Unravelling the Lymph Node–bone Axis: How Immune Crosstalk Shapes Fracture Healing

Bone fracture repair exhibits a unique, scar-free healing process. However, complications such as delayed healing or nonunion formation can arise, influenced by the patient's immune status. The lymphatic system plays a critical role in fluid homeostasis, waste clearance, and immune responses. Recently, bone associated lymphatic vessels have been identified and visualized. While benefits of post-fracture lymphatic drainage and expansion following genotoxic stress are documented, the role of lymphatic vessels and draining lymph nodes (dLNs) in fracture healing, especially in regards to immune – and injury responses, remains unclear. This study investigates the function, capacity, and potential benefit of the lymphatic system and dLNs in fracture settings. This study was approved by the State Office for Health and Social Affairs (LAGeSo). A murine osteotomy model was employed, incorporating mice with varying immune experiences induced by differential exposure to natural antigens. Congenic regulatory T cells (Tregs) were adoptively transferred prior to osteotomy and analyzed on days 3 and 7 post-surgery across multiple tissues. The dLN was surgically removed during or on different timepoints after the osteotomy to assess longitudinal effects on bone healing, as well as on systemic and local immune cells. To do so, histology, high-parameter flow cytometry, micro-computed tomography and RNA bulk sequencing were applied. After osteotomies, lymphatic vessel formation in the surrounding tissue and fracture area, along with swelling of dLNs were observed. Within the dLN, pronounced cell proliferation, inflammatory signaling, cytokine production, and cellular interactions were observed. Adoptively transferred Tregs predominantly migrated to the dLN, with their phenotype and functionality modulated in dependence of the individual immune status of each animal. Removal of the dLN altered bone healing progression and led to increased healing especially in immune experienced animals. Moreover, this affected systemic immune cell regulatory functionality. Collectively, our findings highlight a critical lymph node–bone axis in fracture settings, demonstrating its significance in immune responses during bone healing. Signaling of immune cells within the draining lymph node affects healing onset and progression. Simultaneously, immune cell functionality is affected by a fracture itself but also by individual immune experience. The lymphatic system emerges as a pivotal regulator of immune responses to bone injuries while allowing the crosstalk between bone and lymph node.