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

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.

Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis

“…In addition to the production of pro-inflammatory cytokines, macrophages also produce nitric oxide, which plays a critical role in resistance against African trypanosomes via its cytostatic and cytotoxic effects ( 93 – 95 ). This is not surprising, given that the intracellular signaling pathways that result in the production of pro-inflammatory cytokines also lead to the activation of the iNOS gene and subsequent production of nitric oxide ( 96 ). Recently, we showed that in an IFN-γ-enriched environment, T. congolense -treated immortalized macrophages and BMDMs from the relatively resistant and highly susceptible mice show differential phosphorylation of MAPKs and STATs, suggesting that African trypanosomes induce differential signaling events in macrophages from different strains of mice in vitro ( 96 ).…”

“…This is not surprising, given that the intracellular signaling pathways that result in the production of pro-inflammatory cytokines also lead to the activation of the iNOS gene and subsequent production of nitric oxide ( 96 ). Recently, we showed that in an IFN-γ-enriched environment, T. congolense -treated immortalized macrophages and BMDMs from the relatively resistant and highly susceptible mice show differential phosphorylation of MAPKs and STATs, suggesting that African trypanosomes induce differential signaling events in macrophages from different strains of mice in vitro ( 96 ). In addition to differential MAPK and STAT phosphorylation, T. congolense induces significantly higher levels of NO production in IFN-γ-primed immortalized macrophages and BMDMs the relatively resistant than from highly susceptible mice ( 96 ).…”

“…Recently, we showed that in an IFN-γ-enriched environment, T. congolense -treated immortalized macrophages and BMDMs from the relatively resistant and highly susceptible mice show differential phosphorylation of MAPKs and STATs, suggesting that African trypanosomes induce differential signaling events in macrophages from different strains of mice in vitro ( 96 ). In addition to differential MAPK and STAT phosphorylation, T. congolense induces significantly higher levels of NO production in IFN-γ-primed immortalized macrophages and BMDMs the relatively resistant than from highly susceptible mice ( 96 ). Interestingly, we found that nitric oxide production in macrophages occurred via the activation of MAPK (including p38, Erk1/2, and JNK), and this was significantly inhibited by specific MAPK inhibitors in BALB/c but not in C57BL/6 cells ( 96 ).…”

“…In addition to differential MAPK and STAT phosphorylation, T. congolense induces significantly higher levels of NO production in IFN-γ-primed immortalized macrophages and BMDMs the relatively resistant than from highly susceptible mice ( 96 ). Interestingly, we found that nitric oxide production in macrophages occurred via the activation of MAPK (including p38, Erk1/2, and JNK), and this was significantly inhibited by specific MAPK inhibitors in BALB/c but not in C57BL/6 cells ( 96 ). In addition, T. congolense and IFN-γ-induced NO production was dependent on STAT1 phosphorylation and was totally suppressed by fludarabine (a specific STAT1 inhibitor).…”

African trypanosome control in the insect vector and mammalian host

“…From the invasive step and the ascending phase of parasitemia in the mammalian host ( Figure 2), proliferating parasites face innate resistance mechanisms mainly relying on type I interferon (IFN), NO, and tumor necrosis factor (TNF) released by M1-type myeloid cells [39][40][41][42][43].…”