Asthma Sample Collection Protocol: Defining the Role of Apolipoprotein Pathways in Asthma

哮喘样本采集方案：定义载脂蛋白通路在哮喘中的作用

• 批准号: 10929167
• 负责人: Stewart Levine
• 金额: $ 187.8万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10929167
• 关键词: ADP Ribose Transferases; Airway Disease; Alveolar Macrophages; Apical; Apolipoproteins; Asthma; Attenuated; Blood specimen; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Bronchoscopy; Brush Cell; CD14 gene; Cell membrane; Cells; Chemotaxis; Ciliated Bronchial Epithelial Cell; Clinical Immunology; Collection; Cytokine Receptors; Dendritic Cells; Epithelial Cells; Epithelium; Extrinsic asthma; Fasting; Genes; HLA-DR Antigens; High Density Lipoproteins; Human; Hypersensitivity; ITGAX gene; Immune; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1; Length; Ligands; Link; Lipids; Liquid substance; Lung; MAP3K7 gene; MAP3K8 gene; MAPK8 gene; Macrophage; Mediating; Membrane; Microfluidics; Minor; Modification; Myelogenous; Neutrophil Activation; Nutrient; Pathway interactions; Peripheral Blood Mononuclear Cell; Phenotype; Population; Production; Protocols documentation; Pyroglyphidae; RANTES; Regulation; Research; Role; Sampling; Serum; Severity of illness; Signal Pathway; Signal Transduction; Sputum; Steroids; TLR4 gene; Th2 Cells; Time; Tissue Procurements; Transcript; Triglycerides; Tumor Necrosis Factor Receptor; VLDL receptor; Viral; adaptive immune response; airway epithelium; airway inflammation; asthmatic; asthmatic patient; bronchial epithelium; chemokine; cytokine; eosinophil; eosinophilic asthma; exosome; human RNA sequencing; human neutrophil peptide 1; mRNA Expression; mouse model; nanoscale; neutrophil; particle; patient subsets; peripheral blood; point of care; receptor; sample collection; soluble TNF receptor type I; vesicular release; volunteer

The progress achieved under this protocol is summarized as follows: 1. The structural cell populations obtained by this protocol have been utilized to characterize the expression of ARTS-1 in lung cell populations. Bronchial epithelial cells that have been obtained by bronchial brushings have been utilized to demonstrate co- localization of membrane-associated ARTS-1 and TNFR1 in the apical cell membrane of ciliated bronchial epithelial cells. 2. Bronchoalveolar lavage fluid obtained by this protocol has been utilized to identify exosome-like vesicle release as a new mechanism by which soluble cytokine receptors can be generated, independent of ectodomain cleavage by receptor sheddases. The majority of soluble type I TNF receptor (TNFR1) in bronchoalveolar lavage fluid was found to be full-length, 55-kDa, exosome-associated TNFR1, whereas the 28-kDa cleaved TNFR1 ectodomain represented only a minor fraction. Thus, release of exosome-associated TNFR1 (i.e., eTNFR1) represents an important alternative mechanism for the release of soluble TNF receptors. 3. BAL fluid from asthmatic patients has been utilized in studies investigating the ADP-ribosyltransferase-specific modification of human neutrophil peptide-1. 4. Expression of the VLDL receptor was demonstrated for the first time on circulating CD11c+/CD14-/HLA-DR+ dendritic cells. This confirmed our result in a murine model that the VLDL receptor is expressed by dendritic cells and attenuates house dust mite-induced airway inflammation by suppressing dendritic cell-mediated adaptive immune responses. 5. BAL fluid cells have been used to show that expression of CD163 is reduced on alveolar macrophages from asthmatics as compared to normal subjects. 6. Induced sputum cells have been used to develop components of a nanoscale microfluidic flow cytometer that will liquefy sputum samples for point-of-care inflammatory phenotyping of asthmatic patients. 7. Myeloid dendritic cell subsets from patients with eosinophil-high asthma were shown to have lower levels of LRP-1 expression than those from healthy nonasthmatic subjects and that a negative correlation exists between LRP-1 expression by myeloid dendritic cell subsets and peripheral blood eosinophil counts in asthmatic patients. 8. HDL particles were shown to be negatively correlated, whereas serum triglycerides were shown to be positively correlated, with blood eosinophils in atopic asthmatics. This supports the concept that serum levels of HDL and triglycerides may be linked to systemic type 2 inflammation in atopic asthma. (J Lipid Research 2017 Aug; 58(8): 1713-1721. PMID: 28655726.) 9. Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. (JACI 2018 Oct; 142(4):1066-1079.e6. DOI: 10.1016/j.jaci.2017.10.044. PMID: 29274414.) 10. Peripheral blood mononuclear cells from asthmatics were used to show that prolonged fasting blunts the NLRP3 inflammasome and Th2 cell activation in steroid-naive asthmatics as well as diminishes airway epithelial cell cytokine production. This identifies a potential role for nutrient level-dependent regulation of inflammation in asthma. (J Immunol 2018 Sep 1; 201(5):1382-1388. PMID 30021766). 11. Bronchoalveolar lavage macrophages from asthmatics were used to show that APOE can function as an endogenous, concentration-dependent pulmonary danger signal that primes and activates the NLPR3 inflammasome to secrete IL-1. This might represent a mechanism through which APOE amplifies pulmonary inflammatory responses when concentrations in the lung are increased to greater than normal levels, which can occur during viral exacerbations of HDM-induced asthma characterized by neutrophilic airway inflammation. (J Allergy Clinical Immunology 2019; 144(2):426-441.e3. DOI: https://doi.org/10.1016/jaci.2019.02.027). 12. RNA sequencing of human asthmatic bronchial brushing cells stimulated with APOE identified increased expression of mRNA transcripts encoding multiple proinflammatory genes, including CXCL5 (C-X-C motif chemokine ligand 5), an epithelial-derived chemokine that promotes neutrophil activation and chemotaxis. We subsequently characterized the APOE signaling pathway that induces CXCL5 secretion by human asthmatic small airway epithelial cells (SAECs). We showed that elevated levels of APOE in the airway may activate a TLR4/TAK1/IK/NF-B/TPL2/JNK signaling pathway that induces CXCL5 secretion by human asthmatic SAECs. These findings identify new roles for TLR4 and TPL2 in APOE-mediated proinflammatory responses in asthma. (Am J Resp Cell Mol Biol 2020; 63 (2): 185 - 197.)

在该议定书下取得的进展概述如下： 1.利用该方法获得的结构细胞群已被用来研究ARTS-1在肺细胞群中的表达。通过支气管刷检获得的支气管上皮细胞已被用来证明膜相关的ARTS-1和TNFR1在纤毛支气管上皮细胞的顶端细胞膜上的共定位。 2.通过该方法获得的支气管肺泡灌洗液已被用于鉴定外体样囊泡的释放，这是一种不依赖于受体脱落酶对胞外结构域的切割而产生可溶性细胞因子受体的新机制。支气管肺泡灌洗液中的可溶I型肿瘤坏死因子受体(TNFR1)大部分是55 kDa的外切体相关的全长TNFR1，而28 kDa裂解的TNFR1胞外区只占一小部分。因此，释放外切体相关的TNFR1(即eTNFR1)是释放可溶性肿瘤坏死因子受体的重要替代机制。 3.利用哮喘患者的肺泡灌洗液研究了ADP-核糖基转移酶对人中性粒细胞多肽-1的特异性修饰。 4.首次在循环CD11c+/CD14-/HLA-DR+树突状细胞上发现VLDL受体的表达。这证实了我们在小鼠模型中的结果，即极低密度脂蛋白受体由树突状细胞表达，并通过抑制树突状细胞介导的适应性免疫反应来减轻屋尘螨诱导的呼吸道炎症。 5.BAL液细胞实验表明，哮喘患者肺泡巨噬细胞表面CD163的表达较正常人明显降低。 6.诱导痰细胞已被用于开发纳米级微流控流式细胞仪的部件，该仪器将液化痰样本，用于哮喘患者的护理点炎性表型。 7.哮喘患者外周血嗜酸粒细胞计数与髓系树突状细胞亚群表达LRP-1呈负相关。 8.特应性哮喘患者血中高密度脂蛋白颗粒与嗜酸性粒细胞呈负相关，血清甘油三酯与嗜酸性粒细胞呈正相关。这支持了血清高密度脂蛋白和甘油三酯水平可能与特应性哮喘的系统性2型炎症有关的概念。(J Lipid Research 2017年8月；58(8)：1713-1721。PMID：28655726。) 9.嗜酸粒细胞哮喘患者外周血髓系DC亚群的LRP-1表达低于健康非哮喘患者。(JACI 2018年10月；142(4)：1066-1079.e6.DOI：10.1016/j.Jaci.2017.10.044.PMID：29274414。) 10.哮喘患者外周血单个核细胞的检测结果表明，长期禁食可抑制激素治疗后哮喘患者NLRP3炎性小体和Th2细胞的活化，减少呼吸道上皮细胞细胞因子的产生。这确定了哮喘中依赖营养水平的炎症调节的潜在作用。(《免疫杂志》2018年9月1日；201(5)：1382-1388。PMID 30021766)。 11.哮喘患者肺泡灌洗巨噬细胞实验表明，APOE可作为内源性、浓度依赖的肺危险信号，启动和激活NLPR3炎性小体分泌IL-1。这可能代表了一种机制，通过这种机制，当肺中的浓度增加到高于正常水平时，APOE会放大肺部炎症反应，这可能发生在以中性粒细胞炎症为特征的HDM诱导的哮喘的病毒加重期间。(《过敏临床免疫学》2019；144(2)：426-441.e3.DOI：https://doi.org/10.1016/jaci.2019.02.027). 12.经APOE刺激的人哮喘支气管刷毛细胞的RNA测序表明，编码多种致炎基因的mRNA转录增加，包括CXCL5(C-X-C基序趋化因子配体5)，这是一种促进中性粒细胞激活和趋化的上皮源性趋化因子。随后，我们鉴定了诱导人哮喘小气道上皮细胞(SAECs)分泌CXCL5的APOE信号通路。我们发现，高水平的APOE可能激活TLR4/TAK1/IK/NF-B/Tpl2/JNK信号通路，诱导人哮喘SAEC分泌CXCL5。这些发现确定了TLR4和Tpl2在载脂蛋白E介导的哮喘促炎反应中的新作用。(Am J RESP Cell Mol Biol 2020；63(2)：185-197。)

项目成果

Apolipoprotein E is a concentration-dependent pulmonary danger signal that activates the NLRP3 inflammasome and IL-1β secretion by bronchoalveolar fluid macrophages from asthmatic subjects.

• DOI: 10.1016/j.jaci.2019.02.027
• 发表时间: 2019-08
• 期刊: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
• 影响因子: 14.2
• 作者: Gordon, Elizabeth M.;Yao, Xianglan;Xu, Haitao;Karkowsky, William;Kaler, Maryann;Kalchiem-Dekel, Or;Barochia, Amisha V.;Gao, Meixia;Keeran, Karen J.;Jeffries, Kenneth R.;Levine, Stewart J.
• 通讯作者: Levine, Stewart J.