Molecular Regulation of Trypanosoma congolense-Induced Nitric Oxide Production in Macrophages

Rani Singh,Bruce C Kone,Jude E Uzonna,Abdelilah S Gounni,Pierre Bobé

doi:10.1371/journal.pone.0059631

Abstract

BALB/c mice are highly susceptible while C57BL/6 mice are relatively resistant to experimental Trypanosoma congolense infection. Several reports show that an early interferon-gamma (IFN-γ) response in infected mice is critically important for resistance via the activation of macrophages and production of nitric oxide (NO). NO is a pivotal effector molecule and possesses both cytostatic and cytolytic properties for the parasite. However, the molecular mechanisms leading to T. congolense (TC)-induced NO release from macrophages are not known. In this study, we investigated the signaling pathways induced by trypanosomes in immortalized macrophage cell lines from the highly susceptible BALB/c (BALB.BM) and relatively resistant C57Bl/6 (ANA-1) mice. We found that T. congolense whole cell extract (TC-WCE) induces significantly higher levels of NO production in IFN-γ-primed ANA-1 than BALB.BM cells, which was further confirmed in primary bone marrow-derived macrophage (BMDM) cultures. NO production was dependent on mitogen-activated protein kinase (MAPK, including p38, Erk1/2, and JNK) phosphorylation and was significantly inhibited by specific MAPK inhibitors in BALB.BM, but not in ANA-1 cells. In addition, T. congolense- and IFN-γ-induced NO production in ANA-1 and BALB.BM cells was dependent on STAT1 phosphorylation and was totally suppressed by the use of fludarabine (a specific STAT1 inhibitor). We further show that T. congolense induces differential iNOS transcriptional promoter activation in IFN-γ-primed cells, which is dependent on the activation of both GAS1 and GAS2 transcription factors in BALB.BM but only on GAS1 in ANA-1 cells. Taken together, our findings show the existence of differential signalling events that lead to NO production in macrophages from the highly susceptible and relatively resistant mice following treatment with IFN-γ and T. congolense. Understanding these pathways may help identify immunomodulatory mechanisms that regulate the outcome of infection during Trypanosome infections.

Highlights

African trypanosomiasis, known as sleeping sickness, is a parasitic disease of humans and livestock that is transmitted by various species of tsetse fly belonging to the genus Glossina
We found that unlike ANA-1 cells, T. congolense lysate alone induced detectable levels of nitric oxide (NO) in BALB.BM cells. This effect was not observed in primary bone marrow-derived macrophages from BALB/c mice, suggesting that the immortalization processes may have impacted differently on ANA-1 and BALB.BM cell lines
We showed that mitogen-activated protein kinases (MAPKs) differentially regulate NO production in BALB/c and C57BL/6 macrophages in the presence of IFN-c and T. congolense lysate

Summary

Introduction

Known as sleeping sickness, is a parasitic disease of humans and livestock that is transmitted by various species of tsetse fly belonging to the genus Glossina. The disease causes significant mortality in both humans and livestock and significantly impacts on economic development of sub-Saharan African countries where it is endemic. Trypanotolerance, or the capacity of some indigenous West African cattle breeds such as the N’dama to remain productive despite being infected, is correlated with a genetic capacity to limit parasitemia, anaemia and production of proinflammatory cytokines [5]. C57Bl/6 mice are considered relatively resistant because they can control several waves of parasitemia and survive up to 80–120 days after infection [6]. Macrophages play a critical role in the control of many protozoan parasitic infections including African trypanosomiasis. The parasiticidal activities of macrophages has been shown to correlate with changes in their inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production [7,8]; which is in part related to the levels of interferongamma (IFN-c) production by T cells

Objectives

Methods

Results

Conclusion