Mesenchymal stem cells (MSCs) activate the endogenous immune regulatory system, inducing a therapeutic effect in recipients. MSCs have demonstrated the ability to modulate the differentiation of myeloid cells toward a phagocytic and anti-inflammatory profile. Allogeneic, adipose-derived MSCs (ASCs) have been investigated for the management of complex perianal fistula, with darvadstrocel being the first ASC therapy approved in Europe in March 2018. Additionally, ASCs are being explored as a potential treatment in other indications. Yet, despite these clinical advances, their mechanism of action is only partially understood. Freshly isolated human monocytes from the peripheral blood were differentiated in vitro toward M0 non-polarized macrophages (Mphs), M1 pro-inflammatory Mphs, M2 anti-inflammatory Mphs, or mature dendritic cells (mDCs) in the presence or absence of ASCs, in non-contact conditions. The phenotype and function of the differentiated myeloid populations were determined by flow cytometry, and their secretome was analyzed by OLINK technology. We also investigated the capacity of ASCs to modulate the phenotype and function of terminally differentiated M1 Mphs. The role of soluble factors interleukin (IL)-6 and prostaglandin E2 (PGE2) on the ability of ASCs to modulate myeloid cells was assessed using neutralization assays, CRISPR/Cas9 knock-down of cyclooxygenase 2 (COX-2), and ASC-conditioned medium assays using pro-inflammatory stimulus. Co-culture of monocytes in the presence of ASCs resulted in the polarization of Mphs and mDCs toward an anti-inflammatory and phagocytic phenotype. This was characterized by an increase in phagocytic receptors on the cell surface of Mphs (M0, M1, and M2) and mDCs, as well as modulation of chemokine receptors and reduced expression of pro-inflammatory, co-stimulatory molecules. ASCs also modulated the secretome of Mphs and mDCs, demonstrated by reduced expression of pro-inflammatory factors and increased expression of anti-inflammatory and reparative factors. Chemical inhibition of PGE2 with indomethacin abolished this modulatory effect, whereas treatment with a neutralizing anti-IL-6 antibody resulted in a partial abolishment. The knock-down of COX-2 in ASCs and the use of IL-1β-activated ASC-conditioned media confirmed the key role of PGE2 in ASC-mediated myeloid modulation. In our in vitro experimental settings, ASCs failed to modulate the phenotype and function of terminally polarized M1 Mphs. The results demonstrate that ASCs are able to modulate the in vitro differentiation of myeloid cells toward an anti-inflammatory and reparative profile. This modulatory effect was mediated mainly by PGE2 and, to a lesser extent, IL-6.

中文翻译：

---

人脂肪间充质干细胞调节骨髓细胞朝向抗炎和修复表型：IL-6 和 PGE2 的作用

间充质干细胞 (MSCs) 激活内源性免疫调节系统，在受体中诱导治疗效果。MSCs 已证明能够调节骨髓细胞向吞噬和抗炎特征的分化。异基因脂肪来源的 MSCs (ASCs) 已被研究用于治疗复杂的肛周瘘，darvadstrocel 是 2018 年 3 月在欧洲批准的第一个 ASC 疗法。此外，正在探索 ASCs 作为其他适应症的潜在治疗方法。然而，尽管有这些临床进展，但它们的作用机制只是部分了解。从外周血中新鲜分离的人单核细胞在体外分化为 M0 非极化巨噬细胞 (Mphs)、M1 促炎 Mphs、M2 抗炎 Mphs、在非接触条件下，在存在或不存在 ASC 的情况下或成熟的树突状细胞 (mDC)。流式细胞仪测定分化的髓系群体的表型和功能，并通过OLINK技术分析其分泌组。我们还研究了 ASC 调节终末分化 M1 Mphs 的表型和功能的能力。可溶性因子白细胞介素 (IL)-6 和前列腺素 E2 (PGE2) 对 ASC 调节骨髓细胞能力的作用使用中和试验、CRISPR/Cas9 敲除环氧合酶 2 (COX-2) 和 ASC-使用促炎刺激的条件培养基测定。在 ASCs 存在下单核细胞的共培养导致 Mphs 和 mDCs 向抗炎和吞噬表型极化。其特征是 Mphs（M0、M1 和 M2）和 mDCs 细胞表面的吞噬受体增加，以及趋化因子受体的调节和促炎、共刺激分子的表达减少。ASCs 还调节 Mphs 和 mDCs 的分泌组，通过减少促炎因子的表达和增加抗炎和修复因子的表达来证明。用吲哚美辛对 PGE2 的化学抑制消除了这种调节作用，而用中和性抗 IL-6 抗体治疗导致部分消除。ASC 中 COX-2 的敲低和 IL-1β 激活的 ASC 条件培养基的使用证实了 PGE2 在 ASC 介导的骨髓调节中的关键作用。在我们的体外实验环境中，ASCs 未能调节末端极化 M1 Mphs 的表型和功能。结果表明，ASCs 能够调节骨髓细胞向抗炎和修复特性的体外分化。这种调节作用主要由 PGE2 介导，在较小程度上由 IL-6 介导。