Modeling Pulmonary HIV-Cytokine Interactions

描述(由申请人提供)： 我们的提案致力于定义肺部艾滋病毒感染的发病机制。它描述了一系列体内和体外实验，旨在构建机械性数学模型，以更好地了解细胞因子网络及其在肺部艾滋病毒复制动力学中的作用。破译细胞因子网络及其对HIV-1(艾滋病毒)在肺部复制的不同和综合影响是一个极其复杂的问题。我们的研究清楚地表明，在儿童和成人中，肺白细胞都可以是促炎细胞因子、趋化因子和复制病毒的重要来源，然而，肺参与HIV感染的实际动机尚不清楚。我们之前的研究表明，导致巨噬细胞高度激活的病毒和非病毒条件都有利于艾滋病毒在肺内的复制。这一点，以及促进细胞激活的浓度梯度和细胞因子表达模式的变化，可能是建立这一利基环境的关键。我们之前的研究也表明，在停止联合抗逆转录病毒治疗(ART)后，血液中潜伏感染的CD4vT细胞只贡献了病毒反弹的一部分，其他部位似乎是大部分反弹病毒的来源。肺巨噬细胞寿命相对较长，艾滋病毒感染不是明显的细胞病变，已被认为可能是艾滋病毒复制的持久储存库，包括在长时间的抗逆转录病毒治疗期间。因此，我们建议的更长期目标是更好地确定宿主和促进艾滋病毒在肺部复制的病毒因素之间的关系，特别是在抗逆转录病毒治疗期间。为此，我们将研究HIV阳性患者肺部中HIV特异性宿主细胞的范围与疾病阶段和细胞因子/趋化因子表达谱的关系。这将使用ELISA微阵列技术和原位杂交技术来完成，该技术允许从诱导痰和支气管肺泡灌洗(BAL)中获得表型定义的细胞内特定病毒和细胞因子mRNAs的定位和定量。有了这些数据，我们将构建和评估复制HIV的细胞和表达特定趋化因子和促炎细胞因子的细胞之间相互作用的机械性和非机械性数学模型。我们假设，肺组织或痰/BAL液中HIV RNA浓度最高的人组织和液体中的vRNA+肺泡巨噬细胞会比病毒载量较低的人在每个细胞上产生更多的病毒。我们还假设，在这些人中，肺微环境将发生改变，有利于Th1型细胞因子环境。最后，我们将通过检测ART期间复制病毒的滴度和爆发大小以及病毒衰变动力学来检验肺是从血液中分离出来的病毒学隔间的假设。因此，我们建议肺泡巨噬细胞在HIV复制的持续性和随后的发病机制中发挥中心作用。特别是，我们假设，根据ART，感染在这些细胞中以活跃状态持续存在，我们将通过检查治疗与未治疗患者的病毒序列多样性和分歧来评估这一点。

DESCRIPTION (provided by applicant): Our proposal is devoted to defining the pathogenesis of HIV infection in the lung. It describes a series of in vivo and in vitro experiments oriented towards constructing mechanistic mathematical models to better understand cytokine networks and their role in the dynamics of HIV replication in the lung. Deciphering cytokine networks and their differential and combined effects on HIV-1 (HIV) replication in the lung presents a problem of enormous complexity. Our studies clearly show that pulmonary leukocytes can be a significant source of proinflammatory cytokines, chemokines and replicating virus in both children and adults, however, the actual impetus for lung involvement in HIV infection is not known. Our previous studies suggest that both viral and nonviral conditions that lead to a heightened state of macrophage activation favor the replication of HIV in the lung. This, along with alterations in concentration gradients and patterns of cytokine expression that promote cellular activation, may be crucial for establishing this niche. Our previous studies have also indicated that latently infected CD4vT cells in the blood contribute only a portion of the viral rebound following cessation of combination antiretroviral therapy (ART) and that other sites appear to be the source of much of the rebounding virus. Pulmonary macrophages, which are relatively long-lived and for which HIV infection is not overtly cytopathic, have been suggested as a possible persistent reservoir of HIV replication, including during long periods of ART. Hence, the more long-term objectives of our proposal are to better define the relationship between host and viral factors that promote HIV replication in the lung, particularly during ART. To do this, we will examine the range of HIV-specific host cells in the lungs of HIV-positive persons in relation to the stage of disease and cytokine/chemokine expression profiles. This will be accomplished using ELISA and cDNA microarray technologies and by in situ hybridization techniques that allow for the localization and quantification of specific viral and cytokine mRNAs within phenotypically-defined cells obtained from induced sputum and bronchoalveolar lavage (BAL). With these data we will construct and evaluate mechanistic and nonmechanistic mathematical models for the interaction between cells that replicate HIV and those that express specific chemokine and proinflammatory cytokines in the lung. We hypothesize that persons with the highest concentrations of HIV RNA in lung tissues or sputum/BAL fluids will have more vRNA+ alveolar macrophages in tissues and fluids and these cells will produce more virus on a per-cell basis that persons with lower viral load. We also hypothesize that in such persons the pulmonary microenvironment will be altered in favor of a Thl -type cytokine milieu. Last, we will test the hypothesis that the lung is a separate virologic compartment from blood by examining titers and burst size of replicating virus and viral decay kinetics during ART. As such, we propose a central role for alveolar macrophages in the persistence of HIV replication and subsequent pathogenesis. In particular, we hypothesize that infection persists in these cells in an active state under ART to which we will assess by examining viral sequence diversity and divergence in treated vs. untreated persons.