Wang Yu-Han, Li A-Guo, Wang Hong-Yan, Tu Yong-Sheng
Guangdong Provincial Key Laboratory of Protein Modification and Degradation, Guangzhou Medical University, Guangzhou, China.
Department of Physiology, College of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China.
Front Immunol. 2024 Nov 26;15:1472425. doi: 10.3389/fimmu.2024.1472425. eCollection 2024.
The role of the JAK2-STAT1/PD-L1 pathway in the phagocytic activity of alveolar macrophages (AMs) during LPS-induced acute lung injury in mice remains poorly understood. This study aims to explore whether the JAK2-STAT1/PD-L1 pathway is upregulated on AMs in LPS-induced mice acute lung injury and to further explore the impact of the JAK2-specific inhibitor CEP-33779 on the LPS-induced impairment of AMs phagocytic activity and lung injury.
ALI was induced in mice via intratracheal administration of LPS, followed by intragastric administration of JAK2 inhibitor CEP-33779 suspension. Immunohistochemistry was conducted to assess PD-L1 expression in lung tissue, as well as p-JAK2, p-STAT1, and PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) using immunofluorescence. Levels of TNF-α and IL-6, as well as protein concentration in BALF, were measured using enzyme-linked immunosorbent assay and Bicinchoninic acid assays, respectively. Hematoxylin-eosin staining and lung injury score were employed to evaluate pathological changes in mouse lungs. Total cell count in BALF was determined using a cell counter. Furthermore, western blot and immunofluorescence was conducted to assess the effect of JAK2 and STAT1 inhibitor on JAK2-STAT1 pathway activation and PD-L1 expression, while confocal microscopy with latex beads rabbit IgG FITC complex was used to observe MH-S cells phagocytic ability.
The study revealed that LPS stimulation triggered the activation of the JAK2-STAT1 pathway and an upregulation of PD-L1 on AMs in both LPS-induced acute lung injury mice and MH-S cell lines. Moreover, treatment with the JAK2 and STAT1 inhibitor effectively reduced the activation of JAK2-STAT1 signaling, downregulated PD-L1 expression on AMs in BALF from LPS-induced ALI mice and LPS-stimulated MH-S cells, and significantly improved the LPS-induced reduction in phagocytic activity in MH-S cells. Most notably, CEP-33779 treatment significantly mitigated the pulmonary inflammatory response and lung injury in mice with LPS-induced ALI.
Collectively, these findings imply that the JAK2-STAT1 pathway plays a role in the upregulation of PD-L1, which in turn is associated with the diminished phagocytic activity in LPS-induced AMs as well as lung injury. Furthermore, our study highlights that CEP-33779 treatment can effectively improve the reduced phagocytic activity of AMs and relieve lung injury induced by LPS through suppression of the JAK2-STAT1/PD-L1 pathway.
JAK2-STAT1/PD-L1通路在脂多糖（LPS）诱导的小鼠急性肺损伤过程中对肺泡巨噬细胞（AMs）吞噬活性的作用仍知之甚少。本研究旨在探讨JAK2-STAT1/PD-L1通路在LPS诱导的小鼠急性肺损伤中是否在AMs上上调，并进一步探讨JAK2特异性抑制剂CEP-33779对LPS诱导的AMs吞噬活性损伤和肺损伤的影响。
通过气管内给予LPS诱导小鼠发生急性肺损伤（ALI），随后灌胃给予JAK2抑制剂CEP-33779悬浮液。采用免疫组织化学法评估肺组织中PD-L1的表达，并用免疫荧光法检测支气管肺泡灌洗液（BALF）中AMs上的p-JAK2、p-STAT1和PD-L1表达。分别采用酶联免疫吸附测定法和二喹啉甲酸测定法测量BALF中TNF-α和IL-6的水平以及蛋白质浓度。采用苏木精-伊红染色和肺损伤评分评估小鼠肺组织的病理变化。使用细胞计数器测定BALF中的细胞总数。此外，进行蛋白质免疫印迹法和免疫荧光法评估JAK2和STAT1抑制剂对JAK2-STAT1通路激活和PD-L1表达的影响，同时使用乳胶珠兔IgG FITC复合物共聚焦显微镜观察MH-S细胞的吞噬能力。
研究发现，LPS刺激在LPS诱导的急性肺损伤小鼠和MH-S细胞系中均触发了JAK2-STAT1通路的激活以及AMs上PD-L1的上调。此外，用JAK2和STAT1抑制剂处理可有效降低JAK2-STAT1信号的激活，下调LPS诱导的ALI小鼠BALF中AMs以及LPS刺激的MH-S细胞上的PD-L1表达，并显著改善LPS诱导的MH-S细胞吞噬活性降低的情况。最值得注意的是，CEP-33779处理显著减轻了LPS诱导的ALI小鼠的肺部炎症反应和肺损伤。
总体而言，这些发现表明JAK2-STAT1通路在PD-L1的上调中起作用，而这又与LPS诱导的AMs吞噬活性降低以及肺损伤有关。此外，我们的研究强调，CEP-33779处理可通过抑制JAK2-STAT1/PD-L1通路有效改善AMs降低的吞噬活性并减轻LPS诱导的肺损伤。
