Teleost Fish Phagocytic B cells: Novel Roles in Inflammation and Homeostasis

智力特长：吞噬作用是真核细胞对大颗粒的吞噬。 作为动物免疫系统的组成部分，它在微生物的摄取和破坏以及凋亡和坏死细胞的清除中起着重要作用。微生物和坏死细胞的吞噬作用通常导致炎症，而凋亡细胞以非炎症方式被清除。在哺乳动物中，吞噬作用主要由特定的吞噬细胞类型进行;这些骨髓来源的“专职吞噬细胞”（“白色血细胞”）包括多形核白细胞（PMN，有时也称为粒细胞）、单核细胞和巨噬细胞。直到最近，人们还认为，在鱼类中，情况也是如此，即，粒细胞和巨噬细胞是鱼类的主要吞噬细胞。与这种信念相反，Sunyer博士的实验室在NSF的支持下进行的研究导致了令人兴奋的发现，即鱼类中的B细胞（抗体或免疫球蛋白的来源）具有强大的吞噬能力，并构成了这些物种中“专业吞噬细胞”的重要组成部分。这些令人惊讶的发现发表在《自然免疫学》杂志上，并成为2006年10月刊的封面。本更新项目建立在虹鳟鱼吞噬B细胞研究过程中的三个关键观察结果之上：1）基因表达和抗体染色分析表明，硬骨鱼的B细胞至少可以分为两个主要群体，一个表达IgM，另一个表达IgT（这些鱼特有的免疫球蛋白; 2）所有IgM+ B细胞表达显著水平的IL-1、TNF-α、C3 α R和C5 α R，一组在哺乳动物中限于骨髓来源的先天性炎性白细胞的促炎分子（即，巨噬细胞）; 3）鳟鱼IgM+ B细胞中磷脂酰丝氨酸受体（PSR）的鉴定和表达，这一发现表明这些淋巴细胞参与了凋亡细胞的去除（哺乳动物白细胞中的这种作用限于巨噬细胞和树突细胞）。基于上述观察结果，并且由于鱼类中B细胞的数量异常高，因此假设这些物种中的B细胞代表炎症反应的重要组分，并且通过参与凋亡细胞的清除而有助于体内平衡。因此，本项目的目的是：1，分析IgM+和IgT+淋巴细胞群体之间的表型、吞噬和杀菌差异。将使用血清学方法将IgM+ B细胞与IgT+ B细胞分离，以研究和比较这两种淋巴细胞群的吞噬和杀微生物活性。2，确定吞噬B细胞对炎症反应的贡献。为了解决鱼中的B细胞对炎性刺激有反应的假设，研究了各种促炎介质（例如，白细胞介素、TNF、考克斯-1和考克斯-2）的体内和体外检测将在用内源性（即，C5 a，C3 a）或外源性（即，微生物化合物、细菌）刺激。3.确定吞噬B细胞对清除凋亡和坏死细胞的贡献。为了检验鳟鱼IgM+ B细胞也可能参与凋亡细胞的非炎症清除的假设，将评估IgM+和IgT+ B细胞亚群吞噬鳟鱼凋亡和坏死细胞的能力，并与鳟鱼粒细胞和巨噬细胞的能力进行比较。更广泛的影响：PI将向美国兽医试剂网络提供纯化的IgT，其目标是产生自由分布的单克隆抗体，从而使整个免疫学研究团体受益。该项目将包括培训一名博士后研究员和一名研究生。此外，PI将继续每年参加在佛罗里达国际大学（FIU）举行的比较免疫学研讨会，该大学是一所少数民族服务机构，大多数参加研讨会的学生主要来自代表性不足的群体。在更广泛的层面上，本文提出的研究将通过对水产养殖业的贡献而造福社会，因为更好地了解鱼类免疫系统对于开发新的治疗剂和疫苗以预防鱼类疾病至关重要。PI将继续在国家和国际会议上展示其研究成果。

Intellectual Merit:Phagocytosis is the engulfment of large particles by eukaryotic cells. As an integral part of the immune system in animals, it plays an essential role in the uptake and destruction of microbes as well as in the clearance of apoptotic and necrotic cells. Phagocytosis of microbes and necrotic cells generally leads to inflammation, whereas apoptotic cells are cleared in a non-inflammatory manner. In mammals, phagocytosis is primarily carried out by certain cell types that are specialized for phagocytosis; these ''professional phagocytes,'' of myeloid origin (''white blood cells''), include polymorphonuclear leukocytes(PMNs, also sometimes called granulocytes), monocytes and macrophages. Until recently, it was thought that, in fish, the same was true, i.e., that granulocytes and macrophages were the main phagocytic cells in fish. Contrary to this belief, studies carried out in Dr. Sunyer''s laboratory with prior NSF support led to the exciting finding that B cells (immune cells that are the source of antibodies, or immunoglobulins) in fish have potent phagocytic abilities and comprise a significant fraction of the ''professional phagocytes'' in these species. These surprising findings were reported in Nature Immunology and made the cover of the 2006 October issue. This renewal project builds on three key observations made during the study of phagocytic B cells in rainbow trout: 1) gene expression and antibody staining analysis suggesting that B cells of teleosts can be divided at least into two major populations, one expressing IgM and the other expressing IgT (an immunoglobulin unique to these fish; 2) The finding that all IgM+ B cells express significant levels of Il-1, TNF-a, C3aR and C5aR, a group of pro-inflammatory molecules that in mammals is restricted to innate inflammatory leukocytes of myeloid origin (i.e., macrophages); 3) The identification and expression of the phosphatidylserine receptor (PSR) in trout IgM+ B cells, a finding that suggests the involvement of these lymphocytes in the removal of apoptotic cells (this role in mammalian leukocytes is restricted to macrophages and dendritic cells). Based on the aforementioned observations and because of the unusually high numbers of B cells in fish, it is hypothesized that B cells in these species represent an essential component of the inflammatory response and contribute to homeostasis by participating in the clearance of apoptotic cells. Thus, the aims of this project are: 1, to analyze phenotypic, phagocytic and bactericidal differences between IgM+ and IgT+ lymphocyte populations. Serological methods will be used to separate IgM+ B cells from IgT+ B cells, in order to investigate and compare the phagocytic and microbicidal activities of these two lymphocyte populations.2, to determine the contribution by phagocytic B cells to the inflammatory response. To address the hypothesis that B cells in fish respond to inflammatory stimuli, the expression of various pro-inflammatory mediators (e.g., interleukins, TNFs, COX-1 and COX-2) in vivo an in vitro will be analyzed after stimulation of fish or cells with endogenous (i.e., C5a, C3a) or exogenous (i.e., microbial compounds, bacteria) stimuli. 3, to determine the contribution of phagocytic B cells to the clearance of apoptotic and necrotic cells. To test the hypothesis that trout IgM+ B cells may be involved also in the non-inflammatory clearance of apoptotic cells, the capacity of IgM+ and IgT+ B cell subsets to phagocytose trout apoptotic and necrotic cells will be assessed and compared to that of trout granulocytes and macrophages.Broader Impacts:The PI will make available purified IgT to the US Veterinary Reagent Network with the goal of generating monoclonal antibodies that will be freely distributed, thus benefiting the entire immunology research community. This project will involve the training of a postdoctoral fellow and a graduate student. In addition, the PI will continue his annual participation in the Comparative Immunology Symposium held at Florida International University (FIU), a minority serving institution where most of the attending students are primarily from underrepresented groups. On a broader level, the studies proposed here will benefit society by contributing to the aquaculture industry, since a better knowledge of the fish immune system is essential to the development of new therapeutic agents and vaccines to prevent fish diseases. The PI will continue presenting the results of his research in national and international conferences.