Heterotopic ossification (HO) is the pathologic formation of extra-skeletal bone that occurs in ~20% of patients after hip arthroplasty, musculoskeletal trauma or burns, whereas this incidence increases to over 80% in patients with high energy injuries implicating the role of the innate immune response. Standardized treatment protocols to prevent HO are missing and surgical resection of HO fails to restore pre-injury functional capacity and has a high risk of recurrence. HO, regardless of the inciting event, most commonly forms at sites of mobility. Once HO is diagnosed, physicians restrict movement of the effected joint to limit progression, however, the mechanism behind limiting mobility to alter inflammation and HO progression remains unknown. Further complicating treatment is the fact that there are currently no biomarkers to guide clinicians on which patients are at high HO risk and therefore should receive prophylaxis and when to initiate treatment. Thus, there is a substantial clinical need to develop an effective, inflammatory targeted HO therapy and to validate a biomarker to guide patient selection and precise therapeutic timing. This proposal will generate data sets for those two unmet clinical needs to provide a breakthrough towards more efficient intervention for HO. Recent novel dynamic analyses of HO injuries by our group have identified neutrophil phenotype as central to HO. Specifically, we found that structural components released by neutrophils, known as neutrophil extracellular traps (NETs), play a critical role in HO. This is a novel aspect how the innate immune response contributes to HO. It is reported that tissue injury prompts formation of NETs for prevention of infections (primary NETosis). HO is unique as it forms in sites of mobility which adds a unique force (extrinsic) placed on NETs which has not been studied. Preliminary data demonstrates that motion of a joint disrupts primary NETs to induce propagation of NETs (secondary NETosis), critical to develop HO. We found this HO-specific novel mechanism is mediated by toll-like receptor 9 (TLR9), a known receptor for DNA complexes. Therefore, we propose that TLR9 is a novel target specific to HO.
Aim 1 : Evaluate the role of NETs as a biomarker to predict HO formation. We will evaluate differential NET formation in HO compared to non-HO control in our mouse models and in a well characterized human patient cohort at risk for HO (hip arthroplasty) to examine injury site and systemic NET levels as a HO biomarker.
Aim 2 : Characterize the role and therapeutic potential specific to secondary NET formation through TLR9 signaling in HO formation and progression. We will also assess the ability of pharmacologic TLR9 inhibition and neutrophil specific Tlr9 deletion to mitigate secondary NETosis and HO in proven mouse models.
Traumatic heterotopic ossification (HO) remains a widespread debilitating process for which no early disease biomarker or proven treatment strategy exists. Recent work from my laboratory has revealed that neutrophil phenotype, specifically structural neutrophil extracellular traps (NETs) portend HO formation and that when NETs are disrupted by movement, they stimulate a second wave of NETs (secondary NETosis) through the toll like receptor 9 (TLR9) pathway. This application will address whether NETs can serve as an HO biomarker in a well characterized animal model and in a human hip arthroplasty cohort as well as the ability of TLR9 inhibitors to mitigate secondary NETosis, HO formation and progression.
