Triamcinolone induced folate receptor expression in OA is associated with skewing of macrophages towards an anti-inflammatory phenotype

Purpose: Folate-based radiotracers have been used in patients with cancer and inflammatory diseases to visualize folate receptor expressing cells using PET or SPECT techniques. Activated macrophages express folate receptor beta (FR-β) and this allows specific imaging of these cells in-vivo. From previous work using SPECT imaging to visualize folate receptor expressing macrophages in both animal models and in patients with OA we know that macrophages are present in OA affected joints. However, it remains unclear what role these macrophages play in the different stages of OA and whether they can be influenced by treatment with corticosteroids. Aim of the study. To understand which macrophage subtypes express the folate receptor and can be visualized using SPECT imaging and whether these macrophages are affected by treatment with corticosteroids (Triamcinolone Acetonide, TA). Methods: In vivo. In twenty 16-week-old male Wistar rats severe osteoarthritis was induced using a low dose intra-articular papain injections in their left knee joints combined with exposure to a moderate exercise protocol. Animals were divided over two groups: ten rats served as untreated OA controls and ten rats were treated during the experiment with weekly intra-articular injections of TA (100ug in 70ul saline). After six weeks an in vivo folate SPECT/CT scan and ex vivo EPIC-μCT and histology was obtained. In vitro. We generated macrophages from human peripheral blood monocytes in vitro by culturing them for 7 days in the presence of GM-CSF (M1 proinflammatory phenotype) or M-CSF (M2 anti-inflammatory phenotype). The effects of LPS, cytokines (IL-4, IL-10, IFN-y) or TA (1ug/ml) were evaluated. FR-β (FOLR2) and macrophage marker expression was measured using FACS and mRNA expression analysis. Results: Increased macrophage activation was seen on SPECT-scans in OA induced knees versus control knees. Interestingly, treatment with corticosteroids prevented osteophyte formation but also caused an increase in activated macrophages seen on folate-SPECT imaging (figure1). On human in vitro generated macrophages FRβ expression was high in M2 macrophages and very low in M1 macrophages. The highest expression was observed in M2 macrophages stimulated with IL-4 and IL-10. TA significantly reduced TNF-α production and CD80 expression by M1 macrophages and increased FRβ and M2 markers such as CD163. A time dependent effect of TA exposure on folate receptor and IL-10 gene expression was observed, with the largest effect when TA was added at day 0 (figure 2). Conclusions: Our data indicate that the increase in macrophage activation as seen on SPECT is most likely due to an increase in M2 anti-inflammatory macrophages. Interestingly, this suggests that an increase in M2 macrophages could be associated with reduced osteophyte formation in our animal study. It is known that corticosteroids can drive macrophages towards an M2 phenotype and we indeed observed an upregulation of both IL10 and FOLR2 gene expression when TA was added during maturation. Together our results indicate that anti-inflammatory macrophage subtypes may play a role in OA and require further investigation.