Modeling intestinal dysfunction in HIV infection with organoid technology

利用类器官技术模拟 HIV 感染的肠道功能障碍

• 批准号: 10083740
• 负责人: Melanie Maria Ott
• 金额: $ 78.98万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-10 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083740
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Acute; Address; Affect; Biology; Biology of HIV Infection; Biopsy; Blood Circulation; Cell Death; Cells; Chronic; Clinical; Clinical Research; Coculture Techniques; Colon; Culture Media; Data; Defect; Development; Differentiation and Growth; Disease; Disease Progression; Early treatment; Enterocytes; Enteroendocrine Cell; Epithelial Cells; Equilibrium; Etiology; Experimental Designs; Failure; Formulation; Foundations; Functional disorder; Funding; Gastrointestinal tract structure; Genetic Transcription; Goals; Goblet Cells; Grant; Growth; Growth and Development function; HIV; HIV Infections; Health; Human; Immunologics; Impairment; Individual; Infection; Inflammation; Inflammatory Bowel Diseases; Intervention; Intestines; Laboratories; Large Intestine; Lead; Length; Link; Lipopolysaccharides; Literature; Liver diseases; Longevity; Measures; Mediator of activation protein; Mission; Modeling; Molecular; Molecular Biology; Morphology; Natural regeneration; Observational Study; Organ; Organoids; Outcome; Paneth Cells; Patients; Plasma; Process; Public Health; Publishing; Recovery; Regenerative capacity; Research; Research Support; SIV; Small Intestines; Subgroup; T-Lymphocyte; TNF gene; Technology; Testing; Therapeutic Intervention; United States National Institutes of Health; antiretroviral therapy; cell injury; cell type; clinical infrastructure; clinical predictors; co-infection; comorbidity; epithelium regeneration; experimental study; gastrointestinal; gastrointestinal epithelium; healthspan; immune activation; improved; in vitro Model; in vitro testing; in vivo; inflammatory disease of the intestine; innovation; insight; intestinal epithelium; microbial; molecular targeted therapies; mortality; novel; novel therapeutic intervention; novel therapeutics; patient subsets; persistent symptom; single-cell RNA sequencing; stem cell biology; stem cell function; stem cell proliferation; stem cells; success; systemic inflammatory response; three dimensional structure; virology

ABSTRACT Disruption of intestinal epithelial barrier function is an important cause of HIV-associated chronic immune activation but underlying molecular mechanisms are not known. The central hypothesis of this proposal is that HIV infection impairs the regenerative capacity of intestinal stem cells, resulting in long-lasting barrier dysfunction. This hypothesis was formulated on the basis of available data in the literature showing that in inflammatory bowel diseases, the intestinal stem cell pool and the compositional balance of intestinal cell types is disrupted, and our own published results showing intestinal stem cell proliferation is impaired in a subgroup of HIV+ patients with high systemic inflammation and poor clinical outcome. Intestinal stem cell function can be tested in vitro by expanding them into self-organizing three-dimensional structures termed “organoids”. They can be further differentiated into all intestinal epithelial cell types. This proposal leverages access to intestinal stem cells through intestinal biopsies by Dr. Ma Somsouk, an expert in HIV-associated gut inflammation, and expertise in organoid growth and basic HIV virology present in Dr. Melanie Ott's laboratory to examine intestinal stem cell function and organoid growth in HIV infection. The central hypothesis will be tested in two specific aims: 1) To define how HIV infection influences intestinal stem cell function in T cell: organoid co-culture experiments. The working hypothesis is that early contact between intestinal stem cells and HIV-infected T cells leads to stem cell damage, impaired gut epithelial regeneration and long-lasting barrier dysfunction. This hypothesis will be tested in T cell: organoid co-culture models established in the Ott lab. We will determine the effect of HIV-infected T cells on organoid growth, differentiation, barrier function and transcription at the single-cell level. Candidate factors such as TNF-α or factors emerging from our studies will be added directly to organoid culture media to analyze effects on growth and differentiation. 2) To determine the effects of chronic HIV infection on intestinal stem cell function. Our working hypothesis is that intestinal stem cell function is altered in HIV+ individuals, especially those with late onset of treatment and poor immunological recovery. This hypothesis will be tested by comparing growth and differentiation of organoids grown from HIV-infected individuals with different onset of treatment or uninfected individuals using morphological, functional and single-cell RNA sequencing analysis. This proposal will provide detailed insight into fundamental processes within the gastrointestinal tract that impact infection, persistence, and comorbidities in people living with HIV and is as such well aligned with this RFA. It is innovative because it shifts the focus to intestinal stem cells and their ability to renew the gut epithelium for proper barrier functions. It also uses innovative new organoid technology combined with clinical studies aimed at explaining why barrier defects persist in patients despite ART. Thus, important advances in the basic biology and novel therapeutic approaches towards HIV- associated chronic immune activation are expected.

抽象的 肠上皮屏障功能的破坏是艾滋病毒相关慢性免疫的重要原因 激活但基本的分子机制尚不清楚。该提议的核心假设是 HIV感染会损害肠道干细胞的再生能力，导致持久屏障 功能障碍。该假设是根据文献中的可用数据提出的，表明 炎症性肠病，肠道干细胞库和肠细胞类型的复合平衡 被破坏了，我们自己发表的结果表明，肠道干细胞增殖受到了损害 HIV+全身性炎症且临床不良的患者。肠道干细胞功能可以是 通过将它们扩展到自组织的三维结构中，在体外测试。他们可以 进一步分化为所有肠上皮细胞类型。该建议利用肠干的访问权限 通过肠道活检的细胞Ma Somsouk博士，艾滋病毒相关肠道注射专家，专业知识 在梅兰妮·奥特（Melanie Ott）博士的实验室中存在的器官生长和基本HIV病毒学中，检查肠道干细胞 HIV感染中的功能和器官生长。中央假设将以两个具体的目的进行检验：1） 定义HIV感染如何影响T细胞中的肠道干细胞功能：类器官共培养实验。这 工作假设是，肠道干细胞和HIV感染的T细胞之间的早期接触导致干细胞 损害，肠道上皮再生受损和持久的屏障功能障碍。该假设将进行检验 在T细胞中：OTT实验室中建立的类器官共培养模型。我们将确定HIV感染的T的影响 细胞在单细胞水平上的器官生长，分化，屏障功能和转录。候选人 诸如TNF-α或我们研究中出现的因素等因素将直接添加到Organoid培养基中 分析对生长和分化的影响。 2）确定慢性艾滋病毒感染对肠道的影响 干细胞功能。我们的工作假设是，在HIV+个体中，肠道干细胞功能发生了改变， 特别是那些治疗迟到且免疫恢复差的患者。该假设将通过 比较从具有不同发作的HIV感染者生长的类器官的生长和分化 使用形态，功能和单细胞RNA测序分析的治疗或未感染的个体。 该提案将为胃肠道中的基本过程提供详细的见解，以影响 感染，持久性和合并症在艾滋病毒感染者中，与此RFA如此吻合。这是 创新的，因为它将重点转移到肠道干细胞及其更新肠道上皮的能力 适当的屏障功能。它还使用创新的新器官技术与临床研究相结合 解释为什么障碍在患者目的地艺术中持续存在。这是基本生物学的重要进步 预计对HIV相关的慢性免疫激活的新型治疗方法。