Prostaglandin E2 Receptor 2 Modulates Macrophage Activity for Cardiac Repair

Abstract

Background Prostaglandin E₂ has long been known to be an immune modulator. It is released after tissue injury and plays a role in modulating macrophage activities, which are essential for tissue regeneration. However, the involvement of prostaglandin E₂ receptor 2 ( EP 2)-dependent regulation of macrophages in postischemic heart is unclear. This study aims to evaluate the role of EP 2 in damaged heart. Methods and Results The effect of EP 2 in postischemic heart was evaluated using EP 2-deficient transgenic mice. We demonstrated that cardiac function was worse after myocardial injury on loss of EP 2. Furthermore, EP 2 deficiency also altered proinflammatory response and resulted in a defect in macrophage recruitment to the injured myocardium. Transcriptome analysis revealed that the expression of erythroid differentiation regulator 1 ( Erdr1) was significantly induced in EP 2-deficient macrophages. Knocking down Erdr1 expression restored migration ability of EP 2-deficient cells both in vitro and in vivo. By using a genetic fate-mapping approach, we showed that abolishment of EP 2 expression effectively attenuated cell replenishment. Conclusions The EP 2-dependent signaling pathway plays a critical role in regulating macrophage recruitment to the injured myocardium, thereby exerting a function in modulating the inflammatory microenvironment for cardiac repair.

Keywords: EP2; inflammation; ischemia; macrophage; myocardial; therapy.

Figure 1

Loss of prostaglandin E₂ receptor 2 (EP2) expression worsens cardiac function after injury. A, At 2 months after myocardial infarction, cardiac function of EP2‐null (EP2^−/−) mice (n=15) and their heterozygous (EP2^+/−; n=13) and wild‐type (WT; n=14) littermates was evaluated by echocardiography. Statistical analysis was performed using 1‐way ANOVA with Dunnett's test. **P=0.0023 for ejection fraction (EF); **P=0.0026 for fractional shortening (FS); **P=0.0081 for left ventricular diastolic dimension (LVDd); **P=0.0042 for left ventricular systolic dimension (LVDs). B and C, The infarct region at mid‐LV was evaluated by Masson trichrome staining. WT, n=7; EP2^+/−, n=7; EP2^−/−, n=9. Statistical analysis was performed using 1‐way ANOVA with Dunnett's test. Bar=1 mm. n.s. indicates not significant. **P=0.005.

Figure 2

Prostaglandin E₂ receptor 2 (EP2) knockout mice show less inflammation response and decreased macrophage recruitment at the infarcted myocardium. A, At day 3 after myocardial infarction (MI), the levels of a panel of proinflammatory cytokines in the infarct tissue of EP2‐null (EP2^−/−) mice were compared with those in the wild‐type (WT) animals. Quantification was performed with 5 animals for each genotype. Statistical analysis was performed using an unpaired t test or a nonparametric test. The levels of the rest of cytokines are not statistically different. *P=0.0159 for IL‐18; **P=0.0051 and **P=0.0072 for interleukin‐1β and interleukin‐17A, respectively. B, At day 3 after MI, the infarcted region of injured heart was excised and enzymically digested for quantification of immune cells. The representative flow cytometric results of F4/80⁺ macrophages and Gr1⁺ neutrophils from the infarcted hearts of EP2^−/− and WT mice are shown. C and D, Quantification of F4/80⁺ macrophages (C) and Gr1⁺ neutrophils (D) relative to the weight of injured tissue from EP2^−/− (n=5) and WT (n=6) mice at day 3 after MI. Statistical analysis was performed using an unpaired t test or a nonparametric test. *P=0.0173. E and F, The distribution of F4/80⁺ macrophages in the injured hearts of EP2^−/− and WT mice was analyzed. Quantification was performed with 5 animals for each genotype. Statistical analysis was performed using an unpaired t test. Bar=20 µm. **P=0.0036. G, Levels of anti‐inflammatory cytokines in the injured regions of EP2^−/− and WT animals were compared. Quantification was performed with 5 animals for each genotype. Statistical analysis was performed using an unpaired t test, and no significant difference is observed. GM‐CSF indicates granulocyte‐macrophage colony‐stimulating factor; IFN‐γ, interferon‐γ; n.s., not significant.

Figure 3

Macrophages isolated from prostaglandin E₂ receptor 2 (EP2) knockout mice show attenuated migration ability. A, The effect of EP2 deficiency on the number of monocytes was examined in various tissues. Representative flow cytometric analyses of CD115⁺/Ly6c⁺ monocytes, as indicated in the red boxes, in the bone marrow, spleen, and peripheral blood before and after myocardial infarction (MI) are shown. B, The number of monocytes examined in different tissues of wild‐type (WT) and EP2‐null (EP2^−/−) mice before and after MI was quantified and statistically analyzed. Before MI, n=5 to 6 animals per group. After MI, n=5 to 9 animals per group. Statistical analysis was performed using an unpaired t test or a nonparametric test. C and D, The mobilization ability of macrophages isolated from WT and EP2^−/− mice was examined. C, Representative images showing the macrophages, as presented by 4,6‐diamidino‐2‐phenylindole staining, which migrated in response to different levels of monocyte chemoattractant protein 1 (MCP‐1). The experiment was repeated 4 times. D, The mobilization ability of macrophages was quantified as cell migration index. Statistical analysis was performed using 2‐way ANOVA with Sidak's test. Bar=50 µm. n.s. indicates not significant. **P=0.0040 and ****P<0.0001 at MCP‐1 concentrations of 3 and 30 ng/mL, respectively.

Figure 4

Erythroid differentiation regulator 1 (Erdr1) acts downstream of prostaglandin E₂ receptor 2 (EP2) signaling to regulate macrophage mobilization. A, Transcriptome heat map and gene ontology analysis (right panel) show the pathways that were most affected by loss of EP2. B, Comparison of candidate gene expression in macrophages isolated from EP2‐null (EP2^−/−) and wild‐type (WT) mice; n=6 for each genotype. Statistical analysis was performed using an unpaired t test or a nonparametric test. n.s. indicates not significant. *P=0.0228 for Selp; *P=0.037 for Gata2; *P=0.0169 for Thbs1; *P=0.0343 for Tgf‐β2; and **P=0.0022 for Erdr1. C, In vitro migration assay was performed to examine the mobilization ability of the EP2^−/− macrophages on small hairpin RNA (shRNA)–mediated knockdown of Erdr1. Cells transduced with vector alone were used as the control group. The mobilization ability of cells was evaluated by the migration index. Statistical analysis was performed using 2‐way ANOVA with Dunnett's test. MCP‐1 indicates monocyte chemoattractant protein 1; and n.s., not significant vs vector alone control. ***P=0.0004; ****P=0.0001. D and E, At day 3 after myocardial infarction (MI), cardiomyocyte‐depleted small cells were isolated for quantification of infused F4/80⁺/green fluorescent protein–positive (GFP ⁺) macrophages. Control groups were the mice injected with wild type (WT; WT control) or EP2^−/− (EP2^−/− control) macrophages transduced with vector alone. D, Representative flow cytometric examination of the GFP ⁺ cells in the injured hearts of WT mice with or without cell infusion is shown. E, The number of GFP ⁺ cells in the injured myocardium among groups was quantified. MI alone, n=5; WT control, n=5; EP2^−/− control, n=5; EP2^−/− Erdr1 shRNA, n=5. Statistical analysis was performed using 1‐way ANOVA with Dunnett's test. n.s. indicates not significant vs EP2^−/− control. *P=0.0383; ****P=0.0001.

Figure 5

Cardiomyocyte replenishment is abolished on loss of prostaglandin E₂ receptor 2 (EP2) expression after myocardial infarction (MI). A and B, At day 14 after MI (MI14D), the hearts of MCM/ZEG (MZ) mice with homozygous (EP2^−/−/MZ) or heterozygous (EP2^+/−/MZ) deletion of EP2 expression were harvested for evaluation of adult cardiomyocyte replenishment after MI. Shown are representative images of green fluorescent protein–positive (GFP ⁺) and β‐Gal⁺ cardiomyocytes at the border zone (A, left panels) and the remote area (B, left panels) of the injured hearts from different groups. The percentages of resident GFP ⁺ cardiomyocytes and regenerated β‐Gal⁺ cardiomyocytes were quantified and statistically analyzed at the border zone (A, right panels) and remote region (B, right panels). Wild‐type MZ MI14D, n=7; EP2^−/−/MZ sham, n=4; EP2^−/−/MZ MI14D, n=6; EP2^+/−/MZ MI14D, n=7. Statistical analysis was performed using 1‐way ANOVA with Dunnett's test. Bar=20 µm. n.s. indicates not significant. At the border zone, **P=0.0031 and **P=0.0032 for GFP ⁺ and β‐Gal⁺ cells, respectively. At the remote area, *P=0.0240.