Mechanisms of innate immunity in bony fish

在所有多细胞生物体中，先天免疫系统负责维持体内平衡和抵御病原体。在水产养殖环境中，由于压力和拥挤的条件，鱼类特别容易受到各种病原体的影响。尽管先天免疫在宿主防御病原体方面很重要，但与哺乳动物相比，人们对鱼类先天免疫的机制知之甚少。髓系细胞是天然宿主防御和动态平衡的重要组成部分。对于骨鱼中调节髓系细胞发育(造髓)的因素知之甚少，从而限制了我们对动态平衡过程和免疫系统对病原体反应的破坏的理解。该研究计划的第一个目标是在单个细胞和细胞群体水平上描述早期骨髓生成过程中髓系生长因子、转录因子和microRNAs之间的相互作用。为了实现这一目标，我们建议使用一种独特的金鱼原代肾脏巨噬细胞培养系统和重组金鱼巨噬细胞生长因子及其受体，我们在之前的NSERC发现基金的任期内对其进行了表征。作为对感染的反应，被称为巨噬细胞的天然免疫中心细胞利用许多不同的抗菌机制来抑制微生物复制并有助于消除病原体。与大量关于控制哺乳动物巨噬细胞抗菌反应的机制的信息相比，关于细胞因子对硬骨鱼巨噬细胞抗菌功能的调节知之甚少。拟议研究计划的第二个目标是检查巨噬细胞的抗微生物反应的调节。我们建议研究细胞内(海洋分枝杆菌、沙门氏气单胞菌)和细胞外(卡氏锥虫)病原体的胞液模式识别受体，以及金鱼II型干扰素对金鱼巨噬细胞抗菌反应的调节。我们能够很好地满足这两个目标，因为我们(A)建立了金鱼原代巨噬细胞的产生和培养程序；(B)建立了三个体外和体内金鱼病原体模型系统(海洋分枝杆菌、鲑鱼分枝杆菌和卡氏锥虫)；以及(C)产生和验证金鱼特异性试剂和一组分析，使单细胞和细胞种群能够对基因表达、增殖、分化和功能进行评估。这项研究将有助于我们对硬骨鱼先天宿主防御机制的基础知识，这是设计鱼类传染病预防策略所必不可少的。

In all multicellular organisms, the innate immune system acts to maintain homeostasis and defend against pathogens. In aquaculture setting, fish are especially susceptible to a variety of pathogens due to stress and crowded conditions. Despite the importance of innate immunity in host defense against pathogens, little is known about the mechanisms of fish innate immunity compared to those of mammals. An important component of innate host defence and homeostasis are cells of myeloid lineage. Relatively little is known about the factors that mediate myeloid cell development (myelopoiesis) in bony fish, thus limiting our understanding of homeostatic processes and immune system disruption in response to pathogens. The first objective of the proposed research program is to characterize the interactions between myeloid growth factors, transcription factors and microRNAs during early myelopoiesis at a single cell and at cell population levels. To accomplish this objective, we propose to use a unique goldfish primary kidney macrophage culture system and recombinant goldfish macrophage growth factors and their receptors that we characterized during tenure of a previous NSERC Discovery grant. In response to infection, central cells of innate immunity called macrophages, utilize a number of different antimicrobial mechanisms that inhibit microbial replication and contribute to the elimination of pathogens. In contrast to the large body of information regarding the mechanisms that control mammalian macrophage antimicrobial responses, relatively little is known about cytokine regulation of bony fish macrophage antimicrobial functions. The second objective of the proposed research program is to examine the regulation of antimicrobial responses of macrophages. We propose to examine the recognition of intracellular (Mycobacterium marinum, Aeromonas salmonicida) and extracellular (Trypanosoma carassii) fish pathogens by cytosolic pattern recognition receptors, and regulation of goldfish macrophage antibacterial responses by goldfish Type II interferons.We are in an excellent position to address both objectives because we (a) developed procedures for generation and cultivation of primary macrophages from the goldfish; (b) established three in vitro and in vivo goldfish pathogen model systems (M. marinum, A. salmonicida and T. carassii); and (c) generated and validated goldfish-specific reagents and a panel of assays that enable both single-cell and cell population assessments of gene expression, proliferation, differentiation and function.Due to depletion of natural fish stocks, the aquaculture industry in Canada has been growing at a rate of more that 20% per year. This research will contribute to our basic knowledge of the mechanisms of innate host defense in bony fish that is absolutely required for design of prevention strategies against fish infectious diseases.