Pathogenic Role of NK Cells During Infection-Induced Toxic Shock

描述(申请人提供)：本研究项目的长期目标是阐明NK细胞在感染细胞内细菌时调节T细胞反应的免疫学机制。人单核细胞增多性埃立克体病(HME)是一种由查菲棘球绦虫引起的、在北美高度流行的、危及生命的硬虱传播疾病。这种感染可能是亚临床的，也可能是致命的，类似于中毒性休克综合征。虽然这种疾病的治疗依赖于多西环素，但当药物在感染后期使用时，治疗失败率很高。我们广泛的小鼠研究表明，致死性埃立克体能诱导 休克是由于CD8+T细胞介导的免疫病理。产生干扰素-伽玛-CD4+Th1细胞对消除埃立克体病至关重要；然而，这些细胞在致命疾病期间会受到抑制和晚期凋亡。我们最近证实，在致死性埃利希菌诱导的休克中，NK细胞损害了细菌的有效清除，并介导了组织损伤。然而，NK细胞在埃利希菌诱导的休克中的致病作用的免疫调节机制仍不清楚。我们假设，在致死性埃利希菌诱导的中毒性休克过程中，NK细胞通过抑制CD4+Th1细胞反应，而促进致病CD8+T细胞的诱导和组织损伤，负向调节保护性抗埃立克体免疫反应的产生。我们将使用三个具体目标来检验这一假设。在AIM 1，我们将确定在致死性埃立克病毒感染过程中NK细胞对保护性CD4+Th1细胞功能的影响。在目标2中，我们将确定NK细胞在埃立克体致中毒性休克发病机制中的分子机制。在目标3中，我们将描述人类感染埃立克病毒的整个过程中的NK细胞反应。我们将通过以下方法来实现这些目标：1)检测感染埃立克病毒的NK耗竭小鼠或假对照组中CD4+T细胞和CD8+T细胞的扩增和效应功能的差异；2)比较在存在或不存在埃立克病毒感染的抗原提呈细胞(APC)的情况下，与致死性或非致死性感染小鼠的NK细胞在体外共培养时，效应性CD4+T细胞的增殖和细胞因子产生的差异；3)比较照射小鼠骨髓嵌合体中的CD4+T细胞和CD8+T细胞的反应，其中IL-10仅由非造血细胞或非B/T淋巴细胞固有的造血细胞(包括NK细胞和APC)表达；4)比较严重和轻度埃立克体病患者的NK和T细胞反应，并确定体外去除人NK细胞是否改变这些患者的人CD4+T细胞的增殖和功能。HME不仅是一个新兴的公共卫生问题，而且代表了由其他细菌引起的毒性或感染性休克的模型。因此，所产生的知识将有助于合理开发新的治疗方法来对抗感染所致的脓毒症。

DESCRIPTION (provided by applicant): The long term goal of this research project is to elucidate the immunological mechanisms by which NK cells regulate T cell responses during infections with intracellular bacteria. Human monocytotropic ehrlichiosis (HME) is a highly prevalent life-threatening tick-borne disease in North America caused by E. chaffeensis. The infection can be subclinical or fatal, mimicking toxic shock syndrome. Although management of this disease relies on doxycycline, there is a high rate of treatment failure when the drug is administered late in infection. Our extensive murine studies indicated that fatal Ehrlichia-induced shock is due to CD8+ T cell-mediated immunopathology. IFN-gamma producing-CD4+ Th1 cells are critical for the elimination of Ehrlichia; however, these cells undergo suppression and late apoptosis during fatal disease. We recently demonstrated that NK cells impair effective bacterial elimination and mediate tissue injury during fatal Ehrlichia-induced shock. However, the immunoregulatory mechanisms that account for the pathogenic role of NK cells in Ehrlichia- induced shock remain unknown. We hypothesize that NK cells negatively regulate the generation of protective anti-erhrlichial immune responses by suppressing CD4+ Th1 cell responses, while promoting the induction of pathogenic CD8+ T cells and tissue injury during fatal Ehrlichia-induced toxic shock. We will test this hypothesis using three specific aims. In Aim 1, we will determine the impact of NK cells on the functions of protective CD4+ Th1 cells during fatal ehrlichial infection. In Aim 2, we will identify the molecular mechanisms by which NK cells contribute to the pathogenesis of Ehrlichia-induced toxic shock. In Aim 3, we will characterize the NK cell responses throughout the course of Ehrlichia infection in humans. We will carry out these aims by: 1) measuring the differences in expansion and effector functions of CD4+ T cells and CD8+ T cells in Ehrlichia- infected NK-depleted mice or sham controls; 2) comparing the proliferation and cytokine production of effector CD4+T cells when co-cultured in vitro with NK cells derived from lethally- or non-lethally infected mice in the presence or absence of Ehrlichia-infected antigen presenting cells (APCs); 3) comparing CD4+ T cell and CD8+ T cell responses in irradiated bone marrow mouse chimeras, in which IL-10 will be only expressed by non-hematopoietic cells or non B/T lymphocyte-innate hematopoietic cells including NK cells and APCs; and 4) comparing NK and T cell responses in patients with severe and mild ehrlichiosis and determine whether in vitro depletion of human NK cells alters the proliferation and functions of human CD4+T cells in these patients. HME is not only an emerging public health concern, but represents a model for toxic or septic shock caused by other bacteria. Therefore, knowledge generated herein will facilitate rational development of novel therapeutics against infection-induced sepsis.