The regenerative face of the immune system for musculoskeletal healing
Delayed bone healing is still an unsolved clinical problem as confirmed by interviewing orthopaedic clinicians from Charité’s orthopaedic and trauma department. These clinicians state that further therapeutic options to treat difficult bone healing scenarios would be highly desirable. These difficult healing scenarios are encountered when treating multi-fragmentary open fractures of long bones prone to infection. Likewise, among others, pathological fractures with unknown underlying comorbidities or when confronted with non-unions or in complex endo-prosthetic settings can demand novel therapeutic strategies. With more than 160 million new fractures worldwide each year and over 400 million patients suffering from consequences of a bone injury this very real unsolved clinical problem of supporting bone regeneration is very present 1.
Bone as an organ is one of the rare examples of being capable of complete regeneration in form and function after injury. Thus, bone is ideal to study regeneration in order to harness newly gained knowledge with the goal to transfer that knowledge towards new clinical treatment approaches.
More than 15 years ago, we started to investigate the very early bone healing phase, more precisely the first 60 hours after injury. Using a sheep model, we discovered that among all the cells analysed the CD8 positive T cells of the adaptive immune system showed a clear difference in quantity when comparing normal and delayed healing. In normal healing, this cell population decreased by more than 50% during the first 60 hours while the percentage remained high in our delayed healing model 2,3. This was our first indicator that the adaptive immune system could play a pivotal role in the success or failure of bone regeneration.
A small clinical cohort of patients with a fracture of the tibia was subsequently analysed by Geissler et al.. They used x-ray, functionality tests and blood and hematoma samples to characterise differences in patients with normal or delayed healing. Interestingly, the CD8 positive T cell subset again showed significant differences when comparing the groups. More precisely, the terminally differentiated effector memory CD8 positive T cells could be elucidated to play a major role in delayed healing scenarios 4. Elevated percentages of this cell subset were observed in patients with delayed healing throughout the complete healing progression and remained elevated even after healing was completed. This indicated that high percentages of this very pro-inflammatory effector T cell subset could be a biomarker for disturbed healing. Indeed, this concept has been elaborated on and is currently validated in a multicentre phase III clinical study to confirm this new biomarker that would be able to identify patients in need of additional care in difficult bone healing scenarios (BioBone Study).
Our goal was now to find a way to help patients that have an intrinsic risk for delayed healing due to the high level of effector T cells. These cells accumulate in the bone hematoma after fracture and express high levels of pro-inflammatory cytokines such as TNFa (tumor necrosis factor-alpha) and IFNg (interferon gamma) thus delaying the healing process. In order to investigate potential therapeutic options, we first needed adequate pre-clinical models. A mouse osteotomy model would be preferable for these studies as more analysing tools are available. However, most laboratory animals are being kept under very clean pathogenic-free conditions and thus do not develop effector T cells. Mammals are born with a naïve immune system and during the course of life, while encountering pathogens, an immune memory is established made up of terminally differentiated effector memory T cells. This process is now termed immunoageing and these cells are also pro-inflammatory: inflamm-ageing 5. Thus, we needed to establish a mouse model with an experienced/immunoaged immune system that had a chance to build up an immunological memory. We achieved this by using conventional/environmentally exposed housing and by closely monitoring the immune system 5.
Using these experienced mice, we were able to develop a therapeutical approach to counteract the overarching pro-inflammatory reaction during the early bone healing phase that was identified as being detrimental to the healing success by raising cyclic adenosine monophosphate levels by applying a prostacyclin analogue 6. We were able to show that this dampened the pro-inflammatory reaction of effector T cells, supporting the function of regulatory T cells that counteract inflammation and thus changing the early healing process to become more supportive of bone formation.
However, we have not stopped our endeavour to enhance bone healing at this point and were able to identify another target: interleukin 22. This signalling molecule is overexpressed in individuals with a high percentage of effector memory T cells and was shown to hinder bone formation in our mouse osteotomy model. We are currently exploring routes to locally administer a blocker of interleukin 22 in a time-dependent manner to further this new therapeutic approach to enhance bone formation.
1 Collaborators, G. B. D. F. Global, regional, and national burden of bone fractures in 204 countries and territories, 1990-2019: a systematic analysis from the Global Burden of Disease Study 2019. Lancet Healthy Longev 2, e580-e592, doi:10.1016/S2666-7568(21)00172-0 (2021).
2 Schmidt-Bleek, K. et al. Initial immune reaction and angiogenesis in bone healing. J Tissue Eng Regen Med, doi:10.1002/term.1505 (2012).
3 Schmidt-Bleek, K. et al. Inflammatory phase of bone healing initiates the regenerative healing cascade. Cell Tissue Res 347, 567-573, doi:10.1007/s00441-011-1205-7 (2012).
4 Reinke, S. et al. Terminally Differentiated CD8+ T Cells Negatively Affect Bone Regeneration in Humans. Sci Transl Med 5, 177ra136, doi:10.1126/scitranslmed.3004754
5/177/177ra36 [pii] (2013).
5 Bucher, C. H. et al. Experience in the Adaptive Immunity Impacts Bone Homeostasis, Remodeling, and Healing. Frontiers in immunology 10, 797, doi:10.3389/fimmu.2019.00797 (2019).
6 Wendler, S. et al. Immune Modulation to Enhance Bone Healing-A New Concept to Induce Bone Using Prostacyclin to Locally Modulate Immunity. Frontiers in immunology 10, 713, doi:10.3389/fimmu.2019.00713 (2019).