The Role and Therapeutic Potential of ex-vivo Polarized Monocyte-Macrophages in an in vitro Human Joint Co-culture Model of Osteoarthritis

Chan, Mable Wing Yan 1, 2; Viswanathan, Sowmya 1, 2; Gómez-Aristizábal, Alejandro 1, 2

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Krembil Research Institute, University Health Network

Osteoarthritis (OA) is a degenerative joint disease with rising prevalence in the aging population and no current treatments that can prevent its progression, thus incurring considerable societal impact and healthcare costs. OA is not only a disease of the cartilage, but involves inflammation of the joint. However, single target anti-inflammatory pharmacotherapies against IL-1ß and TNF-a fail to inhibit the progression of OA or provide pain relief, suggesting multimodal therapies may be needed.

Monocyte-macrophages (a heterogeneous mixture of both, represented as Mfs) are immune cells that consist of a spectrum of subtypes, from inflammation-inducing M1-Mfs to inflammation-resolving M2-Mfs. OA patients have elevated levels of Mfs in the diseased joint versus in circulation and the pro-inflammatory subset of Mfs has been found to selectively infiltrate into the OA joint (Gomez-Aristizabal et al., in preparation). Cytokines secreted by Mfs influence both chondrocytes and synoviocytes, thus targeting multiple inflammatory and matrix modifying signalling pathways. We postulate that using ex vivo functionally polarized M2-Mfs would be a multimodal regenerative approach that can modulate both pain and cartilage destruction.

To probe the functionality of human ex vivo polarized Mfs, an ex vivo joint explant co-culture was derived from end-stage OA arthroplasty cartilage and synovial tissue. Mfs were functionally polarized into M1 and M2 subtypes by 48 h standard treatments. Control treatment with OA-associated cytokines 5 ng/mL OSM and IL-1ß produced an OA-like response in cartilage tissue: a 48-fold decrease in ACAN expression compounded with a 4-fold increase of aggrecan loss. Preliminary results show that ex vivo polarized M1-Mfs also decrease ACAN expression 28-fold, but with no observed change in aggrecan loss. In both positive control and M1-Mf treatments, gene expression of key cartilage component COL2A1, ECM components PRG4 and COLX were downregulated by greater than 20-fold. Inflammatory markers CXCL8, IL6, INOS were upregulated by greater than 30-fold. In synovial tissue explants, control treatment with 100 ng/mL IFN-γ induced 17-fold upregulation of INOS, while there was a 6-fold increase in response to the ex-vivo polarized M1-Mf treatment group, paired with a 13-fold increase in ECM protease MMP13. M1-Mfs decreased expression of anti-inflammatory cytokine, IL10, and protease inhibitor, TIMP1, by greater than 10-fold. Ongoing experiments will compare the effect of polarized M2-Mfs and incorporate early OA synovial explants to elucidate treatment timeline.

Our explant model gives a short-term but diverse output on the effects of our minimally manipulated polarized Mfs on OA joint tissue, which together with in vivo evaluations will provide complementary insights into role of Mfs in OA development and progression. These experiments are well positioned to generate proof-of-concept data supporting a novel therapeutic strategy to treat OA.