PROJECT SUMMARY Osteoarthritis (OA) is a painful and debilitating disease, in which pain is the primary symptom that prompts patients to seek medical care. Currently, there are not any safe and effective treatments for OA and associated pain that have reached FDA approval. The association between pain and OA is not well understood, and radiographic images do not correlate with pain severity. I propose to use a novel microphysiological system equipped with cartilage tissue, synovial-like tissue with fibroblasts, and macrophages that are innervated with human neurites to study acute pain associated with neuro-immune interactions that occur during OA. I hypothesize that a positive feedback loop between neuro-immune interactions contributes to the ongoing pain and hypersensitivity associated with OA. To test this hypothesis, I will use our novel microphysiological system to first understand the influence of synovial- like tissue inflammatory molecules on innervating neurons through the treatment of human DRG with different conditioned mediums from OA-like cartilage tissue and inflamed synovial-like tissue. I will then investigate the role of chemokines on nociception, with a focus on chemokine CXCL-1 through a series of experiments such as the exogenous treatment of CXCL-1 and assessment of conditioned medium through proteomics. Second, I will assess the role of neuropeptides SP and CGRP on synovial immune cells. I will begin by assessing the contribution of macrophages to inflammatory output in the synovial-like tissue. Then I will use exogenous treatments of SP and CGRP and innervation to specify mechanisms of inflammation promoted by these neuropeptides. This proposed work will broadly apply to OA pain, mediating therapeutic targets and mechanisms behind synovial-related acute OA pain. Lastly, it can be used to model other musculoskeletal diseases, such as pain in intervertebral disc degeneration. This proposal highlights the contributions made by the PI throughout the training, which will primarily focus on the assessment of chemokines on nociception and how neuropeptides affect immune cells in the synovial-like tissue. Successful completion of this proposal is the critical step to providing a platform for understanding neuro-immune interactions, testing pain-mediating therapeutics on human tissue, and developing disease-modifying osteoarthritis drugs.