The therapeutic use of mesenchymal precursor cells in an ovine collagen induced arthritis model of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease that affects 1% of the population and causes joint destruction, deformity and substantial disability. The current treatment of the disease alleviates the clinical symptoms to some extent but does not affect a cure. Also, the patient’s response to current therapeutic options is highly variable and often incomplete, reflecting the complex involvement of multiple pathways in RA disease progression. Furthermore, continuing anti-inflammatory treatment may suppress immune responses essential in host defense. Thus, despite current therapies, novel therapeutic approaches to RA are required. Mesenchymal stem cells (MSC) are self renewable, multipotent, non-hematopoietic progenitor cells that have the capacity to differentiate into various lineages like adipocytes, osteocytes and chondrocytes. Additionally, they have immunomodulatory properties that are considered as a potential therapy for immune mediated diseases and for RA in particular. To examine the therapeutic effect of these cells, animal models that can mimic the human disease are required in order to investigate the immunomodulatory effects of these cells.

Collagen induced arthritis (CIA) is the most studied and used RA model in animals, as it shares many pathological and immunological features of the human disease. Although CIA has been examined in a number of species including mice, rats and monkeys, it has not been fully investigated in a large animal model that might offer more advantages over the current rodent models and which may more closely resemble humans in joint size and degree of load bearing. The aims of this study were first to develop the CIA model of RA in sheep and confirm its reproducibility and capacity to provide efficient assessment of disease activity before investigating the efficacy of allogeneic ovine mesenchymal precursor cells (MPC) which had been immune-selected and culture-expanded to reduce arthritis in this model. MPC are a later developmental stage of MSC and because of their purification techniques through the isolation using stro1 and stro3 antibodies, they are more homogeneous population of cells than MSC.

Two subcutaneous (s.c.) injection of bovine collagen type II (BCII) in freund’s adjuvants followed with intra-articular (i.a.) injection of the same protein in saline in the hock joints has resulted in establishment of CIA in sheep with arthropathy displaying much of the classical pathology associated with human RA. Further, it provided clinical, histopathological and immunopathological features that can be used in testing biological therapeutics for the treatment of RA, such as stem cells. Following induction of arthritis with collagen, clinical signs of lameness and swelling were evident in all sheep and gross thickening of the synovium surrounding the hock joint and erosion on the cartilage surface was evident at necropsy (2 week and 6 week time points). Leukocyte cell counts and levels of antibodies to BCII were increased in serum and synovial fluid (SF), and there was synovial hyperplasia, thickening of the intimal layer, inflammation and marked angiogenesis in the synovial tissue. There was a large influx of monocytes and both T and B lymphocytes to the synovial tissue, many of which appeared to be in active phases of their cell cycle.

After CIA establishment in sheep, the therapeutic effects of allogeneic ovine MPC were investigated in the early stages of CIA by Intravenous (i.v.) administration of MPC one day after arthritis induction. The development of arthritis was followed for two weeks before the sheep were euthanized. Sheep in the MPC treated group showed significantly reduced clinical signs throughout the trial and also showed significant reductions in the pro-inflammatory markers, IL-6 and acute phase proteins (APP) in blood. Activin A levels in both blood and SF of the treated group were also significantly lower than in the control group. Conversely, blood levels of the anti-inflammatory cytokine IL-10 showed a large spike following the administration of MPC. MPC treatment also lowered the neutrophil response in blood following arthritis induction, and the influx of CD4+ cells and monocytes into synovial tissues. All of these effects were accompanied by a significant reduction in the severity of histopathology in the synovial membranes of the treated hock joints compared to the control ones. These results indicate that the immunomodulatory features of MPC have a dramatic effect on acute inflammatory processes involved in arthritis and could become a first line treatment for reducing inflammation and destruction in the joints of people suffering from severe RA.

The second MPC study in this thesis was to investigate the effects of MPC on later stages of arthritis using three different MPC doses and with i.v. and i.a. routes of administration. MPC were administered two weeks after arthritis induction and the sheep were monitored for four weeks. In the control sheep, histopathological analysis of hock joints demonstrated that the left hock synovium was characterized by leukocyte infiltration, synovial hyperplasia, pro-inflammatory cytokine expression, and cartilage erosion. In comparison with saline treated controls, synovial tissue from arthritic sheep receiving a single i.v. injection of MPC showed reduced histopathology scores and reductions in the expression of the pro-inflammatory cytokines IL-6, TNF-α, and IL-17 and absence of IL-1β expression. This was accompanied by a significant reduction in infiltrating monocytes/macrophages and a slight increase in the levels of the anti-inflammatory cytokine IL-10. The i.a delivery of MPC did not appear to lessen the intensity of the arthritis. These results indicate that a single i.v. injection of MPCs in an ovine model of collagen-induced RA attenuates joint inflammation, involving inhibition of the Th17 T cell subset and monocyte-derived production of multiple pro-inflammatory cytokines. Together, these data suggest that MPCs may have great potential as a first line treatment for joint inflammation and RA disease progression.