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Abstract：
UNASSIGNED: The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1).
UNASSIGNED: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay.
UNASSIGNED: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages.
UNASSIGNED: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.
UNASSIGNED: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay.
UNASSIGNED: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages.
UNASSIGNED: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.
摘要：
■生物钟的破坏与炎症和免疫疾病有关。BMAL2，一种关键的昼夜节律蛋白，与时钟形成二聚体，激活转录。细胞外冷诱导RNA结合蛋白（eCIRP），在脓毒症期间释放，能诱导巨噬细胞内毒素耐受。我们假设eCIRP通过触发在骨髓细胞上表达的受体-1（TREM-1）诱导BMAL2表达并促进巨噬细胞内毒素耐受。
■C57BL/6野生型(WT)雄性小鼠通过盲肠结扎穿孔(CLP)进行脓毒症。通过ELISA评估CLP后20小时的eCIRP血清水平。用各种剂量的重组小鼠(rm)CIRP(eCIRP)处理腹膜巨噬细胞(PerM)24小时。然后用LPS刺激细胞5小时。通过ELISA评估培养上清液中TNF-α和IL-6的水平。PerM用eCIRP处理24小时，并通过qPCR评估PD-L1，IL-10，STAT3，TREM-1和昼夜节律基因如BMAL2，CRY1和PER2的表达。使用来自TREM-1-/-小鼠的PerM通过qPCR测定TREM-1对eCIRP诱导的PerM内毒素耐受性和PD-L1、IL-10和STAT3表达的影响。通过PCR阵列确定eCIRP处理的巨噬细胞中的昼夜节律基因表达谱,并通过qPCR确认。通过转染BMAL2CRISPR活化质粒在骨髓来源的巨噬细胞中进行BMAL2活化的诱导。通过计算建模确定BMAL2在PD-L1启动子中的相互作用，并通过BIAcore测定进行确认。
■与假手术小鼠相比,在败血症小鼠中eCIRP的血清水平增加。用eCIRP预处理的巨噬细胞在LPS攻击后显示TNFα和IL-6释放减少，表明巨噬细胞内毒素耐受。此外,eCIRP增加免疫耐受标志物PD-L1、IL-10和STAT3的表达。有趣的是,TREM-1缺乏可逆转eCIRP诱导的巨噬细胞内毒素耐受，并显著降低PD-L1、IL-10和STAT3表达。用eCIRP处理的腹膜巨噬细胞中昼夜节律基因的PCR阵列筛选显示BMAL2，CRY1和PER2的表达升高。在eCIRP处理的巨噬细胞中，TREM-1缺乏阻止了这些昼夜节律基因的上调。在巨噬细胞中，可诱导的BMAL2表达与PD-L1表达增加相关。在败血症的人类患者中，与健康受试者相比,血液单核细胞显示BMAL2和PD-L1的表达增加.计算建模和BIAcore测定确定了PD-L1启动子中BMAL2的推定结合区，提示BMAL2正调节巨噬细胞中PD-L1的表达。
■eCIRP通过TREM-1上调BMAL2的表达，导致脓毒症中巨噬细胞内毒素耐受。靶向eCIRP以维持昼夜节律可以纠正内毒素耐受性并增强宿主对细菌感染的抵抗力。
■C57BL/6野生型(WT)雄性小鼠通过盲肠结扎穿孔(CLP)进行脓毒症。通过ELISA评估CLP后20小时的eCIRP血清水平。用各种剂量的重组小鼠(rm)CIRP(eCIRP)处理腹膜巨噬细胞(PerM)24小时。然后用LPS刺激细胞5小时。通过ELISA评估培养上清液中TNF-α和IL-6的水平。PerM用eCIRP处理24小时，并通过qPCR评估PD-L1，IL-10，STAT3，TREM-1和昼夜节律基因如BMAL2，CRY1和PER2的表达。使用来自TREM-1-/-小鼠的PerM通过qPCR测定TREM-1对eCIRP诱导的PerM内毒素耐受性和PD-L1、IL-10和STAT3表达的影响。通过PCR阵列确定eCIRP处理的巨噬细胞中的昼夜节律基因表达谱,并通过qPCR确认。通过转染BMAL2CRISPR活化质粒在骨髓来源的巨噬细胞中进行BMAL2活化的诱导。通过计算建模确定BMAL2在PD-L1启动子中的相互作用，并通过BIAcore测定进行确认。
■与假手术小鼠相比,在败血症小鼠中eCIRP的血清水平增加。用eCIRP预处理的巨噬细胞在LPS攻击后显示TNFα和IL-6释放减少，表明巨噬细胞内毒素耐受。此外,eCIRP增加免疫耐受标志物PD-L1、IL-10和STAT3的表达。有趣的是,TREM-1缺乏可逆转eCIRP诱导的巨噬细胞内毒素耐受，并显著降低PD-L1、IL-10和STAT3表达。用eCIRP处理的腹膜巨噬细胞中昼夜节律基因的PCR阵列筛选显示BMAL2，CRY1和PER2的表达升高。在eCIRP处理的巨噬细胞中，TREM-1缺乏阻止了这些昼夜节律基因的上调。在巨噬细胞中，可诱导的BMAL2表达与PD-L1表达增加相关。在败血症的人类患者中，与健康受试者相比,血液单核细胞显示BMAL2和PD-L1的表达增加.计算建模和BIAcore测定确定了PD-L1启动子中BMAL2的推定结合区，提示BMAL2正调节巨噬细胞中PD-L1的表达。
■eCIRP通过TREM-1上调BMAL2的表达，导致脓毒症中巨噬细胞内毒素耐受。靶向eCIRP以维持昼夜节律可以纠正内毒素耐受性并增强宿主对细菌感染的抵抗力。
