BACKGROUND Lymphotoxin β receptor (LTβR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTβR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTβR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTβR-induced NF-κB signaling. METHODS To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-β-cyclodextrin and simvastatin were applied. LTβR localization was studied by confocal microscopy. The activity of LTβR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTβR with its protein partners were investigated by immunoprecipitation. RESULTS We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-β-cyclodextrin impaired LTβR internalization and potentiated LTβR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTβR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTβR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTβR-based therapies. Video abstract.

中文翻译：

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胆固醇可限制淋巴毒素β受体触发的NF-κB信号传导。

背景技术淋巴毒素β受体（LTβR）在免疫系统的发育和免疫应答中起重要作用。在细胞水平，结合配体的LTβR激活促炎性NF-κB途径，但调节其信号转导的详细机制仍不清楚。由于LTβR及其配体是有前途的治疗靶标，因此对它们的了解至关重要。在这里，我们研究了扰动的细胞胆固醇含量对LTβR诱导的NF-κB信号传导的影响。方法为了调节肺癌A549和H2228以及内皮HUVEC细胞中胆固醇的利用率和/或水平，应用了不同的治疗方案，分别是使用菲利普林，甲基-β-环糊精和辛伐他汀。通过共聚焦显微镜研究了LTβR的定位。通过Western印迹测量NF-κB途径抑制剂IκBα的水平和RelA转录因子的磷酸化来评估LTβR诱导的NF-κB途径的活性。分别通过qRT-PCR，ELISA和Western印迹检查NF-κB的转录反应，趋化因子的产生和粘附分子。用荧光法测量了不同类型的原代免疫细胞对上皮A549细胞和内皮HUVEC的粘附。通过免疫沉淀研究了LTβR与其蛋白伴侣的相互作用。结果我们发现，由胆固醇介导的胆固醇螯合或其从质膜中被甲基-β-环糊精清除会损害LTβR的内在化，并增强LTβR依赖性的NF-κB途径的典型分支的活化。后者表现为IκBα抑制剂的降解增强，RelA磷酸化升高，编码已知在免疫反应中起重要作用的细胞因子和粘附分子等的NF-κB靶基因表达大幅增加。紧随其后的是CXCL8的强劲分泌和ICAM1的上调，这有助于免疫细胞（NK和T细胞，嗜中性粒细胞）粘附于A549细胞和HUVEC。从机理上讲，我们表明胆固醇的消耗稳定了配体刺激的LTβR与修饰形式的TRAF2和NEMO蛋白之间的相互作用。结论我们的结果表明，降低胆固醇的质膜含量或将其螯合可强烈增强LTβR引发的信号转导。因此，调节胆固醇水平的药物可能会改善基于LTβR的疗法的疗效。录像摘要。