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.

项目摘要 我们的长期目标是表征在改变的免疫应答过程中影响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，而抗体对强大的适应性免疫至关重要 反应许多研究小组已经确定了成熟阶段，B细胞抗原的分子机制 受体基因重排和受体多样性的产生，以及常规B中自身耐受的机制 细胞支持B细胞祖细胞和成熟B细胞的骨髓（BM）中的微环境已经被研究。 如（2-7）所述，并且似乎来自B细胞的稳健抗体应答的发展受到相对免疫应答的影响。 脾脏和淋巴结的生殖中心的低氧水平（11）。然而，相对而言， 氧水平调节骨髓中B细胞发育的机制。我们的目标是填补科学 对影响成人B细胞亚群的维持和功能的微环境生态位的知识差距。在 在我们研究骨稳态改变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 (Vhl)在骨细胞中有条件地缺失（12）。我们假设骨细胞中的Vhl缺陷导致结构性骨坏死。 以及BM微环境中血管结构的分子变化，这可能会改变氧张力， 支持异常的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 导致脉管系统的渗透性增加。然而，Dmp 1-Cre转基因在大肠杆菌中的脱靶表达， 成骨细胞模糊了这种解释。传统上用于研究骨骼系统的其他组织特异性Cre小鼠显示 脱靶或比预期更广泛的表达，降低了它们在确定特定细胞中基因缺失作用方面的效用 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BM微环境。MSC是骨细胞前体，可支持BM中的B细胞发育（13），但 MSC、OB和OCYs对B细胞成熟的特异性和独特的贡献尚不清楚。因此，我们建议 产生一种新的可诱导的Vhl-条件性敲除小鼠模型，以评估Vhl缺失在小鼠中的细胞外源性作用。 MSC对B细胞发育的影响。我们的目标是将联合收割机移植策略与高分辨率活体（活体） 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，以便识别可能影响B细胞发育的变化，如氧张力、血管张力 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生态位，以确定不同的物理性质如何在 骨变化，并影响作为时间函数的Vhl缺陷骨中B细胞发育缺陷的进展。 这一信息可以应用于未来的研究辐射的影响，清髓性条件反射，或骨建设 骨髓小生境和免疫细胞发育的药物。