Orthopaedic Wear Debris, Vitamin D, and Innate Immunity

描述(由申请人提供)：涉及聚乙烯假体和钴铬股骨假体的全髋关节置换临床失败将在未来20年内影响100多万人。这些假体中的大多数失败的原因是聚乙烯(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的一个或两个组件的无菌性松动。这项建议的总体目标是1]确定剥离的PE颗粒与宿主细胞接触以启动先天免疫反应的方式，以及2)确定在假体周围骨溶解事件中进行有针对性的治疗并且可能是廉价的干预的机会。因此，假设种植体局部释放的PE磨损颗粒激活Toll样受体(TLRs)，TLRs是嵌入巨噬细胞质膜的模式识别受体的子集，以指导导致骨溶解的炎性细胞因子(例如RANKL)的形成。这一建议是由于最近的发现：1)人们现在可以高产量地分离、纯化和表征在生理磨损模拟过程中产生的PE颗粒的全谱；2)人巨噬细胞初级分离株中的先天免疫信号是由TLRs介导的，并受到宿主中维生素D缺乏状态的损害。为了检验上述假设，本文提出了两个目标。具体目标1将寻求确定TLR对暴露于正宗的纯磨损颗粒的反应，这些颗粒来自髋关节模拟器对传统聚乙烯髋臼杯和钴铬股骨球的研究，以单核细胞基因和蛋白质的表达为条件。在这里，表达TLR的小鼠骨髓来源的巨噬细胞来自野生型和来自TLR-和MyD88(TLRs通用适配蛋白)缺陷的小鼠，将被用来通过定量的PCR和/或ELISA法监测TLRs和单因子的表达，它们是已知的介导骨吸收的因子。对涉及的TLR和TLR-TLR合作的初始筛查将利用TLR靶向的慢病毒shRNAmir表达来进行功能丧失的RNA干扰。具体目的2将尝试在小鼠皮下气囊模型中确定体内维生素D系统是否通过1)维生素D代谢物反应调节剂(例如维生素D-羟基酶和维生素D受体[VDR])、2]TLR表达和3)细胞因子对颗粒刺激的反应来影响TLR对穿戴颗粒的反应性。这些生物反应的标志物将在体内定量分析维生素D缺乏、缺乏、从缺乏中拯救以及TLR和MyD88缺乏的条件下。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，包括那些i)已经有失败的假体或ii)是接受髋关节植入物的候选人，那么扭转维生素D不足的状态可能是一种简单、经济、有效的方法来改善特别是髋关节植入物和关节成形术的结果。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年内将发生在100多万人中，原因是植入物释放出聚乙烯(PE)磨损碎片，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，种植体局部脱落的PE磨粒将激活巨噬细胞中依赖维生素D的Toll样受体(TLR)信号通路，指导与骨溶解有关的炎性细胞因子(如RANKL)的产生。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，包括那些i)已经有失败的假体或ii)是接受髋关节植入物的候选人，那么扭转维生素D不足的状态可能是一种简单、经济、有效的方法来改善特别是髋关节植入物和关节成形术的结果。

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.