Jeong Seonghee, Yang Kyungwon, Lee Ye-Ji, Park Joo-Won, Park Eun-Mi, Kang Jihee Lee
Department of Physiology, College of Medicine, Ewha Womans University, Seoul, Republic of Korea.
Inflammation-Cancer Microenvironment Research Center, College of Medicine, Ewha Womans University, Seoul, Republic of Korea.
Front Immunol. 2025 Feb 17;16:1523166. doi: 10.3389/fimmu.2025.1523166. eCollection 2025.
Growth arrest-specific 6 (Gas6) protein signaling plays a critical role in maintaining immune homeostasis and regulating inflammation. However, novel mechanisms for modulating macrophage activity through the Gas6 axis are being identified. Gas6 enhances the production of apoptosis inhibitor of macrophages (AIM), a protein with potent anti-inflammatory properties. This study investigates whether Gas6-induced AIM suppresses acute lung injury (ALI) in mice by modulating key inflammatory pathways, including inflammasome activation, autophagy, reactive oxygen species (ROS) generation, and efferocytosis.
ALI was induced in wild-type (WT) and mice via intratracheal administration of LPS. To evaluate the effects of the Gas6-AIM axis on lung inflammation, recombinant Gas6 (rGas6) was treated intraperitoneally. Inflammatory responses were evaluated using enzyme-linked immunosorbent assay, a cell-sizing analyzer, and Bicinchoninic acid assays. Lung pathology was assessed using hematoxylin-eosin staining. NLRP3 inflammasome activation and autophagy were evaluated using western blot, quantitative real-time PCR, and immunofluorescence. Reactive oxygen species (ROS) levels in alveolar macrophages were measured via fluorescence microscopy, while efferocytosis was assessed in cytospin-stained BAL cells and cultured alveolar macrophages co-cultured with apoptotic Jurkat cells. Additionally, rGas6-mediated effects on NLRP3 inflammasome activation and autophagy were validated in mouse bone marrow-derived macrophages (BMDMs) using siRNAs targeting AIM, Axl, LXRα, or LXRβ.
Proinflammatory cytokine production, neutrophil infiltration, and protein levels in BALF were significantly reduced by rGas6 administration in WT mice but not in mice. Specifically, rGas6 reduced IL-1β and IL-18 levels, caspase-1 activity, and the production of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) in alveolar macrophages. Additionally, rGas6 promoted autophagy and efferocytosis in alveolar macrophages while reducing ROS levels through AIM production. These protective effects were absent in mice. Furthermore, siRNA-mediated silencing of Axl, LXRα, LXRβ, or AIM reversed the inhibitory effects of rGas6 on NLRP3 inflammasome activation in BMDMs, and AIM was essential for rGas6-induced autophagy.
Gas6-induced AIM production protects against LPS-induced ALI by inhibiting NLRP3 inflammasome activation, enhancing autophagy and efferocytosis, and reducing oxidative stress. These findings highlight the Gas6-AIM axis as a potential therapeutic target for mitigating inflammatory lung diseases.
生长停滞特异性6（Gas6）蛋白信号传导在维持免疫稳态和调节炎症中起关键作用。然而，通过Gas6轴调节巨噬细胞活性的新机制正在被发现。Gas6增强巨噬细胞凋亡抑制剂（AIM）的产生，AIM是一种具有强大抗炎特性的蛋白质。本研究调查Gas6诱导的AIM是否通过调节关键炎症途径（包括炎性小体激活、自噬、活性氧（ROS）生成和胞葬作用）来抑制小鼠急性肺损伤（ALI）。
通过气管内给予脂多糖（LPS）在野生型（WT）小鼠和[此处缺失相关基因敲除小鼠信息]小鼠中诱导ALI。为了评估Gas6-AIM轴对肺部炎症的影响，腹腔注射重组Gas6（rGas6）。使用酶联免疫吸附测定、细胞大小分析仪和二辛可宁酸测定法评估炎症反应。使用苏木精-伊红染色评估肺病理学。使用蛋白质免疫印迹法、定量实时聚合酶链反应和免疫荧光评估NLRP3炎性小体激活和自噬。通过荧光显微镜测量肺泡巨噬细胞中的活性氧（ROS）水平，同时在细胞涂片染色的支气管肺泡灌洗（BAL）细胞和与凋亡Jurkat细胞共培养的培养肺泡巨噬细胞中评估胞葬作用。此外，使用靶向AIM、Axl、肝X受体α（LXRα）或肝X受体β（LXRβ）的小干扰RNA（siRNA）在小鼠骨髓来源的巨噬细胞（BMDM）中验证rGas6对NLRP3炎性小体激活和自噬的介导作用。
WT小鼠腹腔注射rGas6后，促炎细胞因子产生、中性粒细胞浸润和BALF中的蛋白质水平显著降低，但[此处缺失相关基因敲除小鼠信息]小鼠中未降低。具体而言，rGas6降低了肺泡巨噬细胞中白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）水平、半胱天冬酶-1活性以及含有半胱天冬酶激活和募集结构域（ASC）的凋亡相关斑点样蛋白的产生。此外，rGas6促进肺泡巨噬细胞中的自噬和胞葬作用，同时通过产生AIM降低ROS水平。这些保护作用在[此处缺失相关基因敲除小鼠信息]小鼠中不存在。此外，siRNA介导的Axl、LXRα、LXRβ或AIM沉默逆转了rGas6对BMDM中NLRP3炎性小体激活的抑制作用，并且AIM对于rGas6诱导的自噬至关重要。
Gas6诱导的AIM产生通过抑制NLRP3炎性小体激活、增强自噬和胞葬作用以及降低氧化应激来预防LPS诱导的ALI。这些发现突出了Gas- AIM轴作为减轻炎症性肺病的潜在治疗靶点。
