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

基本信息

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
项目类别：
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

项目摘要

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破坏是HIV相关慢性免疫的重要原因激活但潜在的分子机制尚不清楚该提议的中心假设是： HIV感染会损害肠道干细胞的再生能力，导致持久的屏障该假设是根据文献中的现有数据提出的，表明在炎症性肠病、肠道干细胞池和肠道细胞类型的组成平衡被破坏，我们自己发表的结果显示肠道干细胞增殖在一个亚组中受到损害全身炎症严重且临床结果不佳的 HIV+ 患者可以进行肠道干细胞功能治疗。通过将它们扩展成称为“类器官”的自组织三维结构来进行体外测试。该提案利用肠干细胞进一步分化为所有肠上皮细胞类型。 HIV 相关肠道炎症专家 Ma Somsouk 博士通过肠道活检对细胞进行了检测 Melanie Ott 博士实验室的类器官生长和基本 HIV 病毒学研究用于检查肠道干细胞 HIV 感染中的功能和类器官生长将在两个具体目标上进行检验：1）定义 HIV 感染如何影响 T 细胞中肠道干细胞的功能：类器官共培养实验。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肠道损伤、上皮再生受损和长期屏障功能障碍将得到检验。 T 细胞：Ott 实验室建立的类器官共培养模型，我们将确定感染 HIV 的 T 细胞的作用。细胞在单细胞水平上对类器官生长、分化、屏障功能和转录的影响。 TNF-α 等因子或我们研究中出现的因子将直接添加到类器官培养基中分析对生长和分化的影响 2) 确定慢性 HIV 感染对肠道的影响。我们的工作假设是肠道干细胞功能存在于 HIV+ 个体中，特别是那些治疗开始较晚且免疫恢复较差的患者，这一假设将得到检验。比较从不同发病时间的 HIV 感染者身上生长的类器官的生长和分化使用形态学、功能和单细胞 RNA 测序分析对治疗或未感染的个体进行分析。该提案将详细介绍影响胃肠道内的基本过程 HIV 感染者的感染、持续性和合并症，因此与 RFA 非常一致。创新是因为它将焦点转移到肠道干细胞及其更新肠道上皮的能力上它还使用创新的新型类器官技术并结合临床研究。解释为什么尽管接受了抗逆转录病毒疗法，患者体内仍然存在屏障缺陷，因此，基础生物学取得了重要进展。预计针对 HIV 相关慢性免疫激活的新治疗方法。