Pentose Phosphate shunt Modulates reactive Oxygen species and nitric Oxide Production controlling Trypanosoma cruzi in Macrophages

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (M phi) anti-microbial response. M phi s infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classical M phi s. How M phi s 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 M phi s elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-alpha expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO by 18 h. Interferon (IFN)-gamma addition enhanced the Tc-induced ROS/ NO release and shut down mitochondrial respiration to the levels noted in classical M phi s. Inhibition of PPAR-alpha 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-gamma stimulated Mfs. 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 M phi. We conclude that IFN-gamma, but not inhibition of PPAR-alpha, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/ NO response in Tc-infected M phi s. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by M phi s.