Defining modes of helper T cell motility within inflamed tissues

描述(申请人提供)：根据周围环境的性质，白细胞已被证明在体外利用不同的运动模式。使用活体双光子显微镜系统对小鼠发炎真皮内的活T细胞进行成像，我们观察到了两种不同的间质爬行表型，这两种表型表现为功能上离散的CD4+T细胞群。Th1细胞以阿米巴样运动快速移动，Th2细胞以间充质样运动爬行。这两种模式表明，这些功能不同的T细胞的运动机制是不同的。考虑到细胞外基质和细胞内信号之间通过表面受体(如整合素)的反馈，这些观察结果与主要利什曼原虫模型有关，在该模型中，我们之前的数据表明，局部感染组织中T细胞存在功能变化，可能限制Th1的积累。因此，我们将检验这样的假设：1)Th1和Th2效应器T细胞在体内的间质爬行过程中利用不同的分子机制进行运动；2)在寄生的大型乳杆菌感染过程中，运动能力被改变。具体目标1：Th1和Th2效应细胞是否利用不同的机制在炎症部位爬行？目前，细胞生物学家已经描述了两种主要的间质爬行模式：整合素依赖(间充质)和整合素非依赖、肌球蛋白II依赖(阿米巴)运动。这个目标的目的是确定不同的辅助T细胞群体在发炎的真皮的间质间隙中爬行的分子手段。候选的分子通路将被抗体阻断、基因缺失和siRNA破坏，并通过活体双光子显微镜确定其对运动的影响。特定目标2：辅助T细胞的迁移和爬行模式能被病原体诱导的局部变化所调节吗？除了伴随慢性感染的免疫反应外，大型乳杆菌等病原体还可以引起当地环境的显著变化。该菌可通过改变局部组织ECM的结构和组成来调节反式T细胞的运动，并通过调节感染宿主细胞表达的趋化因子和黏附分子来调节顺式T细胞的运动。这些变化可能导致局部微环境与稳定状态甚至非寄生性炎症组织截然不同。本研究的目的是评估大型乳杆菌感染的组织微环境对Th1和Th2细胞爬行的影响，以及寄生的抗原提呈细胞与不同效应T细胞亚群相互作用的能力。 公共卫生相关性：使用活体显微镜系统对小鼠发炎真皮内的活T细胞进行成像，我们观察到了两种独特的爬行表型，这两种表型表现为功能上不同的CD4+T细胞群。在目前的研究训练计划中，我们将测试观察到的不同爬行表型是否利用体内独特的分子机制。了解不同功能的效应T细胞所采用的机制可以提供优先操纵组织驻留效应细胞亚群的方法，可能为许多疾病状态提供巨大的治疗潜力。

DESCRIPTION (provided by applicant): Leukocytes have been shown to utilize different modes of motility in vitro, depending on the nature of their surrounding environment. Using a system of intravital 2-photon microscopy to image live T cells within the inflamed dermis of a mouse, we have observed two distinct interstitial crawling phenotypes that were displayed by functionally discrete CD4+ T cell populations. Th1 cells moved rapidly with amoeboid-like motility while Th2 cells crawled with mesenchymal-like motility. These two modes suggest the mechanisms underlying movement of these functionally distinct T cells are distinct. Given the feedback between the extracellular matrix and intracellular signaling through surface receptors (such as integrins), these observations have relevance in a model of Leishmania major, in which our previous data has suggests that there are functional alterations to T cells in the locally infected tissue that may restrict Th1 accumulation. Thus, we will test the hypotheses that 1) Th1 and Th2 effectors T cells utilize different molecular machinery for motility during in vivo interstitial crawling and 2) motility is modified during parasitic L. major infection. Specific Aim 1: Do effector Th1 and Th2 cells utilize distinct mechanisms to crawl in sites of inflammation? Currently, two main modes of interstitial crawling have been described by cell biologists; integrin-dependent (mesenchymal) and integrin-independent, myosin II-dependent (amoeboid) motility. The objective of this aim is to determine the molecular means by which distinct helper T cell populations crawl within the interstitial space of the inflamed dermis. Candidate molecular pathways will be disrupted by antibody-blockade, genetic deletion and siRNA and the effects on motility determined by intravital 2-photon microscopy. Specific Aim 2: Can the migratory and crawling patterns of helper T cells be modulated by local changes induced by a pathogen? In addition to the immunological responses that accompany chronic infection, pathogens such as L. major can induce significant changes in the local milieu. L. major could modulate T cell motility in trans through pathogen-driven changes in architecture and composition of the ECM of the local tissue and in cis through modulating the chemokines and adhesion molecules expressed by the infected host cells. These changes can result local microenvironments that are dramatically different from steady state and even non-parasitized inflamed tissue. The goal of this aim is to evaluate the effects of the L. major-infected tissue microenvironment on Th1 and Th2 cell crawling, as well as the ability of parasitized antigen presenting cells to interact with the different effector T cells subsets. PUBLIC HEALTH RELEVANCE: Using a system of intravital microscopy to image live T cells within the inflamed dermis of a mouse, we have observed two unique crawling phenotypes that were displayed by functionally distinct CD4+ T cell populations. In the current research training plan, we will test whether the different crawling phenotypes observed utilize unique molecular mechanisms in vivo. An understanding of mechanisms employed by functionally distinct effector T cells could present means of preferentially manipulating subsets of tissue-resident effector cells, potentially offering great therapeutic potential for many disease states.