Cao Guoqin, Memida Takumi, Huang Shengyuan, Dalir Abdolahinia Elaheh, Ruiz Sunniva, Hassantash Sahar, Ari Jayant, Shindo Satoru, Lin Jiang, Kawai Toshihisa, Han Xiaozhe
Department of Oral Science and Translational Research, College of Dental Medicine, Nova Southeastern University, 3200 South University Drive, Fort Lauderdale, FL 33328, USA.
Department of Stomatology, Beijing Tongren Hospital, Capital Medical University, Beijing 100005, China.
Int J Mol Sci. 2025 Jun 1;26(11):5332. doi: 10.3390/ijms26115332.
The interaction between immune regulatory cells, such as regulatory B cells (Breg) and pro-resolving macrophages (M2 macrophages), plays an important role in the restoration of immune homeostasis during inflammation. PD-L1 is one of the effector molecules that mediates the immune regulation function of M2 macrophages. The activation of PD-L1/PD-1 signaling promotes the differentiation of Breg. Previous studies have shown that Breg promoted M2 macrophage polarization and enhanced their function, but little is known about the regulatory function of M2 macrophages on Breg differentiation. This study aims to determine the effect of M2 macrophages on Breg induction and the potential mechanism in vitro. Bone-marrow-derived macrophages were isolated from wild-type (WT) mice and polarized into M2 using IL-4/IL-13. To investigate the role of PD-L1/PD-1 in M2 macrophage-induced Breg differentiation, spleen B cells were isolated from WT or PD-1 knockout (KO) mice and co-cultured with either naïve (M0) or M2 macrophages for 48 h with or without trans-well inserts. The expression of IL-10, IL-35, and TGF-β1 in B cells was evaluated by flow cytometry and immunofluorescence staining. Recombinant PD-L1 was used to stimulate activated B cells, followed by the detection of IL-35 and TGF-β1. The results show that there was no significant difference in IL-10 expression among all groups. However, IL-35 and TGF-β1 expression in B cells was significantly increased in the M2+B, but not in M0+B, compared to B cells alone. Notably, such increases were diminished when M2 and B cells were separated by trans-well inserts. IL-35 expression was not significantly changed when B cells from PD-1 KO mice were co-cultured with M2 compared to the control. However, TGF-β1 expression was significantly increased when PD-1 KO B cells were co-cultured with M2 compared to the control. IL-35 expression in activated B cells was increased upon stimulation with PD-L1. However, TGF-β1 expression in activated B cells was increased regardless of the PD-L1 availability. This study demonstrates that pro-resolving macrophage-induced IL-35 but not TGF-β1 regulatory B cell activation requires the PD-L1/PD-1 pathway.
免疫调节细胞（如调节性B细胞（Breg）和促解决巨噬细胞（M2巨噬细胞））之间的相互作用在炎症期间免疫稳态的恢复中起重要作用。PD-L1是介导M2巨噬细胞免疫调节功能的效应分子之一。PD-L1/PD-1信号通路的激活促进Breg的分化。先前的研究表明，Breg促进M2巨噬细胞极化并增强其功能，但关于M2巨噬细胞对Breg分化的调节功能知之甚少。本研究旨在确定M2巨噬细胞对Breg诱导的影响及其体外潜在机制。从野生型（WT）小鼠中分离出骨髓来源的巨噬细胞，并用IL-4/IL-13将其极化为M2。为了研究PD-L1/PD-1在M2巨噬细胞诱导的Breg分化中的作用，从WT或PD-1基因敲除（KO）小鼠中分离出脾脏B细胞，并与未活化的（M0）或M2巨噬细胞共培养48小时，有无Transwell小室。通过流式细胞术和免疫荧光染色评估B细胞中IL-10、IL-35和TGF-β1的表达。用重组PD-L1刺激活化的B细胞，然后检测IL-35和TGF-β1。结果表明，所有组之间IL-10表达无显著差异。然而，与单独的B细胞相比，M2+B组中B细胞的IL-35和TGF-β1表达显著增加，而M0+B组中则未增加。值得注意的是，当M2和B细胞通过Transwell小室分开时，这种增加会减弱。与对照组相比，PD-1 KO小鼠的B细胞与M2共培养时，IL-35表达无显著变化。然而，与对照组相比，PD-1 KO B细胞与M2共培养时，TGF-β1表达显著增加。用PD-L1刺激后，活化B细胞中的IL-35表达增加。然而，无论是否存在PD-L1，活化B细胞中的TGF-β1表达均增加。本研究表明，促解决巨噬细胞诱导的IL-35而非TGF-β1调节性B细胞活化需要PD-L1/PD-1途径。
