Orthopaedic Wear Debris, Vitamin D, and Innate Immunity

描述（由申请人提供）：在未来20年内，涉及聚乙烯髋臼杯和钴铬合金股骨部件的全髋关节置换术的临床失败将影响超过100万人。这些植入物中的大多数失效将由聚乙烯（PE）磨屑的释放、PE颗粒依赖性炎症的出现以及随后髋关节假体的一个或两个组件的无菌性松动引起。该提议的总体目的是1]确定脱落的PE颗粒与宿主细胞接合以启动先天免疫应答的方式，和2]确定假体周围溶骨性事件中的靶向治疗性干预的机会，并且可能是廉价的干预。因此，假设植入物局部脱落的PE磨损颗粒激活Toll样受体（TLR）（一种嵌入巨噬细胞质膜的模式识别受体子集），以指导导致骨质溶解的炎性细胞因子（例如RANKL）的产生。最近的发现使得该提议成为可能，即1]现在可以高收率地分离、纯化和表征生理磨损模拟期间产生的全谱PE颗粒，和2]人巨噬细胞的原代分离物中的先天免疫信号传导由TLR介导，并受到宿主中维生素D不足状态的损害。提出了两个目标来检验上述假设。具体目标1将寻求确定暴露于真实纯磨损颗粒的TLR反应，这些磨损颗粒来源于传统聚乙烯髋臼杯与钴铬合金股骨球在单核因子基因和蛋白质表达方面的髋关节模拟器研究。在此，来自野生型和来自TLR和MyD 88（TLR的通用衔接蛋白）缺陷型小鼠的表达TLR的小鼠骨髓衍生的巨噬细胞将用于通过定量PCR和/或ELISA监测已知介导骨吸收的TLR和单核因子的表达。对所涉及的TLR和TLR-TLR合作的初始筛选将利用TLR靶向的慢病毒shRNAmir表达进行功能丧失RNA干扰。具体目标2将尝试在小鼠皮下气囊模型中体内确定维生素D系统是否影响TLR对磨损颗粒的响应性（1）维生素D代谢物响应调节剂（例如维生素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响应。将在维生素D充足、不足、从不足中拯救以及TLR和MyD 88缺乏的条件下在体内定量分析这些生物应答标志物。考虑到该国的老龄化人口，其中大多数是维生素D不足，包括那些i）已经患有失败的假体或ii）是接受髋关节植入物的候选人，那么逆转维生素D不足的状态可能是一种简单，具有成本效益和有效的方法，特别是髋关节植入物和关节置换术的结果。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年内将发生在超过100万人中，是由植入物释放的聚乙烯（PE）磨损碎屑、PE颗粒依赖性炎症以及随后的假体周围骨质溶解和假体组件松动造成的。假设植入物局部脱落的PE磨损颗粒将激活巨噬细胞中的Toll样受体（TLR）、维生素D依赖性信号传导途径，以指导导致骨质溶解的炎性细胞因子（例如RANKL）的产生。考虑到该国的老龄化人口，其中大多数是维生素D不足，包括那些i）已经患有失败的假体或ii）是接受髋关节植入物的候选人，那么逆转维生素D不足的状态可能是一种简单，具有成本效益和有效的方法，特别是髋关节植入物和关节置换术。

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.