Department of Orthopedic Surgery, 38088Kitasato University School of Medicine, Sagamihara, Japan.
Department of Immunology, 38088Kitasato University School of Medicine, Sagamihara, Japan.
Int J Immunopathol Pharmacol. 2022 Jan-Dec;36:3946320221103792. doi: 10.1177/03946320221103792.
Studies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-β is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-β on M2 polarization using a mice disc-puncture injury model.
To investigate the origin of M2 macrophages, 30 GFP chimeric mice were created by bone marrow transplantation. IVDs were obtained from both groups on pre-puncture (control) and post-puncture days 1, 3, 7, and 14 and CD86 (M1 marker)- and CD206 (M2 marker)-positive cells evaluated by flow cytometry ( = 5 at each time point). To investigate the role of TGF-β on M2 polarization, TGF-β inhibitor (SB431542) was also injected on post-puncture days (PPD) 5 and 6 and CD206 expression was evaluated on day 7 by flow cytometry ( = 5) and real time PCR ( = 10).
The proportion of CD86 Mϕ within the GFP+ population was significantly increased at PPD 1, 3, 7, and 14 compared to control. CD206-positive cells in GFP-populations were significantly increased on PPD 7 and 14. In addition, the percentage of CD206-positive cells was significantly higher in GFP-populations than in GFP+ populations. TGF-β inhibitor reduced CD206-positive cells and expression at 7 days after puncture.
Our findings suggest that M2 Mϕ following IVD injury may originate from resident Mϕ. TGF-β is a key factor for M2 polarization of macrophages following IVD injury.
研究已经确定了 M1 和 M2 巨噬细胞（Mϕ）在受伤的椎间盘（IVD）中的存在。然而，M2 Mϕ的起源和极化调节因子尚未完全阐明。TGF-β 是几种组织中 M2 极化的调节因子。在这里，我们使用小鼠椎间盘穿刺损伤模型研究了 M2 Mϕ的来源以及 TGF-β对 M2 极化的作用。
为了研究 M2 巨噬细胞的起源，通过骨髓移植创建了 30 只 GFP 嵌合小鼠。在穿刺前（对照）和穿刺后第 1、3、7 和 14 天，从两组中获取 IVD，并通过流式细胞术评估 CD86（M1 标志物）和 CD206（M2 标志物）阳性细胞（每个时间点= 5）。为了研究 TGF-β对 M2 极化的作用，也在穿刺后第 5 和 6 天（PPD）注射 TGF-β抑制剂（SB431542），并通过流式细胞术（= 5）和实时 PCR（= 10）在第 7 天评估 CD206 表达。
与对照相比，在 PPD 1、3、7 和 14 时 GFP+群体中的 CD86 Mϕ 比例明显增加。在 GFP 群体中，CD206 阳性细胞在 PPD 7 和 14 时明显增加。此外，GFP 群体中 CD206 阳性细胞的比例明显高于 GFP+群体。TGF-β 抑制剂可减少穿刺后 7 天的 CD206 阳性细胞和 表达。
我们的研究结果表明，IVD 损伤后 M2 Mϕ 可能起源于固有 Mϕ。TGF-β 是 IVD 损伤后巨噬细胞 M2 极化的关键因素。
