Abstract：
BACKGROUND: A growing body of research has shown that the phenotypic change in macrophages from M0 to M1 is essential for the start of the inflammatory process in septic acute respiratory distress syndrome (ARDS). Potential treatment targets might be identified with more knowledge of the molecular regulation of M1 macrophages in septic ARDS.
METHODS: A multi-microarray interrelated analysis of high-throughput experiments from ARDS patients and macrophage polarization was conducted to identify the hub genes associated with macrophage M1 polarization and septic ARDS. Lipopolysaccharide (LPS) and Poly (I:C) were utilized to stimulate bone marrow-derived macrophages (BMDMs) for M1-polarized macrophage model construction. Knock down of the hub genes on BMDMs via shRNAs was used to screen the genes regulating macrophage M1 polarization in vitro. The cecal ligation and puncture (CLP) mouse model was constructed in knockout (KO) mice and wild-type (WT) mice to explore whether the screened genes regulate macrophage M1 polarization in septic ARDS in vivo. ChIP-seq and further experiments on BMDMs were performed to investigate the molecular mechanism.
RESULTS: The bioinformatics analysis of gene expression profiles from a clinical cohort of 26 ARDS patients and macrophage polarization found that the 5 hub genes (IFIH1, IRF1, STAT1, IFIT3, GBP1) may have a synergistic effect on macrophage M1 polarization in septic ARDS. Further in vivo investigations indicated that IFIH1, STAT1 and IRF1 contribute to macrophage M1 polarization. The histological evaluation and immunohistochemistry of the lungs from the IRF1-/- and WT mice indicated that knockout of IRF1 markedly alleviated CLP-induced lung injury and M1-polarized infiltration. Moreover, the molecular mechanism investigations indicated that knockdown of IFIH1 markedly promoted IRF1 translocation into the nucleus. Knockout of IRF1 significantly decreases the expression of STAT1. ChIP-seq and PCR further confirmed that IRF1, as a transcription factor of STAT1, binds to the promoter region of STAT1.
CONCLUSIONS: IRF1 was identified as the key molecule that regulates macrophage M1polarization and septic ARDS development in vivo and in vitro. Moreover, as the adaptor in response to infection mimics irritants, IFIH1 promotes IRF1 (transcription factor) translocation into the nucleus to initiate STAT1 transcription.
METHODS: A multi-microarray interrelated analysis of high-throughput experiments from ARDS patients and macrophage polarization was conducted to identify the hub genes associated with macrophage M1 polarization and septic ARDS. Lipopolysaccharide (LPS) and Poly (I:C) were utilized to stimulate bone marrow-derived macrophages (BMDMs) for M1-polarized macrophage model construction. Knock down of the hub genes on BMDMs via shRNAs was used to screen the genes regulating macrophage M1 polarization in vitro. The cecal ligation and puncture (CLP) mouse model was constructed in knockout (KO) mice and wild-type (WT) mice to explore whether the screened genes regulate macrophage M1 polarization in septic ARDS in vivo. ChIP-seq and further experiments on BMDMs were performed to investigate the molecular mechanism.
RESULTS: The bioinformatics analysis of gene expression profiles from a clinical cohort of 26 ARDS patients and macrophage polarization found that the 5 hub genes (IFIH1, IRF1, STAT1, IFIT3, GBP1) may have a synergistic effect on macrophage M1 polarization in septic ARDS. Further in vivo investigations indicated that IFIH1, STAT1 and IRF1 contribute to macrophage M1 polarization. The histological evaluation and immunohistochemistry of the lungs from the IRF1-/- and WT mice indicated that knockout of IRF1 markedly alleviated CLP-induced lung injury and M1-polarized infiltration. Moreover, the molecular mechanism investigations indicated that knockdown of IFIH1 markedly promoted IRF1 translocation into the nucleus. Knockout of IRF1 significantly decreases the expression of STAT1. ChIP-seq and PCR further confirmed that IRF1, as a transcription factor of STAT1, binds to the promoter region of STAT1.
CONCLUSIONS: IRF1 was identified as the key molecule that regulates macrophage M1polarization and septic ARDS development in vivo and in vitro. Moreover, as the adaptor in response to infection mimics irritants, IFIH1 promotes IRF1 (transcription factor) translocation into the nucleus to initiate STAT1 transcription.
摘要：
背景：越来越多的研究表明，巨噬细胞从M0到M1的表型变化对于脓毒症急性呼吸窘迫综合征（ARDS）的炎症过程的开始至关重要。通过对脓毒症ARDS中M1巨噬细胞的分子调控的更多了解，可以确定潜在的治疗目标。
方法：对ARDS患者的高通量实验和巨噬细胞极化进行多微阵列相关分析，以鉴定与巨噬细胞M1极化和脓毒症ARDS相关的hub基因。利用脂多糖(LPS)和聚(I:C)刺激骨髓来源的巨噬细胞(BMDM)以构建M1极化的巨噬细胞模型。通过shRNA敲低BMDM上的hub基因用于体外筛选调节巨噬细胞M1极化的基因。在敲除（KO）小鼠和野生型（WT）小鼠中构建盲肠结扎和穿孔（CLP）小鼠模型，以探讨筛选的基因是否在体内调节脓毒症ARDS的巨噬细胞M1极化。进行ChIP-seq和对BMDM的进一步实验以研究分子机制。
结果：对26例ARDS患者临床队列的基因表达谱和巨噬细胞极化的生物信息学分析发现，5个hub基因（IFIH1，IRF1，STAT1，IFIT3，GBP1）可能对脓毒症ARDS的巨噬细胞M1极化具有协同作用。进一步的体内研究表明，IFIH1，STAT1和IRF1有助于巨噬细胞M1极化。来自IRF1-/-和WT小鼠的肺的组织学评估和免疫组织化学表明，IRF1的敲除可显着减轻CLP诱导的肺损伤和M1极化的浸润。此外，分子机制研究表明，IFIH1的敲除显着促进IRF1易位进入细胞核。IRF1的敲除显著降低STAT1的表达。ChIP-seq和PCR进一步证实IRF1作为STAT1的转录因子与STAT1的启动子区结合。
结论：IRF1被确定为在体内和体外调节巨噬细胞M1极化和感染性ARDS发展的关键分子。此外，作为对感染的反应的适配器模仿刺激物，IFIH1促进IRF1(转录因子)易位进入细胞核以启动STAT1转录。
方法：对ARDS患者的高通量实验和巨噬细胞极化进行多微阵列相关分析，以鉴定与巨噬细胞M1极化和脓毒症ARDS相关的hub基因。利用脂多糖(LPS)和聚(I:C)刺激骨髓来源的巨噬细胞(BMDM)以构建M1极化的巨噬细胞模型。通过shRNA敲低BMDM上的hub基因用于体外筛选调节巨噬细胞M1极化的基因。在敲除（KO）小鼠和野生型（WT）小鼠中构建盲肠结扎和穿孔（CLP）小鼠模型，以探讨筛选的基因是否在体内调节脓毒症ARDS的巨噬细胞M1极化。进行ChIP-seq和对BMDM的进一步实验以研究分子机制。
结果：对26例ARDS患者临床队列的基因表达谱和巨噬细胞极化的生物信息学分析发现，5个hub基因（IFIH1，IRF1，STAT1，IFIT3，GBP1）可能对脓毒症ARDS的巨噬细胞M1极化具有协同作用。进一步的体内研究表明，IFIH1，STAT1和IRF1有助于巨噬细胞M1极化。来自IRF1-/-和WT小鼠的肺的组织学评估和免疫组织化学表明，IRF1的敲除可显着减轻CLP诱导的肺损伤和M1极化的浸润。此外，分子机制研究表明，IFIH1的敲除显着促进IRF1易位进入细胞核。IRF1的敲除显著降低STAT1的表达。ChIP-seq和PCR进一步证实IRF1作为STAT1的转录因子与STAT1的启动子区结合。
结论：IRF1被确定为在体内和体外调节巨噬细胞M1极化和感染性ARDS发展的关键分子。此外，作为对感染的反应的适配器模仿刺激物，IFIH1促进IRF1(转录因子)易位进入细胞核以启动STAT1转录。
