Viral transmission and pathogenesis in human tissues

人体组织中的病毒传播和发病机制

• 批准号: 10007512
• 负责人: Leonid B. Margolis
• 金额: $ 204.05万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/10007512
• 关键词: Acute; Acute myocardial infarction; Antigens; Antiviral Agents; Arterial Fatty Streak; Atherosclerosis; Atypical lymphocyte; Binding; Biogenesis; Blood; Blood Platelets; CD14 Antigen; CD14 gene; CD4 Positive T Lymphocytes; Cardiovascular Diseases; Cardiovascular system; Cell Communication; Cell Cycle; Cells; Characteristics; Chronic; Collaborations; Complex; Complication; Coronary; Coronary Arteriosclerosis; Cytomegalovirus; Cytomegalovirus Infections; DNA; Data; Defect; Development; Disease; Disease Marker; Endothelium; Europe; Event; Experimental Models; Exposure to; FCGR3B gene; Failure; Flow Cytometry; Frequencies; Functional disorder; Gene Expression Profiling; Genetic Transcription; Goals; HIV; HIV Infections; HIV-1; Health; Herpesviridae; Human; Human Pathology; Hypertension; Immune; Immune System Diseases; Immune system; Impairment; In Vitro; Incubated; Individual; Infection; Interleukin-15; Laboratories; Lead; Lymphoid Tissue; Maintenance; Mediating; Mitochondria; Modeling; Myocardial Infarction; Natural regeneration; Pathogenesis; Pathology; Patients; Peroxisome Proliferator-Activated Receptors; Phenotype; Phosphatidylserines; Physiological; Plasma; Platelet Activation; Play; Polymerase Chain Reaction; Population Control; Prevalence; Process; Production; RNA; Regulatory T-Lymphocyte; Residual state; Risk; Ritonavir; Role; Rupture; Stains; Syndrome; System; T-Lymphocyte; Testing; Time; Tissues; Transforming Growth Factor beta; Unstable angina; Up-Regulation; Vesicle; Viral; Viral Pathogenesis; Virus; Virus Replication; Work; acute coronary syndrome; antiretroviral therapy; cytokine; driving force; endothelial dysfunction; experimental study; extracellular vesicles; healthy volunteer; human disease; human model; human pathogen; human tissue; immune activation; in vivo; mitochondrial fitness; monocyte; mtTF1 transcription factor; nervous system disorder; novel therapeutic intervention; particle; pathogen; prevent; progression marker; receptor; reconstitution; response; restoration; vesicular release; viral transmission; volunteer

HIV-1 tissue pathogenesis after antiretroviral therapy Immune activation is now considered to be a driving force of various human pathologies, including HIV-1 disease. Currently, a combination of antiretroviral therapy (ART) has proven to be efficient in suppressing HIV-1 replication. However, lengthy suppression of HIV-1 replication by ART is associated with an increased risk of complications, including neurological and cardiovascular diseases. These diseases seem to be related to the residual immune activation in patients undergoing ART. Cytokines may play an important role in this residual immune activation. To investigate mechanisms of his phenomenon it is necessary to develop an ex vivo laboratory-controlled system reflecting what happens in vivo. We have developed such a system. As an experimental model we used ex vivo human lymphoid tissues where critical events in HIV-1 infection occur in vivo. In the present work we evaluated 33 cytokines released by donor matched human lymphoid tissues ex vivo productively infected with HIV-1 over 16 days of infection and treated or not with the antiviral, ritonavir. We evaluated soluble and EV-associated cytokines. We found that HIV-1 infection led to upregulation of several key cytokines. EV-associated cytokines were more likely to be elevated than soluble ones. Antivirals suppressed HIV in infected tissues. However, most of the cytokines upregulated upon HIV infection remained upregulated in the absence of HIV replication. EV-associated cytokines were more likely to remain elevated in spite of ART. In conclusion, the phenomenon of residual immune activation after successful ART is reproduced in a laboratory experimental system with human lymphoid tissue ex vivo opening a way to study this phenomenon under controlled laboratory conditions. Another complication in HIV-1 infected individuals undergoing ART is a failure to reconstitute the CD4+ T cells (immune non-responders, INR). Here, we show that cells with the phenotype and transcriptional profile of Tregs were enriched among cycling cells in health and in HIV infection. Yet there were diminished frequencies and numbers of Tregs among cycling CD4+ T cells in INRs, and cycling CD4+ T cells from INR subjects displayed transcriptional profiles associated with the impaired development and maintenance of functional Tregs. Flow cytometric assessment of TGF-beta activity confirmed the dysfunction of Tregs in INR subjects. Transcriptional profiling and flow cytometry revealed diminished mitochondrial fitness in Tregs among INRs, and cycling Tregs from INRs had low expression of the mitochondrial biogenesis regulators peroxisome proliferator activated receptor coactivator 1- (PGC1) and transcription factor A for mitochondria. In vitro exposure to IL-15 allowed cells to complete division, restored the expression of PGC1, and regenerated mitochondrial fitness in the cycling Tregs of INRs. Our data suggest that rescuing mitochondrial function could correct the immune dysfunction characteristic of Tregs in INRs and enhance immune restoration in these subjects. In conclusion, our analysis showed that HIV-1 infected lymphoid tissues ex vivo upregulated production of many cytokines, both free and EV-associated, and the majority of these cytokines remained upregulated despite suppression of viral replication by ART. Also, in spite of ART a number of patients do not reconstitute their immune system. Mechanisms of both of these phenomena can now be investigated under controlled laboratory conditions resulting in the development of new therapeutic strategies. 2. Cytomegalovirus and EVs in atherosclerosis: Immune activation Acute cardiovascular syndrome (ACS) is associated with a general activation of the immune system that includes activation of many cells, in particular monocytes and platelets, leading to destabilization and rupture of coronary atherosclerotic plaques and to acute myocardial infarction (AMI). Recently, it was found that immune activation is associated with the release of EVs by activated cells that mediate cell-cell communication and play an important role in immune activation. One of the manifestations of the activation of monocytes and platelets is the formation of monocyte-platelet complexes (MPC). We analyzed MPC in vivo and in vitro and investigated the abilities of different monocyte subclasses to form MPC, characteristics of the cells and EVs involved in MPC formation, and MPC changes in AMI. We identified MPC by co-staining for platelet antigen CD41a and monocyte antigens CD14 and CD16. Platelet activation was evaluated by expression of phosphatidylserine (PS). Monocytes of different classes disproportionately formed MPC: although classical monocytes (CD14++CD16-) constituted the majority, MPC were preferentially formed by intermediate monocytes (CD14++CD16+). CD41-positive events in MPC exposed more PS than the circulating ones. AMI was associated with a 50% increase in circulating monocytes and with a threefold increase in MPC, in particular in those formed by classical monocytes. In AMI patients, MPC formed by intermediate monocytes contained more CD41a-positive events than MPC with other monocyte subsets, whereas in controls, MPC formed by classical monocytes carried more platelets than other MPC. The sizes of some of the CD41a+ events in MPC were smaller than those of regular platelets and may represent platelet-derived EV. Some of the aggregates seem to consist of monocytes and platelet-derived EVs. Binding of EVs to monocytes was confirmed in in vitro experiments when monocytes were co-incubated with platelet-generated EVs. There was association between the increase in MPC and their composition, and in-patient complications of AMI. Aggregation of EVs and platelets with monocytes in AMI patients is another manifestation of immune activation associated with atherosclerosis that plays an important role in this pathology and can be used as an AMI correlate. However, it remains to be understood what causes this immune activation. Our results as well as other data may indicate the presence of a specific antigen(s) towards which these lymphocytes are reactive. Herpesviruses, in particular CMV, are one of the main candidates causing persistent immunoactivation within plaques due to their ubiquity and ability to cycle between dormancy and replication, as well as by their ability to cause endothelial dysfunction. In our previous work, we showed that productive cytomegalovirus infection is more common in patients with acute coronary syndrome than in patients with chronic coronary artery disease and healthy volunteers. Moreover, in that work we also found a positive correlation between the cytomegaloviral DNA load and T-lymphocyte differentiation within the atherosclerotic plaques of patients with cardiovascular diseases Now, we analyzed the presence of cytomegaloviral DNA in plasma and endothelial function in 33 patients with ST-elevation myocardial infarction (STEMI) and 33 volunteers without cardiovascular diseases, using real-time polymerase chain reaction and a noninvasive test of flow-mediated dilation. We found that the presence of cytomegaloviral DNA in plasma of STEMI patients was significantly higher than in volunteers without cardiovascular diseases. Also, we found a significant prevalence in the number of copies of cytomegaloviral DNA in older hypertensive patients, indicating that one of the possible mechanisms of the development of hypertension in patients with cytomegalovirus infection could be the development of endothelial dysfunction triggered by cytomegalovirus. In summary, we found that cytomegalovirus infection is strongly associated with endothelial dysfunction in STEMI patients. Therefore, it is conceivable that preventing the reactivation of cytomegalovirus may enhance endothelial functions

抗逆转录病毒治疗后的 HIV-1 组织发病机制 免疫激活现在被认为是各种人类疾病的驱动力，包括 HIV-1 疾病。目前，抗逆转录病毒治疗 (ART) 组合已被证明可有效抑制 HIV-1 复制。然而，ART 长期抑制 HIV-1 复制会增加并发症的风险，包括神经系统和心血管疾病。这些疾病似乎与接受 ART 的患者体内残留的免疫激活有关。细胞因子可能在这种残余免疫激活中发挥重要作用。为了研究这种现象的机制，有必要开发一个反映体内发生情况的离体实验室控制系统。我们开发了这样一个系统。 作为实验模型，我们使用离体人类淋巴组织，其中 HIV-1 感染的关键事件发生在体内。在目前的工作中，我们评估了捐献者匹配的人淋巴组织释放的 33 种细胞因子，这些组织在感染 HIV-1 的 16 天内有效地离体感染，并接受或未接受抗病毒药物利托那韦治疗。我们评估了可溶性细胞因子和 EV 相关细胞因子。 我们发现 HIV-1 感染导致几种关键细胞因子的上调。 EV 相关细胞因子比可溶性细胞因子更有可能升高。抗病毒药物抑制受感染组织中的艾滋病毒。然而，大多数在 HIV 感染时上调的细胞因子在没有 HIV 复制的情况下仍然上调。 尽管进行了 ART，EV 相关细胞因子更有可能保持升高。 总之，成功 ART 后残留免疫激活的现象在实验室实验系统中重现，人体淋巴组织离体，为在受控实验室条件下研究这种现象开辟了道路。 接受 ART 的 HIV-1 感染者的另一个并发症是无法重建 CD4+ T 细胞（免疫无反应细胞，INR）。在这里，我们发现具有 Tregs 表型和转录谱的细胞在健康和 HIV 感染的循环细胞中富集。然而，INR 中循环 CD4+ T 细胞中 Treg 的频率和数量减少，并且来自 INR 受试者的循环 CD4+ T 细胞显示出与功能性 Tregs 发育和维持受损相关的转录谱。 TGF-β 活性的流式细胞术评估证实了 INR 受试者中 Tregs 的功能障碍。转录分析和流式细胞术显示 INR 中 Tregs 的线粒体适应性降低，并且 INR 中的循环 Tregs 线粒体生物发生调节剂过氧化物酶体增殖物激活受体辅激活因子 1- (PGC1) 和线粒体转录因子 A 的表达较低。体外暴露于 IL-15 使细胞能够完成分裂，恢复 PGC1 的表达，并恢复 INR 循环 Tregs 中的线粒体适应性。我们的数据表明，挽救线粒体功能可以纠正 INR 中 Tregs 的免疫功能障碍特征，并增强这些受试者的免疫恢复。 总之，我们的分析表明，HIV-1感染的淋巴组织离体上调了许多细胞因子的产生，包括游离的和EV相关的，并且尽管ART抑制了病毒复制，但大多数细胞因子仍然上调。此外，尽管接受了抗逆转录病毒疗法，许多患者仍无法重建其免疫系统。现在可以在受控实验室条件下研究这两种现象的机制，从而开发新的治疗策略。 2. 动脉粥样硬化中的巨细胞病毒和 EV：免疫激活 急性心血管综合征（ACS）与免疫系统的全面激活有关，包括许多细胞，特别是单核细胞和血小板的激活，导致冠状动脉粥样硬化斑块不稳定和破裂，并导致急性心肌梗死（AMI）。最近发现，免疫激活与激活细胞释放EVs有关，介导细胞间通讯，在免疫激活中发挥重要作用。单核细胞和血小板活化的表现之一是形成单核细胞-血小板复合物（MPC）。 我们分析了体内和体外的 MPC，并研究了不同单核细胞亚类形成 MPC 的能力、参与 MPC 形成的细胞和 EV 的特征以及 AMI 中 MPC 的变化。 我们通过血小板抗原 CD41a 和单核细胞抗原 CD14 和 CD16 共染色来鉴定 MPC。通过磷脂酰丝氨酸（PS）的表达来评估血小板活化。不同类别的单核细胞不成比例地形成MPC：虽然经典单核细胞（CD14++CD16-）占大多数，但MPC优先由中间单核细胞（CD14++CD16+）形成。 MPC 中的 CD41 阳性事件比循环事件暴露更多的 PS。 AMI 与循环单核细胞增加 50% 以及 MPC 增加三倍相关，特别是由经典单核细胞形成的单核细胞。在 AMI 患者中，由中间单核细胞形成的 MPC 比由其他单核细胞亚群形成的 MPC 含有更多的 CD41a 阳性事件，而在对照组中，由经典单核细胞形成的 MPC 比其他 MPC 携带更多的血小板。 MPC 中一些 CD41a+ 事件的大小小于常规血小板的大小，可能代表血小板衍生的 EV。一些聚集物似乎由单核细胞和血小板衍生的 EV 组成。当单核细胞与血小板生成的 EV 共孵育时，体外实验证实了 EV 与单核细胞的结合。 MPC 及其组成的增加与 AMI 住院并发症之间存在关联。 AMI 患者中 EV 和血小板与单核细胞的聚集是与动脉粥样硬化相关的免疫激活的另一种表现，在这种病理学中发挥着重要作用，可用作 AMI 的相关因素。 然而，导致这种免疫激活的原因仍有待了解。我们的结果以及其他数据可能表明这些淋巴细胞对其有反应的特定抗原的存在。疱疹病毒，特别是巨细胞病毒，是引起斑块内持续免疫激活的主要候选病毒之一，因为它们普遍存在并且能够在休眠和复制之间循环，并且能够引起内皮功能障碍。 在我们之前的工作中，我们表明，与慢性冠状动脉疾病患者和健康志愿者相比，急性冠状动脉综合征患者中生产性巨细胞病毒感染更常见。此外，在这项工作中我们还发现心血管疾病患者动脉粥样硬化斑块内的巨细胞病毒DNA载量与T淋巴细胞分化呈正相关 现在，我们使用实时聚合酶链反应和血流介导扩张的无创测试，分析了 33 名 ST 段抬高型心肌梗死 (STEMI) 患者和 33 名无心血管疾病的志愿者血浆中巨细胞病毒 DNA 的存在和内皮功能。 我们发现STEMI患者血浆中巨细胞病毒DNA的含量显着高于没有心血管疾病的志愿者。此外，我们发现老年高血压患者巨细胞病毒DNA拷贝数显着普遍，表明巨细胞病毒感染患者发生高血压的可能机制之一可能是巨细胞病毒引发的内皮功能障碍。 总之，我们发现巨细胞病毒感染与 STEMI 患者的内皮功能障碍密切相关。因此，可以想象，阻止巨细胞病毒的重新激活可能会增强内皮功能