Generation of Novel Osteolineage VHL Conditional Knockout Mice to Study B Cell Microenvironments

生成新型骨谱系 VHL 条件敲除小鼠以研究 B 细胞微环境

• 批准号: 10368064
• 负责人: JENNIFER O MANILAY
• 金额: $ 18.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-08 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10368064
• 关键词: Adoptive Transfer; Adult; Affect; Aging; Anatomy; Antibody Formation; Antibody Response; Architecture; Autoimmunity; B-Cell Antigen Receptor; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Biology; Biomedical Engineering; Blood Vessels; Blood flow; Bone Marrow; Bone Matrix; Calvaria; Cell Communication; Cell Maturation; Cells; Chemicals; Chimera organism; Communities; Data; Defect; Development; Endothelium; Flow Cytometry; Fluorescence; Future; Gene Deletion; Gene Rearrangement; Generations; Genes; Hematopoietic; Hematopoietic stem cells; Homeostasis; Hypoxia; Image; Immune; Immunology; Impairment; Knockout Mice; Knowledge; Label; Location; Longevity; LoxP-flanked allele; Lymphocyte; Maintenance; Mature B-Lymphocyte; Measures; Mesenchymal Stem Cells; Microscopy; Molecular; Morphology; Mus; Osteoblasts; Osteocytes; Oxygen; Pathway interactions; Pattern; Permeability; Pharmaceutical Preparations; Proteins; Reporter; Resolution; Role; Self Tolerance; Skeletal system; Sorting - Cell Movement; Specificity; Spleen; Stromal Cells; Structure; Structure of germinal center of lymph node; Supporting Cell; System; Tamoxifen; Terminator Codon; Time; Tissues; Transgenes; Transplantation; VHL gene; Work; adaptive immune response; applied biomedical research; base; bone; bone cell; bone imaging; bone mass; cell motility; cell type; conditional knockout; conditioning; dentin matrix protein 1; ex vivo imaging; high resolution imaging; in vivo imaging; in vivo monitoring; intravital imaging; intravital microscopy; irradiation; long bone; lymph nodes; microCT; mouse model; novel; physical property; postnatal; progenitor; protein expression; receptor; response; skeletal; stem cell survival; stem cells; three-dimensional modeling; tool; two-photon

PROJECT SUMMARY Our long-term objective is to characterize the microenvironments that influence B lymphocyte development during altered bone homeostasis. B lymphocytes are required for the production of antibodies that are crucial for a robust adaptive immune response. Studies from many groups have determined the stages of maturation, the molecular mechanisms of B cell antigen receptor gene rearrangements and generation of receptor diversity, and mechanisms of self-tolerance in conventional B cells. The microenvironments in the bone marrow (BM) that support B cell progenitors and mature B cells have been described (2-7), and it appears that development of robust antibody responses from B cells is influenced by the relatively low oxygen levels in the germinal centers of the spleen and lymph nodes (11). However, relatively little is known about the mechanisms by which oxygen levels regulate B cell development in the bone marrow. We aim to fill the scientific knowledge gap on the microenvironmental niches that influence the maintenance and function of adult B cell subsets. In the course of our studies to examine the effects of altered bone homeostasis on immune cell development, we discovered that B cell development was severely impaired in mice in which a hypoxia response pathway gene, von-Hippel Lindau (Vhl), is conditionally deleted in osteocytes (12). We hypothesized that Vhl-deficiency in osteocytes results in structural and molecular changes in the vascular architecture in the BM microenvironments, which may alter oxygen tension to levels that support aberrant B development. In support of this, our preliminary analyses suggest that deletion of Vhl in osteocytes results in increased permeability in the vasculature. However, off-target expression of the Dmp1-Cre transgene in osteoblasts clouds this interpretation. Other tissue-specific Cre mice traditionally used to study the skeletal system display off-target or broader expression than expected, diminishing their utility in defining the role of gene deletion in specific cell types. In addition, current mouse models do not permit the study of Vhl-deficiency on immune cell development in postnatal and adult BM microenvironments. MSCs are osteocyte precursors, can support B cell development in the BM (13) but the specific and unique contributions of the MSCs, OBs and OCYs to B cell maturation is unclear. Therefore, we propose to generate a novel inducible Vhl-conditional knockout mouse model, to assess the cell-extrinsic effects of Vhl deletion in MSCs on B cell development. We aim to combine transplantation strategies with high-resolution intravital (live) microscopy of the calvaria and ex vivo long bone imaging to characterize the Vhl-deficient bone marrow microenvironment in order to identify changes that may influence B cell development, such as oxygen tension, blood vessel type and structure, the presence and location of stromal cells or hematopoietic cells, and changes in bone morphology. Finally, we aim to analyze the niches longitudinally over time, in order to determine how the distinct physical properties in the bone change and affects the progression of B cell developmental defects in Vhl-deficient bones as a function of time. This information could be applied to future studies of the effects of irradiation, myeloablative conditioning, or bone-building drugs on bone marrow niches and immune cell development.

项目概要 我们的长期目标是表征改变过程中影响 B 淋巴细胞发育的微环境。 骨稳态。 B 淋巴细胞是产生抗体所必需的，而抗体对于强大的适应性免疫至关重要 回复。许多团体的研究已经确定了 B 细胞抗原的成熟阶段、分子机制 受体基因重排和受体多样性的产生以及传统B中的自我耐受机制 细胞。骨髓 (BM) 中支持 B 细胞祖细胞和成熟 B 细胞的微环境 描述 (2-7)，并且 B 细胞产生的强大抗体反应似乎受到相对 脾脏和淋巴结生发中心的氧含量低 (11)。然而，人们对此知之甚少 氧气水平调节骨髓中 B 细胞发育的机制。我们的目标是填补科学 影响成体 B 细胞亚群维持和功能的微环境生态位的知识差距。在 在我们研究改变骨稳态对免疫细胞发育的影响的过程中，我们发现 缺氧反应途径基因 von-Hippel Lindau 的小鼠 B 细胞发育严重受损 (Vhl) 在骨细胞中被有条件删除 (12)。我们假设骨细胞中的 Vhl 缺陷会导致结构性损伤 以及骨髓微环境中血管结构的分子变化，这可能会改变氧张力的水平 支持异常 B 发育。为了支持这一点，我们的初步分析表明，骨细胞中 Vhl 的缺失 导致脉管系统的通透性增加。然而，Dmp1-Cre 转基因的脱靶表达 成骨细胞掩盖了这种解释。其他传统上用于研究骨骼系统的组织特异性 Cre 小鼠 脱靶或比预期更广泛的表达，降低了它们在定义特定细胞中基因缺失的作用方面的效用 类型。此外，目前的小鼠模型不允许研究 Vhl 缺陷对出生后免疫细胞发育的影响 和成人骨髓微环境。 MSC 是骨细胞前体，可以支持 BM 中的 B 细胞发育 (13)，但 MSC、OB 和 OCY 对 B 细胞成熟的具体和独特贡献尚不清楚。因此，我们建议 生成一种新型诱导型 Vhl 条件敲除小鼠模型，以评估 Vhl 缺失的细胞外在效应 MSC 对 B 细胞发育的影响。我们的目标是将移植策略与高分辨率活体（实时）结合起来 颅骨显微镜检查和离体长骨成像以表征 Vhl 缺陷骨髓 微环境，以确定可能影响 B 细胞发育的变化，例如氧压、血管 类型和结构、基质细胞或造血细胞的存在和位置以及骨形态的变化。 最后，我们的目标是随着时间的推移纵向分析利基，以确定不同的物理特性如何 骨骼变化并影响 Vhl 缺陷骨骼中 B 细胞发育缺陷的进展，作为时间的函数。 该信息可应用于未来对辐射、清髓性调节或骨构建效果的研究 影响骨髓生态位和免疫细胞发育的药物。