Pathogenesis and Treatment of HIV Infection

HIV感染的发病机制和治疗

基本信息

批准号：
9552528
负责人：
H. Clifford Lane
金额：
$ 153.29万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9552528
关键词：
Adaptor Signaling Protein Biological Assay Biological Response Modifiers Blood CD4 Positive T Lymphocytes Cell Count Cell Line Cell Nucleus Cells Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Complex Conflict (Psychology)Confocal Microscopy Cytoplasm DNA DNA Binding Domain DNA Repair Gene DNA Virus Infections DNA Viruses Data Dendritic Cells Embryo Environment Enzymes Exposure to Family Fibrin fragment D G22P1 gene Generations Genetic Transcription HIV HIV Infections HIV-1 HIV-2 Homeostasis Human Human Herpesvirus 2 Immune Immune response Immunologic Deficiency Syndromes Immunosuppression Infection Inflammation Inflammatory Response Innate Immune Response Interferon Type I Interferon-alpha Interferon-beta Interferons Interleukin-12 Interleukin-15 Interleukins Kidney Knock-out Knowledge Measures Mediating NADPH Oxidase Nucleic Acids Pathogenesis Patients Phosphorylation Plasma Play Production Proviruses RNA Reactive Oxygen Species Reporting Role Simplexvirus Superoxides System Time Transfection Viral Viral Load result Viremia Virus Diseases Western Blotting Work cohort cytokine genome editing immune activation immune system function inflammatory marker influenzavirus innovation knock-down macrophage member monocyte protein complex response sensor

项目摘要

Work over the past year in this project has largely focused on the role of non-specific mediators of the immune and inflammatory responses in the pathogenesis of HIV-1 infection. We had previously demonstrated that Interleukin (IL)-27, a member of the IL-12 cytokine family that plays an important and diverse role in the function of the immune system, is an anti-viral cytokine capable of inhibiting HIV-1, HIV-2, Influenza virus and herpes simplex virus infection. In attempting to discern the mechanism underlying this anti-viral effect we looked at the ability of IL-27 to enhance the generation of reactive oxygen species (ROS) during the differentiation of monocytes to macrophages. Real time PCR, western blot and knock down assays demonstrated that IL-27 was able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells in association with the induction and phosphorylation of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase. Prior work from our lab identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic nucleic acid sensor that induces the production of interferon-lambda (IFN-L) by human primary cells and cell lines. IFN-L is a type III IFN and has similar activity to that of the type I IFNs (IFN-alpha and IFN-beta). The type I interferons are known to be a significant component of HIV-1 associated immune activation. Given our other recent observation that incomplete proviruses may be capable of leading to transcription of RNA we sought to further study the relationships between nucleic acid sensing in the cytoplasm and induction of inflammatory responses. We observed that human embryonic kidney (HEK) 293T cells, that were deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-L in response to DNA. Conversely, parental HEK 293 cells produced IFN-1 after they were exposed to exogenous DNA; however, when STING was knocked out in these cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. The DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-L in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-1 innate immune response to exogenous DNA or DNA virus infection. Studies are underway to see if the same occurs in the presence of defective HIV-1 proviruses. IL-15 is a pleiotropic cytokine capable of inducing the activation, proliferation and differentiation of a variety of immune cells. It has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. Over the past year we measured the levels of IL-15 in a cohort of 501 well-characterized HIV-1 infected patients and correlated the levels with plasma levels of HIV-1 and other well-known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14. IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 1.53 pg/ml) compared to either uninfected controls (1.69 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001). Thus, a significant direct correlation was noted between IL-15 levels and HIV-1 viremia and an inverse correlation was notd between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.

该项目过去一年的工作主要集中在免疫和炎症反应的非特异性介质在 HIV-1 感染发病机制中的作用。我们之前已经证明，白细胞介素 (IL)-27 是 IL-12 细胞因子家族的成员，在免疫系统功能中发挥着重要而多样的作用，是一种能够抑制 HIV-1、HIV 的抗病毒细胞因子。 -2、流感病毒和单纯疱疹病毒感染。为了探究这种抗病毒作用的机制，我们研究了 IL-27 在单核细胞分化为巨噬细胞过程中增强活性氧 (ROS) 生成的能力。实时 PCR、蛋白质印迹和敲低测定表明，IL-27 不仅能够在分化过程中增强超氧化物产生的潜力，而且还能在终末分化巨噬细胞和未成熟树突细胞中增强超氧化物产生的潜力，这与 p47phox 的诱导和磷酸化有关。 ROS 产生酶 NADPH 氧化酶的胞质成分。我们实验室之前的工作确定了 Ku70（DNA 修复蛋白复合物的一个亚基）作为一种胞质核酸传感器，可诱导人类原代细胞和细胞系产生干扰素 lambda (IFN-L)。 IFN-L 是一种 III 型 IFN，具有与 I 型 IFN（IFN-α 和 IFN-β）相似的活性。已知 I 型干扰素是 HIV-1 相关免疫激活的重要组成部分。鉴于我们最近的另一项观察结果，即不完整的原病毒可能能够导致 RNA 转录，我们试图进一步研究细胞质中的核酸感应与炎症反应诱导之间的关系。我们观察到，人类胚胎肾 (HEK) 293T 细胞缺乏先天免疫接头蛋白 STING（IFN 基因刺激剂），因此不会响应 DNA 产生 IFN-L。相反，亲代 HEK 293 细胞在接触外源 DNA 后会产生 IFN-1；然而，当通过 CRISPR（成簇规则间隔短回文重复）/Cas9 基因组编辑系统在这些细胞中敲除 STING 时，它们失去了这种反应。通过共聚焦显微镜，我们证明内源性 Ku70 位于细胞核中，然后在 DNA 暴露后易位到细胞质，与 STING 形成复合物。 Ku70 的 DNA 结合域对于 Ku70-STING 复合物的形成至关重要。敲除原代人类巨噬细胞中的 STING 会抑制它们响应 DNA 转染或 DNA 病毒 HSV-2（单纯疱疹病毒-2）感染而产生 IFN-L 的能力。总之，这些数据表明 STING 介导 Ku70 介导的 IFN-1 对外源 DNA 或 DNA 病毒感染的先天免疫反应。目前正在进行研究，看看在存在缺陷的 HIV-1 原病毒的情况下是否会发生同样的情况。 IL-15是一种多效细胞因子，能够诱导多种免疫细胞的活化、增殖和分化。它被认为在 HIV-1 感染中发挥重要作用，但关于其在这些患者中的表达水平存在相互矛盾的报道。在过去的一年里，我们测量了 501 名 HIV-1 感染患者的 IL-15 水平，并将该水平与 HIV-1 血浆水平和其他众所周知的炎症标志物（包括 CRP、D-二聚体、sCD163 和 sCD14。与未感染对照（1.69±0.37pg/ml，p<0.001）或病毒载量<50拷贝/ml的患者相比，病毒载量>100,000拷贝/ml（3.02±1.53pg/ml）的患者的IL-15水平显着较高。 ml (1.59 0.40 pg/ml (p<0.001)。HIV-1 与病毒血症和 IL-15 水平之间（Spearman r = 0.54，p<0.001）以及 CD4+ T 细胞计数和 IL-15 水平之间（Spearman r = -0.56，p<0.001）因此，注意到 IL-15 之间存在显着的直接相关性。 15 水平与 HIV-1 病毒血症之间存在负相关关系，并且 IL-15 水平与 CD4+ T 细胞计数之间存在负相关性，这些数据支持了其潜在作用。 IL-15 在 HIV 相关免疫激活发病机制中的作用。