Epigenetic regulation of the PGE2 pathway modulates macrophage phenotype in normal and pathologic wound repair
Frank M. Davis, … , Bethany B. Moore, Katherine A. Gallagher
Frank M. Davis, … , Bethany B. Moore, Katherine A. Gallagher
Published September 3, 2020
Citation Information: JCI Insight. 2020;5(17):e138443. https://doi.org/10.1172/jci.insight.138443.
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Research Article Endocrinology Inflammation

Epigenetic regulation of the PGE2 pathway modulates macrophage phenotype in normal and pathologic wound repair

Abstract

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB–mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-β–induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b–mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.

Authors

Frank M. Davis, Lam C. Tsoi, Rachael Wasikowski, Aaron denDekker, Amrita Joshi, Carol Wilke, Hongping Deng, Sonya Wolf, Andrea Obi, Steven Huang, Allison C. Billi, Scott Robinson, Jay Lipinski, William J. Melvin, Christopher O. Audu, Stephan Weidinger, Steven L. Kunkel, Andrew Smith, Johann E. Gudjonsson, Bethany B. Moore, Katherine A. Gallagher

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Figure 2

TGF-β–induced mir29b increases COX-2 production via inhibition of DNMT3-mediated methylation in wound macrophages.

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TGF-β–induced mir29b increases COX-2 production via inhibition of DNMT3-...
(A) Whole wounds from DIO and control mice were isolated on days 3 and 7 postwounding and analyzed for TGF-β1 protein levels by Bioplex. (n = 3/group, repeated once.) (B) Wound monocyte/macrophages and BMDMs were isolated on day 5 postwounding and analyzed for miR29b expression. (n = 3/group, repeated in triplicate.) (C) Wound monocyte/macrophages from DIO mice were sorted on days 3 and 5 postwounding via MACS and miR29b levels were analyzed. (D and E) BMDMs were isolated and stimulated with media, TGF-β (2 μg/mL), or TGF-β (2 μg/mL)+A8301 (0.5 μM) for 4 hours and analyzed for miR29b (D) and Cox-2 (E) expression. (n = 3/group, repeated in triplicate.) (F and G) BMDMs were isolated from Alk5fl/fl Lyz2Cre– and Alk5fl/fl Lyz2Cre+ and stimulated with TGF-β (2 or 20 μg/mL) for 4 hours and analyzed for miR29b (F) and Cox-2 (G) expression. (n = 3/group, repeated in triplicate.) (H) BMDMs were isolated and stimulated with media, TGF-β (2 μg/mL), or TGF-β (2 μg/mL)+A8301 (0.5 μM) for 4 hours and analyzed for Dnmt3b expression. (n = 3/group, repeated in triplicate.) (I and J) Wound monocyte/macrophages from DIO and db/db mice and their respective controls were isolated on day 3 and analyzed for DNMT3a and DNMT3b expression (n = 3/group, repeated twice). (K) Bisulfite sequencing was performed on DIO and control wound monocyte/macrophages treated with miR29b or a negative miR control (30 nM) and the Cox-2 promoter was analyzed for DNA methylation (n = 3/group). *P < 0.05, **P < 0.01. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed normality test, 2-tailed Student’s t test for 2 groups and 2-way ANOVA for multiple groups was used.