Yang Jianhua, Zhang Handan, Wang Wenzhe, Yin Qiqi, He Xiaoning, Tao Dihao, Wang Hanzhe, Liu Wenhao, Wang Yiming, Dong Zhiwei, Chen Xin, Li Bei
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi'an, People's Republic of China.
Department of Chemical Engineering, Shaanxi Key Laboratory of Energy Chemical Process Intensification, Institute of Polymer Science in Chemical Engineering, School of Chemical Engineering and Technology, Xi'an Jiaotong University, Xi'an, 710049, People's Republic of China.
Int J Nanomedicine. 2025 May 21;20:6379-6398. doi: 10.2147/IJN.S517357. eCollection 2025.
Aberrant interaction between innate immune and adaptive immune cells can disrupt tissue homeostasis, consequently triggering chronic inflammatory diseases such as rheumatoid arthritis (RA) and periodontitis (PD). Pro-inflammatory macrophages serve as critical mediators in the early immune response, constituting a major population of CD80 cells, while anti-inflammatory macrophages modulating inflammatory processes through the secretion of transforming growth factor-beta (TGF-β). This cytokine facilitates the differentiation of peripheral regulatory T cells (Tregs) and contributes to the establishment of immune tolerance. However, there are no definitive therapies to reshape the tissue homeostasis between innate immune and adaptive immune cells.
(1) anti-CD80-MTX-EVs was obtained by gradient centrifugation, which were characterized by TEM and DLS, and the associated membrane proteins were identified by Western Blot. (2) The mouse bone marrow-derived macrophages were co-cultured separately with EVs, anti-CD80-EVs, and anti-CD80-MTX-EVs in vitro, and the expression of CD80 on the macrophages surface as well as the proportion of Treg cell generation were detected. (3) EVs, anti-CD80-EVs and anti-CD80-MTX-EVs were injected into mice models of arthritis and periodontitis for treatment, the therapeutic effect was evaluated by the expressions of related cytokines, staining of HE, the proportion of CD80 macrophages and the phenotypic differentiation of T cells in the tissues.
We successfully constructed engineered EVs (anti-CD80-MTX-EVs) targeting inflammatory macrophages for intracellular MTX delivering, which inducing the anti-inflammatory transformation while upregulating the expression of TGF-β of macrophages. Furthermore, our findings demonstrate that anti-CD80-MTX-EVs effectively reduce CD80 macrophage levels, promote Treg cell generation, and inhibit Th1 cell production in vivo.
In this study, the anti-CD80-MTX-Evs demonstrated significant therapeutic effects in both rheumatoid arthritis and periodontitis models through a triple mechanism: reducing CD80 macrophage population, enhancing Treg cell differentiation, and suppressing Th1 cell development. Overall, this study presents an innovative strategy for resolving inflammation within chronic inflammatory diseases.
先天性免疫细胞与适应性免疫细胞之间的异常相互作用会破坏组织稳态，从而引发类风湿性关节炎（RA）和牙周炎（PD）等慢性炎症性疾病。促炎性巨噬细胞在早期免疫反应中起关键介质作用，构成CD80细胞的主要群体，而抗炎性巨噬细胞通过分泌转化生长因子-β（TGF-β）调节炎症过程。这种细胞因子促进外周调节性T细胞（Tregs）的分化，并有助于建立免疫耐受。然而，目前尚无明确的疗法来重塑先天性免疫细胞与适应性免疫细胞之间的组织稳态。
（1）通过梯度离心获得抗CD80-MTX-EVs，用透射电子显微镜（TEM）和动态光散射（DLS）对其进行表征，并用蛋白质免疫印迹法鉴定相关膜蛋白。（2）将小鼠骨髓来源的巨噬细胞分别与EVs、抗CD80-EVs和抗CD80-MTX-EVs在体外共培养，检测巨噬细胞表面CD80的表达以及Treg细胞生成的比例。（3）将EVs、抗CD80-EVs和抗CD80-MTX-EVs注射到关节炎和牙周炎小鼠模型中进行治疗，通过相关细胞因子的表达、苏木精-伊红（HE）染色、CD80巨噬细胞的比例以及组织中T细胞的表型分化来评估治疗效果。
我们成功构建了靶向炎性巨噬细胞用于细胞内递送甲氨蝶呤（MTX）的工程化细胞外囊泡（抗CD80-MTX-EVs），其可诱导抗炎转化，同时上调巨噬细胞TGF-β的表达。此外，我们的研究结果表明，抗CD80-MTX-EVs在体内可有效降低CD80巨噬细胞水平，促进Treg细胞生成，并抑制Th1细胞产生。
在本研究中，抗CD80-MTX-Evs通过减少CD80巨噬细胞群体、增强Treg细胞分化和抑制Th1细胞发育的三重机制，在类风湿性关节炎和牙周炎模型中均显示出显著的治疗效果。总体而言，本研究提出了一种解决慢性炎症性疾病中炎症的创新策略。
