Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two‐photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia‐inducible factor activity. We observe that hypoxia decreases ten‐eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen‐dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.

Oxygen is a key regulator of both development and homeostasis. To study the role of oxygen, a variety of in vitro and ex vivo cell and tissue models have been used in biomedical research. However, because of ambiguity surrounding the level of oxygen that cells experience in vivo, the cellular pathway related to oxygenation state and hypoxia have been inadequately studied in many of these models. Here, we devised a method to determine the oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy with the cell-penetrating phosphorescent probe, BTPDM1. Phosphorescence lifetime imaging revealed the physiological level of oxygen tension in monocytes to be 5.3% in live mice exposed to normal air. When the mice inhaled hypoxic air, the level of oxygen tension in bone marrow monocytes decreased to 2.4%. By performing in vitro cell culture experiment within the physiological range of oxygen tension, hypoxia changed the molecular phenotype of monocytes, leading to enhanced the expression of CD169 and CD206, which are markers of a unique subset of macrophages in bone marrow, osteal macrophages. This current study enables the determination of the physiological range of oxygen tension in bone marrow with spatial resolution at a cellular level and application of this information on oxygen tension in vivo to in vitro assays. Quantifying oxygen tension in tissues can provide invaluable information on metabolism under physiological and pathophyisological conditions. This method will open new avenues for research on oxygen biology.

Osteoclasts are derived from the monocyte-macrophage lineage of the bone marrow and are bone-resorbing cells essential for bone homeostasis. Osteoclasts contain more mitochondria and require more oxygen to support the energy demands associated with bone resorption. However, local oxygen distribution within bone marrow has not been completely understood. Intravital two-photon microscopy is a powerful tool for investigating biological processes in live animals. In this study, we performed two-photon phosphorescence lifetime imaging to characterize the oxygen tension in osteoclasts.Two-photon-enhanced phosphorescent probe, iridium complex (BTPDM1), was injected into knock-in mice in which EGFP is expressed in osteoclast lineage cells. The emissive triplet state of the BTPDM1 is sensitive to local oxygen tension. By measuring the phosphorescence from EGFP positive cells, oxygen tension in osteoclasts was determined. Furthermore, we quantitatively analyzed changes in oxygen tension in osteoclasts by measuring the phosphorescence decay time in EGFP positive cells. Thus, we have succeeded in detecting oxygen tension in vivo. This method is applicable to various cell types for determining local oxygen tension inside bone marrow.