Impact of the adaptive immune system in bone fracture healing

Bone fracture healing is a highly complex process involving different cell and tissue types. It consists of different but overlapping phases that have to be well organized and regulated for a successful healing outcome. Bone tissue possesses the remarkable capacity to fully regenerate after injury. However, in 10–15 % of patients, unsuccessful bone repair is still a preset problem that has to be considered. The bone and the immune system are highly interconnected. Components of the adaptive immune system play an indispensable role in bone regeneration, probably due to their cellular interactions and their secreted cytokine profile. For CD8+ terminally differentiated effector memory T cells (TEMRA), a negative correlation of an increased amount and an impaired bone repair process was already shown in patients. The here presented PhD thesis focused on a deeper analysis of the impact of the adaptive immunity in bone regeneration in order to better understand their influence in tissue repair. More precisely, the interaction of CD4+ regulatory T cells (TReg) during fracture healing was evaluated. TReg are a promising candidate for playing a positive modulating role in bone repair. However, their sole influence in bone regeneration was not analyzed so far. In a murine osteotomy model of a non-critical size defect, the spatial and temporal distribution of distinct immune and bone cell subsets was analyzed within the healing tissue of the osteotomized bone. Cells of the innate (macrophages) and adaptive (T and B cells) immunity are detectable throughout the whole healing cascade in the injured area. The appearance of anti-inflammatory M2 macrophages coincided with the formation of new vessels which reestablished the necessary oxygen and nutrition supply. M2 macrophages further co-localized with osteoblasts in the fracture gap indicating an orchestrating function of macrophages for the production of bone by osteoblasts. T and B cells also directly co-localized with osteoblasts and osteoclasts, respectively, in the early fracture healing phase. These results highlight the interconnectivity of immune and bone cells during regeneration which was not analyzed and shown in any other study before. By adoptive transfer of freshly isolated murine regulatory T cells prior to osteotomy, an immunomodulatory approach to improve bone healing was conducted. The activation status of the adaptive immunity prior to surgery was included into this analysis. Mice possessing an unexperienced immune system (housing in an SPF environment) showed a consistent improved healing outcome after adoptive transfer of TReg in comparison to the control group 21 days post-osteotomy. However, mice with a more experienced immune system (semi-sterile housing environment) receiving an adoptive TReg transfer demonstrated a controversial healing outcome after 21 days: half of the mice showed a significantly improved and the other half a significantly poorer healing outcome. In the mice with a poorer healing outcome, a higher ratio between CD8+ effector T cells and TReg was observed. In a following proof of concept study, a pre-osteotomy defined ratio of CD8+ effector T cells and TReg could predict the healing outcome 21 days after adoptive transfer of TReg and osteotomy. A potential positive impact of TReg in bone repair was confirmed in this study. However, the tremendous impact of the environment and thereby of the immune status prior to immunomodulation was also clearly demonstrated. Transferring these findings to the clinical situation reveals how individually each patient might react to the same immunomodulatory therapy and how this therapy could affect the results of the treatment in an unfavorable way. Hence, for the clinic, it is even more important to develop and to apply patient based treatment approaches in the field of immunotherapy considering the individual immune status of each patient prior to treatment.