Project 1

项目1

• 批准号: 10425157
• 负责人: Anuradha Ray
• 金额: $ 53.92万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10425157
• 关键词: Address; Adrenal Cortex Hormones; Aftercare; Algorithms; Aspergillus; Asthma; Bioinformatics; Biological; Biological Markers; Biological Products; Bronchoalveolar Lavage; CD8-Positive T-Lymphocytes; CD8B1 gene; CXCL10 gene; Cell Aging; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Chemotactic Factors; Clinic; Complex; Cytometry; Data; Dinucleoside Phosphates; Disease; Enrollment; Epithelial; Epithelial Cells; Event; Experimental Models; Flow Cytometry; Funding; Gene Expression; Gene Expression Profile; Genes; Glucocorticoid Receptor; Goals; Grant; Hematopoiesis; Heterogeneity; Human; IL7 gene; Immune; Immune System Diseases; Immune response; Immunologics; In Situ; Innate Immune Response; Interferon Type II; Interleukin-10; Interleukin-4; Interleukin-5; Lymphocyte; Memory; Methods; Modeling; Molecular; Molecular Abnormality; Mus; Outcome Measure; Pathogenesis; Pathway interactions; Patients; Peptide Hydrolases; Periodicity; Phenotype; Population; Production; Refractory; Regulation; Reporting; Research; Resolution; Role; STAT1 gene; Signal Transduction; Steroids; T cell receptor repertoire sequencing; T-Lymphocyte; Time; Tissues; Work; adaptive immune response; airway epithelium; airway hyperresponsiveness; airway inflammation; asthmatic airway; asthmatic patient; cell type; cohort; cytokine; disease phenotype; high dimensionality; insight; mouse model; mucosal site; novel; operation; profiles in patients; programs; response; symptomatic improvement; targeted treatment; therapeutic development; transcriptome; transcriptome sequencing; transcriptomics; treatment response

Despite many advancements in the treatment of severe asthma (SA) with the advent of biologics, there still remain challenges because of incomplete understanding of the underlying immune and molecular aberrations. Towards this end, we analyzed the bronchoalveolar lavage (BAL) cells of patients enrolled in the severe asthma research program (SARP) and also of those in the P01-funded IMSA (Immune Mechanisms in Severe Asthma) program. In addition, we studied the transcriptome of these cells using bulk RNA-seq methods. Initial studies of the SARP cohort revealed that the BAL cells in 35-40% of SA patients harbor a high Type 1 (T1)/IFN-g immune signature and implicated IFN-g-activated STAT1 in steroid insensitivity due to co-operation with the glucocorticoid receptor (GR). Subjecting the BAL cells of 41 subjects in the IMSA cohort to mass cytometry/CyTOF and downstream bioinformatic analysis allowed clustering of the SA patients into two groups (PGs), PG2 and PG3, displaying distinct immune profiles. The PG2 group showed a heightened innate immune response dominated by FceRI+IL-4+ cells while PG3 was a lymphocyte-dominated group characterized by increased numbers of IFN-g+ CD4+ and CD8+ T cells, which included tissue-resident memory (TRM) cells. The immune cells were associated with gene modules by deconvolution of their transcriptomic data using our novel algorithm, ICLite. Module-associated gene expression suggests importance of FceRI- and IL-7-signaling in cytokine production by innate cells in PG2 and of co-stimulatory molecules in positive regulation of IFN-g production from TRMs in PG3. In the clinic, response to treatment of SA patients with the biologic, dupilumab, also highlights heterogeneity despite similar biomarker profiles of the treated patients. Thus, given the novel insight gained about distinct immune phenotypes of SA patients with specific transcriptomic signatures, we now have the opportunity to determine in fine resolution, immunologically and molecularly, how dupilumab differentially impacts the local immune (Proj. 1) and epithelial (Proj. 2) phenotypes in patients who show similar biomarker profiles and yet do not respond similarly to treatment. Collectively, these data allow us to hypothesize that two distinct immune mechanisms, one regulated by FceRI+ innate immune cells in conjunction with airway epithelial cells, and the second by T cells, with their impact on epithelial cells, collectively determines SA. To address this hypothesis, the specific aims of Project 1 are to: Aim 1. Characterize airway immune cells in high-resolution by CITE-seq and TCR-seq and determine impact of dupilumab on immune phenotype. Aim 2. Determine mechanisms that induce innate immune cell-driven SA phenotype using a novel fungal protease-driven model. Aim 3. Determine mechanisms that regulate a T1high adaptive immune response using an established HDM+cyclic dinucleotide-driven model. Collectively, the findings will qualify heterogeneity in immune response and response to therapy at the single cell level and establish key molecules/pathways that drive distinct immune phenotypes using novel experimental models.

尽管随着生物制剂的出现，严重哮喘（SA）的治疗取得了许多进展，但仍然存在严重的哮喘。 由于对潜在的免疫和分子畸变的不完全理解， 为此，我们分析了入选重度哮喘患者的支气管肺泡灌洗（BAL）细胞， 哮喘研究计划（SARP）和P01资助的IMSA（严重哮喘患者的免疫机制） 哮喘）程序。此外，我们使用批量RNA-seq方法研究了这些细胞的转录组。初始 SARP队列的研究显示，35-40%的SA患者的BAL细胞具有高的1型 (T1)/IFN-g免疫特征和由于合作导致的类固醇不敏感性中的IFN-g激活的STAT 1 糖皮质激素受体（GR）。对IMSA队列中41名受试者的BAL细胞进行质量分析， 流式细胞术/CyTOF和下游生物信息学分析允许将SA患者聚类为两组 (PGs)、PG 2和PG 3，显示出不同的免疫谱。PG 2组表现出更高的先天性 免疫应答以FceRI+IL-4+细胞为主，而PG 3是淋巴细胞为主的组 其特征是IFN-γ + CD 4+和CD 8 + T细胞数量增加，包括组织驻留记忆 (TRM)细胞免疫细胞通过其转录组的去卷积与基因模块相关联， 数据使用我们的新算法，ICLite。模块相关的基因表达表明FceRI-和 IL-7-信号在PG 2先天细胞产生细胞因子和阳性细胞共刺激分子中的作用 调节PG 3中TRM产生IFN-g。在临床上，SA患者对 生物制剂dupilumab也突出了异质性，尽管治疗患者的生物标志物特征相似。 因此，鉴于获得了关于SA患者的特异性免疫表型的新见解， 转录组签名，我们现在有机会确定在精细分辨率，免疫学和 分子上，dupilumab如何差异性地影响局部免疫（Proj. 1)和上皮（Proj. 2)表型 在显示类似生物标志物特征但对治疗没有类似反应的患者中。总的来说， 这些数据使我们能够假设两种不同的免疫机制，一种是由FceRI+先天免疫调节的， 免疫细胞与气道上皮细胞结合，第二种是T细胞，它们对 上皮细胞，共同决定SA。为了解决这一假设，项目1的具体目标是： 目标1。通过CITE-seq和TCR-seq以高分辨率表征气道免疫细胞并确定其影响 dupilumab对免疫表型的影响。目标2.确定诱导先天免疫细胞驱动的SA的机制 表型使用一种新的真菌蛋白酶驱动的模型。目标3.确定调节T1高的机制 使用已建立的HDM+环二核苷酸驱动模型的适应性免疫应答。统称 这些发现将在单细胞水平上鉴定免疫应答和治疗应答的异质性， 使用新的实验模型建立驱动不同免疫表型的关键分子/途径。