Macrophages Promote Oxidative Metabolism To Drive Nitric Oxide Generation in Response to Trypanosoma cruzi

Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (m phi s), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) m phi s were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent m phi s timulation and infection states, respectively. M phi s incubated with lipopolysaccharide and gamma interferon (LPS/IFN-gamma) and with interleukin-4 (IL-4) were used as controls. We monitored the cytokine profile (using enzyme-linked immunosorbent assay [ELISA]), reactive oxygen species (ROS; fluorescent probes), nitric oxide (center dot NO; Griess assay), and metabolic state using a custom-designed mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-gamma treatment of m phi s elicited a potent increase in production of tumor necrosis alpha (TNF-alpha) at 3 h and of ROS and center dot NO by 18 h. Upon SYL infection, murine m phi s elicited an inflammatory cytokine profile (TNF-alpha >> TGF-beta + IL-10) and low levels of center dot NO and ROS production. LPS/IFN-gamma treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in m phi s, while IL-4-treated m phi s utilized oxidative metabolism to meet energy demands. SYL infection resulted in an intermediate functional metabolic state with increased mitoxosome gene expression and glycolysis, and IFN-gamma addition shut down the oxidative metabolism in SYL-infected m phi s. Further, TCC-and SYL-stimulated m phi s exhibited similar levels of cell proliferation and production of TNF-alpha and ROS, while TCC-stimulated m phi s exhibited up to 2-fold-higher levels of oxidative metabolism and center dot NO production than SYL-infected m phi s. Inhibiting ATP-coupled O-2 consumption suppressed the center dot NO generation in SYL-infected m phi s. Mitochondrial oxygen consumption constitutes a mechanism for stimulating center dot NO production in m phi s during T. cruzi infection. Enhancing the oxidative metabolism provides an opportunity for increased center dot NO production and pathogen clearance by m phi s to limit disease progression.