Guiding synovial inflammation by macrophage phenotype modulation: an in vitro study towards a therapy for osteoarthritis L. Utomo, G.J.V.M. van Osch,

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Guiding synovial inflammation by macrophage phenotype modulation: an in vitro study towards a therapy for osteoarthritis L. Utomo, G.J.V.M. van Osch, Y. Bayon, J.A.N. Verhaar, Y.M. Bastiaansen-Jenniskens Osteoarthritis and Cartilage Volume 24, Issue 9, Pages 1629-1638 (September 2016) DOI: 10.1016/j.joca.2016.04.013 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Gene expression profile of osteoarthritic synovium with and without IFNγ + TNFα stimulation. (A) Expression of genes encoding for pro-inflammatory proteins (IL1B, IL6 and TNFA) and genes encoding for anti-inflammatory proteins (IL1RA, CCL18, CD206 and CD163) relative to the expression of GAPDH. (B) An M1/M2-index based on expression of all measured pro-inflammatory genes and anti-inflammatory genes to provide an overall assessment of the inflammatory state of the synovium with or without IFNγ + TNFα stimulation. Data is presented as boxplots with whiskers from minimum to maximum for n = 4 donors in triplicate. Osteoarthritis and Cartilage 2016 24, 1629-1638DOI: (10.1016/j.joca.2016.04.013) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Modulation of stimulated and non-stimulated OA synovial tissue. (A) Gene expression profiles of IFNγ + TNFα stimulated OA synovium, and (B) non-stimulated OA synovium after treatment with dexamethasone, rapamycin, BMP-7 or pravastatin relative to the expression levels of the vehicle controls as represented by the dotted line. 0.01% DMSO was used as vehicle control for dexamethasone and rapamycin and X-VIVO medium was used as vehicle control for BMP-7 and pravastatin. The M1/M2-index provides an overview of the overall inflammatory state of the synovial tissue after treatment with the compounds based on expression of all the measured pro-inflammatory genes and anti-inflammatory genes. Data is presented in boxplots with whiskers from minimum to maximum for n = 4 donors in triplicate. Osteoarthritis and Cartilage 2016 24, 1629-1638DOI: (10.1016/j.joca.2016.04.013) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Characterization of primary human macrophages stimulated with IFNγ and TNFα (M(IFNγ + TNFα)), IL-4 (M(IL-4)) and IL-10 (M(IL-10)). (A) Gene expression profile relative to GAPDH expression and (B) protein production of IL-6, CCL18 and sCD163 corrected for amount of DNA. Data is shown as mean ± SD for n = 3 donors in 5-fold. The error bars represent the variation between donors. Osteoarthritis and Cartilage 2016 24, 1629-1638DOI: (10.1016/j.joca.2016.04.013) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Modulation of primary polarized macrophages. IL-6, CCL18 and sCD163 protein production of M(IFNγ + TNFα), M(IL-4) and M(IL-10) macrophages after modulation with (A) dexamethasone, (B) rapamycin, (C) BMP-7, and (D) pravastatin. Data is presented for n = 3 independent experiments in triplicate as boxplots with whiskers from minimum to maximum and relative to the compound vehicle controls represented as a dotted line. 0.01% DMSO was used as vehicle control for dexamethasone and rapamycin and X-VIVO medium was used as vehicle control for BMP-7 and pravastatin. Osteoarthritis and Cartilage 2016 24, 1629-1638DOI: (10.1016/j.joca.2016.04.013) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions