Development of an HLA-DP2 Transgenic Murine Model of Chronic Beryllium Disease

慢性铍病 HLA-DP2 转基因小鼠模型的建立

• 批准号: 8223751
• 负责人: Andrew P. Fontenot
• 金额: $ 21.63万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223751
• 关键词: Alleles; Alveolitis; Amino Acids; Animal Disease Models; Animal Model; Animals; Antigens; Beryllium; Binding; Binding Sites; Blood; Breathing; C3H/HeJ Mouse; C57BL/6 Mouse; CD4 Positive T Lymphocytes; Cells; Charge; Chronic berylliosis; Data; Development; Disease; Disease susceptibility; Exposure to; FVB/N Mouse; Fibrosis; Generations; Genetic Predisposition to Disease; Glutamic Acid; Granulomatous; HLA-DP Antigens; HLA-DP2; HLA-DP4; HLA-DPB1 gene; Hilar; Histocompatibility Antigens Class II; Human; Immune System Diseases; Immune response; Inbred Strains Mice; Individual; Inflammation; Inflammatory Response; Knowledge; Lead; Link; Lung; Lung diseases; Lymphocyte; Lysine; Mediating; Metals; Modeling; Mononuclear; Mus; Nature; Oxides; Pathology; Patients; Positioning Attribute; Public Health; Publishing; Research Personnel; Risk; Sampling; Site; Spleen; Structure; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Threonine; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Workplace; animal model development; base; cytokine; human disease; human subject; interstitial; lymph nodes; lymphocyte proliferation; mouse model; response

DESCRIPTION (provided by applicant): Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium exposure in the workplace and is characterized by the accumulation of beryllium-specific CD4+ T cells in the lung. Disease susceptibility has been linked to HLA-DPB1 alleles, including DPB1*0201, that possess a glutamic acid residue at position 69 of the HLA-DP2 2-chain (2Glu69). We have shown that beryllium directly binds to HLA-DP2 molecules and that the majority of beryllium-specific CD4+ T cells recognize beryllium in the context of HLA- DP. Importantly, the DP molecules that present beryllium match those implicated in disease susceptibility, confirming that the HLA contribution to disease is based on the ability of those molecules to bind and present beryllium to T cells. We have recently crystallized HLA-DP2 and identified a potential beryllium binding site involving ¿Glu69 and two other invariant glutamic acids at positions ¿26 and ¿68. Functional studies confirmed that beryllium recognition was dependent on this cluster of negative charges. To date, no animal model of beryllium-induced disease exists. Previous attempts to generate a murine model failed to document a beryllium-specific adaptive immune response. Importantly, mice lack a major histocompatibility complex class II (MHCII) molecule of similar sequence and structure to HLA-DP2. Thus, we hypothesize that the generation of a humanized transgenic mouse containing the cluster of negative charges unique to HLA-DP2 will result in a disease-specific murine model with pathology reflective of the human disease. HLA-DP2 and -DP4 transgenic mice on an FVB/N background have been generated by researchers at NIOSH. However, these mice express murine MHC and lack human CD4, an important coreceptor for optimal T cell activation. No studies characterizing the response of these mice to inhaled beryllium have been published. Preliminary data show peribronchovascular mononuclear cell infiltrates occurring only in beryllium-exposed HLA-DP2 transgenic mice after beryllium oxide (BeO) exposure, strongly suggesting that the inflammatory response is due to the presence of the HLA-DP2 transgene and supporting our hypothesis that ¿Glu69-containing HLA-DP molecules are required for the generation of a murine model of beryllium-induced disease. In the first specific aim, we will re-derive the HLA-DP2 transgene in C57BL/6 mice lacking murine MHCII molecules and expressing human CD4. In the second aim, we will directly compare the beryllium-induced adaptive immune response in these animals and DP2-expressing FVB/N mice, testing the hypothesis that stabilization of the interaction between HLA-DP2 and T cell receptor with the human CD4 coreceptor and elimination of the confounding effects of mouse MHCII will optimize the beryllium-specific immune response. Together, these studies will lead to a disease-specific murine model, targeting the exact molecule necessary for the development of a human disease. PUBLIC HEALTH RELEVANCE: The development of an HLA-DP2 transgenic mouse model of beryllium-induced disease will enhance our ability to define the precise environmental antigen causing a human granulomatous lung disease and to determine how metal antigens trigger an immune response.

描述(申请人提供)：慢性铍病(CBD)是一种因工作场所接触铍而引起的肺部肉芽肿性疾病，其特征是特定于铍的CD4+T细胞在肺内积聚。疾病的易感性与包括DPB1*0201在内的人类白细胞抗原-DPB1等位基因有关，该等位基因在人类白细胞抗原-DP2 2-链(2Glu69)第69位具有谷氨酸残基。我们已经证明，铍直接与人类白细胞抗原DP2分子结合，并且大多数铍特异的CD4+T细胞在人类白细胞抗原-二聚体的背景下识别铍。重要的是，呈现铍的DP分子与那些与疾病易感性有关的分子相匹配，证实了人类白细胞抗原对疾病的贡献是基于这些分子与T细胞结合并呈现铍的能力。我们最近结晶了人类白细胞抗原-DP2，并确定了一个潜在的铍结合部位，包括26位和68位的Glu69和另外两个不变谷氨酸。功能研究证实，铍的识别依赖于这一簇负电荷。到目前为止，还没有铍诱导的疾病的动物模型。之前试图建立小鼠模型的尝试未能证明铍特异的适应性免疫反应。重要的是，小鼠缺乏一种主要的组织相容性复合体II(MHCII)分子，其序列和结构与人类白细胞抗原DP2相似。因此，我们假设，含有人类白细胞抗原-DP2特有的负电荷簇的人源化转基因小鼠的产生将导致具有反映人类疾病的病理学的疾病特异性小鼠模型。NIOSH的研究人员已经培育出了FVB/N背景下的HL-DP2和-DP4转基因小鼠。然而，这些小鼠表达小鼠的MHC，并且缺乏人的CD4，这是一种重要的辅助受体，用于最佳T细胞的激活。还没有关于这些小鼠对吸入铍的反应的研究发表。初步数据显示，只有在铍暴露后的转基因小鼠中才会出现支气管血管周围单个核细胞的浸润，这有力地表明炎症反应是由于存在转基因的HLA-DP2，并支持了我们的假设，即含有Glu69的HLA-DP分子是建立铍诱导的疾病小鼠模型所必需的。在第一个特定目标中，我们将在缺乏小鼠MHCII分子和表达人CD4的C57BL/6小鼠中重新获得HLA-DP2转基因。在第二个目标中，我们将直接比较铍在这些动物和表达DP2的FVB/N小鼠中诱导的适应性免疫反应，验证这样的假设：稳定人类白细胞抗原-DP2和T细胞受体与人CD4共受体之间的相互作用，消除小鼠MHCII的混杂效应将优化铍特异的免疫反应。总而言之，这些研究将导致一种疾病特异性小鼠模型，瞄准人类疾病发展所需的确切分子。 公共卫生相关性：铍诱导疾病的人类白细胞抗原-DP2转基因小鼠模型的发展将增强我们确定导致人类肉芽肿性肺部疾病的准确环境抗原的能力，并确定金属抗原如何触发免疫反应。