Leishmania Parasites Differently Regulate Antioxidant Genes in Macrophages Derived From Resistant and Susceptible Mice

Abstract

Macrophage-Leishmania interactions are central to parasite growth and disease outcome. Macrophages have developed various strategies to fight invaders, including oxidative burst. While some microorganisms seem to survive and even thrive in an oxidative environment, others are susceptible and get killed. To counter oxidative stress, macrophages switch the expressions of cytoprotective and detoxifying enzymes, which are downstream targets of the nuclear factor erythroid 2-related factor 2 (Nrf2), to enhance cell survival. We have explored the transcription of NRF2 and of its target genes and compared the effect of the parasite on their transcription in bone marrow-derived macrophages (BMdMs) from Leishmania-resistant and Leishmania-susceptible mice. While heme oxygenase 1 (HO-1) transcription is independent of the genetic background, the transcription of glutathione reductase (Gsr) and of cysteine/glutamate exchange transporter (Slc7a11), involved in glutathione accumulation, was differentially regulated in BMdMs from both mouse strains. We also show that, except for HO-1, known to favor the survival of the parasite, the transcription of the selected genes, including Gsr, CD36, and catalase (CAT), was actively repressed, if not at all time points at least at the later ones, by the parasite, especially in Balb/c BMdMs. Consistent with these results, we found that the silencing of NRF2 in this study increases the survival and multiplication of the parasite.

Keywords: Leishmania; NRF2; antioxidants; macrophage; resistance; susceptibility.

Figure 1

Leishmania major induced the expression of HO-1 at both the mRNA and protein levels. Balb/c bone marrow-derived macrophages (BMdMs) and/or Raw264.7 cells were infected for the indicated time periods with L. major promastigotes. (A) The extracted mRNAs were used to perform RT-PCR. (B) The expression of heme oxygenase 1 (HO-1) was evaluated at the protein level by immunoblotting. CoCl₂ stimulation (250 ng/ml) for 3h was used as control. (C) Densitometric quantification of HO-1 protein levels using ImageJ. All data are expressed as the mean ± SD from three independent experiments. Statistical significance was placed at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. non-stimulated cells (two-way ANOVA).

Figure 2

Stimulation and nuclear accumulation of Nrf2 in response to Leishmania major infection. Balb/c bone marrow-derived macrophages (BMdMs) and/or Raw264.7 cells were infected for the indicated time period with L. major promastigotes. (A) The extracted mRNAs were used to perform RT-PCR. (B) Total Nrf2 expression was evaluated at the protein level by immunoblotting. (C) Densitometric quantification of the NRF2 protein levels using ImageJ. All data are expressed as the mean ± SD from two independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. non-stimulated BMdMs (two-way ANOVA). (D) Total protein extracts were fractionated into cytoplasmic and nuclear extracts and immunoblots performed with an anti-Nrf2 antibody. ERK1/2 and PARP were used to confirm the efficacy of cytosol and nuclear fractionation.

Figure 3

Leishmania major induced HO-1 expression through PI3K/Akt and the Nrf2 transcription factor. (A, C) Raw264.7 cells were infected for the indicated time periods with L. major promastigotes and the expressions of Akt and HO-1 assessed using Western blot. CoCl₂ stimulation for 3 h (250 ng/ml) was used as the control. (B, D) Macrophages were pre-incubated with wortmannin (200 mM) for 3 h, followed by infection with L. major promastigotes for different times. Akt phosphorylation and HO-1 expression were determined by immunoblotting. (E) Raw264.7 cells were transfected (24 h) with either Nrf2 (Si) or control siRNA (NT-pLKO), followed by infection with lipopolysaccharide (LPS) for 18 h, lysed, and examined by Western blot for Nrf2 expression. (F) NRF2 ^−/− cells were infected for different times with L. major promastigotes, lysed, and examined by Western blot for HO-1 expression.

Figure 4

NRF2 and HO-1 mRNAs were induced in Leishmania-susceptible and Leishmania-resistant bone marrow-derived macrophages (BMdMs). Balb/c and C57Bl6 BMdMs were infected by live parasites (P) (blue filled circle) or heat-inactivated (Kp) (red filled square) L. major promastigotes for different times. RT-PCR targeting Nrf2 and HO-1 was performed. The graphs show fold change results expressed as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. non-stimulated BMdMs (two-way ANOVA).

Figure 5

Transcription of different antioxidant genes was repressed by parasites. Balb/c bone marrow-derived macrophages (BMdMs) were infected with live parasites (P) (blue filled circle or heat-inactivated (Kp) (red filled square) L. major promastigotes for different times. RT-PCR targeting Slc7a11, GCLc, GCLm, Gsr, CAT, and CD36 was performed. The graphs show fold change results expressed as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. non-stimulated BMdMs (two-way ANOVA).

Figure 6

Slc7a11 and Gsr were differentially regulated by Leishmania parasites in Balb/c and C57Bl6 bone marrow-derived macrophages (BMdMs). Balb/c and C57Bl6 BMdMs were infected by live parasites (P) (blue filled circle) or heat-inactivated (Kp) (red filled square) L. major promastigotes for different times. RT-PCR targeting Slc7a11 and Gsr was performed. The graphs show fold change results expressed as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. non-stimulated BMdMs (two-way ANOVA).

Figure 7

Silencing of NRF2 promoted parasite survival. NT-pLKO or si-NRF2 Raw264.7 cells were infected with Ds-Red promastigotes. Macrophages (MΦs) parasitized with Ds-Red parasites were analyzed 72 h post-infection by flow cytometry. The percentages of Ds-Red fluorescence corresponding to the multiplication of amastigotes in parasitized MΦs were compared in NT-pLKO and si-NRF2-infected macrophages.