Orthopaedic Wear Debris, Vitamin D, and Innate Immunity

描述（由申请人提供）：临床失败的全髋关节置换术包括聚乙烯杯和钴铬股骨假体在未来20年期间将影响超过100万人。大多数此类植入物的失败是由于聚乙烯（PE）磨损碎片的释放，PE颗粒依赖性炎症的出现以及随之而来的髋关节假体一个或两个组件的无菌松动。本建议的总体目标是：1]确定剥离的PE颗粒与宿主细胞结合以启动先天免疫反应的方法；2]确定在假体周围溶骨事件中进行靶向治疗（可能是廉价的）干预的机会。因此，假设植入物局部脱落的PE磨损颗粒激活toll样受体（TLRs），这是嵌入巨噬细胞质膜中的模式识别受体的一个子集，以指导负责骨溶解的炎症细胞因子（例如RANKL）的加工。最近的发现使这一提议成为可能：1]人们现在可以高产量地分离、纯化和表征生理磨损模拟过程中产生的全谱PE颗粒；2]人类巨噬细胞原代分离物中的先天免疫信号是由tlr介导的，并受到宿主维生素d不足状态的损害。为了检验上述假设，提出了两个目标。特异性目标1将寻求确定TLR对暴露于真实的纯磨损颗粒的反应，这些颗粒来自髋关节模拟器研究，传统聚乙烯髋臼杯与钴铬股骨球在单因子基因和蛋白质表达方面的差异。本研究将利用野生型、TLR-和MyD88 （TLR的通用适配蛋白）缺失小鼠的TLR表达小鼠骨髓源性巨噬细胞，通过定量PCR和/或ELISA法监测已知介导骨吸收的TLR和单因子的表达。初步筛选涉及的tlr和TLR-TLR合作将利用tlr靶向慢病毒shRNAmir表达功能丧失RNA干扰。特异性目标2将试图在小鼠皮下气囊模型中确定维生素D系统是否会影响TLR对磨损颗粒的反应：1]维生素D代谢物反应调节剂（如维生素D羟化酶和维生素D受体[VDR]）， 2] TLR表达和3]细胞因子对颗粒刺激的反应。这些生物反应标志物将在维生素D充足、不足、不足恢复、TLR和MyD88缺乏的条件下在体内定量分析。考虑到这个国家的老龄化人口，其中大多数是维生素D不足的，包括那些i)已经有一个失败的假体或ii)正在接受髋关节植入的候选人，那么维生素D不足状态的逆转可能是一种简单，成本效益和有效的方法，特别是改善髋关节植入和一般关节置换术的结果。公共卫生相关性：现有全髋关节置换术的临床失败，估计在未来20年期间将有超过100万人发生，其原因是植入物中聚乙烯（PE）磨损碎片的释放，PE颗粒依赖性炎症以及随之而来的假体周围骨溶解和假体部件松动。据推测，植入物局部脱落的PE磨损颗粒会激活巨噬细胞中依赖维生素d的toll样受体（TLR）信号通路，从而指导导致骨溶解的炎症细胞因子的合成（例如RANKL）。考虑到这个国家的老龄化人口，其中大多数是维生素d不足的，包括那些i)已经有一个失败的假体或ii)正在接受髋关节植入的候选人，那么维生素d不足状态的逆转可能是一个简单，经济有效的方法，特别是在髋关节植入和一般的关节置换术中改善结果。

DESCRIPTION (provided by applicant): Clinical failure of total hip replacements involving polyethylene cups and cobalt-chrome femoral components will affect more than a million individuals in the coming 20-year period. Failure in most of these implants will result from the release of polyethylene (PE) wear debris, the appearance of PE particle-dependent inflammation and consequent, aseptic loosening of one or both of the components of the hip prosthesis. The overall aim of this proposal is 1] to ascertain the means by which the exfoliated PE particles engage cells of the host to initiate the innate immune response and 2] to identify opportunities for targeted therapeutic, and perhaps inexpensive, intervention in periprosthetic osteolytic events. As such, it is hypothesized that PE wear particles shed locally by the implant activate Toll-like receptors (TLRs), a subset of pattern recognition receptors embedded in the plasma membrane of macrophages, to direct the elaboration of inflammatory cytokines responsible for osteolysis (e.g. RANKL). This proposal is made possible by the recent discoveries that 1] one can now isolate, purify and characterize in high yield the full spectrum of PE particles generated during physiologic wear simulation and 2] innate immune signaling in primary isolates of human macrophages is mediated by TLRs and impaired by a vitamin D-insufficient status in the host. Two aims are proposed to test the above-stated hypothesis. Specific Aim 1 will seek to determine the TLR response to exposure to authentic, pure wear particles derived from hip simulator studies of conventional polyethylene acetabular cups against cobalt-chrome femoral balls in terms of monokine gene and protein expression. Here TLR-expressing mouse bone marrow-derived macrophages from wild-type and from TLR- and MyD88 (universal adapter protein for TLRs)-deficient mice will be employed to monitor by quantitative PCR and/or ELISA expression of the TLRs and monokines which are known to mediate bone resorption. Initial screens for involved TLRs and TLR-TLR cooperation will utilize TLR-targeted lentiviral shRNAmir expression for loss-of-function RNA interference. Specific Aim 2 will attempt to ascertain in vivo in the mouse subcutaneous air pouch model whether the vitamin D system influences TLR responsivity to wear particles in terms of 1] vitamin D metabolite response modifiers (e.g. the vitamin D-hydroxylases and vitamin D receptor [VDR]), 2] TLR expression and 3] cytokine responses to particle stimulation. These markers of bioresponse will be analyzed quantitatively in vivo under conditions of vitamin D sufficiency, insufficiency, rescue from insufficiency, and TLR and MyD88 deficiency. Considering that the aging population in this country, the majority of which are vitamin D- insufficient, encompasses those who i) already harbor a failing prosthesis or ii) are candidates to receive a hip implant, then reversal of the vitamin D-insufficient state may represent a simple, cost-efficient and effective means of improving outcomes with hip implants in particular and in joint arthroplasties in general. PUBLIC HEALTH RELEVANCE: Clinical failure of existing total hip replacements, estimated to occur in more than a million individuals in the coming 20-year period, results from the release of polyethylene (PE) wear debris from the implant, PE particle- dependent inflammation and consequent periprosthetic osteolysis and loosening of the components of the prosthesis. It is hypothesized that PE wear particles shed locally by the implant will activate the Toll-like receptor (TLR), vitamin D-dependent signaling pathway in macrophages to direct the elaboration of inflammatory cytokines responsible for osteolysis (e.g. RANKL). Considering that the aging population in this country, the majority of which are vitamin D-insufficient, encompasses those who i) already harbor a failing prosthesis or ii) are candidates to receive a hip implant, then reversal of the vitamin D-insufficient state may represent a simple, cost-efficient and effective means of improving outcomes with hip implants in particular and in joint arthroplasties in general.