Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during Trypanosoma cruzi Infection and Chagas Disease.

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.

SIRT1-PGC1α-NFκB Pathway of Oxidative and Inflammatory Stress during Trypanosoma cruzi Infection: Benefits of SIRT1-Targeted Therapy in Improving Heart Function in Chagas Disease.

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.