Wang Yana, Fan Min, Chen Linjian, Gill Patrick Spencer, Wang Xiaohui, Ha Tuanzhu, Williams David L, Li Chuanfu, Yang Kun
Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States.
Center of Excellence in Inflammation, Infectious Disease and Immunity, East Tennessee State University, Johnson City, TN, United States.
Front Immunol. 2025 May 15;16:1587898. doi: 10.3389/fimmu.2025.1587898. eCollection 2025.
Cardiac macrophages are essential mediators of inflammation and tissue remodeling following myocardial infarction (MI). Endothelial cell-specific heat shock protein A12B (eHSPA12B) has emerged as a key vascular regulator, but its role in modulating immune cell responses after MI remains unknown. This study investigates whether eHSPA12B regulates monocyte infiltration following MI injury.
We used endothelial cell-specific Hspa12b knockout (e ) and wild-type (WT) mice to assess cardiac function and monocyte infiltration following MI. Cardiac resident macrophages and infiltrating monocytes were examined by flow cytometry 3 days post-MI. Plasma levels of pro-inflammatory cytokines were evaluated by ELISA following MI. To investigate the mechanism by which Hspa12b regulates immune response of macrophages, endothelial cells were transduced with adenovirus expressing HSPA12B followed by hypoxia challenge. In a separate experiment, endothelial cell-derived exosomes were prepared. Macrophages, Raw 264.7 or bone marrow derived macrophages (BMDMs) were incubated with endothelial cell conditioned medium or endothelial cell-derived exosomes. Macrophage phenotypes were examined by immunofluorescence staining, ELISA and qPCR. Protein degradation of toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) in macrophages was assessed by immunoprecipitation and Western blotting.
e mice exhibited significantly worsened cardiac function and increased infiltration of monocytes compared to WT controls at 3 days post-MI. Conditioned medium from HSPA12B-overexpressing endothelial cells promoted a pro-regenerative macrophage phenotype, characterized by reduced pro-inflammatory and increased anti-inflammatory cytokine production. HSPA12B was secreted via exosomes from endothelial cells, and these exosomes were sufficient to induce macrophage polarization. Mechanistically, uptake of HSPA12B-containing exosomes promotes the degradation of TLR4 and MyD88 in macrophages.
Endothelial HSPA12B plays a novel immunomodulatory role in controlling monocyte infiltration and immune activation following MI.
心脏巨噬细胞是心肌梗死（MI）后炎症和组织重塑的重要介质。内皮细胞特异性热休克蛋白A12B（eHSPA12B）已成为关键的血管调节因子，但其在MI后调节免疫细胞反应中的作用尚不清楚。本研究调查eHSPA12B是否在MI损伤后调节单核细胞浸润。
我们使用内皮细胞特异性Hspa12b基因敲除（e ）和野生型（WT）小鼠来评估MI后的心脏功能和单核细胞浸润。MI后3天通过流式细胞术检测心脏驻留巨噬细胞和浸润单核细胞。MI后通过酶联免疫吸附测定法评估促炎细胞因子的血浆水平。为了研究Hspa12b调节巨噬细胞免疫反应的机制，用表达HSPA12B的腺病毒转导内皮细胞，随后进行缺氧刺激。在另一个实验中，制备内皮细胞衍生的外泌体。将巨噬细胞、Raw 264.7或骨髓来源的巨噬细胞（BMDM）与内皮细胞条件培养基或内皮细胞衍生的外泌体一起孵育。通过免疫荧光染色、酶联免疫吸附测定法和定量聚合酶链反应检测巨噬细胞表型。通过免疫沉淀和蛋白质印迹法评估巨噬细胞中Toll样受体4（TLR4）和髓样分化初级反应88（MyD88）的蛋白质降解情况。
与WT对照组相比，e 小鼠在MI后3天表现出明显恶化的心脏功能和增加的单核细胞浸润。过表达HSPA12B的内皮细胞的条件培养基促进了促再生巨噬细胞表型，其特征在于促炎细胞因子产生减少和抗炎细胞因子产生增加。HSPA12B通过内皮细胞分泌的外泌体分泌，并且这些外泌体足以诱导巨噬细胞极化。从机制上讲，摄取含HSPA12B的外泌体促进巨噬细胞中TLR4和MyD88的降解。
内皮HSPA12B在控制MI后的单核细胞浸润和免疫激活中发挥新的免疫调节作用。
