Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages

Abstract

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHⁱⁿT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1β induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

Keywords: glia; immune suppression; keto acids; macrophages; trypanosomes.

Figure 1

Trypanosoma brucei secreted factors induce HO-1 expression & suppress pro-inflammatory cytokines in mixed glia. (A) Primary murine mixed glia were cultured with different concentrations of Trypanosoma brucei for 24 h. Expression of HO-1 was measured by Western blot. Representative blot of three independent experiments is shown. (B) Supernatant from Trypanosoma brucei cultures was added to primary murine mixed glia. Expression of HO-1 was measured after 24 h by Western blot. Representative blot of three independent experiments is shown. Densitometric analysis of 3 immunoblots was performed using ImageLab (Bio-Rad) software. Results shown are mean ± SEM of the relative expression of HO-1: β-actin from 3 independent experiments. (C) Supernatant from Trypanosoma brucei cultures was added to primary murine mixed glia for 30 min prior to stimulation with LPS (100 ng/ml). Concentrations of TNFα and IL-6 in culture supernatants was measured by ELISA after 24 h. Results shown are mean ± SD concentrations from a triplicate culture, and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Full length blots are presented in Supplementary Figure 4.

Figure 2

Trypanosoma brucei produce and secrete aromatic ketoacids which are non-toxic and induce HO-1 expression in mixed glia and BMDM. Primary murine mixed glia and BMDM were incubated with indole pyruvate, hydroxyl-phenyl pyruvate, or phenylpyruvate (0.25–1 mM) for 24 h. (A) Viability of mixed glia was determined by alamarBlue reduction, and is expressed as a percentage of the untreated control. Results shown are mean ± SEM of the percentage viabilities of mixed glia from three independent experiments. (B) Expression of HO-1 in mixed glia was determined by Western blot. Blots shown are representative of three independent experiments. (C) Viability of BMDM was determined by alamarBlue reduction, and is expressed as a percentage of the untreated control. Results shown are mean ± SEM of the percentage viabilities of BMDM from four independent experiments. (D) Expression of HO-1 in BMDM was measured by Western blot. Blots shown are representative of two independent experiments. (E) Schematic depicting metabolism of aromatic ketoacids to aromatic hydroxyacids by AHADH. (F) Bloodstream form AHADHⁱⁿ cells were found to grow at similar rates to wild type MITat 1.2 T. brucei (▴), whether induced (°) or non-induced (•). (G) Secreted aromatic ketoacid concentrations in HMI-9 culture media, as measured via AHADH assay after 48 h. AHADH cells were either non-induced or induced with 2 mg/ml tetracycline. Results shown are mean ± SD of triplicate measurements, and are representative of three independent experiments. (H) Supernatants from WT and AHADHⁱⁿ T. brucei were added to mixed glia for 24 h. HO-1 expression was measured by Western blot. Representative blot of three independent experiments is shown. Full length blots are presented in Supplementary Figure 5.

Figure 3

Ketoacids inhibit pro-IL-1β expression in LPS-stimulated mixed glia, microglia, and BMDM. Primary murine mixed glia, microglia and BMDM were treated with indole pyruvate (IP), hydroxy-phenylpyruvate (HPP), or phenylpyruvate (PP) (0.25–1 mM) for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. Expression of pro-IL-1β in (A) mixed glia, (B) microglia, and (C) BMDM was measured by Western blot. Blots shown are representative of two to three independent experiments. Expression of iNOS in (D) BMDM was measured by Western blot. Blots shown are representative of three independent experiments. Full length blots are presented in Supplementary Figure 6.

Figure 4

Indole pyruvate and hydroxyl-phenyl pyruvate inhibit pro-inflammatory cytokine production in LPS-stimulated microglia but not BMDM. Primary murine microglia and BMDM were treated with indole pyruvate, hydroxy-phenylpyruvate, or phenylpyruvate (0.25–1 mM) for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. Concentrations of TNFα and IL-6 in (A) mixed glia and (B) BMDM supernatants was measured by ELISA. Results shown are mean ± SEM concentrations of IL-6 and TNFα from three to six independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 5

Ketoacids induce Nrf2 expression and upregulate Nrf-2 dependent genes in mixed glia and BMDM. (A) Primary murine mixed glia were treated with indole pyruvate, hydroxy-phenylpyruvate, or phenylpyruvate (0.25–1 mM) for 24 h. Expression of Nrf2 was measured by Western blot. Blots shown are representative of three independent experiments. (B) BMDM were treated with indole pyruvate, hydroxy-phenylpyruvate or phenylpyruvate (0.25–1 mM) for 24 h. Expression of Nrf2 was measured by Western blot. Blots shown are representative of three independent experiments. (C) Primary murine mixed glia were treated with indole pyruvate (IP), hydroxy-phenylpyruvate (HPP), or phenylpyruvate (PP) (0.5 mM) for 24 h. mRNA expression of NQO-1 and GSR was measured by RT-PCR. (D) BMDM were treated with indole pyruvate, hydroxy-phenylpyruvate, or phenylpyruvate (1 mM) for 24 h. mRNA expression of NQO-1 and GSR was measured by RT-PCR. Results shown are mean ± SEM fold expression of NQO-1 and GSR from three independent experiments. ***p < 0.001, *p < 0.05. Full length blots are presented in Supplementary Figure 7.