Diabetes primes neutrophils to undergo NETosis, which impairs wound healing

Journal name:
Nature Medicine
Volume:
21,
Pages:
815–819
Year published:
DOI:
doi:10.1038/nm.3887
Received
Accepted
Published online

Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins1. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4−/− mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4−/− mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.

At a glance

Figures

  1. Diabetes or high glucose concentrations in vitro prime human and mouse neutrophils to undergo NETosis.
    Figure 1: Diabetes or high glucose concentrations in vitro prime human and mouse neutrophils to undergo NETosis.

    (a,b) Combined (a) and separate (b) data showing the percentage of NET production by unstimulated and ionomycin-stimulated peripheral neutrophils isolated from fresh whole blood of healthy individuals (black circles, n = 10) and individuals with diabetes mellitus (DM) (pink circles, type 1 DM, n = 5; purple squares, type 2 DM, n = 5). (c) Western blot analysis of PAD4 expression in neutrophils from healthy or diabetic individuals (top) and quantification of PAD4 expression, normalized to GAPDH expression (bottom). AU, arbitrary units. n = 6 for healthy control, n = 6 for diabetic individuals. (d) Percentage of NET production by neutrophils from healthy individuals that were exposed to normal glucose (NG, 5.5 mM), high glucose (HG, 22 mM) and mannitol (M, 16.5 mM plus 5.5 mM glucose) in vitro. n = 5 per condition. (e,g,h) Percentage of cells that were hypercitrullinated at histone H3 (H3Cithigh, left) and produced NETs (right) in neutrophils isolated from streptozotocin (STZ)-induced diabetic mice (n = 12 for vehicle, n = 10 for STZ) (e), db/db diabetic mice (n = 7 for m+/db; n = 8 for db/db) (g) and normoglycemic WT mice whose neutrophils were exposed to different glucose concentrations in vitro (n = 10 per medium condition) (h). US, unstimulated. (f) Representative immunofluorescence images of isolated neutrophils from vehicle- or STZ-treated mice. Neutrophils were exposed to LPS (25 μg/ml) for 2.5 h. Yellow arrows indicate NETs. Scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (a–c,g) Mann-Whitney test; (d,h) repeated-measures analysis of variance (ANOVA) followed by Bonferroni's post-test; (e) Student's t-test. Data are means = s.e.m.

  2. NETs are present in the wounds of WT mice.
    Figure 2: NETs are present in the wounds of WT mice.

    (a) Representative western blot of the time course for H3Cit appearance in wounds after skin injury (left) and quantification of levels of H3Cit to histone H3 (right). AU, arbitrary units. Ctrl, control unwounded skin; H3, histone H3. **P < 0.01 versus Ctrl, Student's t-test, n = 3 for Ctrl, 1 and 4 h; n = 5 for 1, 3, 7 and 14 d. Blot is representative of three independent experiments. Data are mean = s.e.m. (b) Immunofluorescence images of the wound bed immediately beneath the scab 3 d after injury. Scale bar, 50 μm. (c) Representative confocal images of four wounds 3 d after injury. Area enclosed by the yellow box is magnified and shown on the right. Scale bars, 100 μm (left), 50 μm (right). (d) Western blots of wounds collected 3 d after injury from mice with defective leukocyte recruitment (Cd18−/−, left) and mice depleted of neutrophils using an anti-Ly6G antibody (right, representative of n = 7). IgG, IgG isotype control for the anti-Ly6G antibody. GADPH serves as a loading control.

  3. PAD4 deficiency facilitates wound repair in normoglycemic mice.
    Figure 3: PAD4 deficiency facilitates wound repair in normoglycemic mice.

    (a) Images of H&E staining (top) and confocal microscopy (bottom) of wounds from WT and Padi4−/− mice 3 d after injury. Scale bars, 50 μm. The presence of extracellular DNA (blue streaks) in the scab of WT mice is indicated by yellow arrows, whereas intact neutrophils in that of Padi4−/− mice are indicated by yellow arrowheads in the H&E images. (b) Representative (of five) western blots of wounds from WT (+/+) and Padi4−/− (–/–) mice. GADPH serves as a loading control. See Supplementary Figure 8 for quantifications. (c) Photographs of healing wounds of WT and Padi4−/− mice up to 7 d after wounding. Scale bar, 5 mm. (d) Changes in wound area compared to day 0. Per order in the bar chart, n = 16, 16, 15, 12 for WT groups; n = 12, 12, 12, 9 for Padi4−/− groups; *P < 0.05, **P < 0.01, ***P < 0.001 versus WT, Student's t-test. (e) Percent of WT and Padi4−/− mice that completed wound healing on day 13 and 14 after injury. Day 13: WT (2/11) versus Padi4−/− (6/9), P = 0.065; Day 14: WT (4/16) versus Padi4−/− (10/13), **P < 0.01; two-tailed Fisher′s exact test. (f) Re-epithelialization as determined from H&E staining on wounds from WT and Padi4−/− mice 3 d after wounding. See Supplementary Figure 9 for histology of wounds. n = 9 for WT; n = 6 for Padi4−/−; ***P < 0.001, Student's t-test. Data are mean = s.e.m.

  4. PAD4 deficiency or DNase 1 treatment enhances wound healing in diabetic mice.
    Figure 4: PAD4 deficiency or DNase 1 treatment enhances wound healing in diabetic mice.

    (a–f) Data from all groups were obtained simultaneously in multiple experiments but split into three graphs (ac and df) to facilitate comparison. n = 7 for WT vehicle, n = 9 for WT STZ, n = 5 for Padi4−/− vehicle, n = 6 for Padi4−/− STZ. *P < 0.05, **P < 0.01, ***P < 0.001 and NS, non-significant between groups on respective post-wounding day (ac, Student's t-test) or between curves (df, log-rank test). (a–c) Changes in wound area compared to day 0. (d–f) Percentage of mice with open wounds recorded after injury up to day 19. (a,d) Normoglycemic and diabetic WT mice were compared. (b,e) Diabetic WT and Padi4−/− mice were compared. (c,f) normoglycemic and diabetic Padi4−/− mice were compared. (g) Representative western blots of H3Cit levels in wounds 1 d after wounding from vehicle-treated normoglycemic and STZ-induced diabetic mice (top) and quantification (compared to mean of vehicle after normalization to respective H3 level) (bottom). n = 5 per group, *P < 0.05, Mann-Whitney test. (h,i) Wound area reduction (top) and re-epithelialization (bottom) with DNase 1 (dornase alfa) treatment in diabetic WT and Padi4−/− mice (h) and normoglycemic WT mice (i). (h) Per order in the bar chart, n = 6, 8, 9 for the WT groups; n = 5, 6, 8 for the Padi4−/− groups; *P < 0.05, ***P < 0.001 and NS, non-significant using Kruskal-Wallis test followed by Dunn′s post-test; #P < 0.05, ##P < 0.01 using Mann-Whitney test. (i) n = 9 without DNase 1, n = 10 with DNase 1, *P < 0.05, Student's t-test. Data are mean = s.e.m.

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Author information

Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Siu Ling Wong,
    • Melanie Demers,
    • Kimberly Martinod,
    • Maureen Gallant &
    • Denisa D Wagner
  2. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

    • Siu Ling Wong,
    • Melanie Demers,
    • Kimberly Martinod &
    • Denisa D Wagner
  3. Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA.

    • Yanming Wang
  4. Section of Clinical Research, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA.

    • Allison B Goldfine
  5. Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA.

    • C Ronald Kahn
  6. Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Denisa D Wagner

Contributions

S.L.W. designed the study, performed the majority of the experiments, analyzed the data and wrote the manuscript; M.D. and K.M. performed experiments and analyzed data; M.G. provided expert technical assistance; Y.W. provided Padi4−/− mice and critical discussion of the work; A.B.G. provided clinical advice and selected diabetic patients for in vitro NETosis assays; C.R.K. provided helpful suggestions on experimental design and critical reading of the manuscript; D.D.W. designed the study, supervised the project and co-wrote the manuscript.

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The authors declare no competing financial interests.

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