New Animal Model for Studies of mucosal Immunity and IgA Nephropathy

描述（由申请人提供）：其特征是系膜IgA1免疫沉积，起源于含有异常糖基化IgA1的循环免疫复合物，即IgA1与半乳糖（Gal）缺陷的o -聚糖。一些证据表明，在异常糖基化、含有异常糖基化IgA1的免疫复合物的形成、它们在系膜中的沉积和IgAN的肾损伤之间存在直接的因果关系。我们确定IgAN患者的IgA1生成细胞分泌的IgA1具有gal缺陷的O-聚糖，这种异常是由于特异性糖基转移酶的表达/活性失调。这些发现表明，缺乏IgA1在IgAN的发病机制中起着关键作用。了解IgAN中IgA1碳水化合物含量变化的分子基础，对于定义导致IgAN中异常聚糖合成的基本缺陷至关重要。然而，由于IgA1仅存在于人类和类人猿灵长类动物中，因此无法从人类粘膜组织、淋巴结或骨髓中获得足够的感兴趣细胞，并且缺乏动物模型，这些研究一直受到挫折。作为开发小鼠模型的第一步，我们现在已经证明，在体外由人缺乏gal的IgA1和抗聚糖IgG制备并静脉注射到裸鼠体内的免疫复合物在系膜中沉积并诱导血尿和蛋白尿。由于小鼠不具有带有铰链区o -聚糖的IgA，我们假设，产生含有人类铰链区IgA的转基因小鼠将导致小鼠含有o -链聚糖的IgA，可以被操纵成类似于异常的人类IgA1。这种转基因小鼠品系将为研究o -聚糖在IgAN和粘膜免疫中的作用提供新的工具。我们拟利用人IgA1的铰链区构建一种产生IgA的敲入转基因小鼠菌株，评估其o糖基化，并通过抑制关键酶- 21,3-半乳糖基转移酶，在小鼠转基因IgA上产生缺乏gal的o聚糖。此外，我们将确定由小鼠转基因gal缺陷IgA和甘聚糖特异性IgG组成的免疫复合物是否沉积在小鼠肾系膜中引起肾病。我们将通过对肾脏组织进行组织学分析以及对尿液和血清进行实验室分析，进一步发展和验证这一真正反映人类疾病的第一个动物模型。这种转基因小鼠将为检测异常O糖基化IgA的产生及其系膜沉积和清除的遗传和生化机制开辟新的可能性。相关性：IgAN是最常见的原发性肾小球肾炎，20% - 40%的患者可导致终末期肾功能衰竭。目前对IgAN发病机制的理解存在空白，这是发展IgAN特异性治疗的主要障碍。本研究将为今后IgAN相关生理动物模型的建立奠定基础，促进对人类IgAN发病机制的认识。

DESCRIPTION (provided by applicant): is characterized by mesangial IgA1 immune deposits that originate from circulating immune complexes containing aberrantly-glycosylated IgA1, i.e., IgA1 with galactose (Gal)-deficient O-glycans. Several lines of evidence suggest a direct causal relationship between aberrant glycosylation, the formation of immune complexes containing aberrantly glycosylated IgA1, their deposition in the mesangium, and renal injury in IgAN. We determined that IgA1-producing cells of IgAN patients secrete IgA1 with Gal-deficient O- glycans and that this aberrancy is due to dysregulation in expression/activity of specific glycosyltransferases. These findings indicate that Gal-deficient IgA1 plays a pivotal role in the pathogenesis of IgAN. An understanding of the molecular basis for the variations in the carbohydrate content of IgA1 in IgAN is essential for the definition of the fundamental defects that result in the synthesis of the aberrant glycans in IgAN. Such studies have been frustrated, however, by the fact that it is not feasible to obtain sufficient cells of interest from human mucosal tissues, lymph nodes, or bone marrow and by the lack of animal models, as IgA1 is present exclusively in humans and hominoid primates. As a first step toward developing a murine model, we have now shown that immune complexes, prepared in vitro between human Gal-deficient IgA1 and anti-glycan IgG and i.v.-injected to nude mice, deposit in the mesangium and induce hematuria and proteinuria. Because mice do not have IgA with hinge-region O-glycans, we hypothesized that generating transgenic mice with IgA containing the human hinge region would result in murine IgA containing O-linked glycans that can be manipulated to resemble the aberrant human IgA1. Such a transgenic mouse strain would represent a new tool for studies of the role of the O-glycans in IgAN and in mucosal immunity. We propose to generate a knock-in transgenic mouse strain producing IgA with hinge region from human IgA1, assess its O-glycosylation, and, by inhibiting the key enzyme - 21,3-galactosyltransferase, to generate Gal-deficient O-glycans on the murine transgenic IgA. Furthermore, we will determine whether the immune complexes composed of murine transgenic Gal-deficient IgA and glycan-specific IgG deposit in renal mesangium of mice causing nephropathy. We will further develop and validate this first animal model truly reflecting the human disease by performing histological analysis of renal tissue and laboratory analysis of urine and serum. This transgenic mouse will open new possibilities for testing genetic and biochemical mechanisms involved in production of aberrantly O- glycosylated IgA and its mesangial deposition and clearance. Relevance: IgAN is the most common primary glomerulonephritis and leads to end-stage renal failure in 20% to 40% of patients. The current gaps in the understanding of the pathogenesis of IgAN represent a major barrier to the development of IgAN-specific treatments. The proposed studies will provide the foundation for the future development of a relevant physiological animal model of IgAN that will advance the understanding of the pathogenesis of human IgAN. PUBLIC HEALTH RELEVANCE: We propose to develop a murine model of IgAN. We will generate transgenic mice in which IgA will include the human hinge region containing O-linked glycans. We will further generate Gal-deficient O-glycans on the murine transgenic IgA and use anti-glycan IgG to form pathogenic complexes that will deposit in the kidneys.