Pathogenesis and Treatment of HIV Infection

HIV感染的发病机制和治疗

• 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
• 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

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.

在过去的一年中，该项目的工作主要集中在免疫和炎症反应中非特异性介体在HIV-1感染发病机理中的作用。 我们先前曾证明，介体（IL）-27是IL-12细胞因子家族的成员，在免疫系统的功能中起重要而多样化的作用，是一种能够抑制HIV-1，HIV-1，HIV-1，HIV-2，流感病毒和单纯性疱疹病毒感染的抗病毒细胞因子。 为了辨别这种抗病毒作用的机制，我们研究了IL-27在分化单核细胞为巨噬细胞的过程中增强活性氧（ROS）产生的能力。实时PCR，Western印迹和敲除测定表明，IL-27不仅能够在分化过程中，而且在末端分化的巨噬细胞和未成熟的树突状细胞中增强了超氧化物的产生潜力，并与p47phox的诱导和磷酸化相关，这是一种玫瑰糖酶的细胞增多剂。 我们实验室的先前工作确定了ku70，即DNA修复蛋白复合物的亚基，是一种胞质核酸传感器，可通过人类原代细胞和细胞系诱导干扰素 - lambda（IFN-L）的产生。 IFN-L是III型IFN，其活性与I型IFN（IFN-Alpha和IFN-BETA）的活性相似。 I型干扰素已知是HIV-1相关免疫激活的重要组成部分。 鉴于我们最近的观察结果是，不完全的病毒可能能够导致RNA转录，我们试图进一步研究细胞质中核酸传感与炎症反应诱导之间的关系。 我们观察到，在先天免疫衔接蛋白刺激（IFN基因的刺激剂）中缺乏缺乏的人类胚胎肾脏（HEK）293T细胞并未响应DNA产生IFN-L。相反，父母HEK 293细胞在暴露于外源DNA后产生IFN-1。但是，当通过CRISPR将刺激在这些细胞中（群集定期间隔短的圆柱体重复序列）/CAS9基因组编辑系统时，他们失去了这一响应。通过共聚焦显微镜，我们证明了内源性ku70位于核中，然后在DNA暴露时转移到细胞质中，形成带有刺的复合物。 KU70的DNA结合结构域对于形成Ku70-sting复合物至关重要。在原代人巨噬细胞中敲低刺激性抑制了它们在用DNA转染或用DNA病毒HSV-2感染的转染后产生IFN-L的能力（单纯疱疹病毒-2）。总之，这些数据表明STIN介导KU70介导的IFN-1先天性免疫反应对外源DNA或DNA病毒感染。 正在进行研究，以查看在HIV-1病毒缺陷的存在下是否同样发生这种情况。 IL-15是一种多效细胞因子，能够诱导多种免疫细胞的激活，增殖和分化。 据推测，它在HIV-1感染中发挥了重要作用，但有关其表达水平在这些患者中的报道矛盾。在过去的一年中，我们测量了501例特征良好的HIV-1感染患者的IL-15水平，并将其与血浆HIV-1的水平和其他众所周知的炎症标记相关，包括CRP，D-Dimer，SCD163和SCD14。 与未感染的对照组（1.69 0.37 pg/ml，p <0.001）相比，病毒载荷> 100,000拷贝/mL（3.02 1.53 pg/ml）的患者的IL-15水平明显更高（3.02 1.53 pg/ml）。 （Spearman r = 0.54，p <0.001）和CD4+ T细胞计数和IL-15水平之间的水平（Spearman r = -0.56，p <0.001）。 HIV相关的免疫激活。