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.
Highlights
Trypanosoma cruzi is the etiologic agent of Chagas disease
Our data showed the myocardial level of 4-HNE (Fig. 1B.b, score 3) and carbonyl (Fig. 1B.d & f, score 2 and 3) protein modifications were significantly increased in chagasic patients in comparison to that noted in healthy controls (Fig. 1B.a,c,e)
We demonstrated that myocardial levels of oxidative modifications (4-HNE, carbonyls, MDA, and 3-NT adducts) were significantly increased in chagasic patients and experimental animals, and affected cardiomyocytes viability
Summary
Trypanosoma cruzi is the etiologic agent of Chagas disease. According to World Health Organization estimates, the overall prevalence of human T. cruzi infection is at ,16–18 million cases, and ,120 million people are at risk of infection [1]. Upon exposure to T. cruzi, hosts elicit strong B and T cell-mediated immune responses, resulting in control of acute parasitemia (reviewed in [2,3]). Immune-mediated cytotoxic reactions are the source of reactive oxygen species (ROS, e.g. O2N2, H2O2, NOH) during acute T. cruzi infection [2,4]. ROS and secondary by-products of oxidative stress induce many different types of protein modifications [11]. Tyrosine residues on proteins are susceptible to attack by peroxynitrite and result in stable polypeptide-bound 3-nitrotyrosine (3-NT) residues [14]. These oxidative modifications of proteins, if not appropriately prevented, can have cytotoxic effects on the host
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