Sustained induction of IP-10 by MRP8/14 via the IFNβ-IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice.
Sustained induction of IP-10 by MRP8/14 via the IFNβ-IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice.
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DOI：
10.1093/burnst/tkad006
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发表时间：
2023
期刊：
影响因子：
5.3
通讯作者：
Jiang, Yong
中科院分区：
文献类型：
--
作者：
Wang, Juan;Chen, Guiming;Li, Lei;Luo, Sidan;Hu, Bingrong;Xu, Jia;Luo, Haihua;Li, Shan;Jiang, Yong
关键词：
髓样相关蛋白8/14（MRP8/14）异源二聚体作为一种损伤相关的分子模式，介导多种炎症性疾病，如脓毒症。然而，MRP8/14如何通过调节内毒素血症期间的炎症反应来促进肺损伤仍然在很大程度上未知。本研究旨在阐明MRP8/14在内毒素血症中的病理作用。用野生型和骨髓细胞特异性Mrp8缺失（Mrp8 Δ MC）小鼠制备内毒素血症模型，以评价血浆细胞因子水平。通过苏木素和伊红（H & E）染色、损伤评分和湿干重（W/D）比评价肺损伤。采用实时荧光定量聚合酶链反应（qPCR）检测巨噬细胞MRP 8/14诱导的干扰素γ（IFN γ）诱导蛋白10（IP-10）mRNA表达的动态变化。使用免疫印迹来评估由JAK-STAT信号传导途径的活化诱导的IP-10水平的增加。荧光素酶报告基因检测IRF7在Ip-10基因转录中的作用。进行体内气囊实验以确定由MRP8/14诱导的IP-10的生物学功能。Mrp8 Δ MC小鼠实验表明，MRP8/14可促进支气管肺泡灌洗液（BALF）中IP-10等细胞因子的产生，并可引起内毒素小鼠肺损伤。qPCR结果显示，MRP 8/14处理12 h后，巨噬细胞中仍有IP-10 mRNA表达。中和实验表明，MRP 8/14诱导的RAW264.7细胞表达Ip-10是由细胞外IFN β介导的。磷酸化特异性抗体的蛋白质印迹显示，MRP8/14处理的RAW264. 7细胞中JAK1/TYK2-STAT1信号通路被激活，导致Ip-10基因表达上调。IRF7被进一步鉴定为JAK-STAT途径的下游调节剂，其介导用MRP 8/14处理的巨噬细胞中的Ip-10基因表达。体内气囊实验证实，IFN β-JAK1/TYK2-STAT1-IRF7通路是趋化因子（C-X-C基序）受体3（CXCR3）+T淋巴细胞迁移所必需的，其在内毒素血症的背景下促进肺损伤。总之，我们的研究表明，MRP8/14通过IFN β-JAK1/TYK2-STAT1-IRF7通路诱导IP-10的持续产生，以吸引CXCR3 + T淋巴细胞进入肺组织，并最终导致内毒素血症背景下过度炎症反应导致肺损伤。
As a damage-associated molecular pattern, the myeloid-related protein 8/14 (MRP8/14) heterodimer mediates various inflammatory diseases, such as sepsis. However, how MRP8/14 promotes lung injury by regulating the inflammatory response during endotoxemia remains largely unknown. This study aims at illuminating the pathological functions of MRP8/14 in endotoxemia. An endotoxemic model was prepared with wild-type and myeloid cell-specific Mrp8 deletion (Mrp8ΔMC) mice for evaluating plasma cytokine levels. Lung injury was evaluated by hematoxylin and eosin (H&E) staining, injury scoring and wet-to-dry weight (W/D) ratio. The dynamic profile of interferon γ (IFNγ)-inducible protein 10 (IP-10) mRNA expression induced by macrophage MRP8/14 was determined by quantitative real-time polymerase chain reaction (qPCR). Immunoblotting was used to evaluate the increase in IP-10 level induced by activation of the JAK–STAT signaling pathway. Luciferase reporter assay was performed to detect the involvement of IRF7 in Ip-10 gene transcription. In vivo air pouch experiments were performed to determine the biological function of IP-10 induced by MRP8/14. Experiments with Mrp8ΔMC mice showed that MRP8/14 promoted the production of cytokines, including IP-10, in the bronchoalveolar lavage fluid (BALF) and lung injury in endotoxic mice. The result of qPCR showed sustained expression of Ip-10 mRNA in macrophages after treatment with MRP8/14 for 12 h. Neutralization experiments showed that the MRP8/14-induced Ip-10 expression in RAW264.7 cells was mediated by extracellular IFNβ. Western blotting with phosphorylation-specific antibodies showed that the JAK1/TYK2-STAT1 signaling pathway was activated in MRP8/14-treated RAW264.7 cells, leading to the upregulation of Ip-10 gene expression. IRF7 was further identified as a downstream regulator of the JAK–STAT pathway that mediated Ip-10 gene expression in macrophages treated with MRP8/14. In vivo air pouch experiments confirmed that the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway was required for chemokine (C-X-C motif) receptor 3 (CXCR3)+ T lymphocyte migration, which promoted lung injury in the context of endotoxemia. In summary, our study demonstrates that MRP8/14 induces sustained production of IP-10 via the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway to attract CXCR3+ T lymphocytes into lung tissues and ultimately results in lung injury by an excessive inflammatory response in the context of endotoxemia.
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