Mechanisms of HIV-associated epithelial intestinal stem cell (ISC) dysfunction

摘要 胃肠道(GI)是人类免疫缺陷病毒(HIV)复制的主要部位，导致 肠道炎症、CD4+T细胞耗竭和肠上皮屏障功能障碍。这些缺陷被认为是 通过允许管腔微生物产品转移到循环中来推动艾滋病毒疾病的进展，这 触发慢性系统免疫激活和疾病进展。尽管抗逆转录病毒疗法(ART) 有效地抑制血液中的病毒复制，它不能恢复肠道中的CD4+T细胞水平；即使是个体 几十年来病毒载量受到抑制，具有持续性的肠道屏障损害和持续的微生物 易位。肠上皮干细胞(ISCs)负责补充肠上皮 在正常的动态平衡周转和协调损伤后的上皮修复期间。当前的研究现状 HIV的肠道上皮功能障碍是有限的，主要依赖于基于 组织学或外周生物标记物。我们的建议通过以下方式解决知识中的关键差距：1)深入执行 HIV感染者体内ISC增殖、自我更新和分化的特征 未感染的个体；2)利用来自撒哈拉以南非洲独特的个体队列的组织样本， 艾滋病毒负担最高和最具破坏性的地方；3)应用尖端技术揭示分子 参与HIV诱导的上皮功能障碍的信号通路；以及4)彻底测试机制 假说使用相关的小鼠模型和体外有机物培养系统。凭借我们在以下领域的综合专业知识 艾滋病毒疾病、粘膜免疫学和胃肠道病理学，我们处于有利地位，可以揭示特定的机制 这是HIV感染中肠道上皮功能障碍的基础。了解这些机制是至关重要的。 促进开发新的治疗方法，以改善艾滋病毒携带者的健康结果。 在这个方案中，我们假设HIV感染导致了ISCs的过度增殖，异常的ISC 分化，增加上皮细胞的死亡，以及上皮细胞连接完整性的功能障碍。另外， 根据我们的数据，我们假设这种与HIV相关的ISC功能障碍可以通过两种机制发生： 1)肠道CD4+T细胞耗尽和促进肠道上皮细胞生长的特异性T辅助细胞因子的丧失 动态平衡和2)HIV感染者ISCs内的I型和II型干扰素信号。要解决这些问题 假设我们提出了两个互补的目标。Aim 1将使用最先进的技术来 直接在体外在肠夹层活检组织中表征ISC的功能 我们在南非德班的阿尔伯特·卢图利中心医院建立了队列。在AIM 2中将使用活体小鼠 建立CD4+T细胞耗竭模型，研究其对ISC增殖、自我更新和分化的影响。 并利用体外人体器官模型研究I型和II型干扰素途径对ISC功能的影响。 这项建议将解决ISC生物学的重要未知机制，这些机制可能有助于 逆转艾滋病毒感染中肠道屏障缺陷的新策略。

SUMMARY The gastrointestinal (GI) tract is a major site of Human Immunodeficiency Virus (HIV) replication, which leads to intestinal inflammation, CD4+ T cell depletion and gut epithelial barrier dysfunction. These defects are thought to drive HIV disease progression by allowing translocation of luminal microbial products into the circulation, which triggers chronic systemic immune activation and disease progression. Although antiretroviral therapy (ART) effectively suppress viral replication in the blood, it does not restore CD4+ T cell levels in the gut; even individuals with suppressed viral loads for decades have persistent gut barrier impairment and continued microbial translocation. Epithelial intestinal stem cells (ISCs) are responsible for replenishing the intestinal epithelium during normal homeostatic turnover and orchestrating epithelial repair following injury. Current studies of intestinal epithelial dysfunction in HIV are limited and have relied primarily on correlative observations based on histology or peripheral biomarkers. Our proposal addresses critical gaps in knowledge by 1) performing in-depth characterization of ISC proliferation, self-renewal and differentiation ex vivo and in vivo in HIV-infected and uninfected individuals; 2) utilizing tissue samples from a unique cohort of individuals in sub-Saharan Africa, where HIV burden is highest and most devastating; 3) applying cutting-edge techniques to uncover molecular signalling pathways involved in HIV-induced epithelial dysfunction; and 4) thoroughly testing mechanistic hypotheses using relevant mouse models and ex vivo organoid culture systems. With our combined expertise in HIV disease, mucosal immunology and GI pathology, we are well positioned to uncover specific mechanisms that underlie intestinal epithelial dysfunction in HIV infection. An understanding of these mechanisms is critical to facilitate development of novel therapeutic approaches to improve health outcomes of people living with HIV. In this proposal, we hypothesize that HIV infection results in hyperproliferation of ISCs, aberrant ISC differentiation, increase epithelial death, and dysfunction of epithelial cellular junction integrity. Additionally, based upon our data, we hypothesize that this HIV-associated ISC dysfunction can occur via two mechanisms: 1) depletion of intestinal CD4+ T cells and loss of specific T helper cytokines that promote intestinal epithelial homeostasis and 2) type I and II interferon signalling within ISCs in HIV-infected individuals. To address these hypotheses we are proposing two complementary aims. Aim 1 will use state-of-the-art technologies to characterise ISC function directly ex vivo in gut pinch biopsies sampled throughout the intestinal mucosa from our established cohort at Albert Luthuli Central Hospital in Durban, South Africa. In Aim 2 will use in vivo mouse models of CD4+ T cell depletion and examine the impact on ISC proliferation, self-renewal and differentiation, and use ex vivo human organoid models to study the impact of type I and II interferon pathways on ISC function. This proposal will address important unknown mechanisms of ISC biology that may help in the development of new strategies to reverse gut barrier defects in HIV infection.