Center for Tumor Diagnosis and Therapy, Jinshan Hospital, Fudan University, Shanghai, China.
Department of Hematology, Zhongshan Hospital Qingpu Branch, Fudan University, Shanghai, China.
Clin Transl Med. 2023 Jul;13(7):e1344. doi: 10.1002/ctm2.1344.
The first-line therapy is effective for the treatment of primary immune thrombocytopenia (ITP); however, maintaining the long-term responses remains challenging. Low-dose decitabine (DAC) has been adopted to treat refractory ITP, while its role in macrophage polarization has not been fully understood. We aimed to investigate the mechanistic role of DAC in M2 macrophage polarization and evaluated its therapeutic effect in ITP.
The M2 monocytes were identified by flow cytometry from peripheral blood mononuclear cells in healthy controls (HCs) and ITP patients. The expression of PPARγ, Arg-1, DNMT3b and NLRP3, together with IL-10 plasma levels was measured to examine its function. Bisulfite-sequencing PCR was used to evaluate the methylation status of PPARγ promoter, and the binding affinity of KLF4 was measured by Cut&Tag. A sh-PPARγ THP-1 cell line was created to verify if low-dose DAC-modulated M2 macrophage polarization was PPARγ-dependent. The passive ITP models were used to investigate the therapeutic effects of low-dose DAC and its role in modulating polarization and immunomodulatory function of macrophages. NLRP3 inflammasome and reactive oxygen species were also tested to understand the downstream of PPARγ.
The M2 monocytes with impaired immunoregulation were observed in ITP. After high-dose dexamethasone (HD-DXM) treatment, M2 monocytes increased significantly with the elevated expression of PPARγ, Arg-1 and IL-10 in CR patients. Low-dose DAC promoted M2 macrophage polarization in a PPARγ-dependent way via demethylating the promoter of PPARγ, especially the KLF4 binding sites. Low-dose DAC alleviated ITP mice by restoring the M1/M2 balance and fine-tuning immunomodulatory function of macrophages. The downstream of the PPARγ modulation of M2 macrophage polarization might physiologically antagonize NLRP3 inflammasome.
Low-dose DAC promoted M2 macrophage polarization due to the demethylation within the promoter of PPARγ, thus enhanced the KLF4 binding affinity in ITP.
一线治疗对原发性免疫性血小板减少症（ITP）的治疗有效；然而，维持长期反应仍然具有挑战性。低剂量地西他滨（DAC）已被用于治疗难治性 ITP，但它在巨噬细胞极化中的作用尚未完全了解。我们旨在研究 DAC 在 M2 巨噬细胞极化中的机制作用，并评估其在 ITP 中的治疗效果。
通过流式细胞术从健康对照（HC）和 ITP 患者的外周血单核细胞中鉴定 M2 单核细胞。测量 PPARγ、Arg-1、DNMT3b 和 NLRP3 的表达以及 IL-10 血浆水平，以研究其功能。亚硫酸氢盐测序 PCR 用于评估 PPARγ 启动子的甲基化状态，并通过 Cut&Tag 测量 KLF4 的结合亲和力。创建 sh-PPARγ THP-1 细胞系以验证低剂量 DAC 调节的 M2 巨噬细胞极化是否依赖于 PPARγ。使用被动 ITP 模型研究低剂量 DAC 的治疗效果及其在调节极化和巨噬细胞免疫调节功能中的作用。还测试了 NLRP3 炎性小体和活性氧以了解 PPARγ 的下游。
在 ITP 中观察到免疫调节受损的 M2 单核细胞。在 CR 患者中，经大剂量地塞米松（HD-DXM）治疗后，M2 单核细胞明显增加，同时 PPARγ、Arg-1 和 IL-10 的表达水平升高。低剂量 DAC 通过去甲基化 PPARγ 启动子，特别是 KLF4 结合位点，以 PPARγ 依赖的方式促进 M2 巨噬细胞极化。低剂量 DAC 通过恢复 M1/M2 平衡和微调巨噬细胞的免疫调节功能来缓解 ITP 小鼠。PPARγ 调节 M2 巨噬细胞极化的下游可能在生理上拮抗 NLRP3 炎性小体。
低剂量 DAC 通过去甲基化 PPARγ 启动子促进 M2 巨噬细胞极化，从而增强了 ITP 中 KLF4 的结合亲和力。
