Department of Obstetrics and Gynecology, Second Affiliated Hospital of Harbin Medical University, 148 Baojian Road, Harbin, 150086, China.
Department of Obstetrics and Gynecology, Luohe Central Hospital, Luohe, China.
Stem Cell Res Ther. 2022 Jul 15;13(1):294. doi: 10.1186/s13287-022-02981-2.
Endometriosis (EMs) is a common benign gynecological disease that affects approximately 10% of females of reproductive age. Endometriosis ectopic lesions could recruit macrophages, which in turn facilitates endometriosis progression. Several studies have indicated that CCL20 derived from macrophages activates the expression of CCR6 in several cells and induces cell proliferation and migration. However, the function of the CCL20/CCR6 axis in the interactions between macrophages and endometriotic stromal cells (ESCs) in EMs has yet to be elucidated.
Ectopic and normal endometrial tissues were collected from 35 ovarian endometriosis patients and 21 control participants for immunohistochemical staining. It was confirmed that macrophages secreted CCL20 to promote CCR6 activation of ESCs during co-culture by ELISA, qRT-PCR and western blot analysis. CCK8 and Edu assays were used to detect cell proliferation, and wound healing and Transwell assay were used to detect cell migration. Autophagic flux was detected by measuring the protein expression levels of LC3 and P62by western blot and analyzing the red/yellow puncta after ESCs were transfected with mRFP-GFP-LC3 double fluorescence adenovirus (Ad-LC3). Lysosomal function was tested by quantifying the fluorescent intensities of Lyso-tracker and Gal3 and activity of acid phosphatase. In addition, co-IP experiments verified the binding relationship between CCR6 and TFEB. Finally, the suppressive effect of CCL20-NAb on endometriosis lesions in vivo was demonstrated in mice models.
We demonstrated that macrophages secreted CCL20 to promote CCR6 activation of ESCs during co-culture, which further induced the proliferation and migration of ESCs. We observed that the CCL20/CCR6 axis impaired lysosomal function and then blocked the autolysosome degradation process of autophagic flux in ESCs. The combination of CCR6 and TFEB to inhibit TFEB nuclear translocation mediates the role of the CCL20/CCR6 axis in the above process. We also found that co-culture with ESCs upregulated the production and secretion of CCL20 by macrophages. The suppression effect of CCL20-NAb on endometriosis lesions in vivo was demonstrated in mice models.
Our data indicate that macrophages block TFEB-mediated autolysosome degradation process of autophagic flux in ESCs via the CCL20/CCR6 axis, thereby promoting ESC proliferation and migration.
子宫内膜异位症（EMs）是一种常见的良性妇科疾病，影响大约 10%的育龄女性。异位内膜病灶可招募巨噬细胞，进而促进子宫内膜异位症的进展。有几项研究表明，巨噬细胞衍生的 CCL20 激活了几种细胞中 CCR6 的表达，诱导细胞增殖和迁移。然而，CCL20/CCR6 轴在 EMs 中巨噬细胞与子宫内膜间质细胞（ESCs）相互作用中的功能仍有待阐明。
收集 35 例卵巢子宫内膜异位症患者和 21 例对照参与者的异位和正常子宫内膜组织，进行免疫组织化学染色。通过 ELISA、qRT-PCR 和 Western blot 分析证实，在共培养过程中，巨噬细胞分泌 CCL20 促进 ESCs 的 CCR6 激活。CCK8 和 Edu 检测用于检测细胞增殖，划痕愈合和 Transwell 检测用于检测细胞迁移。通过 Western blot 分析和转染 mRFP-GFP-LC3 双荧光腺病毒（Ad-LC3）后红色/黄色斑点分析来检测自噬流，通过定量测定 Lyso-tracker 和 Gal3 的荧光强度和酸性磷酸酶的活性来检测溶酶体功能。此外，通过 co-IP 实验验证了 CCR6 和 TFEB 之间的结合关系。最后，在小鼠模型中证明了 CCL20-NAb 对子宫内膜异位症病变的抑制作用。
我们证明巨噬细胞在共培养过程中分泌 CCL20 以促进 ESCs 的 CCR6 激活，进而诱导 ESCs 的增殖和迁移。我们观察到 CCL20/CCR6 轴损害了溶酶体功能，然后阻断了 ESCs 中自噬流的自噬溶酶体降解过程。CCR6 和 TFEB 的结合抑制 TFEB 核易位介导了 CCL20/CCR6 轴在上述过程中的作用。我们还发现与 ESCs 共培养可上调巨噬细胞 CCL20 的产生和分泌。在小鼠模型中证明了 CCL20-NAb 对子宫内膜异位症病变的抑制作用。
我们的数据表明，巨噬细胞通过 CCL20/CCR6 轴阻断 TFEB 介导的自噬流的自噬溶酶体降解过程，从而促进 ESCs 的增殖和迁移。
