Liu Yi, Cai Yu Chun, Chen Jia Xu, Chen Shao Hong, Yu Ying Fang
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research); National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; National Health Commission Key Laboratory On Parasite and Vector Biology, WHO Collaborating Centre for Tropical Diseases, National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai, 200025, China.
Shanghai University of Medicine & Health Science, The College of Medical Technology, Shanghai, 201318, China.
Parasit Vectors. 2025 Jun 1;18(1):199. doi: 10.1186/s13071-025-06802-2.
Trichinella spiralis, in its newborn larva (NBL) stage, invades the host bloodstream and disseminates throughout the body. Concurrently, M1 macrophages undergo transformation into M2 macrophages. In our previous studies, we demonstrated that extracellular vesicles secreted by NBL (NBL-EVs) significantly express the microRNA (miRNA) cel-let-7-5p. In this study, we investigated the immunomodulatory effects and mechanisms of action of EVs derived from T. spiralis NBL and the influence of their key miRNA, cel-let-7-5p, on M1 macrophages.
This study investigates the impact of T. spiralis NBL-EVs and cel-let-7-5p on RAW264.7 macrophages through in vitro co-culture, followed by a dual luciferase assay to confirm C/EBPδ as the target of cel-let-7-5p. M1-polarized RAW264.7 cells were subsequently transfected with various agents, including NBL-EVs, cel-let-7-5p mimic, C/EBPδ small interfering RNA (siRNA), and so forth. The cell functions, surface molecule expression, transcription, and cytokine release were analyzed using flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) to elucidate the regulatory mechanisms of NBL-EVs and cel-let-7-5p on macrophage polarization.
Results show that cel-let-7-5p transported by T. spiralis NBL-EVs inhibited the functional activity of M1 RAW264.7 macrophages by targeting C/EBPδ. This inhibition was validated by reduced CD86 and increased CD206 expression, along with decreased nitric oxide (NO) synthesis and downregulation of the M1 marker genes interleukin-12 (IL-12) and inducible nitric oxide synthase (iNOS). In contrast, the messenger RNA (mRNA) levels of IL-10 and arginase-1 (Arg1), which are M2 characteristic genes, were significantly enhanced. However, the release of M1 pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-alpha (TNF-α), and IL-1β, was decreased proportionally. Notably, introducing a cel-let-7-5p inhibitor effectively reversed the suppressive effect of NBL-EVs on M1 macrophage function and partially mitigated their transition to the M2 phenotype, notably impacting Arg1 gene expression. However, no significant changes were observed in CD206 protein expression or IL-10 mRNA levels.
The findings of this study reveal that cel-let-7-5p in T. spiralis NBL-EVs can inhibit the function of M1-type RAW264.7 macrophages by targeting C/EBPδ.
旋毛虫新生幼虫（NBL）阶段侵入宿主血液并播散至全身。同时，M1巨噬细胞会转变为M2巨噬细胞。在我们之前的研究中，我们证明了NBL分泌的细胞外囊泡（NBL-EVs）显著表达微小RNA（miRNA）cel-let-7-5p。在本研究中，我们调查了旋毛虫NBL来源的细胞外囊泡的免疫调节作用及作用机制，以及其关键miRNA cel-let-7-5p对M1巨噬细胞的影响。
本研究通过体外共培养研究旋毛虫NBL-EVs和cel-let-7-5p对RAW264.7巨噬细胞的影响，随后进行双荧光素酶测定以确认C/EBPδ是cel-let-7-5p的靶标。随后，用包括NBL-EVs、cel-let-7-模拟物、C/EBPδ小干扰RNA（siRNA）等各种试剂转染M1极化的RAW264.7细胞。使用流式细胞术、逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法和酶联免疫吸附测定（ELISA）分析细胞功能、表面分子表达、转录和细胞因子释放，以阐明NBL-EVs和cel-let-7-5p对巨噬细胞极化的调节机制。
结果表明，旋毛虫NBL-EVs转运的cel-let-7-5p通过靶向C/EBPδ抑制M1 RAW264.7巨噬细胞的功能活性。通过降低CD86表达和增加CD206表达，以及减少一氧化氮（NO）合成和下调M1标志物基因白细胞介素-12（IL-12）和诱导型一氧化氮合酶（iNOS），验证了这种抑制作用。相反，M2特征基因白细胞介素-10（IL-10）和精氨酸酶-1（Arg1）的信使核糖核酸（mRNA）水平显著增强。然而，M1促炎细胞因子如IL-6、肿瘤坏死因子-α（TNF-α）和IL-1β的释放成比例下降。值得注意的是，引入cel-let-7-5p抑制剂有效地逆转了NBL-EVs对M1巨噬细胞功能的抑制作用，并部分减轻了它们向M2表型的转变，尤其影响Arg1基因表达。然而，CD206蛋白表达或IL-10 mRNA水平未观察到显著变化。
本研究结果表明，旋毛虫NBL-EVs中的cel-let-7-5p可通过靶向C/EBPδ抑制M1型RAW264.7巨噬细胞的功能。
