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 technique...