Table 1.
RT-qPCR primer list.
Table 2.
Antibodies and reagents used for flow cytometry.
Fig 1.
Expression of endogenous Nrf2 and its target genes in keratinocytes and immune cells.
Immune cells from the blood or the wound tissue were isolated by FACS based on the following markers: CD45+CD11b- (lymphocytes), CD45+CD11b+Ly6G+ (neutrophils), CD45+CD11b+F4/80+ (mainly macrophages, but also some Langerhans cells and monocytes) and CD45+CD11b+Ly6C+ (monocytes/inflammatory macrophages). (A) RT-qPCR analysis using RNA from sorted cells of 5-day wounds (dw) from C57BL/6 mice for Nrf2, Gclc and Nqo1 relative to Rps29. (B) Blood was collected prior to wounding (0dw) and at day 5 after wounding (5dw) of C57BL/6 mice, and RNA from FACS-sorted immune cells was analyzed by RT-qPCR analysis for Nrf2 and Gclc. (C) RT-qPCR analysis using RNA from sorted immune cells and keratinocytes (CD49f+CD140a-CD45-CD31-) of 5-day wounds (dw) from C57BL/6 x FVB/N1 mice for Nrf2, Gclc and Nqo1 relative to Rps29. N = 3–6 mice. Bars indicate median with 95% confidence interval (CI). Each data point represents the result from one mouse. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (one-way ANOVA for multiple group comparisons and Student’s t-test for two-group comparison).
Fig 2.
Characterization of deletion efficiency of LysM-Cre mice in blood and wound leukocytes.
Flow cytometry analysis to quantify the percentage of red fluorescent protein positive (RFP+) cells among all leukocytes or among neutrophils, monocytes or macrophages (A) in the blood of non-injured mice (N = 6 mice) or (B) in 5-day wounds (n = 4 wounds obtained from two different mice (N = 2)). Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (one-way ANOVA).
Fig 3.
Genetic activation of Nrf2 in myeloid cells.
(A) Schematic representation of the transgenes used for the generation of LysM-Cre CMVcaNrf2 mice. (B) Left upper panel: RNA from sorted neutrophils or macrophages from 5dw of control (tg/wt) and LysM-cre CMVcaNrf2 mice (tg/tg) were analyzed for caNrf2 transgene expression and for Rps29 by RT-qPCR. PCR products were analyzed by agarose gel electrophoresis. Other panels: RT-qPCR using RNA from lymphocytes, neutrophils and macrophages isolated from 5d wounds for the Nrf2 target genes Gclc, Srxn1, Slpi, and Nqo1 relative to Rps29. N = 3 mice. Bars indicate median with 95% CI. (C) Flow cytometry analysis of 5dw and 13dw of tg/wt and tg/tg mice for total leukocytes, myeloid cells, neutrophils and macrophages. N = 3–5 mice, n = 5–10 wounds. Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (Mann-Whitney U test).
Fig 4.
Characterization of LysM-Cre Nrf2-ko mice.
(A) Schematic representation of the breeding strategy for LysM-Cre Nrf2-ko mice. (B) RNA from different immune cells sorted from 5dw of control (tg/wt) and LysM-Cre Nrf2-ko (tg/ko) mice were analyzed by RT-qPCR for Nrf2 (exon5), Nqo1 and Gclc relative to Rps29. N = 4 mice per genotype. *P ≤0.05 (Mann-Whitney U test). (C) Bone marrow derived cells (BMDCs) from tg/wt and tg/ko mice were pre-incubated for 15 min with 5 μM DCFH-DA and subsequently stimulated with glucose oxidase (GO; 85 mU) or left untreated (control) for another 15 min. Cells were analyzed by flow cytometry based on DCF fluorescence (reflecting ROS levels). Results shown are representatives of at least three independent experiments. N = 2–3 mice per experiment, two data points represent each mouse. (D) Survival of BMDCs after GO treatment. The percentage of dead cells as determined by flow cytometry is shown. (E) RNA from sorted neutrophils of 5dw was analyzed by RT-qPCR for Tnfa, Il6 and Vegfa relative to Rps29. N = 4 mice. Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (Mann-Whitney U test).
Fig 5.
Wound healing is not affected in LysM-Cre Nrf2-ko mice.
(A) Representative pictures of Herovici-stained sections from 5dw of tg/wt and tg/ko mice. Scale bar: 500 μm. D: Dermis; E: Epidermis, Es: Eschar; G: Granulation tissue; HE: Hyperproliferative wound epidermis. Quantitative data from a morphometric analysis of 5dw of tg/wt and tg/ko mice for wound closure, area and length of the granulation tissue, and area and length of wound epithelium are shown below (N = 3–4 mice, n = 6–8 wounds). The raw data of the morphometric analysis of the wounds are shown in S1 Table. (B) Flow cytometry analysis of 1dw, 5dw and 14dw of tg/wt and tg/ko mice for total leukocytes, myeloid cells, neutrophils, monocytes and macrophages. N = 4–6 mice, n = 8–12 wounds. (C) RNA from 1dw and 14dw from control and conditional knockout mice was analyzed for Tnfa, Il1b and Il6 relative to Rps29. Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (Mann-Whitney U test).