Osteopetrosis is an inherited metabolic disease, characterized by increased bone density and narrow marrow cavity. Patients with severe osteopetrosis exhibit abnormal bone brittleness, anemia, and infection complications, which commonly cause death within the first decade of life. Pathologically, osteopetrosis impairs not only the skeletal system, but also the hemopoietic and immune systems during development, while the underlying osteoimmunological mechanisms remain unclear. Osteoclastic mutations are regarded as the major causes of osteopetrosis, while osteoclast non-autonomous theories have been proposed in recent years with unclear underlying mechanisms. Retinoic acid (RA), the metabolite of Vitamin A, is an essential requirement for skeletal and hematopoietic development, through the activation of retinoic acid signaling. RA can relieve osteopetrosis symptoms in some animal models, while its effect on bone health is still controversial and the underlying mechanisms remain unclear. In this study, we constructed an osteoblast-specific inhibitory retinoic acid signaling mouse model and surprisingly found it mimicked the symptoms of osteopetrosis found in clinical cases: dwarfism, increased imperfectly-formed trabecular bone deposition with a reduced marrow cavity, thin cortical bone with a brittle skeleton, and hematopoietic and immune dysfunction. Micro-CT, the three-point bending test, and histological analysis drew a landscape of poor bone quality. Single-cell RNA sequencing (scRNA-seq) of the femur and RNA-seq of osteoblasts uncovered an atlas of pathological skeletal metabolism dysfunction in the mutant mice showing that osteogenesis was impaired in a cell-autonomous manner and osteoclastogenesis was impaired via osteoblast-osteoclast crosstalk. Moreover, scRNA-seq of bone marrow and flow cytometry of peripheral blood, spleen, and bone marrow uncovered pathology in the hematopoietic and immune systems in the mutant mice, mimicking human osteopetrosis. Results showed that hematopoietic progenitors and B lymphocyte differentiation were affected and the osteoblast-dominated cell crosstalk was impaired, which may result from transcriptional impairment of the ligands Pdgfd and Sema4d. In summary, we uncovered previously unreported pathogenesis of osteopetrosis-like disorder in mice with skeletal, hematopoietic, and immune system dysfunction, which was induced by the inhibition of retinoic acid signaling in osteoblasts, and sheds new insights into a potential treatment for osteopetrosis.

骨硬化症是一种遗传性代谢性疾病，其特征是骨密度增加和骨髓腔狭窄。严重骨硬化症患者表现出异常的骨脆性、贫血和感染并发症，这通常会导致在生命的最初十年内死亡。从病理学上讲，骨硬化症不仅损害骨骼系统，还损害发育过程中的造血和免疫系统，而潜在的骨免疫机制仍不清楚。破骨细胞突变被认为是硬化骨症的主要原因，而近年来提出了破骨细胞非自主性理论，但其潜在机制尚不清楚。视黄酸 （RA） 是维生素 A 的代谢物，通过激活视黄酸信号传导，是骨骼和造血发育的基本需求。RA 可以缓解一些动物模型中的骨硬化症状，但其对骨骼健康的影响仍存在争议，潜在机制仍不清楚。在这项研究中，我们构建了一个成骨细胞特异性抑制性视黄酸信号小鼠模型，并令人惊讶地发现它模拟了临床病例中发现的骨硬化症症状：侏儒症、未完美形成的小梁骨沉积增加伴骨髓腔缩小、皮质骨薄且骨骼脆性，以及造血和免疫功能障碍。显微 CT、三点弯曲试验和组织学分析得出了骨质量差的景象。股骨单细胞 RNA 测序 （scRNA-seq） 和成骨细胞 RNA-seq 揭示了突变小鼠病理骨骼代谢功能障碍的图谱，显示成骨以细胞自主方式受损，而破骨细胞生成通过成骨细胞-破骨细胞串扰受损。 此外，骨髓的 scRNA-seq 和外周血、脾脏和骨髓的流式细胞术揭示了突变小鼠造血和免疫系统的病理，类似于人类骨硬化症。结果显示，造血祖细胞和 B 淋巴细胞分化受到影响，成骨细胞显性细胞串扰受损，这可能是由于配体 Pdgfd 和 Sema4d 的转录受损所致。总之，我们发现了以前未报道的骨骼、造血和免疫系统功能障碍小鼠中骨硬化样疾病的发病机制，这是由成骨细胞中视黄酸信号的抑制诱导的，并为骨硬化症的潜在治疗方法提供了新的见解。