Hemocyte Transcriptome and Immunity in Aedes aegypti

描述（由申请人提供）：蚊子先天免疫应答是控制载体和病原体之间相互作用的主要因素;因此，它是载体能力的主要决定因素。蚊子和其他昆虫的先天免疫采用细胞和体液成分来应对入侵的病原体，但先天免疫的体液方面的研究占主导地位，主要强调抗菌肽（AMP）的鉴定和参与其生产的信号通路。对血细胞和细胞免疫反应的重视程度要低得多，但最近的数据清楚地证实，细胞反应可能在通过吞噬作用、黑化或两种过程的组合来清除微生物病原体方面发挥关键作用。准确定义调控过程、血细胞产生的效应分子以及这些细胞和细胞产物在先天免疫中的作用所需的数据中存在空白。同样清楚的是，AMP是针对入侵微生物的先天免疫应答中的重要因素，但尚不清楚细胞和基于AMP的体液应答如何协同作用以清除微生物感染。我们认为，一个还原论的方法，研究一个基因，或一小部分基因，与表型，有局限性，以确定复杂的相互关系，无疑存在于蚊子的先天免疫反应。因此，为了启动更全面的实验方法来研究蚊子的先天免疫，我们开发了一个大的表达序列标签（EST）数据集从埃及伊蚊免疫激活的血细胞和互补的基于荧光素的微阵列作为基板，以评估蚊子的细胞，以及体液，免疫反应。本文提出的研究的一般假设是，蚊子先天免疫应答是一个高度复杂的过程网络，其采用不同的但不相互排斥的吞噬作用和黑化反应，与AMP协同作用，以清除受感染蚊子血淋巴中的细菌感染。为了验证这一假设，我们将使用蚊子Ae。埃及伊蚊和许多细菌物种在（1）高通量分子方法以分析基因转录并鉴定作为吞噬作用和黑化的抗菌应答基础的不同模式，（2）利用微阵列分析来评估与不同细菌物种相关的转录谱变化（其致病性和引发应答的能力不同），以便更好地剖析影响细胞免疫的机制，和（3）采用微阵列分析，合成肽，RNAi方法和表型评估，以更好地定义AMP的作用，并选择未知的基因，在先天免疫中发挥作用。所有来自微阵列分析的数据，包括使用的实验方案，将在包含我们的血细胞EST数据的交互式网站上公开。

DESCRIPTION (provided by applicant): The mosquito innate immune response is a major factor governing the interaction between vector and pathogen; consequently, it is a primary determinant of vector competence. Innate immunity in mosquitoes and other insects employs both cellular and humoral components in response to invading pathogens, but studies of the humoral aspects of innate immunity have dominated, with a primary emphasis on the identification of antimicrobial peptides (AMPs) and the signaling pathways involved in their production. Far less emphasis has been placed on hemocytes and cellular immune responses, but recent data clearly verify that cellular responses likely play a critical role in clearing microbial pathogens by phagocytosis, melanization, or by both processes in combination. There is a void in the data required to accurately define regulatory processes, hemocyte-produced effector molecules, and the role these cells and cell products play in innate immunity. It also is clear that AMPs are significant factors in innate immune responses against invading microbes, but it is not known how cellular and AMP-based humoral responses function in concert to clear microbial infections. We believe that a reductionist approach, that studies one gene, or a small set of genes, in relation to a phenotype, has limitations for determining the complex interrelationships that undoubtedly exist in mosquito innate immune responses. Therefore, in order to initate more holistic experimental approaches for studying innate immunity in mosquitoes, we developed a large expressed sequence tag (EST) data set from Aedes aegypti immune-activated hemocytes and complementary oligonucleotide-based microarrays as substrates to evaluate mosquito cellular, as well as humoral, immune responses. The general hypothesis for the research proposed herein is that mosquito innate immune responses are a highly complex network of processes that employ the distinct, but not mutually exclusive, responses of phagocytosis and melanization, in concert with AMPs, to clear bacteria infections from the hemolymph of infected mosquitoes. To test this hypothesis, we will use the mosquito Ae. aegypti and a number of bacteria species in (1) high-throughput molecular approaches to profile gene transcription and to identify distinct patterns underlying the anti-bacterial responses of phagocytosis and melanization, (2) utilize microarray analyses to assess transcription profile changes in relation to different bacterial species (that vary in their pathogenicity and ability to elicit a response) in order to better dissect mechanisms influencing cellular immunity, and (3) employ microarray analyses, synthetic peptides, RNAi methodologies, and phenotype evaluations to better define the role AMPs, and select unknown genes, play in innate immunity. All data resulting from microarray analyses, including experimental protocols used, will be made publicly available on the interactive website that contains our hemocyte EST data.