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

总结 胃肠道（GI）是人类免疫缺陷病毒（HIV）复制的主要部位，这导致 肠道炎症、CD 4 + T细胞耗竭和肠上皮屏障功能障碍。这些缺陷被认为 通过允许管腔微生物产物易位到循环中来驱动HIV疾病进展， 引发慢性全身免疫激活和疾病进展。虽然抗逆转录病毒疗法（ART） 有效抑制血液中的病毒复制，但不会恢复肠道中的CD 4 + T细胞水平;即使是个人 几十年来抑制病毒载量的患者具有持续的肠道屏障损伤和持续的微生物感染， 易位肠上皮干细胞（ISCs）负责补充肠上皮细胞 在正常的稳态转换和损伤后的上皮修复过程中。目前的研究 HIV中肠上皮功能障碍是有限的，主要依赖于基于以下的相关观察： 组织学或外周生物标志物。我们的建议通过以下方式解决知识方面的关键差距：1）深入开展 在HIV感染的小鼠中离体和体内鉴定ISC增殖、自我更新和分化， 未感染的个体; 2）利用来自撒哈拉以南非洲的一组独特个体的组织样品， 在艾滋病毒负担最高和最具破坏性的地方; 3）应用尖端技术， 参与HIV诱导的上皮功能障碍的信号通路;和4）彻底测试机制 使用相关小鼠模型和离体类器官培养系统的假设。凭借我们在以下方面的综合专长： 艾滋病，粘膜免疫学和胃肠道病理学，我们处于有利地位，以揭示特定的机制 这是HIV感染中肠上皮功能障碍的基础。了解这些机制至关重要 促进开发新的治疗方法，以改善艾滋病毒感染者的健康状况。 在这个建议中，我们假设HIV感染导致ISCs过度增殖，异常的ISCs 分化，增加上皮死亡和上皮细胞连接完整性的功能障碍。此外，本发明还 基于我们的数据，我们假设这种HIV相关的ISC功能障碍可以通过两种机制发生： 1)肠道CD 4 + T细胞的耗竭和促进肠上皮细胞增殖的特异性T辅助细胞因子的丧失 稳态和2）HIV感染个体中ISC内的I型和II型干扰素信号传导。解决这些 假设我们提出两个互补的目标。Aim 1将使用最先进的技术， 在从结肠癌患者的整个肠粘膜取样的肠夹活检中， 我们在南非德班的Albert Luthuli中心医院建立的队列。目标2将使用体内小鼠 CD 4 + T细胞耗竭模型，并检查对ISC增殖、自我更新和分化的影响， 并使用离体人类器官模型来研究I型和II型干扰素途径对ISC功能的影响。 这项提案将解决重要的未知机制的ISC生物学，可能有助于发展， 逆转HIV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.