Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

Abstract

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease.

A change of heart: oxidative stress in governing muscle function?

“…Nitrated peptides derived from titin and actin are found in the plasma of patients with Chagas' disease, and may be useful as biomarkers for the disease (Dhiman et al 2008). In an experimental setting, oxidation of recombinant titin and actin enhanced their immunogenicity and recognition by sera antibodies from chagasic rats and patients (Dhiman et al 2012). Treatment with antioxidants (phenylbutylnitrone, vitamin C, and vitamin E) also consistently attenuated oxidative effects in mice (Wen et al 2006b) and patients with Chagas' disease (Maçao et al 2007).…”

AKT Network of Genes and Impaired Myocardial Contractility During Murine Acute Chagasic Myocarditis

“…20,21 Other differentially regulated networks, centered around HLA-DR and ubiquitin C may suggest important differences in antigen recognition and intracellular parasitic protein amastigote assembly and degradation and may increase immune recognition of cardiac oxidized antigens. 22,23 However, differences in HLA-DR might be a reflection of its great genetic variability and therefore, difficult to interpret. Although previous studies have been done for heart gene expression during T. cruzi infection, most of them analyzed chronic myocardial changes and compared them with noninfected groups.…”

TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice

“…Selected spots (Ͻ1 mm) on the 2D gels containing the protein of interest were excised and trypsin digested as described previously by us (29,30). Briefly, gel spots were incubated at 37°C for 30 min in 50 mM NH 4 HCO 3 and dehydrated twice for 5 min each time in 100 l of acetonitrile, and the proteins were digested in gel at 37°C overnight with 10 l of trypsin solution (1% trypsin in 25 mM ammonium bicarbonate).…”