since all these pathogens are zoonotic, our findings should warn regional health authorities to implement sanitary programs, to better prevent and control vectors associated to these pathogens. On the other hand, human and veterinarian doctors, should consider that patients with a cardiac infection condition could be suffering co-infections with two or more vector transmitted pathogens.

2.
mBio ; 11(6)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33172999

RESUMO

Chagas disease (CD), caused by Trypanosoma cruzi, is a degenerative heart condition. In the present study, we investigated the role of poly [ADP-ribose] polymerase 1/activator protein 1 (PARP1/AP-1) in upregulation of profibrotic macrophages (Mϕ) and subsequent development of cardiac fibrosis in CD. We used in vitro and in vivo models of T. cruzi infection and chemical and genetic inhibition of Parp1 to examine the molecular mechanisms by which Mϕ might augment profibrotic events in CD. Cultured (RAW 264.7 and THP-1) Mϕ infected with T. cruzi and primary cardiac and splenic Mϕ of chronically infected mice exhibited a significant increase in the expression, activity, and release of metalloproteinases (MMP2, MMP9, and MMP12) and the cytokine transforming growth factor ß (TGF-ß). Mϕ release of MMPs and TGF-ß signaled the cardiac fibroblast to myofibroblast differentiation, as evidenced by a shift from S100A4 to alpha smooth muscle actin (α-SMA) expression. Incubation of infected Mϕ with MMP2 and MMP9 inhibitors resulted in 60 to 74% decline in TGF-ß release, and MMP9 and PARP1 inhibitors resulted in 57 to 70% decline in Mϕ TGF-ß-driven cardiac fibroblast differentiation. Likewise, histological studies showed a 12- to 16-fold increase in myocardial expression of CD68 (Mϕ marker) and its colocalization with MMP9/TGF-ß, galectin-3, and vimentin in wild-type mice with CD. In comparison, chronically infected Parp1 -/- mice exhibited a >50% decline in myocardial levels of Mϕ and associated fibrosis markers. Further study showed that PARP1 synergized with c-Fos and JunB AP-1 family members for transcriptional activation of profibrotic response after T. cruzi infection. We conclude that PARP1 inhibition offers a potential therapy for controlling the T. cruzi-driven fibroblast differentiation in CD through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-ß pathway.IMPORTANCE Cardiomyopathy is the most important clinical manifestation of T. cruzi-driven CD. Recent studies have suggested the detrimental role of the matrix metalloproteinases MMP2 and MMP9 in extracellular matrix (ECM) degradation during cardiac remodeling in T. cruzi infection. Peripheral TGF-ß levels are increased in clinically symptomatic CD patients over those in clinically asymptomatic seropositive individuals. We provide the first evidence that during T. cruzi infection, Mϕ release of MMP2 and MMP9 plays an active role in activation of TGF-ß signaling of ECM remodeling and cardiac fibroblast-to-myofibroblast differentiation. We also determined that PARP1 signals c-Fos- and JunB-mediated AP-1 transcriptional activation of profibrotic gene expression and demonstrated the significance of PARP1 inhibition in controlling chronic fibrosis in Chagas disease. Our study provides a promising therapeutic approach for controlling T. cruzi-driven fibroblast differentiation in CD by PARP1 inhibitors through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-ß pathway.

3.
PLoS Pathog ; 16(4): e1008474, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32315358

RESUMO

Trypanosoma cruzi (T. cruzi) is the etiological agent of Chagas cardiomyopathy. In the present study, we investigated the role of extracellular vesicles (Ev) in shaping the macrophage (Mφ) response in progressive Chagas disease (CD). We purified T. cruzi Ev (TcEv) from axenic parasite cultures, and T. cruzi-induced Ev (TEv) from the supernatants of infected cells and plasma of acutely and chronically infected wild-type and Parp1-/- mice. Cultured (Raw 264.7) and bone-marrow Mφ responded to TcEV and TEv with a profound increase in the expression and release of TNF-α, IL-6, and IL-1ß cytokines. TEv produced by both immune (Mφ) and non-immune (muscle) cells were proinflammatory. Chemical inhibition or genetic deletion of PARP1 (a DNA repair enzyme) significantly depressed the TEv-induced transcriptional and translational activation of proinflammatory Mφ response. Oxidized DNA encapsulated by TEv was necessary for PARP1-dependent proinflammatory Mφ response. Inhibition studies suggested that DNA-sensing innate immune receptors (cGAS>>TLR9) synergized with PARP1 in signaling the NFκB activation, and inhibition of PARP1 and cGAS resulted in >80% inhibition of TEv-induced NFκB activity. Histochemical studies showed intense inflammatory infiltrate associated with profound increase in CD11b+CD68+TNF-α+ Mφ in the myocardium of CD wild-type mice. In comparison, chronically infected Parp1-/- mice exhibited low-to-moderate tissue inflammation, >80% decline in myocardial infiltration of TNF-α+ Mφ, and no change in immunoregulatory IL-10+ Mφ. We conclude that oxidized DNA released with TEv signal the PARP1-cGAS-NF-κB pathway of proinflammatory Mφ activation and worsens the chronic inflammatory pathology in CD. Small molecule antagonists of PARP1-cGAS signaling pathway would potentially be useful in reprogramming the Mφ activation and controlling the chronic inflammation in CD.


Assuntos
Doença de Chagas/metabolismo , Vesículas Extracelulares/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , Nucleotidiltransferases/imunologia , Poli(ADP-Ribose) Polimerase-1/imunologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
4.
Vaccines (Basel) ; 8(1)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098116

RESUMO

Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). We identified two candidate antigens (TcG2 and TcG4) that elicit antibodies and T cell responses in naturally infected diverse hosts. In this study, we cloned TcG2 and TcG4 in a nanovector and evaluated whether nano-immunotherapy (referred as nano2/4) offers resistance to chronic Chagas disease. For this, C57BL/6 mice were infected with Tc and given nano2/4 at 21 and 42 days post-infection (pi). Non-infected, infected, and infected mice treated with pcDNA3.1 expression plasmid encoding TcG2/TcG4 (referred as p2/4) were used as controls. All mice responded to Tc infection with expansion and functional activation of splenic lymphocytes. Flow cytometry showed that frequency of splenic, poly-functional CD4+ and CD8+ T cells expressing interferon-γ, perforin, and granzyme B were increased by immunotherapy (Tc.nano2/4 > Tc.p2/4) and associated with 88%-99.7% decline in cardiac and skeletal (SK) tissue levels of parasite burden (Tc.nano2/4 > Tc.p2/4) in Chagas mice. Subsequently, Tc.nano2/4 mice exhibited a significant decline in peripheral and tissues levels of oxidative stress (e.g., 4-hydroxynonenal, protein carbonyls) and inflammatory infiltrate that otherwise were pronounced in Chagas mice. Further, nano2/4 therapy was effective in controlling the tissue infiltration of pro-fibrotic macrophages and established a balanced environment controlling the expression of collagens, metalloproteinases, and other markers of cardiomyopathy and improving the expression of Myh7 (encodes ß myosin heavy chain) and Gsk3b (encodes glycogen synthase kinase 3) required for maintaining cardiac contractility in Chagas heart. We conclude that nano2/4 enhances the systemic T cell immunity that improves the host's ability to control chronic parasite persistence and Chagas cardiomyopathy.

5.
Front Immunol ; 11: 595039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33414785

RESUMO

A parasitic protozoan Trypanosoma cruzi (T. cruzi) is the etiologic agent of Chagas disease. Previously, we have identified T. cruzi antigens TcG2 and TcG4 as potential vaccine candidates, cloned in eukaryotic expression vector pCDNA3.1 (referred as p2/4) and tested their ability to elicit protection from T. cruzi infection. In the present study, we subcloned the two antigens in a nanoplasmid that is optimized for delivery, antigen expression, and regulatory compliance standards, and evaluated the nanovaccine (referred as nano2/4) for prophylactic protection against repeat T. cruzi infections. For this, C57BL/6 mice were immunized with two doses of p2/4 or nano2/4 at 21 days interval, challenged with T. cruzi 21 days after 2nd immunization, and euthanized at 10- and 21-days post-infection (pi) corresponding to parasite dissemination and replication phase, respectively. Some mice were re-challenged 21 days pi and monitored at 7 days after re-infection. Without the help of a vaccine, T. cruzi elicited delayed and sub-par T cell activation and low levels of effector molecules that failed to control tissue dissemination and replication of the parasite and provided no protection against repeat challenge infection. The nano2/4 was most effective in eliciting an early activation and production of IFN-γ by CD4+T effector/effector memory (TEM) cells and cytolytic perforin (PFN) and granzyme B (GZB) molecules by CD4+ and CD8+ TEM subsets at 10 days pi that was followed by robust expansion of CD4+ and CD8+ TEM and TCM cells with further increase in IFN-γ production at 21 days pi. Consequently, nano2/4-immunized mice exhibited potent control of parasite dissemination at 10 days pi, and tissue parasite burden and tissue inflammatory infiltrate and necrosis were barely detectable at 21 days pi. Furthermore, nano2/4-immunized mice responded to re-challenge infection with high levels of effector molecules production by CD4+ and CD8+ TEM subpopulations that offered even better control of tissue parasite burden than was observed after 1st infection. In comparison, non-vaccinated/infected mice exhibited clinical features of sickness and 59% mortality within 7 days after re-infection. In conclusion, we show that delivery of TcG2 and TcG4 in nanoplasmid offers excellent, protective T cell immunity against repeat T. cruzi infections.

6.
Front Immunol ; 11: 622602, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33679710

RESUMO

Innate immune cells play the first line of defense against pathogens. Phagocytosis or invasion by pathogens can affect mitochondrial metabolism in macrophages by diverse mechanisms and shape the macrophage response (proinflammatory vs. immunomodulatory) against pathogens. Besides ß-nicotinamide adenine dinucleotide 2'-phosphate, reduced (NADPH) oxidase, mitochondrial electron transport chain complexes release superoxide for direct killing of the pathogen. Mitochondria that are injured are removed by mitophagy, and this process can be critical for regulating macrophage activation. For example, impaired mitophagy can result in cytosolic leakage of mitochondrial DNA (mtDNA) that can lead to activation of cGAS-STING signaling pathway of macrophage proinflammatory response. In this review, we will discuss how metabolism, mtDNA, mitophagy, and cGAS-STING pathway shape the macrophage response to infectious agents.

7.
Front Immunol ; 10: 1456, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293599

RESUMO

Background: Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). Two antigenic candidates, TcG2 and TcG4, are recognized by antibodies in naturally infected dogs and humans; and these vaccine candidates provided protection from Tc infection in mice and dogs. Trypanosoma rangeli (Tr) is non-pathogenic to mammals and shown to elicit cross-reactive anti-Tc antibodies. In this study, we investigated if fixed Tr (fTr) can further enhance the efficacy of the TcG2/TcG4 DNA vaccine. Methods and Results: C57BL/6 mice were immunized with TcG2/TcG4 DNA vaccine and fTr (delivered as an adjuvant or in prime-boost approach), and challenged with Tc. Serology studies showed that fTr (±quil-A) elicited Tc- and Tr-reactive IgGs that otherwise were not stimulated by TcG2/TcG4 vaccine only, and quil-A had suppressive effects on fTr-induced IgGs. After challenge infection, TcG2/TcG4-vaccinated mice exhibited potent expansion of antigen- and Tc-specific IgGs that were not boosted by fTr±quil-A. Flow cytometry analysis showed that TcG2/TcG4-induced dendritic cells (DC) and macrophages (Mφ) responded to challenge infection by expression of markers of antigen uptake, processing, and presentation, and production of pro-inflammatory cytokines. TcG2/TcG4-induced CD4+T cells acquired Th1 phenotype and expressed markers that orchestrate adaptive immunity. A fraction of vaccine-induced CD4+T cells exhibited iTreg phenotype responsible for aversion of self-injurious immune responses. Further, TcG2/TcG4-vaccinated mice exhibited potent expansion of poly-functional CD8+T cells with TNF-α/IFN-γ production and cytolytic phenotype post-infection. Subsequently, tissue parasites and pathology were hardly detectable in TcG2/TcG4-vaccinated/infected mice. Inclusion of fTr±quil-A had no clear additive effects in improving the Tc-specific adaptive immunity and parasite control than was noted in mice vaccinated with TcG2/TcG4 alone. Non-vaccinated mice lacked sufficient activation of Th1 CD4+/CD8+T cells, and exhibited >10-fold higher levels of tissue parasite burden than was noted in vaccinated/infected mice. Conclusion: TcG2/TcG4 vaccine elicits highly effective immunity, and inclusion of fTr is not required to improve the efficacy of DNA vaccine against acute Tc infection in mice.


Assuntos
Antígenos de Protozoários/farmacologia , Doença de Chagas/prevenção & controle , Imunidade Celular/efeitos dos fármacos , Imunização Secundária , Vacinas Protozoárias/farmacologia , Células Th1/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/farmacologia , Animais , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/patologia , Feminino , Camundongos , Vacinas Protozoárias/imunologia , Células Th1/patologia , Vacinas de DNA/imunologia
8.
Cells ; 9(1)2019 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-31905606

RESUMO

BACKGROUND: Trypanosoma cruzi (Tc) causes Chagas disease (CD) that is the most frequent cause of heart failure in Latin America. TNF-α+ monocytes/macrophages (Mo/Mφ) are associated with inflammatory pathology in chronic CD. In this study, we determined the progenitor lineage of Mo/Mφ contributing to inflammation and examined the regulatory role of SIRT1 in modulating the Mo/Mφ response in Chagas disease. METHODS AND RESULTS: C57BL/6 mice were infected with Tc, treated with SIRT1 agonist (SRT1720) after control of acute parasitemia, and monitored during chronic phase (150 days post-infection). Flow cytometry studies showed an increase in maturation of bone marrow hematopoietic stem cell (HSC)-derived Mo of proinflammatory and anti-inflammatory phenotype in acutely- and chronically-infected mice; however, these cells were not increased in splenic compartment of infected mice. Instead, yolk-sac-derived CD11b+ F4/80+ Mo/Mφ were increased in sinusoidal compartment of Chagas mice. The splenic CD11b+ F4/80+ Mo/Mφ of Chagas (vs. control) mice exhibited increased mRNA, protein, and surface expression of markers of proinflammatory phenotype (CD80+/CD64+ > CD200+/CD206+) associated with proinflammatory cytokines response (IL-6+TNF-α >> Arg-1+IL-10), and these were also detected in the myocardium of chronically infected mice. Infected mice treated with SRT1720 (vs. infected/untreated) exhibited decreased splenic expansion and myocardial infiltration of proinflammatory Mo/Mφ. SRT1720 did not alter the inherent capability of splenic Mo/Mφ of Chagas mice to respond to pathogen stimulus. Instead, SRT1720 dampened the Tc-induced increase in the expression and/or phosphorylation of focal adhesion kinase (FAK) and downstream transcription factors (Pu.1, c-Myb, and Runx1) involved in Mφ proliferation and migration and Notch1 involved in functional activation. Studies in cultured Mφ confirmed the agonistic effects of SIRT1 in controlling the Tc-induced, FAK-dependent increase in the expression of transcription factors and showed that SIRT1 agonist and FAK inhibitor abrogated the NF-κB transcriptional activity and inflammatory cytokine gene expression in Tc-infected Mφ. CONCLUSIONS: The proinflammatory Mo/Mφ of yolk sac origin drive the splenic and tissue inflammatory response in chronic CD. SRT1720 reprogrammed the Tc-induced FAK-dependent transcription factors involved in Mφ proliferation and proinflammatory activation in Chagas disease.


Assuntos
Doença de Chagas/complicações , Doença de Chagas/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Biomarcadores , Doença de Chagas/mortalidade , Doença de Chagas/parasitologia , Doença Crônica , Citocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imunofenotipagem , Inflamação/parasitologia , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/metabolismo
9.
Vaccine ; 37(2): 248-257, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30497833

RESUMO

The efforts for the development and testing of vaccines against Trypanosoma cruzi infection have increased during the past years. We have designed a TcVac series of vaccines composed of T. cruzi derived, GPI-anchored membrane antigens. The TcVac vaccines have been shown to elicit humoral and cellular mediated immune responses and provide significant (but not complete) control of experimental infection in mice and dogs. Herein, we aimed to test two immunization protocols for the delivery of DNA-prime/DNA-boost vaccine (TcVac1) composed of TcG2 and TcG4 antigens in a BALB/c mouse model. Mice were immunized with TcVac1 through intradermal/electroporation (IDE) or intramuscular (IM) routes, challenged with T. cruzi, and evaluated during acute phase of infection. The humoral immune response was evaluated through the assessment of anti-TcG2 and anti-TcG4 IgG subtypes by using an ELISA. Cellular immune response was assessed through a lymphocyte proliferation assay. Finally, clinical and morphopathological aspects were evaluated for all experimental animals. Our results demonstrated that when comparing TcVac1 IDE delivery vs IM delivery, the former induced significantly higher level of antigen-specific antibody response (IgG2a + IgG2b > IgG1) and lymphocyte proliferation, which expanded in response to challenge infection. Histological evaluation after challenge infection showed infiltration of inflammatory cells (macrophages and lymphocytes) in the heart and skeletal tissue of all infected mice. However, the largest increase in inflammatory infiltrate was observed in TcVac1_IDE/Tc mice when compared with TcVac1_IM/Tc or non-vaccinated/infected mice. The extent of tissue inflammatory infiltrate was directly associated with the control of tissue amastigote nests in vaccinated/infected (vs. non-vaccinated/infected) mice. Our results suggest that IDE delivery improves the protective efficacy of TcVac1 vaccine against T. cruzi infection in mice when compared with IM delivery of the vaccine.


Assuntos
Doença de Chagas/prevenção & controle , Eletroporação/métodos , Vacinas Protozoárias/administração & dosagem , Vacinação/métodos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunidade Celular , Imunização Secundária , Imunoglobulina G/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologia , Absorção Cutânea , Trypanosoma cruzi/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
10.
PLoS Negl Trop Dis ; 12(7): e0006687, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30044789

RESUMO

In this study, we have investigated the effects of manganese superoxide dismutase (SOD2 or MnSOD) deficiency on mitochondrial function and oxidative stress during Chagas disease. For this, C57BL/6 wild type (WT) and MnSOD+/- mice were infected with Trypanosoma cruzi (Tc), and evaluated at 150 days' post-infection that corresponded to chronic disease phase. Genetic deletion of SOD2 decreased the expression and activity of MnSOD, but it had no effect on the expression of other members of the SOD family. The myocardial expression and activity of MnSOD were significantly decreased in chronically infected WT mice, and it was further worsened in MnSOD+/- mice. Chronic T. cruzi infection led to a decline in mitochondrial complex I and complex II driven, ADP-coupled respiration and ATP synthesis in the myocardium of WT mice. The baseline oxidative phosphorylation (OXPHOS) capacity in MnSOD+/- mice was decreased, and it had an additive effect on mitochondrial dysregulation of ATP synthesis capacity in chagasic myocardium. Further, MnSOD deficiency exacerbated the mitochondrial rate of reactive oxygen species (ROS) production and myocardial oxidative stress (H2O2, protein carbonyls, malondialdehyde, and 4-hydroxynonenal) in Chagas disease. Peripheral and myocardial parasite burden and inflammatory response (myeloperoxidase, IL-6, lactate dehydrogenase, inflammatory infiltrate) were increased in all chagasic WT and MnSOD+/- mice. We conclude that MnSOD deficiency exacerbates the loss in mitochondrial function and OXPHOS capacity and enhances the myocardial oxidative damage in chagasic cardiomyopathy. Mitochondria targeted, small molecule mitigators of MnSOD deficiency will offer potential benefits in averting the mitochondrial dysfunction and chronic oxidative stress in Chagas disease.


Assuntos
Cardiomiopatia Chagásica/enzimologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/deficiência , Animais , Cardiomiopatia Chagásica/genética , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/parasitologia , Feminino , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Miocárdio/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Superóxido Dismutase/genética , Trypanosoma cruzi/fisiologia
11.
PLoS Pathog ; 14(5): e1007065, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29851986

RESUMO

Chagasic cardiomyopathy is caused by Trypanosoma cruzi infection. Poly(ADP-ribose) polymerase 1 (PARP1) is known for its function in nuclear DNA repair. In this study, we have employed genetic deletion and chemical inhibition approaches to determine the role of PARP1 in maintaining mtDNA dependent mitochondrial function in Chagas disease. Our data show that expression of PARP1 and protein PARylation were increased by >2-fold and >16-fold, respectively, in the cytosolic, nuclear, and mitochondrial fractions of the human cardiac myocytes and the myocardium of wildtype (WT) mice chronically infected with T. cruzi. The nuclear and cytosolic PARP1/PAR did not interfere with the transcription and translation of the components of the mtDNA replisome machinery in infected cardiomyocytes and chagasic murine myocardium. However, PARP1 binding to Polymerase γ and mtDNA in mitochondria were increased, and associated with a loss in mtDNA content, mtDNA-encoded gene expression, and oxidative phosphorylation (OXPHOS) capacity, and an increase in mitochondrial ROS production in cells and heart of WT mice infected with T. cruzi. Subsequently, an increase in oxidative stress, and cardiac collagen deposition, and a decline in LV function was noted in chagasic mice. Genetic deletion of PARP1 or treatment with selective inhibitor of PARP1 (PJ34) improved the mtDNA content, mitochondrial function, and oxidant/antioxidant balance in human cardiomyocytes and chronically infected mice. Further, PARP1 inhibition was beneficial in preserving the cardiac structure and left ventricular function in chagasic mice. We conclude that PARP1 overexpression is associated with a decline in Pol γ-dependent maintenance of mtDNA content, mtDNA-encoded gene expression, and mitochondrial respiratory function, and subsequently contributes to an increase in mtROS and oxidative stress in chagasic myocardium. Inhibition of mitochondrial PARP1/PAR offers a novel therapy in preserving the mitochondrial and LV function in chronic Chagas disease.


Assuntos
Cardiomiopatia Chagásica/fisiopatologia , Polimerase do DNA Mitocondrial/genética , DNA Mitocondrial/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Antioxidantes/metabolismo , Células Cultivadas , Cardiomiopatia Chagásica/genética , Imunoprecipitação da Cromatina , DNA de Protozoário/fisiologia , Células HeLa , Coração/fisiologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Células Musculares/metabolismo , Miócitos Cardíacos/citologia , Estresse Oxidativo , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/genética , Função Ventricular Esquerda/fisiologia
12.
Front Immunol ; 9: 202, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29503646

RESUMO

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (Mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical Mφs. How Mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived Mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classical Mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc + IFN-γ stimulated Mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in Mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected Mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by Mφs.


Assuntos
Cardiomiopatia Chagásica/imunologia , Interações Hospedeiro-Parasita/imunologia , Macrófagos/imunologia , Via de Pentose Fosfato/imunologia , Trypanosoma cruzi/imunologia , Animais , Cardiomiopatia Chagásica/parasitologia , Modelos Animais de Doenças , Humanos , Interferon gama/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Knockout , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , PPAR alfa/genética , PPAR alfa/imunologia , Cultura Primária de Células , Células RAW 264.7 , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
13.
Front Microbiol ; 9: 3320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30697201

RESUMO

Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc-) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05 t-test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10-14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc-/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.

14.
Antioxid Redox Signal ; 27(9): 550-566, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28132522

RESUMO

AIMS: We investigated the effects of mitochondrial reactive oxygen species (mtROS) on nuclear factor (erythroid 2)-like 2 (NFE2L2) transcription factor activity during Trypanosoma cruzi (Tc) infection and determined whether enhancing the mtROS scavenging capacity preserved the heart function in Chagas disease. RESULTS: C57BL/6 wild type (WT, female) mice infected with Tc exhibited myocardial loss of mitochondrial membrane potential, complex II (CII)-driven coupled respiration, and ninefold increase in mtROS production. In vitro and in vivo studies showed that Tc infection resulted in an ROS-dependent decline in the expression, nuclear translocation, antioxidant response element (ARE) binding, and activity of NFE2L2, and 35-99% decline in antioxidants' (gamma-glutamyl cysteine synthase [γGCS], heme oxygenase-1 [HO1], glutamate-cysteine ligase modifier subunit [GCLM], thioredoxin (Trx), glutathione S transferase [GST], and NAD(P)H dehydrogenase, quinone 1 [NQO1]) expression. An increase in myocardial and mitochondrial oxidative adducts, myocardial interventricular septum thickness, and left ventricle (LV) mass, a decline in LV posterior wall thickness, and disproportionate synthesis of collagens (COLI/COLIII), αSMA, and SM22α were noted in WT.Tc mice. Overexpression of manganese superoxide dismutase (MnSOD) in cultured cells (HeLa or cardiomyocytes) and MnSODtg mice preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and cardiac oxidative and fibrotic pathology were significantly decreased in MnSODtg.Tc mice. Importantly, echocardiography finding of a decline in LV systolic (stroke volume, cardiac output, ejection fraction) and diastolic (early/late peak filling ratio, myocardial performance index) function in WT.Tc mice was abolished in MnSODtg.Tc mice. Innovation and Conclusion: The mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic cardiomyopathy. Preserving the NFE2L2 activity arrested the mitochondrial and cardiac oxidative stress, cardiac fibrosis, and heart failure in Chagas disease. Antioxid. Redox Signal. 27, 550-566.


Assuntos
Cardiomiopatias/fisiopatologia , Doença de Chagas/complicações , Regulação para Baixo , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Animais , Elementos de Resposta Antioxidante , Antioxidantes/metabolismo , Cardiomiopatias/genética , Doença de Chagas/genética , Modelos Animais de Doenças , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Superóxido Dismutase/metabolismo , Disfunção Ventricular Esquerda/genética
15.
Trends Cardiovasc Med ; 27(2): 92-94, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27686273
16.
J Innate Immun ; 9(2): 203-216, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27902980

RESUMO

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença de Chagas/imunologia , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/imunologia , Vacinas/imunologia , Animais , Doenças Assintomáticas , Linhagem Celular , Micropartículas Derivadas de Células/imunologia , Citocinas/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vacinação
17.
JACC Basic Transl Sci ; 1(4): 235-250, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27747306

RESUMO

BACKGROUND: Chagasic cardiomyopathy (CCM) caused by Trypanosoma cruzi (Tc) infection is prevalent in Latin America and recognized as an emerging infectious heart disease in the US. The NO-cGMP-PKG1α pathway maintains cardiac homeostasis and inotropy and may be disturbed due to phosphodiesterase (PDE5) mediated cGMP catabolism in CCM. METHODS AND RESULTS: C57BL/6 mice were infected with Tc, and at the end of acute parasitemia (i.e. 45 days post-infection), treated with sildenafil (SIL, 1 mg/kg) twice per week for 3 weeks. Mice were monitored at 150 days post-infection corresponding to chronic disease phase. The cGMP/PKG activity was decreased by 2-fold and PDE5 expression was increased by 1.4-fold and 100% at RNA and protein levels, respectively, in chagasic myocardium. Transthoracic echocardiography showed the left ventricular (LV) systolic function, i.e., stroke volume, cardiac output, and ejection fraction, were significantly decreased in chagasic mice. Sildenafil treatment resulted in normal levels of PDE5 and cGMP/PKG activity and preserved the LV function in chagasic mice. The cardioprotective effects of SIL were provided through inhibition of cardiac collagenosis and chronic inflammation that otherwise were pronounced in CCM. Further, mtDNA-encoded gene expression and ADP-coupled mitochondrial respiration were decreased and mitochondrial oxidative stress and cellular oxidative damage (lipid hydroperoxides and protein carbonyls) were increased in chagasic myocardium. SIL treatment restored the mtDNA-encoded gene expression and complex I (but not complex II) dependent ADP-coupled respiration, and established the oxidant/antioxidant balance in chagasic myocardium. In vitro studies in cardiomyocytes verified that SIL conserved the redox metabolic state and cellular health via maintaining the antioxidant status that otherwise was compromised in response to Tc infection. CONCLUSION: SIL therapy was useful in controlling the LV dysfunction and chronic pathology in CCM.

18.
PLoS Pathog ; 12(10): e1005954, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27764247

RESUMO

Chronic chagasic cardiomyopathy (CCM) is presented by increased oxidative/inflammatory stress and decreased mitochondrial bioenergetics. SIRT1 senses the redox changes and integrates mitochondrial metabolism and inflammation; and SIRT1 deficiency may be a major determinant in CCM. To test this, C57BL/6 mice were infected with Trypanosoma cruzi (Tc), treated with SIRT1 agonists (resveratrol or SRT1720), and monitored during chronic phase (~150 days post-infection). Resveratrol treatment was partially beneficial in controlling the pathologic processes in Chagas disease. The 3-weeks SRT1720 therapy provided significant benefits in restoring the left ventricular (LV) function (stroke volume, cardiac output, ejection fraction etc.) in chagasic mice, though cardiac hypertrophy presented by increased thickness of the interventricular septum and LV posterior wall, increased LV mass, and disproportionate synthesis of collagens was not controlled. SRT1720 treatment preserved the myocardial SIRT1 activity and PGC1α deacetylation (active-form) that were decreased by 53% and 9-fold respectively, in chagasic mice. Yet, SIRT1/PGC1α-dependent mitochondrial biogenesis (i.e., mitochondrial DNA content, and expression of subunits of the respiratory complexes and mtDNA replication machinery) was not improved in chronically-infected/SRT1720-treated mice. Instead, SRT1720 therapy resulted in 2-10-fold inhibition of Tc-induced oxidative (H2O2 and advanced oxidation protein products), nitrosative (inducible nitric oxide synthase, 4-hydroxynonenal, 3-nitrotyrosine), and inflammatory (IFNγ, IL1ß, IL6 and TNFα) stress and inflammatory infiltrate in chagasic myocardium. These benefits were delivered through SIRT1-dependent inhibition of NFκB transcriptional activity. We conclude that Tc inhibition of SIRT1/PGC1α activity was not a key mechanism in mitochondrial biogenesis defects during Chagas disease. SRT1720-dependent SIRT1 activation led to suppression of NFκB transcriptional activity, and subsequently, oxidative/nitrosative and inflammatory pathology were subdued, and antioxidant status and LV function were enhanced in chronic chagasic cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Animais , Antioxidantes/farmacologia , Western Blotting , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Resveratrol , Transdução de Sinais/fisiologia , Sirtuína 1/efeitos dos fármacos , Transcriptoma , Trypanosoma cruzi
19.
Int J Proteomics ; 2016: 1384523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27635260

RESUMO

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

20.
PLoS Negl Trop Dis ; 10(2): e0004490, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26919708

RESUMO

Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Leucócitos Mononucleares/química , Proteínas/química , Proteoma/química , Cardiomiopatia Chagásica/parasitologia , Doença Crônica , Eletroforese em Gel Bidimensional , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Trypanosoma cruzi/fisiologia
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