Modeling intestinal dysfunction in HIV infection with organoid technology

摘要 肠上皮屏障功能障碍是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。这项提议的中心假设是 HIV感染削弱了肠道干细胞的再生能力，导致了持久的屏障 功能障碍。这一假设是基于文献中现有的数据提出的，这些数据表明，在 炎症性肠病、肠道干细胞库和肠道细胞类型的组成平衡 我们自己发表的结果表明，在一组 HIV患者全身炎症程度高，临床转归差。肠道干细胞的功能可以 通过将它们扩展成自组织的三维结构来进行体外测试，这种结构被称为“有机类化合物”。他们可以 进一步分化为所有类型的肠道上皮细胞。这一提议利用了对肠干的访问 由艾滋病毒相关肠道炎症专家Ma Somourk博士通过肠道活组织检查获得的细胞，以及专业知识 在器官生长和基础艾滋病毒病毒学方面，Melanie Ott博士的实验室正在检查肠道干细胞 HIV感染中的功能和有机物生长。中心假设将在两个具体目标中得到检验：1) 确定艾滋病毒感染如何影响T细胞中的肠道干细胞功能：有机共培养实验。这个 工作假说是肠道干细胞和感染艾滋病毒的T细胞之间的早期接触导致干细胞 损伤、肠上皮再生受损和持久的屏障功能障碍。这一假设将得到检验。 T细胞：OTT实验室建立的有机共培养模型。我们将确定感染HIV的T细胞的影响 细胞在单细胞水平上对类器官的生长、分化、屏障功能和转录有影响。侯选人 如肿瘤坏死因子-α或从我们的研究中出现的因子将直接添加到有机培养基中 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 干细胞的功能。我们的工作假设是艾滋病毒患者的肠道干细胞功能发生了变化， 尤其是起病晚、免疫功能恢复差的患者。这一假设将通过以下方式检验 不同发病时间HIV感染者体内有机化合物生长分化的比较 治疗或未感染个体采用形态、功能和单细胞RNA测序分析。 这项提案将提供对胃肠道内影响 艾滋病毒携带者的感染、持久性和共病，因此与本RFA非常一致。它是 创新是因为它将重点转移到肠道干细胞及其更新肠上皮细胞的能力上 适当的屏障函数。它还使用创新的有机化合物技术结合临床研究，旨在 解释为什么在抗逆转录病毒治疗的情况下，患者仍然存在屏障缺陷。因此，基础生物学的重要进展 以及针对艾滋病毒相关的慢性免疫激活的新的治疗方法是可望的。

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.