Fig 1.
Nrf2 endogenous expression is high in PMNs and increases in response to SFN and zymosan.
Freshly obtained BM cells were stained with anti-Ly6G and rabbit anti-Nrf2 IgG or with corresponding isotype, then with goat anti-rabbit Alexa Fluor 488 Ab. Isolated PMNs were incubated for 4 h alone (NS), with 1 μM of SFN before staining or with zymosan 5 μg/ml. (A) Endogenous Nrf2 was quantified in BM cells using intracellular staining followed by flow cytometry. PMNs were identified as Ly6G+, while Ly6G- cells represents the rest of BM cells. Nrf2 expression was quantified as the mean fluorescence intensity (MFI) and compared between Ly6G+ and Ly6G- cells (#p<0.05 and ##p<0.01, Kruskal-Wallis test). (B) Nrf2 accumulation in isolated PMNs after incubation with SFN 1 μM or zymosan 5 μg/ml, was measured using flow cytometry. The ratio of Nrf2 expression was calculated by dividing the MFI of treated PMNs by the MFI of untreated PMNs (NS). Ratios greater than 1 indicate Nrf2 accumulation in stimulated PMNs (Mann-Whitney test). Results are the mean ± SEM of samples from 4 independent experiments, n = 4.
Fig 2.
Nrf2 upregulates the transcription of 3 main target genes in response to SFN and zymosan.
WT and Nrf2 KO PMNs were incubated or not (NS) with SFN 1μM or zymosan 5 μg/ml for 4 h. mRNA expression of Nqo1, Hmox-1 and Cat was quantified using RT-qPCR. Results are expressed as fold increase normalized to WT NS and corrected by the expression of the housekeeping genes β-actin and gapdh in all RT-qPCR experiments. Results are the mean ± SEM of samples from 4 independent experiments, n = 4 (* indicates a significant difference between WT and Nrf2 KO PMNs throughout the study, *p<0.05, Mann-Whitney test).
Fig 3.
Nrf2 activation decreases PMN ROS production in response to zymosan.
WT and Nrf2 KO PMNs were stimulated or not with increasing concentrations of zymosan. The sum of intra- and extracellular ROS was monitored for 60 min by the luminol-amplified chemiluminescence assay. (A) Graphs from one representative experiment show the kinetic of ROS production in unstimulated PMNs (left panel) or in response to zymosan 10 μg/ml (right panel). (B) The area under curve (AUC) from the kinetic curves of 6 independent experiments was used to calculate the ratio of ROS production (KO AUC/WT AUC). Ratios greater than 1 correspond to higher ROS production in KO PMNs (**p<0.01, Mann-Whitney test). (C) The expression of TLR2 was assessed on freshly isolated WT and Nrf2 KO PMNs (grey filled curves) in comparison to corresponding isotypes (black unfilled curves). The associated graph represents the mean ± SEM of TLR2 MFI, n = 4. (D) To ensure an important Nrf2 accumulation, PMNs were pretreated or not (NT) with SFN 1 μM for 4 h then stimulated with 5 μg/ml of zymosan. ROS production was then measured for 60 min by the luminol-amplified chemiluminescence assay. Graphs show the kinetics of ROS production from one representative experiment (left panel) and the mean ± SEM of AUC from the kinetic curves of 6 independent experiments (right panel, n = 6; *p<0.05, Mann-Whitney test).
Fig 4.
Nrf2 modulates Ccl3, Cxcl2 and Tnfα transcription.
WT and Nrf2 KO PMNs were incubated or not with zymosan 5 μg/ml for 4 h. mRNA expression of Ccl3, Cxcl2, Cxcl1, Tnfα, Il-1β and Il-6, were measured using RT-qPCR. All results are expressed as fold increase normalized to WT NS and corrected by the expression of the housekeeping genes β-actin and gapdh (4 independent experiments, n = 4; *p<0.05 Mann-Whitney test).
Fig 5.
The lack of Nrf2 does not affect PMN phagocytic capacity.
Cells were incubated for 90 min alone or with the determined concentrations of pHRodo Red Zymosan Bioparticles. (A) Flow cytometry-based analysis was used to evaluate PMN zymosan uptake. PMNs were subjected to a one-color analysis for the percent of zymosan (25 μg/ml) positive events. (B) Graph representing the percentage of PMNs that phagocytosed increasing concentrations of zymosan (5, 25 and 50 μg/ml). PMNs were pretreated for 30 min with 20 μM of cytochalasin D for negative control. All data are presented as the mean ± SEM, 4 independent experiments, n = 4.
Fig 6.
Nrf2 is necessary for optimal PMN migration toward CXCL12 and CXCL2 without regulating receptors expression.
(A) WT and Nrf2 KO PMNs were allowed to migrate for 1 h across transwell filters spontaneously or toward fMLP 1 μM, CXCL12 or CXCL2. The negative control in CXCL12-dependent migration was obtained by pretreating PMNs with 50 μM of AMD300. Chemotactic indexes were then calculated by dividing the number of PMNs that were counted in the chemokine-stimulated well by the number of PMNs that were counted in the input well (*p<0.05 **p<0.01 Mann-Whitney test). (B) The expression of CXCR2 (B, left panel) and CXCR4 (B, right panel) were assessed on freshly isolated WT PMNs (solid grey line) and Nrf2 KO PMNs (dashed black line) in comparison to corresponding isotypes (unfilled curves). The associated graph represents the mean ± SEM of CXCR2 and CXCR4 MFI, n = 4. (C) The MFI fold variation of CD11b was assessed on PMNs that have migrated through the transwell in comparison to PMNs in the input well. (D) ATP levels in freshly isolated PMNs were quantified using an ATP luminescent assay. The expression levels from 6 independent experiments are shown as the mean of MFI ± SEM (n = 6).