Neutrophil Biomarker and neutrophil targeted therapy to predict and prevent heterotopic ossification

项目摘要 异位骨化（HO）是骨骼外骨的病理形成，发生在约20%的 髋关节置换术后的患者，肌肉骨骼创伤或烧伤，而这一发病率增加到80%以上 在高能量损伤的患者中，先天免疫反应的作用。标准化 缺乏预防HO的治疗方案，HO的手术切除未能恢复损伤前的功能 容量大，复发风险高。HO，无论激发事件如何，最常见的形成地点 流动性。一旦诊断出HO，医生限制受影响关节的运动以限制进展， 然而，限制活动性以改变炎症和HO进展的机制仍然未知。 更复杂的治疗是，目前没有生物标志物来指导临床医生， 患者处于高HO风险，因此应接受预防以及何时开始治疗。因此，在本发明中， 临床上需要开发有效的炎症靶向HO治疗并验证其有效性， 生物标志物来指导患者选择和精确的治疗时机。该提案将产生数据集， 这两个未满足的临床需求为HO的更有效干预提供了突破。 我们小组最近对HO损伤的新的动态分析已经确定了中性粒细胞表型是HO损伤的中心机制。 是HO。具体来说，我们发现中性粒细胞释放的结构成分，称为中性粒细胞， 细胞外陷阱（NET）在HO中起关键作用。这是一个新颖的方面， 为HO做贡献。据报道，组织损伤促进NET的形成以预防感染 （原发性NETosis）。HO是独特的，因为它形成于移动性位点，这增加了一种独特的力（外在的）， NET，尚未被研究。初步数据表明，关节运动会破坏原发性NET 诱导NET的繁殖（继发性NETosis），这对HO的发展至关重要。我们找到了这本HO专用小说 该机制由Toll样受体9（TLR9）介导，TLR9是DNA复合物的已知受体。所以我们 提出TLR9是HO特异性的新靶点。 目的1：评估NET作为生物标志物预测HO形成的作用。我们将评估差分NET 在我们的小鼠模型和充分表征的人类患者中，与非HO对照相比， HO（髋关节置换术）风险队列检查损伤部位和全身NET水平作为HO生物标志物。 目的2：通过TLR9表征继发性NET形成的作用和治疗潜力 HO形成和进展的信号传导。我们还将评估药理学TLR9抑制的能力， 和中性粒细胞特异性Tlr9缺失以减轻经证实的小鼠模型中的继发性NETosis和HO。

Project Summary 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.