Characterization of LysM-Cre Nrf2-ko mice.

<p><b>(A)</b> Schematic representation of the breeding strategy for LysM-Cre Nrf2-ko mice. <b>(B)</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 <i>Nrf2</i> (exon5), <i>Nqo1</i> and <i>Gclc</i> relative to <i>Rps29</i>. N = 4 mice per genotype. *P ≤0.05 (Mann-Whitney U test). <b>(C)</b> 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. <b>(D)</b> Survival of BMDCs after GO treatment. The percentage of dead cells as determined by flow cytometry is shown. <b>(E)</b> RNA from sorted neutrophils of 5dw was analyzed by RT-qPCR for <i>Tnfa</i>, <i>Il6</i> and <i>Vegfa</i> relative to <i>Rps29</i>. N = 4 mice. Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (Mann-Whitney U test).</p

RT-qPCR primer list.

<p>RT-qPCR primer list.</p

Nrf2 is highly expressed in neutrophils, but myeloid cell-derived Nrf2 is dispensable for wound healing in mice

<div><p>Immune cells of the myeloid lineage are key players in skin wound healing, since they secrete various cytokines and growth factors that orchestrate the repair process. In addition, they are crucial for the defense against invading pathogens through their capacity to produce high levels of reactive oxygen species (ROS). To limit the toxicity of ROS, cells have developed antioxidant defense strategies, including expression of the cytoprotective NRF2 transcription factor. Here we show that murine neutrophils and to a lesser extent macrophages strongly express Nrf2 already when present in the circulation and in particular at the wound site. To determine the role of Nrf2 in neutrophils and macrophages for wound repair, we generated mice with a gain- or loss-of-function of this transcription factor in the myeloid cell lineage. Expression of a constitutively active Nrf2 mutant in myeloid cells did not further enhance the overall Nrf2 activity in these cells due to the already high steady-state activity of endogenous Nrf2. Surprisingly, deletion of Nrf2 in myeloid cells only mildly affected the levels of ROS and the expression of pro-inflammatory cytokines by these cells. In particular, various parameters of wound healing, including wound closure, reepithelialization, wound contraction and the presence of myeloid cells at the wound site were not affected. These results reveal that Nrf2 in myeloid cells is dispensable for wound healing and suggest the presence of additional antioxidant defense strategies of these cells that compensate for the loss of Nrf2, even in the harsh environment of skin wounds.</p></div

RT-qPCR primer list.

<p>RT-qPCR primer list.</p

Wound healing is not affected in LysM-Cre Nrf2-ko mice.

<p><b>(A)</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187162#pone.0187162.s006" target="_blank">S1 Table</a>. <b>(B)</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. <b>(C)</b> RNA from 1dw and 14dw from control and conditional knockout mice was analyzed for <i>Tnfa</i>, <i>Il1b</i> and <i>Il6</i> relative to <i>Rps29</i>. Bars indicate mean ±SD. *P ≤0.05; **P ≤0.01; ***P ≤0.001, **** P ≤0.0001 (Mann-Whitney U test).</p

Antibodies and reagents used for flow cytometry.

<p>Antibodies and reagents used for flow cytometry.</p

Genetic activation of Nrf2 in myeloid cells.

<p><b>(A)</b> Schematic representation of the transgenes used for the generation of LysM-Cre CMVcaNrf2 mice. <b>(B)</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 <i>caNrf2</i> transgene expression and for <i>Rps29</i> 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 <i>Gclc</i>, <i>Srxn1</i>, <i>Slpi</i>, and <i>Nqo1</i> relative to <i>Rps29</i>. N = 3 mice. Bars indicate median with 95% CI. <b>(C)</b> 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).</p

Expression of endogenous Nrf2 and its target genes in keratinocytes and immune cells.

<p>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 <i>Nrf2</i>, <i>Gclc</i> and <i>Nqo1</i> relative to <i>Rps29</i>. <b>(B)</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 <i>Nrf2</i> and <i>Gclc</i>. <b>(C)</b> 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 <i>Nrf2</i>, <i>Gclc</i> and <i>Nqo1</i> relative to <i>Rps29</i>. 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).</p

Gclc deficiency in keratinocytes causes cell damage through increased levels of ROS and RNS.

<p>(A,B) Representative histograms and quantification of relative median of relative fluorescence units (RFU) of (A) H₂DCF-DA (N = 10/6) and (B) DAF-FM-DA (N = 13/11) assay of primary keratinocytes from <i>ctrl</i> and <i>ko</i>^<i>G</i> mice. (C) Quantification of RFU of H₂DCF-DA assay of primary keratinocytes from <i>ctrl</i>, <i>ko</i>^<i>G</i> and <i>ko</i>^<i>G/N</i> mice. N = 11/4/6. (D) Representative histogram and quantification of a lipid peroxidation assay with the lipid peroxidation sensor C11-BODIPY^581/591 (N = 8/5). (E-G) Immunofluorescence staining of primary keratinocytes and quantification of Ki-67 (E; N = 14/7/7), cleaved caspase-3 (F; N = 16/7/5)) and γH2AX (G; N = 15/11/4) positive cells. Scale bar: 50 μm. (H) Schematic representation of the experimental setup for (I,J). (I) Percentage of primary keratinocytes from individual mice after 24 h, 48 h and 96 h of cultivation in normal medium relative to number of cells at t = 0. N = 6. (J) AlamarBlue cell viability assay after treatment of primary keratinocytes from control or <i>ko</i>^<i>G</i> mice with DMSO (vehicle), Z-VAD, Fer-1, Nec-1 or 3MA in DMSO for 24 h. N = 12/11, analyzed in three independent experiments. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001. (K) Electron microscopy of the epidermis at 3W. Left and middle panel: Keratinocytes with signs of ferroptotic cell death, including dysmorphic mitochondria and small mitochondria with increased membrane density and/or membrane accumulation. Right panel: Keratinocyte with signs of necroptotic cell death, including nuclear condensation, vacuolization and presence of autophagosomes. N = nucleus; arrowheads: autophagosomes; arrow: mitochondria. Scale bars: 5 μm (left panel), 700 nm (middle panel), or 3 μm (right panel).</p

Cell damage and apoptosis in the hyperproliferative wound epithelium of Gclc-deficient mice.

<p>(A,B) Immunofluorescence-stained sections of 2-, 3- and 5-day wounds and quantification of (A) cleaved caspase-3 (arrowheads; arrows: positive cells in the adjacent epidermis), and (B) γH2AX positive cells per area HE. White dotted lines show the area of HE in which positive cells were counted. Each picture includes a magnification of the indicated square to show positive cells (if existing). Scale bar: 100 μm. For (A): 2dw N = 6; 3dw N = 6; 5dw N = 10/8. For (B): 2dw N = 4; 3dw N = 7/8; 5dw N = 5. (C) OxyBlot of lysates (pooled from 2 wounds of individual mice) from 3-day wounds. Equal loading was confirmed by Ponceau S staining of the membrane. (D) H&E staining on sections from 3-day wounds. Scale bar: 200 μm. D: dermis Es: eschar, G: granulation tissue, HF: hair follicle, HE: hyperproliferative epithelium. (E-G) Morphometric analysis of (E) percentage wound closure, (F) length HE and (G) area HE of 3-day wounds of <i>ko</i>^<i>G/N</i> and control mice. For (E): N = 6/7; for (F): N = 7/8; for (G): N = 7/8. (H-J) Quantification of (H) Ki-67 (N = 4), (I) cleaved caspase-3 (N = 3/4), and (J) γH2AX (N = 6/5) positive cells per area HE. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05. n.d.: not detectable.</p