The teleost melano-macrophage center response to immunization and helminth-mediated immunosuppression

摘要 非小鼠模型可能是发现以前未被评价的免疫学范例的肥沃土壤 并为脊椎动物免疫提供了新的视角。拟议的工作解决了一个根本问题 硬骨鱼的免疫：黑素巨噬细胞中心(MMC)是鱼类B细胞增殖的场所吗 并产生与哺乳动物生发中心(GC)同源的高亲和力抗体？要回答这么长的问题-- 悬而未决的问题，我们将表征三刺鱼的MMC反应。 阐明MMC的功能将对理解鱼类体液适应性免疫的基础有重要的意义。 脊椎动物适应性免疫的进化起源。本项目的第一个目标是研究MMC的功能 以回应免疫接种。为了确定MMCs是否是B细胞增殖的场所，免疫FISH将 脉冲标记BrdU，以便对增殖的BrdU+细胞进行定位。接下来，为了澄清躯体是否 MMC中存在超突变，我们将对MMC邻近和非MMC中的免疫球蛋白基因突变进行量化 相邻的B细胞。为了确定MMC函数是否确实类似GC，我们将MMC响应与 T依赖(NP-CGG)和T非依赖(NP-葡聚糖)抗原免疫。这第一次的结果是 AIM将确定MMC作为硬骨鱼体液适应性免疫的生物标志物的适用性，以及 MMC是否是“进化上原始的”GC。初步数据有力地支持了我们的假设 MMC类似于GC。因此，在我们的第二个目标中，我们将把我们对MMC函数的知识应用到 研究蠕虫寄生虫--苏氏血吸虫的免疫调节作用。通过组合 实验感染和免疫我们将确定这种绦虫是否抑制硬骨适应性 豁免权。我们发现，一些刺背基因对这种免疫抑制是无效的。使用 QTL定位、群体基因组学和基因RNAseq分析相结合 表达我们将定位负责可变寄生虫介导的免疫调节的宿主基因座。我们会 然后使用CRISPR/Cas9系统对培养的MMC中的这些基因进行修饰，并评估它们在MMC中的作用 在体外发挥作用。在最终目标中，我们将确定抑制MMC活性的蠕虫衍生因子。 通过QTL定位，我们将确定与MMC抑制有关的绦虫特异性基因座。至 作为对这一方法的补充，我们将评估蠕虫排泄/分泌产品是否直接调节 MMC基因表达。阐明蠕虫介导的鱼类免疫调节的机制基础可能 揭示调节脊椎动物免疫功能的新方法。这项工作还将产生一个新的和广泛的 适用的硬骨鱼免疫测定，有可能用于研究不同的鱼类(和其他 变温动物)物种。除了扩展我们对脊椎动物免疫生物学的看法外，这项工作还对 疾病生态学、宿主-病原体共同进化和疫苗开发的学科。

Abstract Non-mouse models can be fertile ground for the discovery of previously unappreciated immunologic paradigms and provide new perspectives of vertebrate immunity. The proposed work addresses a fundamental question of bony fish (teleost) immunity: are melano-macrophage centers (MMC) the sites where fish B cells proliferate and generate high affinity antibody, homologous to germinal centers (GC) in mammals? To answer this long- standing question, we will characterize the threespine stickleback (Gasterosteus aculeatus) MMC response. Clarifying MMC function will yield important insights into the basis of humoral adaptive immunity in fish, and the evolutionary origins of vertebrate adaptive immunity. The first Aim of this project will investigate MMC function in response to immunization. To determine if MMCs are the site of B cell proliferation, immunized fish will be pulse labeled with BrdU so that proliferating BrdU+ cells can be localized. Next, to clarify whether somatic hypermutation occurs in MMCs, we will quantify immunoglobulin gene mutations in MMC-adjacent and non- adjacent B cells. To resolve if MMC function is indeed GC-like, we will compare the MMC response to immunization with T-dependent (NP-CGG) and T-independent (NP-dextran) antigens. The results of this first Aim will determine the suitability of the MMC as a biomarker of teleost humoral adaptive immunity, and whether the MMC is an “evolutionarily primitive” GC. Preliminary data strongly support our hypothesis that MMCs are GC-like. Consequently, in our second Aim we will apply our knowledge of MMC function to investigate the immuno-modulatory effects of a helminth parasite, Schistocephalus solidus. By combining experimental infection and immunization we will determine whether this tapeworm suppresses teleost adaptive immunity. We find that some stickleback genotypes are refractory to this immunosuppression. Using a combination of Quantitative Trait Locus (QTL) mapping, population genomics, and RNAseq analysis of gene expression we will locate the host loci responsible for variable parasite-mediated immune-modulation. We will then use the CRISPR/Cas9 system to modify these genes in cultured MMCs and evaluate their role in MMC function in vitro. In the final Aim, we will identify the helminth-derived factors that dampen MMC activity. Through QTL mapping we will identify the tapeworm-specific loci responsible for MMC suppression. To complement this approach, we will assess whether helminth excretory/secretory products directly modulate MMC gene expression. Clarifying the mechanistic basis of helminth-mediated immunomodulation in fish may reveal new approaches to regulate vertebrate immune function. This work will also generate a new and broadly applicable assay of teleost immunity, which has the potential to be used in the study of diverse fish (and other poikilotherm) species. Beyond expanding our view of vertebrate immunobiology, this work has implications for the disciplines of disease ecology, host-pathogen co-evolution, and vaccine development.