Cartilage Microbial Products as Novel Drivers of Knee Osteoarthritis Epigenetic Dysregulation

项目摘要/摘要 这项拟议的研究的目的是更好地了解与年龄和肥胖相关的肠道 微生物组的变化反映在膝关节软骨微生物组中，以及这些变化可能是如何发生的 与膝骨性关节炎(OA)相关，特别关注表观遗传学。办公自动化是一个主要原因 在退伍军人中，残疾的比例大约是平民的5倍。骨质疏松症的发病率随着 年龄和肥胖的增加，以及之前发表的研究概述了肠道微生物组的变化 年龄和肥胖都是如此。此外，通过饮食可以减少与肥胖相关的小鼠的OA 重塑微生物组的干预措施，我们最近在 人类和小鼠随着骨性关节炎的发展而改变。我们的实验室之前已经详细检查了 与骨性关节炎相关的软骨、软骨下骨和外周血中的表观遗传学改变 我们已经产生了从人类骨性关节炎中扩增出的微生物DNA的初步数据 软骨在体外可以诱导软骨细胞发生类似的表观遗传学变化。在这个项目中，我们的第一个目标是 确定与年龄和肥胖相关的人类骨性关节炎患者肠道微生物群的变化 健康对照组在各种关节微生物群生态位上也有类似的变化，包括软骨， 软骨下骨和滑膜。为此，我们将从末端获取盲肠和软骨的配对样本。 接受全膝关节置换术的骨性关节炎患者和匹配的对照组身体样本 NDRI。然后，我们将使用16S细菌基因下一代测序来描述微生物群。我们会 然后生成与正常衰老和肥胖相关的微生物变化的机器学习模型 将这些与骨质疏松症和骨质疏松症进行比较。我们的第二个目标将决定年龄和/或 肥胖相关软骨微生物群变化对骨关节炎预后和软骨微生物群的影响 具体地说，使用来自患有和不患有骨性关节炎患者的粪便微生物组移植(FMT) 将衰老和肥胖转化为无菌小鼠，并评估DMM手术后的OA结果。我们还将 检查与微生物组移植的差异相关的全身和局部炎症 使用CyTOF预先指定时间点。我们的第三个目标将评估关节内的表观遗传学变化 肠道微生物群差异诱导的组织和炎症细胞，使用相同的移植 拟议的工作很重要，因为我们还不完全了解为什么年龄 肥胖与骨关节炎风险的增加有关，我们也不了解软骨微生物组是如何 影响骨性关节炎风险。我们的工作在使用配对的肠道和软骨微生物样本方面具有相当的创新性， 用于评估微生物组的下一代技术，以及我们使用的无菌小鼠 微生物组移植以评估骨性关节炎的结果。我们还将是第一个应用全基因组 用亚硫酸氢盐测序技术研究人和小鼠软骨的表观遗传学变化 它们被当地的微生物群改变，并与OA的发展有关。最后，我们将是第一个 评估肠道微生物组移植是否可用作改变年龄和年龄的治疗剂 肥胖相关的骨性关节炎风险。我们的建议的成功可能会为OA衰老研究开辟一条新的途径，并可能 为骨性关节炎提供一种新的治疗策略。

Project Summary / Abstract The objective of the proposed research is to better understand how age- and obesity-related gut microbiome changes are reflected in the knee cartilage microbiome and how these changes may be associated with knee osteoarthritis (OA) with a particular focus on epigenetics. OA is a leading cause of disability among Veterans and occurs at roughly 5x the rate of civilians. The incidence of OA rises with increases in age and obesity, and previously published studies have outlined gut microbiome changes with both age and obesity. Furthermore, OA in mice related to obesity can be lessened through dietary interventions that reshape the microbiome, and we have recently described a novel cartilage microbiome in humans and mice that changes with OA development. Our laboratory has previously examined in detail the epigenetic changes within cartilage, subchondral bone, and peripheral blood that are associated with OA development, and we have generated preliminary data that microbial DNA amplified from human OA cartilage can induce similar epigenetic changes in chondrocytes in vitro. In this project, our first Aim is to determine whether age- and obesity-related changes in the gut microbiome in human OA patients and healthy controls are reflected in similar changes in various joint microbiome niches, including cartilage, subchondral bone, and synovium. To do this, we will obtain paired cecal and cartilage samples from end- stage OA patients undergoing total knee replacement and matched control cadaveric samples from the NDRI. We will then profile microbiomes using 16s bacterial gene next-generation sequencing. We will then generate machine learning models of microbial changes associated with normal aging and obesity and compare these with OA-aging and OA-obesity. Our second Aim will determine whether age- and/or obesity-related cartilage microbiome changes impact OA outcomes and the cartilage microbiome specifically, using fecal microbiome transplantation (FMT) from human OA patients with and without aging and obesity into germ-free mice, and evaluating OA outcomes following DMM surgery. We will also examine both systemic and local inflammation associated with differences in microbiome transplants at prespecified timepoints using CyTOF. Our third Aim will evaluate epigenetic changes both within joint tissues and inflammatory cells induced by differences in the gut microbiome, using the same transplantation groups as in Aim 2. The proposed work is important, as we do not have a full understanding of why age and obesity are associated with increases in OA risk, nor do we understand how the cartilage microbiome influences OA risk. Our work is quite innovative in its use of paired gut and cartilage microbial samples, the next-generation techniques used to evaluate the microbiome, and our use of germ-free mouse microbiome transplantation to evaluate OA outcomes. We will also be the first to apply whole-genome bisulfite sequencing techniques to investigate the epigenetic changes within human and mouse cartilage that are altered by the local microbiome and associated with OA development. Finally, we will be the first to evaluate whether gut microbiome transplantation may be used as a therapeutic agent to alter age- and obesity-related OA risk. Success in our proposal may open a new avenue for OA aging research and may offer a novel treatment strategy for OA.