The Mast cell - IL-33 Axis in Post-traumatic Osteoarthritis - Investigators: Abigail Loucks, MS (PhD Student), Aimee Colbath, VMD, PhD, DACVS-LA (Sponsor), Tristan Maerz, PhD (Co-Sponsor). Contributors: Adam Moeser, MS, PhD, DVM; Dana Orange, MD, M.Sc. Context: With identification of synovitis as a driver of osteoarthritis (OA), recent studies have placed an increasing focus on the synovium and the role of intra-articular inflammation in OA pathogenesis. Mast cells (MCs) are long-lived tissue-resident immune cells that act as early responders to infection or injury. Here we are looking to evaluate the temporal role of MCs in post-traumatic osteoarthritis (PTOA) progression and a potential mechanism via IL-33 signaling to alter MC phenotype to mitigate disease severity following an injury. In PTOA patients, OA may be initiated by an isolated event; therefore, a window may exist for therapeutic intervention by immune modulation before the disease progresses. Therefore, the overall objective of this proposal is to further elucidate the phenotype and functions of MCs in the acute and chronic phases of PTOA. Our central hypothesis is that MCs are pro-inflammatory in the acute phase following injury, but prolonged stimulation of MCs by IL-33 results in an immunomodulatory phenotype, mitigating joint inflammation. Specific Aims: To test our hypothesis and achieve our long-term goal to enhance endogenous immunomodulatory activity of MCs following joint injury through manipulation of IL-33 signaling we aim to (i) identify the temporal role of MCs in PTOA and (ii) determine the role of IL-33 in mediating MC phenotypes during disease progression. Research Plan: Aim 1) The effect of MCs on PTOA progression during the acute and chronic phase of the disease will be evaluated by ablating MCs in an MCPT5-Cre x iDTR mouse model 24hrs prior or 10 days after mice undergo a non-invasive anterior cruciate ligament rupture (ACLR). Histology grading of hind limbs will be used to evaluate PTOA severity and single cell RNA sequencing to unbiasedly assess transcriptional changes surrounding MCs in PTOA. Aim 2) We will assess the long- and short-term effect of IL-33 on MC phenotype in both murine bone marrow derived mast cells (mBMMCs) and a human mast cell line (LADR) by culturing cells with recombinant IL-33 for either 24hrs or 7 days followed by stimulation with both IgG and IgE or IgE, respectively. Expression of a known activation marker, CD63, and expression of well-known MC-associated genes will be analyzed via flow cytometry and RT-qPCR, respectively. Media will be analyzed for cytokine and chemokine release via multiplex assay. An IL-33 conditional knockout (Pdgfra-CreER; IL33 f/f) will be utilized to study the progression of PTOA with or without IL-33 released by synovial fibroblasts (SFs) 24hrs prior or 10 days after mice undergo (ACLR) procedure based on histology grading of PTOA severity. Bulk RNA-seq of flow sorted MCs from synovium of these mice will identify alterations in biological processes related to immune cell recruitment and synovitis as associated with MCs present within the joint.
