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.)

根据该议定书取得的进展概述如下： 1. 通过该方案获得的结构细胞群体已被用于表征肺细胞群体中ARTS-1的表达。 通过支气管刷拭获得的支气管上皮细胞已被用于证明共- 纤毛支气管上皮细胞顶端细胞膜中膜相关ARTS-1和TNFR 1的定位。 2. 通过该方案获得的支气管肺泡灌洗液已被用于鉴定外泌体样囊泡释放作为一种新的机制，通过该机制可以产生可溶性细胞因子受体，而不依赖于受体脱落酶的胞外域切割。 支气管肺泡灌洗液中的大多数可溶性I型TNF受体（TNFR 1）被发现是全长，55 kDa，外泌体相关的TNFR 1，而28 kDa裂解的TNFR 1胞外域仅代表一小部分。 因此，释放 外泌体相关的TNFR 1（即，eTNFR 1）代表了可溶性TNF受体释放的重要替代机制。 3. 来自哮喘患者的BAL液已被用于研究人中性粒细胞肽-1的ADP-核糖基转移酶特异性修饰的研究。 4. 首次在循环CD 11 c +/CD 14-/HLA-DR+树突状细胞上证实了VLDL受体的表达。 这证实了我们在小鼠模型中的结果，即VLDL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应答来减弱屋尘螨诱导的气道炎症。 5. BAL液细胞已被用于表明，与正常受试者相比，哮喘患者肺泡巨噬细胞上的CD 163表达减少。 6. 诱导痰细胞已被用于开发纳米级微流控流式细胞仪的组件，该组件将用于哮喘患者的即时炎症表型分析的痰样本。 7. 嗜酸性粒细胞高哮喘患者的骨髓树突状细胞亚群LRP-1表达水平低于健康非哮喘受试者，并且哮喘患者骨髓树突状细胞亚群LRP-1表达与外周血嗜酸性粒细胞计数之间存在负相关。 8. 高密度脂蛋白颗粒呈负相关，而血清甘油三酯呈正相关，与血液嗜酸性粒细胞在特应性哮喘。这支持了血清HDL和甘油三酯水平可能与特应性哮喘中的全身性2型炎症相关的概念。 （J Lipid Research 2017年8月; 58（8）：1713-1721。 PMID：28655726。） 9. 嗜酸性粒细胞性哮喘患者外周血髓系DC亚群LRP-1表达低于健康非哮喘受试者。 (JACI 2018年10月; 142（4）：1066-1079.e6。DOI：10.1016/j.jaci.2017.10.044. PMID：29274414。） 10. 来自哮喘患者的外周血单核细胞用于显示长期禁食减弱了类固醇初治哮喘患者中的NLRP 3炎性体和Th 2细胞活化，以及减少了气道上皮细胞细胞因子的产生。这确定了哮喘中营养水平依赖性炎症调节的潜在作用。（J Immunol 2018 Sep 1; 201（5）：1382-1388。 PMID 30021766）。 11. 来自哮喘患者的支气管肺泡灌洗巨噬细胞用于显示APOE可以作为内源性的浓度依赖性肺危险信号起作用，其引发并激活NLPR 3炎性体以分泌IL-1。这可能代表了当肺中的浓度增加至高于正常水平时APOE放大肺部炎症反应的机制，这可能发生在以嗜肺性气道炎症为特征的HDM诱导的哮喘的病毒加重期间。（J Allergy Clinical Immunology 2019; 144（2）：426-441.e3。 DOI：https：//doi.org/10.1016/jaci.2019.02.027）。 12. 对经APOE刺激的人哮喘支气管刷拭细胞进行RNA测序，发现编码多种促炎基因的mRNA转录本表达增加，包括CXCL 5（C-X-C基序趋化因子配体5），一种促进中性粒细胞活化和趋化性的上皮衍生趋化因子。我们随后表征了诱导人哮喘小气道上皮细胞（SAEC）分泌CXCL 5的APOE信号通路。 我们发现气道中APOE水平升高可能激活TLR 4/TAK 1/IK/NF-B/TPL 2/JNK信号通路，从而诱导人哮喘SAEC分泌CXCL 5。这些发现确定了TLR 4和TPL 2在哮喘APOE介导的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.