Cartilage Microbial Products as Novel Drivers of Knee Osteoarthritis Epigenetic Dysregulation

项目总结/摘要 这项研究的目的是更好地了解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 与膝关节骨关节炎（OA）相关，特别关注表观遗传学。OA是导致 退伍军人中的残疾，发生率约为平民的5倍。OA的发病率随着 年龄和肥胖的增加，以前发表的研究概述了肠道微生物组的变化， 年龄和肥胖都有。此外，与肥胖相关的小鼠中的OA可以通过饮食来减轻。 我们最近描述了一种新的软骨微生物组， 人类和小鼠的变化与OA的发展。我们的实验室以前已经详细检查了 与OA相关的软骨、软骨下骨和外周血中的表观遗传变化 发展，我们已经产生了初步的数据，微生物DNA扩增从人类OA 软骨可以在体外诱导软骨细胞中类似的表观遗传变化。在这个项目中，我们的第一个目标是 确定人类OA患者肠道微生物组中是否存在与年龄和肥胖相关的变化， 健康对照反映在各种关节微生物组小生境，包括软骨， 软骨下骨和滑膜。为了做到这一点，我们将获得配对盲肠和软骨样本，从结束- 接受全膝关节置换术的OA期患者和来自 NDRI。然后，我们将使用16 s细菌基因下一代测序来分析微生物组。我们将 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OA老化和OA肥胖进行比较。我们的第二个目标将决定年龄和/或 肥胖相关的软骨微生物组变化影响OA结局和软骨微生物组 具体地，使用来自患有和不患有OA的人类OA患者的粪便微生物组移植（FMT）， 衰老和肥胖的小鼠，并评估DMM手术后的OA结果。我们还将 检查与微生物组移植差异相关的全身和局部炎症， 预先设定的时间点使用CyTOF。我们的第三个目标是评估关节内和关节外的表观遗传变化， 组织和炎症细胞诱导的肠道微生物组的差异，使用相同的移植 目标2中的组。拟议的工作是重要的，因为我们没有充分了解为什么年龄 和肥胖与OA风险的增加有关，我们也不了解软骨微生物组是如何 影响OA风险。我们的工作在使用成对的肠道和软骨微生物样本方面非常创新， 用于评估微生物组的下一代技术，以及我们使用无菌小鼠 微生物组移植评估OA结果。我们也将是第一个将全基因组 亚硫酸氢盐测序技术研究人类和小鼠软骨内的表观遗传变化 被当地微生物改变并与OA发展相关。最后，我们将成为第一个 评估肠道微生物组移植是否可以用作治疗剂来改变年龄-以及 肥胖相关的OA风险。我们提案的成功可能为OA衰老研究开辟一条新的途径， 为OA提供了一种新的治疗策略。

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.