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Role of Cytosolic DNA-multiprotein Interactome in Allergic Airway Inflammation

胞浆 DNA-多蛋白相互作用组在过敏性气道炎症中的作用

基本信息

批准号：
10328563
负责人：
TAPAS K HAZRA
金额：
$ 78.97万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-10 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10328563
关键词：
Address Allergens Allergic Allergic Disease Allergic inflammation Allergic rhinitis Ambrosia Antibodies Attenuated Binding Biopsy CCL11 gene Cell Nucleus Cells ChIP-seq Chronic Disease Complex Curette Cyclic GMP Cytology Cytoplasm DNA DNA Repair DNA Sequence DNA glycosylase Disease Engineering Eotaxin Epithelial Extrinsic asthma Future Gelshift Analysis Gene Expression Genome Human IRF3 gene Immunoprecipitation Innate Immune Response Intranasal Administration Knowledge Lead Length Location Lung Mass Spectrum Analysis Messenger RNA Monitor Mus Names Nose Nuclear Nuclear Extract Oligonucleotides Outcome Peptides Pharmacology Pollen Proteins Pulmonary Inflammation Punch Biopsy Radiolabeled Reaction Recombinants Reporting Respiratory Mucosa Role Sampling Signaling Protein Site Stimulator of Interferon Genes Structure of mucous membrane of nose TANK-binding kinase 1 Testing Time Up-Regulation airway epithelium allergic airway inflammation cost crosslink cytokine ds-DNA extracellular human subject inhibitor novel oxidative DNA damage prevent promoter public health relevance small molecule small molecule inhibitor transcriptome sequencing

项目摘要

PROJECT SUMMARY/ABSTRACT Allergic rhinitis and asthma are highly prevalent diseases characterized by allergic airway inflammation. The airway epithelium is the first line of defense against allergens. We reported that allergenic extracts stimulate oxidative DNA damage and innate immune responses in these cells. Here we show that cytosolic double strand (ds)DNA forms a complex with several proteins in the nasal mucosa of humans with allergic rhinitis and lungs of allergic mice, but not in non-sensitized humans and mice. We named this cytoplasmic DNA- interactome “Allergosome”. The DNA-interacting proteins in the Allergosome are: interferon regulatory factor 3 (IRF3), the DNA glycosylase Nei-Like 2 (Neil2), and Cyclic GMP-AMP synthase (cGAS). Notably, these factors associate with Stimulator of Interferon Genes (STING), TANK-binding kinase-1 (TBK1), and the pro-allergic cytokine IL-33 in the Allergosome. The formation and role of the Allergosome in allergic inflammation are gaps in knowledge that we address in this proposal. The central hypothesis of this proposal is that “Allergic humans and mice develop an Allergosome in the cytoplasm of the airway mucosa and lungs that stimulates allergic airway inflammation”. In Aim 1 we will test the hypothesis that human subjects with allergic rhinitis, but not healthy control subjects, develop an Allergosome with sequestered IL-33 in nasal mucosal cells. Cytosolic and crosslinked nuclear extracts will be prepared from the nasal punch biopsies of ragweed-allergic and healthy subjects. The cytosolic extract of the nasal biopsies will be subjected to immunoprecipitation (IP) with anti-IRF3 antibody, and analyzed for associated DNA sequences by Chip-seq, and for associated proteins in the Allergosome. In the second Aim, we will test the hypothesis that sequestration of Neil2 in the Allergosome reduces its ability to protect promoter sites in the genome from binding NFκB and stimulating allergic airway inflammation. Cytosolic and nuclear extracts will be prepared as in Aim 1. The cytosolic extracts will be subjected to IP with anti-Neil2 antibody, and analyzed as in Aim 1. The nuclear extracts will be subjected to ChIP-Seq of the IP’d DNA with anti-Neil2 and anti-NFκB antibodies to determine their promoter occupancy, and gel shift analysis to detect NFκB binding. Recombinant WT and catalytically inactive Neil2 will be delivered to the airways of sensitized WT mice by engineered peptide carriers, and allergic inflammation will be reassessed. In Aim 3, we will test the hypothesis that STING and cGAS stabilize the Allergosome and stimulate allergic airway inflammation. Allergosome formation and CDE-induced allergic inflammation in WT mice will be compared to that of StingKO mice, lung epithelium-specific inducible cGasKO mice, and WT mice treated with pharmacologic cGAS inhibitors. These studies will define, for the first time, the central role of the Allergosome in allergic airway inflammation. We will provide proof of principle that targeting components of the Allergosome reduces allergic inflammation. In future studies, these strategies may develop into therapies that mitigate allergic inflammation in allergic rhinitis and asthma.

项目总结/摘要变应性鼻炎和哮喘是以变应性气道炎症为特征的高度流行的疾病。的呼吸道上皮是抵抗过敏原的第一道防线。我们报道了过敏性提取物刺激氧化DNA损伤和先天性免疫反应。这里我们展示了胞质双链（ds）DNA与患有过敏性鼻炎的人的鼻粘膜中的几种蛋白质形成复合物，肺过敏小鼠，但不是在非致敏人类和小鼠。我们把这种细胞质DNA命名为- 相互作用体“过敏体”。过敏小体中的DNA相互作用蛋白质是：干扰素调节因子3 在一些实施方案中，所述糖基化酶包括糖基化酶Nei样2（Neil 2）、糖基化酶IRF 3、DNA糖基化酶Nei样2（Neil 2）和环GMP-AMP合酶（cGAS）。值得注意的是，这些因素与干扰素基因刺激因子（STING）、TANK结合激酶-1（TBK 1）和促过敏原相关。过敏小体中的细胞因子IL-33。过敏原体在过敏性炎症中的形成和作用是空白我们在这份提案中提到的知识。这个提议的核心假设是“过敏的人小鼠的气道粘膜和肺部的细胞质中产生了过敏小体，气道炎症”。在目标1中，我们将检验以下假设：患有过敏性鼻炎但未患过敏性鼻炎的人类受试者，健康对照受试者在鼻粘膜细胞中形成具有隔离的IL-33的过敏体。细胞溶质和交联核提取物将从豚草过敏和健康的鼻穿孔活检中制备科目将鼻活检的胞质提取物与抗IRF 3进行免疫沉淀（IP）抗体，并通过Chip-seq分析相关的DNA序列，并分析抗体中的相关蛋白质。过敏体在第二个目的中，我们将测试Neil 2在过敏体中的隔离的假设。降低其保护基因组中启动子位点不与NFκB结合和刺激过敏性气道的能力炎症将按照目标1制备细胞溶质和细胞核提取物。将细胞溶质提取物用抗Neil 2抗体进行IP，并如目的1所述进行分析。核提取物将被用抗Neil 2和抗NF κB抗体对IP处理的DNA进行ChIP-Seq，以确定其启动子占有率，凝胶位移分析检测NFκB结合。重组WT和无催化活性的Neil 2将被递送致敏WT小鼠的气道，过敏性炎症将被抑制。重新评估。在目标3中，我们将检验STING和cGAS稳定过敏体和刺激过敏性气道炎症。WT中过敏小体形成和CDE诱导的过敏性炎症小鼠将与StingKO小鼠、肺上皮特异性诱导型cGasKO小鼠和WT小鼠进行比较用药理学cGAS抑制剂治疗。这些研究将首次确定过敏性气道炎症中的变态反应体。我们将提供原则证明，过敏体减少过敏性炎症。在未来的研究中，这些策略可能会发展成为治疗方法，减轻过敏性鼻炎和哮喘的过敏性炎症。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Intrapulmonary administration of purified NEIL2 abrogates NF-κB-mediated inflammation.

DOI：
10.1016/j.jbc.2021.100723
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Tapryal N;Shahabi S;Chakraborty A;Hosoki K;Wakamiya M;Sarkar G;Sharma G;Cardenas VJ;Boldogh I;Sur S;Ghosh G;Hazra TK
通讯作者：
Hazra TK

Excision release of 5?hydroxycytosine oxidatively induced DNA base lesions from the lung genome by cat dander extract challenge stimulates allergic airway inflammation.

DOI：
10.1111/cea.13284
发表时间：
2018-12
期刊：
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology
影响因子：
0
作者：
Hosoki K;Jaruga P;Itazawa T;Aguilera-Aguirre L;Coskun E;Hazra TK;Boldogh I;Dizdaroglu M;Sur S
通讯作者：
Sur S

Reply.

回复。

DOI：
10.1002/art.40923
发表时间：
2019
期刊：
Arthritis & rheumatology (Hoboken, N.J.)
影响因子：
0
作者：
Kim,AlfredHJ;Strand,Vibeke;Atkinson,JohnP
通讯作者：
Atkinson,JohnP

Nei-like DNA glycosylase 2 selectively antagonizes interferon-β expression upon respiratory syncytial virus infection.

DOI：
10.1016/j.jbc.2023.105028
发表时间：
2023-08
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Pan, Lang;Xue, Yaoyao;Wang, Ke;Zheng, Xu;Islam, Azharul;Tapryal, Nisha;Chakraborty, Anirban;Bacsi, Attila;Ba, Xueqing;Hazra, Tapas K.;Boldogh, Istvan
通讯作者：
Boldogh, Istvan

Protocols to Measure Oxidative Stress and DNA Damage in Asthma.

测量哮喘氧化应激和 DNA 损伤的方案。