Up-Regulation of IRAK-M is Essential for Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccha ride in Kupffer Cells
Up-Regulation of IRAK-M is Essential for Endotoxin Tolerance Induced by a Low Dose of Lipopolysaccha ride in Kupffer Cells
复制标题
IRAK-M 的上调对于库普弗细胞中低剂量脂多糖诱导的内毒素耐受至关重要。
DOI：
10.1016/j.jss.2007.12.759
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发表时间：
2008-11-01
影响因子：
2.2
通讯作者：
Gong, Jian-Ping
中科院分区：
文献类型：
--
作者：
Liu, Zuo-Jin;Yan, Lu-Nan;Gong, Jian-Ping
背景。内毒素耐受（ET）是维持库普弗细胞（KCs）稳态的重要机制，因为KCs持续暴露于各种病原体相关的分子模式，包括脂多糖（LPS）。ET涉及细胞信号转导通路的多种变化；然而，并不是所有的信号通路都下调，一些蛋白质是上调的。后一种蛋白可能是反调控的，包括白细胞介素-1受体相关激酶M （ERAK-M）的表达。本研究旨在阐明HUK-M是否通过抑制核因子κ B （nf - κ B）介导的途径参与KCs ET的机制。材料和方法。将雄性C57BL/6J小鼠分离的KCs以1 × 10(6)个细胞/孔的速度接种于24孔板中，转染前培养过夜，随机分为两组：IRAK-M短发夹RNA （shRNA）组（转染IRAK-M shRNA）和对照组（转染对照载体）；转染24 h后，再将两组随机分为无内毒素预处理组（在添加10%胎牛血清的Dulbecco's modified Eagle's培养基[Invitrogen， Carlsbad， CA]中孵育）和内毒素预处理组（在含有10 ng/mL LPS的相同培养基中孵育），分别命名为pERAK-M-EP、pIRAR-M-NEP、pCV-EP和pCV-NEP。每个子组包含6口井；24 h后，在每个亚组中加入含有LPS （100 ng/mL）的新鲜培养基，再孵育3 h。Western blot检测IRAK-M基因的表达和蛋白水平，电泳迁移位移法和酶联免疫吸附法检测NF-kappa B的活性，酶联免疫吸附法检测上清中肿瘤坏死因子α的水平。pIRAK-M-shRNA重组质粒转染KCs后可特异性抑制IRAK-M的表达。在加入100 ng/mL LPS后3 h， pCV-EP中IRAK-M的表达明显高于pCV-NEP；而pIRAK-M-NEP组与pIRAK-M-EP组间差异无统计学意义，pCV-EP组NF-kappa B活性和肿瘤坏死因子α水平最低，其余三组均显著升高（P < 0.01）。虽然第一次低剂量的LPS刺激明显减弱了KCs对第二次LPS刺激的反应，但pIRAK-M-EP的抑制作用比pCV-EP的抑制作用部分折射，表明IRAK-M的缺失导致炎症效应异常增强。IRAK-M通过抑制NF-kappa B介导的通路负性调节toll样受体信号，参与KCs ET的机制；因此，它可能是这一重要控制系统的关键组成部分，也是临床治疗败血症的新靶点。(c) 2008爱思唯尔公司版权所有。
Background. Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptor-associated kinase M (ERAK-M) expression. The aim of this study is to clarify weather or not HUK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-kappa B) mediated pathway.Materials and methods. KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 x 10(6) cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-M-short hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco's modified Eagle's medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pERAK-M-EP, pIRAR-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined Western blot, the activities of NF-kappa B were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzyme-linked immunosorbent assay.Results. The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-kappa B activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01).Conclusions. Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-kappa B mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis. (c) 2008 Elsevier Inc. All rights reserved.