Elevated heme impairs macrophage phagocytosis in endometriosis

in Reproduction
Correspondence should be addressed to M-Q Li or X-Y Zhu; Email: mqli@fudan.edu.cn or zhuxiaoyong@fudan.edu.cn

*(Y-Y Liu and Y-K Liu contributed equally to this work)

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Endometriosis (EMS) is a chronic inflammatory disease characterized by the presence of extrauterine endometrial tissues. It has been previously reported that the refluxed blood containing viable endometrial tissues and the defective elimination of peritoneal macrophages in the pelvic cavity may involve in EMS pathogenesis. However, the mechanism by which macrophages exhibit attenuated phagocytic capability in EMS remains undetermined. Herein, we found that heme, the byproduct of lysed erythrocytes, accumulated abnormally in the peritoneal fluid (PF) of patients with EMS (14.22 μmol/L, 95% confidence interval (CI): 12.54–16.71), compared with the EMS-free group (9.517 μmol/L, 95% CI: 8.891–10.1053). This abnormal accumulation was not associated with the color of PF, phase of the menstrual cycle or severity of the disease. The reduced phagocytic ability of peritoneal macrophages (pMφs) was observed in the EMS group. Consistently, a high-concentration (30 μmol/L) heme treatment impaired EMS-pMφs phagocytosis more than a low-concentration (10 μmol/L) heme treatment. A similar phenomenon was observed in the EMS-free control pMφs (Ctrl-pMφs) and the CD14+ peripheral monocytes (CD14+ Mos). These results indicated that a high heme concentration exhibits a negative effect on macrophage phagocytosis, which supplements the mechanism of impaired scavenger function of pMφs in EMS.

 

    Society for Reproduction and Fertility

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    The concentration of heme is higher in the peritoneal fluid of endometriosis patients. (A) Peritoneal heme level in control group (n = 31) and endometriosis group (n = 31). (B) Heme level in yellowish peritoneal fluid (n = 17) and reddish peritoneal fluid (n = 45). (C) Heme level in yellowish peritoneal fluid of the control group (n = 10) and endometriosis group (n = 7), in reddish peritoneal fluid of the control group (n = 21) and endometriosis group (n = 24). (D) Peritoneal heme level of the control group in the proliferative phrase (n = 14) and in the secretary phrase (n = 17), of endometriosis group in the proliferative phrase (n = 13) and in the secretary phrase (n = 18). (E) Peritoneal heme level in the control (n = 31) and the endometriosis group of I/II stages (n = 8) and III/IV stages (n = 23). In (A, B, C, D and E), data are presented as the median with 95% CI. Mann–Whitney test (A, B, C and D), Kruskal–Wallis test with Dunn’s multiple comparison test (E); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, no significance.

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    Heme metabolism molecules are elevated in both the ectopic ESCs and the pMφs of EMS. (A) A schematic diagram shows the molecules that are associated with heme metabolism. ‘↑’: the direction of heme transport. (B and C) Relative mRNA expression of heme metabolism molecules in (B) ESCs (n = 8) and (C) pMφ (n = 6). Data are presented as the mean ± s.e.m.; two-tailed, unpaired t-test; *P < 0.05, **P < 0.01.

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    Phagocytosis of pMφs is reduced in patients with EMS. (A) Representative image of the purity of magnetically isolated pMφs using FCM. The number is the percentage of CD14+CD45+ cells. (B) Representative image of phagocytosis ratio of pMφs in women with or without EMS verified by flow cytometric analysis. Phagocytosis ratio = CFSE+CD45+cells/CD45+cells. (C) Quantification of phagocytosis ratio of pMφs of women with or without EMS; n = 12. Data are presented as the mean ± s.e.m.; two-tailed, unpaired t-test; ***P < 0.001.

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    High level of heme impairs the phagocytic ability of macrophage. (A, C and E) Representative flow cytometric analysis of phagocytosis ratio of EMS-pMφ (A), Ctrl-pMφ (C) and CD14+Mo (E). (B, D and F) Flow cytometric quantification of phagocytosis ratio of EMS-pMφ (B), Ctrl-pMφ (D) and CD14+Mo (F), treated with DMSO or heme (10 μmol/L, 30 μmol/L); n = 12. Data are presented as the mean ± s.e.m./; one-way ANOVA with Turkey’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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    Schematic diagram of the key findings. This study has shown that patients with and without EMS have a particular concentration of heme in the peritoneal fluid but the concentration was higher in the EMS group. Under normal conditions, both peritoneal macrophages and recruited peripheral monocytes will eliminate the ESCs, avoiding the presence of ectopic endometrial lesions. However, when the reflux of large amounts of menstrual blood and the hemorrhage from endometriotic lesions occurred repeatedly, heme is accumulated and a high level of heme impairs the phagocytosis of macrophages. Shed ESCs are able to survive and implant, leading to EMS.

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