Fig 1.
The infection of macrophages with Leishmania spp. leads to upregulation of NRF2 pathway.
A) Western blot analysis of WT and Nrf2-/- BMDMs infected with Lmj, Ltr, Lae, Lmex, Lam, LgyLRV1+, LgyLRV1-, Lbr, Lpa, Linf S1, Linf S2 or Ldo parasites for 4 hrs. Analysis of non-treated (Ø) or tBHQ-treated (10 μM) cells were performed concomitantly. Cells lysates were tested with anti-NRF2 and anti-γTUBULIN antibodies. B) WT BMDMs infected with LgyLRV1+ or LgyLRV1- parasites for 1, 2, 4 or 8 hrs, or stimulated with medium (Ø), or tBHQ (10 μM) were analyzed by immunoblotting with anti-NRF2 and anti-γTUBULIN antibodies. C) Immunofluorescence and quantification of NRF2 nuclear translocation expressed as nucleus/cytoplasm (N/C) ratio in WT BMDMs. Cells were stained with NRF2 (red), DAPI (blue) after 8 hrs of infection with LgyLRV1+ or LgyLRV1- parasites, or stimulated with medium (Ø), or tBHQ (10 μM) and imaged at 63x using a confocal microscope. Scale bar represents 10 and 40 μm for merged and enhanced images, respectively. NRF2 nuclear translocation was quantified for each cell (N = 100–160) using IMARIS software. D) Intracellular levels of ROS in CFSE labelled WT and Nrf2-/- cells infected with LgyLRV1+ or LgyLRV1- parasites or treated with medium (Ø) assessed using DHR 123 time-lapse microscopy. Cells were imaged at 20x for 14 hrs. ROS quantification was determined by Image J software using CFSE and DHR 123 staining. E) and F) Intracellular parasite load in WT and Nrf2-/- cells infected with LgyLRV1+ or LgyLRV1- parasites at 8 hrs (E) or 24 hrs (F) stained with DAPI and imaged at 40x using a high-content microscope. Parasite and cell quantification were assessed using MetaXpress software. Representative blots (A-B) from three independent experiments are shown. Representative images (C) and quantification (C and D) from one of three independent experiments are shown. Data are expressed as mean ± SEM. Statistical significance was calculated by performing two-way ANOVA analysis with Bonferroni’s post-test (D) and unpaired Student’s t test (C and F). * p < 0.05, ** p < 0.01 and **** p < 0.0001. See also S1 Fig.
Fig 2.
NRF2 participates to lesion development in WT mice and controls disease severity in immunocompromised mice infected with LgyLRV1+.
WT, Nrf2-/-, Ifng-/- or IfngxNrf2 dKO (dKO) mice were infected in both hind footpads with 1x106 of either LgyLRV1+ or LgyLRV1- parasites containing the bioluminescent luciferase gene. A) and D) Footpad swelling evolution was measured weekly as a proxy for disease progression for WT and Nrf2-/- mice (A) and Ifng-/- and dKO mice (D). B and E) ROS levels were measured by luminol-generated bioluminescence at week 4 for WT and Nrf2 -/- mice (B) or at week 5 for Ifng -/- and dKO mice (E). C and F) Parasite burden was quantified by luciferin detecting parasite bioluminescence either at week 4 for WT and Nrf2-/- mice (C) or at week 5 for Ifng-/- and dKO mice (F). G) Representative X-Ray images indicating tail tissue and bone destruction at week 8 for Ifng-/- and dKO mice. H) Representative images of Masson’s trichrome staining of footpad and tail sections at week 8 for Ifng-/- and dKO mice at 20x on an automated slide scanning microscope. Collagen (blue), nuclei (black), muscle and cytoplasm (red) are represented. Scale bar represents 50 μm or 200 μm for footpad and tail images, respectively. In vivo bioluminescence measurements were performed using Xtreme II (BRUKER) imaging system. Data represents the mean ± SEM or representative images from one representative experiment (n = 3–5 mice) of three independent experiments. Statistical significance was assessed by two-way ANOVA analysis with Bonferroni’s post-test (A, D, E and F) and unpaired Student’s t test (B and C). Not significant (ns), * p < 0.05, ** p < 0.01 and *** p < 0.001. See also S2 Fig.
Fig 3.
NRF2 expression controls inflammation in LgyLRV1+ infected macrophages.
WT, Tlr3-/- and Nrf2-/-, BMDMs were infected with either LgyLRV1+ or LgyLRV1- parasites. Negative and positive controls of non-treated (Ø), tBHQ (10 μM), poly I:C (I:C, 2 μg/ml), TLR3 agonist, were performed concurrently. A) Secreted levels of TNF-α into the supernatant at 24 hr time-point by ELISA. B) Immunofluorescence of nuclear translocation of P65 subunit of NF-κB complex expressed as nucleus/cytoplasm (N/C) ratio. Cells were stained with NRF2 (red), DNA (blue) at 24 hrs and imaged at 63x using a confocal microscope. Scale bar represents 10 μm. C) Quantification of P65 nuclear translocation at 24 hrs. P65 nuclear translocation was quantified for each cell (N = 50–90) by IMARIS software. Representative images from two independent experiments are shown. Data show mean ± SEM from a pool of three (A) and two (C) independent experiments. Unpaired Student’s t test was used to assess statistical significance. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 4.
NOX2 promotes NRF2 signaling through ROS in LgyLRV1+ and LgyLRV1- infections.
BMDMs from WT, Nox2-/-, Inos-/- and Nrf2-/- mice were either non-stimulated (Ø), or infected with LgyLRV1+ or LgyLRV1- parasites, or stimulated with tBHQ (10 μM). A) Cells lysates were collected at 4 hrs post-infection and NRF2 protein levels were assessed by Western Blot using anti-NRF2 and anti-γTUBULIN antibodies. Relative NRF2 levels were determined by band quantification using Image J software and given as NRF2 over γTUBULIN. B) Nqo1 relative RNA expression levels measured at 8 hrs post-infection by RT-qPCR and normalized to L32 housekeeping gene. C) Immunofluorescence and quantification of NRF2 nuclear translocation expressed as nucleus/cytoplasm (N/C) ratio in WT, Nox2-/- and Inos-/- BMDMs. Cells were stained with NRF2 (red), DNA (blue) at 8 hrs and imaged at 63x using a confocal microscope. Scale bar represents 10 μm. NRF2 nuclear translocation was quantified for each cell (N = 50–160) by IMARIS software. D and E) Cells were cultured with medium only (Ø) (D) or infected with LgyLRV1+ (E) for 6 hrs and treated with three different concentrations (250 μM, 100 μM, 50 μM) of the ROS donor, H2O2 for 2 hrs. Cell lysates were analyzed by Western blot for assessing NRF2 protein expression, γTUBULIN served as positive control. Relative NRF2 levels were determined by band quantification using Image J software and given as NRF2 over γTUBULIN. Representative blots and images and their quantification from three independent experiments. Data is expressed as mean ± SEM (B) from a pool of three independent experiments. Significance was calculated by Student’s t test. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also S4 Fig.
Fig 5.
Decreasing phagocytosis did not block NRF2 activation in Lgy infection.
A-D) WT, Nox2-/- or Nrf2-/- cells were pretreated with DMSO or Cytochalasin D (Cyt D, 40 μM) for 1 hr and infected with LgyLRV1+ or LgyLRV1- parasites or stimulated with tBHQ (10 μM), or medium-treated (Ø). A) Cells lysates were immunoblotted with anti-NRF2 and anti-γTUBULIN antibodies. B) WT cells were stained with NRF2 and DAPI for assessing NRF2 nuclear translocation expressed as nucleus/cytoplasm (N/C) ratio at 8 hrs at 40x using a high-content microscope. NRF2 nuclear translocation was quantified using MetaXpress software and normalized to non-infected (Ø). C) Nqo1 and Hmox1 relative RNA levels were measured by RT-qPCR at 8 hrs post-infection and normalized to L32 housekeeping gene. D) Intracellular parasite load was quantified by DAPI staining at 8 hrs at 40x using a high-content microscope. Parasite load was quantified using MetaXpress software. Representative blots are shown from three independent experiments. The graphs show pooled data expressed as mean ± SEM from two independent experiments. Unpaired Student’s t test was used to measure statistical significance. Not significant (ns), * p < 0.05, ** p < 0.01 and **** p < 0.0001. See also S5 Fig.
Fig 6.
Initial contact of Leishmania with its host cell activated the NRF2 pathway and parasite entry strongly induced metabolic reprogramming.
A) WT cells were infected with live, or heat-killed, or UV-treated or PFA-fixed LgyLRV1+ parasites, or were non-infected (Ø) for 8 hrs. Intracellular parasite load was quantified by DAPI staining at 40x using a high-content microscope. Parasite load was quantified using MetaXpress software. B) WT, Nox2-/- or Nrf2-/- cells were either infected with live or heat-killed or UV-treated or PFA-fixed LgyLRV1+ parasites. Non-treated (Ø) or tBHQ-treated (10 μM), or β-glucan peptide (BGP)-treated (100 μg/ml) cells were performed concurrently. Cell lysates after 4 hrs post-infection were analyzed by Western Blot using anti-NRF2 and anti-γTUBULIN antibodies. Relative NRF2 levels were determined by band quantification using Image J software and given as NRF2 over γTUBULIN. C and D) WT cells were either infected with live, or heat-killed, or UV-treated or PFA-fixed LgyLRV1+ parasites, or non-infected (Ø) for 8 hrs. Nrf2 (C) or Hmox1 (D) relative RNA levels were measured by RT-qPCR and normalized to the L32 housekeeping gene. E) WT and Nrf2-/- cells LgyLRV1+ parasites or non-infected (Ø) for 15 min and basal respiration represented as OCR was measured on Seahorse XFe96 Analyzer, adjusted to protein concentration per condition. F) WT cells were either infected with live, or heat-killed, or UV-treated or PFA-fixed LgyLRV1+ parasites, or non-infected (Ø) for 15 min and basal respiration represented as OCR was measured on Seahorse XFe96 Analyzer, adjusted to protein concentration per condition. G) WT cells were either infected with Linf S1 or Linf S3 or LgyLRV1+ or LgyLRV1- parasites, or non-infected (Ø) for 15 min and basal respiration represented as OCR was measured on Seahorse XFe96 Analyzer, adjusted to protein concentration per condition. H) WT cells were infected with LgyLRV1+ parasites, or non-infected (Ø) zymosan particles-treated (1 μg/ml) or β-glucan peptide (BGP)-treated (100 μg/ml) cells for 15 min and basal respiration represented as OCR was assessed on Seahorse XFe96 Analyzer, adjusted to protein concentration per condition. Representative blots and their quantification from two independent experiments. The graphs show pooled data expressed as mean ± SEM from three independent experiments. Unpaired Student’s t test was used to measure statistical significance. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. See also S6 Fig.
Fig 7.
Leishmania activates the host cell NRF2 pathway through an SRC family kinase signaling cascade.
A-C) WT, Nox2-/- or Nrf2-/- cells were pretreated with DMSO or PP2 (100 μM) for 1 hr and infected with LgyLRV1+ or LgyLRV1- parasites. Non-treated (Ø) or tBHQ-treated (10 μM) cells were performed concurrently. A) Cell lysates after 4 hrs post-infection were analyzed by Western Blot using anti-phosho-NRF2 (S40), anti-NRF2, anti-phospho-PKCδ (S311), anti-PKCδ, anti-phospho-SFK (Y416), anti-SFK and anti-γTUBULIN antibodies. B) Cells were stained with NRF2 and DAPI for assessing NRF2 nuclear translocation expressed as nucleus/cytoplasm (C/N) ratio at 8 hrs with a 40x using a high-content microscope. NRF2 nuclear translocation was quantified using MetaXpress software and normalized to non-infected (Ø). C) Nqo1 relative RNA levels were measured by RT-qPCR and normalized to L32 housekeeping gene. D) WT cells were pretreated with DMSO (-) or PP2 (100 μM) or KB SRC 4 (100 μM) for 1 hr and infected with LgyLRV1+ or LgyLRV1- parasites. Non-treated (Ø) cells were performed concurrently. Cell lysates after 4 hrs post-infection were analyzed by Western Blot using anti-phosho-NRF2 (S40), anti-NRF2, anti-phospho-PKCδ (S311), anti-PKCδ, anti-phospho-SFK (Y416), anti-SFK and anti-γTUBULIN antibodies. E-G) WT cells were pretreated with DMSO (-) or PP2 (100 μM) or KB SRC 4 (100 μM) for 1 hr and infected with LgyLRV1+. E) Nqo1 relative RNA levels were measured at an 8 hr time-point by RT-qPCR and normalized to L32 housekeeping gene. F) Intracellular parasite load was quantified by DAPI staining at 40x using a high-content microscope at 8 hrs post-infection. Parasite load quantified using MetaXpress software. G) Secreted levels of TNF-α into the supernatant at 24 hr time-point by ELISA. H-J) WT, Nox2-/- or Nrf2-/- cells were pretreated with DMSO or MK2206 (5 μM) for 1 hr and infected with either LgyLRV1+ or LgyLRV1- parasites, or non-treated (Ø). H) Cell lysates after 4 hrs post-infection were analyzed by Western Blot using anti-NRF2, anti-phospho-AKT (T308), anti-AKT and anti-γTUBULIN antibodies. I) Intracellular parasite load was quantified by DAPI staining at 40x using a high-content microscope at 8 hrs post-infection. Parasite load quantified using MetaXpress software. H) Representative image of live cell imaging performed to visualize parasite internalization at 20x by confocal microscopy using mChLgyLRV1+ (red) parasites. Scale bar: 40 μm. Anti-SFK and anti-phospho-SFK antibodies recognize SFK members including Lyn, Fyn, Lck, Yes and Hck proteins and their corresponding phosphorylation sites. Representative blots and images from two (D and I) and three (A) independent experiments are shown. The graphs show pool data expressed as mean ± SEM from two independent experiments. Unpaired Student’s t test was used to calculate statistical significance. Not significant (ns), * p < 0.05 and ** p < 0.01. See also S7 Fig.
Fig 8.
NRF2 protects from oxidative stress and hyperinflammation in Lgy infection.
During infection with Lgy parasites, the NRF2 pathway is activated independently of LRV1 by the production of oxidative species by NOX2. NOX2 activation triggers PKCδ phosphorylation by SFK resulting in the release of NRF2 from its negative regulator KEAP1. Stabilized NRF2 translocates to the nucleus favoring the antioxidant response. Concomitantly, stabilized NRF2 blocks the induced pro-inflammatory response activated by LRV1. Thus, NRF2 activation results in activation of the antioxidant response and restriction of the pro-inflammatory response in Lgy infection.