Achieving Sustained Control of Inflammation to Prevent Post-Traumatic Osteoarthritis (PTOA)

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Knee Osteoarthritis (KOA) is highly prevalent in U.S. military service members and Veterans due to the impact of joint trauma and overuse injury. Its socioeconomic impact is estimated at $60 billion per year, and no disease-modifying treatments exist. Joint inflammation is linked to severity and progression of disease 4, but current anti-inflammatory medications do not protect against progression, and have substantial side effects. The purpose of this project is to develop and employ a novel method to modulate the local inflammatory response in the joint after an injury in a sustained manner, to prevent post-traumatic osteoarthritis (PTOA). Specifically, we hypothesize that modulation of Toll-like receptor (TLR) activation, via blockade of the TLR co-receptor CD14, will reduce development of PTOA pathology and functional decline after knee injury. We were the first to discover high levels of CD14 in OA patients and show that it augmented TLR- mediated inflammatory responses. We have now demonstrated that genetic deficiency of CD14 reduces signs of disability and prevents progression of cartilage damage in a murine model of PTOA; Our preliminary data in this same model shows that pharmacologic blockade for therapeutic purposes is possible. We propose to establish blockade of TLR co-receptor CD14 as an effective strategy to prevent PTOA pathology and functional decline, using a large-animal (mini-pig) model of knee OA developed by our team. We will optimize intra- articular (IA) delivery of a clinically developed CD14 blocking antibody to achieve sustained release during the post-injury period, by encapsulating the antibody in innovative Mechanically Activated Microcapsules (MAMCs). Finally, we will test whether TLR activation markers in Veterans with KOA can predict clinical response to an existing anti-inflammatory therapeutic, with a focus on functional recovery, in order to optimize patient stratification for future clinical trials of TLR-targeted anti-inflammatory therapies. In Aim 1, Yucatan mini-pigs will undergo bilateral arthroscopic destabilization of the medial meniscus (DMM) surgery of both hind limbs. Starting one week after surgery, a neutralizing anti-CD14 monoclonal antibody (Implicit Biosciences) will be delivered IA to one knee; the contra-lateral side will receive isotype-matched antibody control. Functional outcomes will include activity and knee joint flexion angles measured every two weeks using a wearable motion-sensor device. Synovial fluid (SF) and serum will be collected prior to DMM and at endpoints for molecular/cellular analysis of effects of treatment. Groups of animals will be euthanized at endpoints up to six months after surgery, and PTOA pathology evaluated using novel MR imaging techniques allowing clinically-relevant early cartilage molecular changes to be detected, and imaging data will be validated using standardized histopathology. In Aim 2 we will optimize encapsulation of anti-CD14 in MAMCs using microfluidic double emulsion technology, and confirm sustained delivery of active drug after encapsulation using in vitro and ex vivo analyses. Encapsulated anti-CD14 will then be delivered IA to one hind knee joint of DMM-operated mini-pigs, and encapsulated control antibody to the contra-lateral side. A single dose will be given one week after DMM. The effects of encapsulated anti-CD14 vs. unencapsulated anti-CD14 on pathology and functional outcomes will be compared. Retention and distribution of MAMCs in the joint will be assessed by IVIS imaging. Finally, in Aim 3, we will leverage the availability of SF samples from participants in a VA-funded clinical trial (MOVE-OK study) testing the efficacy of IA corticosteroids in Veterans with knee OA. Biomarkers related to TLR activity (including sCD14, sTLR4, LBP) will be measured in SF collected and stored as part of the MOVE-OK protocol, using ELISA/multiplex assays. Associations between biomarker levels, and change in activity levels and disability scores with therapy will be tested using linear regression analysis. Pursuit of this project will result in the development of an innovative method to control joint inflammation after injury, and inform patient selection for future clinical trials of TLR-targeted immunomodulatory approaches.