Systems Approach Reveals Nuclear Factor Erythroid 2-Related Factor 2/Protein Kinase R Crosstalk in Human Cutaneous Leishmaniasis

Abstract

Leishmania parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo, and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.

Keywords: Leishmania; PKR; Sod1; macrophage; nuclear factor erythroid 2-related factor 2.

Figure 1

Leishmania amazonensis induced nuclear factor erythroid 2-related factor 2 (Nrf2) expression and nuclear translocation in a protein kinase R (PKR)-dependent manner. Peritoneal macrophages from wild-type or PKR-ko 129/sv mice were infected with stationary promastigotes forms of L. amazonensis for 2 h (A) or 6 h (B). Western-blot was carried out for nuclear or total protein extract, respectively, and then assay was performed using Nrf2 antibody. (C) THP-1 cells were infected with Leishmania amazonensis or treated with IFN-α or PolyI:C for 2 h for nuclear extract or 6 h for total protein extract, before western-blot analysis with Nrf2 antibody. (D) RAW-WT-PKR and RAW-DN-PKR cells were infected with stationary promastigotes forms of L. amazonensis for 4 h and then submitted to chromatin immunoprecipitation assay (ChIP) using Nrf2 ChIP-antibody. (E) RAW 264.7 cells were transiently transfected with p3xARE- or pNrf2-promoter-luciferase reporter plasmids constructs and infected with L. amazonensis 24 h post-transfection. Whole-cell lysates were analyzed for luciferase activity 24 h later. Results are representative of three independent experiments. *p < 0.05.

Figure 2

Sod1 regulation through nuclear factor erythroid 2-related factor 2 (Nrf2) and protein kinase R (PKR) signaling. (A) RAW 264.7 WT or DN-PKR cells were transiently transfected with Sod1-Luc plasmids and infected with stationary promastigotes of Leishmania amazonensis for additional 24 h before luciferase activity assay. (B) The same cells were also infected with stationary promastigotes of L. amazonensis for 18 h before total protein extract for western-blot analyzes with Sod1 and α-tubulin antibodies. (C) RAW 264.7 cells were infected with stationary promastigotes forms of L. amazonensis for 4 h and then submitted to chromatin immunoprecipitation assay (ChIP) using Nrf2 ChIP-antibody. shNrf2 or shControl THP-1 cells were infected with stationary promastigotes of L. amazonensis, (D) Sod1 protein expression was analyzed, and (E) infection index was evaluated. Results are representative of three independent experiments. *p < 0.05.

Figure 3

Protein kinase R (PKR)-dependent PIK3/Akt signaling activation controls positively the nuclear factor erythroid 2-related factor 2 (Nrf2) functions in Leishmania-infected macrophages. (A) Peritoneal macrophages from wild-type or PKR-ko 129/sv mice were infected with stationary promastigotes forms of Leishmania amazonensis at indicated times. Western-blot was carried out for total protein extract with anti-phospho-GSK3 and anti-GSK3. THP-1 cells stably knocked-down for Akt1 expression (B,C) and treated with PI3K/Akt inhibitors (LY294002, Wortmannin and Akt-inhibitor-VIII) (D,E) were infected with stationary promastigotes forms of Leishmania amazonensis at indicate times. Nuclear and total protein extracts were analyzed using Nrf2 antibody. (F) THP-1 cells were transiently transfected with p3xARE- or pNrf2-promoter Luciferase reporter plasmids. Twenty-four hours post-transfection, cells were differentiated into macrophages with phorbol-12 myristate-13 acetate (PMA) treatment for 6 days. The cells were infected with stationary promastigotes forms of L. amazonensis and/or treated with PI3K/Akt inhibitors for additional 24 h. Whole-cell lysates were analyzed for luciferase activity 24 h later. THP-1 cells were infected with stationary promastigotes forms of L. amazonensis and/or treated with Akt-inhibitor-VIII for 4 h and then submitted for chromatin immunoprecipitation assay (ChIP) using Nrf2 ChIP-antibody and primers for Nrf2 (G) and Sod1 (I) promoters. (H) Western-blot for total protein extract analyses with Sod1 antibody was performed at same conditions of infection and treatment. Results are representative of three independent experiments. *p < 0.05.

Figure 4

The reactive oxygen species (ROS) were enhanced upon Leishmania amazonensis infection in nuclear factor erythroid 2-related factor 2 (NRF2)/protein kinase R (PKR)/Akt-deficient macrophages. (A) shNrf2 or shControl and (B) wild-type THP-1 cells treated with PKR-inhibitor or Akt-inhibitor-VIII were infected with stationary promastigotes forms of L. amazonensis at indicated times together with probes for quantifying peroxynitrite (OONO), nitric oxide (NO), and ROS, and then analyzed as described in material and methods. (C) THP-1 transiently knocked-down for Nrf2 expression or shControl cells were infected with stationary promastigotes forms of L. amazonensis for 24 h and treated for additional 24 h with sulforaphane (SFN) or NAC (N-acetylcysteine) before the analysis of infection index. The asterisk means the statistic significant differences between the groups. Results are representative of three independent experiments. *p < 0.05.

Figure 4

The reactive oxygen species (ROS) were enhanced upon Leishmania amazonensis infection in nuclear factor erythroid 2-related factor 2 (NRF2)/protein kinase R (PKR)/Akt-deficient macrophages. (A) shNrf2 or shControl and (B) wild-type THP-1 cells treated with PKR-inhibitor or Akt-inhibitor-VIII were infected with stationary promastigotes forms of L. amazonensis at indicated times together with probes for quantifying peroxynitrite (OONO), nitric oxide (NO), and ROS, and then analyzed as described in material and methods. (C) THP-1 transiently knocked-down for Nrf2 expression or shControl cells were infected with stationary promastigotes forms of L. amazonensis for 24 h and treated for additional 24 h with sulforaphane (SFN) or NAC (N-acetylcysteine) before the analysis of infection index. The asterisk means the statistic significant differences between the groups. Results are representative of three independent experiments. *p < 0.05.

Figure 5

The nuclear factor erythroid 2-related factor 2 (Nrf2)-inhibitor Kelch-like ECH-associated protein 1 (Keap1) is modulated negatively through protein kinase R (PKR) signaling and p62 autophagy-dependent manner in Leishmania infection. (A) RAW-WT-PKR and RAW-DN-PKR cells were infected with stationary promastigotes forms of Leishmania amazonensis for 2 or 4 h and then western-blot assay were performed with total protein extract using Keap1 antibody. (B) THP-1 cells were infected with stationary promastigotes forms of L. amazonensis at indicate times and/or treated with chloroquine and the total or nuclear protein extracts were analyzed using Keap1 and Nrf2 antibodies. (C) THP-1 cells treated with chloroquine were infected with stationary promastigotes forms of L. amazonensis at indicated times together with probes for quantifying OONO, NO, and ROS. (D) THP-1 cells were infected with stationary promastigotes forms of L. amazonensis and western-blot for LC3-I/II protein was performed. RAW-WT-PKR and RAW-DN-PKR cells (E), and shNrf2 or shControl THP-1 cells (F) were infected with stationary promastigotes forms of L. amazonensis and then the total protein extract was analyzed by western-blot assay with p62 antibody. (G) RAW-WT-PKR and RAW-DN-PKR were infected with stationary promastigotes forms of L. amazonensis for 4 h and then submitted for ChIP assay using Nrf2 ChIP-antibody and primers for p62 promoter. Results are representative of three independent experiments. *p < 0.05.

Figure 6

Leishmania braziliensis and different strains of Leishmania amazonensis infections also modulated positively the protein kinase R (PKR)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis pathway. (A) L. amazonensis strains from LCL or diffuse cutaneous leishmaniasis (DCL) patients were used to infect THP-1 cells. Total or nuclear protein extracts were processed and then analyzed by western-blot with phospho-PKR and Nrf2 antibodies, respectively. (B) THP-1 cells were infected with stationary promastigotes forms of L. braziliensis at indicate times and then performed for western-blot with phospho-PKR and Nrf2 antibodies, and 3xARE-promoter Luciferase assays. Peritoneal macrophages of wild-type or PKR-ko mice were infected with L. braziliensis and infection index assays were then analyzed. (C) RAW-WT-PKR and RAW-DN-PKR cells were infected with L. braziliensis and western-blot with anti-Nrf2 was then analyzed. (D) shNrf2 or shControl THP-1 cells were infected with stationary promastigotes forms of L. amazonensis for 24 h before treatment with NAC or polyI:C for additional 24 h. After this time, the cells were fixed and the infection index was evaluated. (E) THP-1 transiently knocked-down for Nrf2 expression or shControl cells were infected with stationary promastigotes forms of L. amazonensis for 4 or 6 h before total protein extract for western-blot analyzes with p62 and Sod1 antibodies. (F) Histological sections from biopsies obtained from lesions of patients with DCL (n = 4) or with LCL (n = 5) were submitted to immunohistochemical reaction with primary antibodies against Nrf2 or Keap1 as previously described. All sections were counterstained with hematoxylin. Digital images (400× magnification) were captured using a Nikon E600 microscope and an Olympus Q-Color 1 digital camera with the Image Pro Plus program. Bars represent 10 µm. Positive cell density was obtained. Graph represents the analysis of reactive positive cells for Nrf2 and Keap1 compared with isotype controls as percentage of positive stained area per total tissue area. Results are representative of three independent experiments. *p < 0.05.

Figure 7

Nuclear factor erythroid 2-related factor 2 (Nrf2) transcriptome-wide correlations confirm the links between IFN/protein kinase R (PKR), antioxidant response element (ARE), PIK3, and autophagy signaling pathways in situ. (A) Transcriptome-wide correlation analysis was applied to Nrf2 transcript levels in microarray data (Illumina) obtained from skin biopsies from LCL patients (n = 20, red) and healthy controls (n = 10, green). Spearman correlation coefficient and p-values are shown for individual transcripts, 95% confidence intervals are shown in gray for patients only. (B) Validation of increased PKR/Nrf2 signaling pathway members by digital mRNA quantification. Heat map and hierarchical cluster analysis (Eucledian distance) of selected genes quantified by nCounter digital transcriptomics (Nanostring) in skin biopsies from normal donors (ND, n = 4) and LCL patients (n = 6), normalized according to PTPRC (CD45) expression levels, to account for differences in tissue leukocyte infiltration.

Figure 8

Proposed model for the protein kinase R (PKR)-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) activation in Leishmania infection. Internalized parasite signals through the endosomal compartment via TLR2 and induce activation of PKR by dimerization and subsequent autophosphorylation. Subsequently, we found that GSK3 phosphorylation is dependent of PKR signaling, allowing that not occur inhibition of Nrf2 through Neh6 inhibitory domain. This activation of Nrf2 is also dependent of Keap1 inhibition through of autophagic and PKR pathways. These mechanisms induce nuclear translocation Nrf2, increasing the gene expression of Sod1, Nrf2, and p62. The sequestosome-1 (p62) could be recruiting, together with processed LC3-II and Keap1 for autophagic vacuoles, allowing greater Nrf2 activation and inhibition of oxidative stress through antioxidant enzymes.