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

ABSTRACT: Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (m?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?s were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent m? stimulation and infection states, respectively. M?s incubated with lipopolysaccharide and gamma interferon (LPS/IFN-?) 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 (·NO; Griess assay), and metabolic state using a custom-designed mitoxosome array and Seahorse XF24 Analyzer. LPS/IFN-? treatment of m?s elicited a potent increase in production of tumor necrosis alpha (TNF-?) at 3 h and of ROS and ·NO by 18 h. Upon SYL infection, murine m?s elicited an inflammatory cytokine profile (TNF-? ? TGF-? + IL-10) and low levels of ·NO and ROS production. LPS/IFN-? treatment resulted in the inhibition of oxidative metabolism at the gene expression and functional levels and a switch to the glycolytic pathway in m?s, while IL-4-treated m?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-? addition shut down the oxidative metabolism in SYL-infected m?s. Further, TCC- and SYL-stimulated m?s exhibited similar levels of cell proliferation and production of TNF-? and ROS, while TCC-stimulated m?s exhibited up to 2-fold-higher levels of oxidative metabolism and ·NO production than SYL-infected m?s. Inhibiting ATP-coupled O2 consumption suppressed the ·NO generation in SYL-infected m?s. Mitochondrial oxygen consumption constitutes a mechanism for stimulating ·NO production in m?s during T. cruzi infection. Enhancing the oxidative metabolism provides an opportunity for increased ·NO production and pathogen clearance by m?s to limit disease progression.

SUBMITTER: Koo SJ

PROVIDER: S-EPMC5116729 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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

Koo Sue-Jie SJ Chowdhury Imran H IH Szczesny Bartosz B Wan Xianxiu X Garg Nisha J NJ

Infection and immunity 20161118 12

Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (mφs), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) mφs were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent mφ stimulation and infection states, respectively. Mφs incubated with lipopolysaccharide and gamma interferon (LPS/IFN-γ) and with inter ...[more]

PMID: 27698021

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Project description:BackgroundThe factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/-)) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection.MethodologyRhesus monkeys and C57BL/6 and Nos2(-/-) mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+) cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue.ResultsChronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+) cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/-) mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue.ConclusionT. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas' heart disease.

Project description:During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), β-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/β-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/β-catenin pathway and then activating the Wnt/Ca+2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of β-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.

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

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

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