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 3

Diabetic wound macrophages exhibit increased EP2 and EP4, resulting in increased inflammation and impaired phagocytosis.

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Diabetic wound macrophages exhibit increased EP2 and EP4, resulting in i...
(A) Wound monocyte/macrophages from DIO and control mice were sorted on day 5 postwounding and analyzed for Ep2 and Ep4 receptor expression (n = 3/group, repeated in triplicate). (B) Relative expression of Ep2 versus Ep4 in day 5 wound monocyte/macrophages (MФs) (n = 3/group, repeated in triplicate). (C) BMDMs from controls were stimulated with media, PGE2 (1000 nM), or PGE2 (1000 nM) + AH6809 (10 μM), and expression of Il1b, Il12, and Nos2 was analyzed by qPCR (n = 3/group, run in triplicate). (D) Wound monocyte/macrophages from DIO mice or controls were incubated with fluorescently labeled P. aeruginosa for 2 hours, and the percentage of cells ingesting bacteria was determined in relative fluorescence units (RFU) (n = 4/group, repeated twice). (E) Wound monocyte/macrophages from DIO and control mice were isolated on day 5 postwounding and analyzed for Marco receptor expression (n = 3/group, run in triplicate). (F) Wound monocyte/macrophages from controls were incubated with media, PGE2 (1000 nM), or PGE2 (1000 nM) + AH6809 (10 μM) for 6 hours and then were exposed to fluorescently labeled P. aeruginosa for 2 hours, and the percentage of cells ingesting bacteria was determined in RFU (n = 3/group, repeated twice). (G) BMDMs from DIO mice or controls were infected with P. aeruginosa for 30 minutes before extracellular bacteria were removed. Cells were allowed to kill ingested bacteria for 2 hours before cells were lysed and CFU determined. Higher CFU/mL indicate impaired intracellular killing (n = 3/group, repeated twice). 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.