Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation C. Manferdini,

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Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation C. Manferdini, F. Paolella, E. Gabusi, L. Gambari, A. Piacentini, G. Filardo, S. Fleury-Cappellesso, A. Barbero, M. Murphy, G. Lisignoli Osteoarthritis and Cartilage Volume 25, Issue 7, Pages 1161-1171 (July 2017) DOI: 10.1016/j.joca.2017.01.011 Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Characterization of synovial tissue in moderate grade OA synovial tissues. A. Immunohistochemical analysis of CD68, CD80 and CD206 in a representative sample. Bars a–d 50 μm; e–h 10 μm. Negative control (Control). B. Percentage of positive cells for CD68, CD80, CD206 in five representative cases with moderate grade synovitis. Data were expressed as mean ± SD. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 ASC migration to M1- and M2-like macrophages. ASC migration was tested after 18 h of co-culture with M1- and M2-like macrophages, SFs and positive control (culture medium with 50 ng/ml PDGF-BB) (N = 4). Data are expressed as mean with 95% confidence interval of the fold increase vs control = 1. Statistical analysis was performed and ∗P < 0.05 was considered statistically significant using Wilcoxon signed rank test. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Fig. 3 Modulation of the expression of activated-M1-like factors by ASC co-cultured in transwell or in direct contact. A. The expression of IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9 was analyzed on activated-M1-like, ASC alone and on activated-M1-like co-cultured with ASC in transwell (A-M1 + ASC Indirect) or in contact (A-M1 + ASC Direct) (N = 10). Data were expressed as median with 95% confidence interval. B. The expression of IL10, CD163, CD206 was analyzed on activated-M1-like, ASC alone and on activated-M1-like co-cultured with ASC in transwell (A-M1 + ASC Indirect) or in contact (A-M1 + ASC Direct) (N = 10). Data were expressed as median with 95% confidence interval. Statistical analysis was performed with the Kruskal–Wallis & Dunn's post hoc for unpaired data, ∗P < 0.01 indicates if the presence of ASC influenced the effect of M1-like macrophages by comparing the M1-like alone group with the ASC alone group and M1-like treated with the ASC group; oP < 0.01 indicates significant effects of culture conditions (transwell and cell-contact) by comparing M1-like group with M1-like treated with ASC in transwell group and M1-like treated with ASC in contact group. ND = not detected. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Fig. 4 Factors released by activated-M1-like macrophages modulated by ASC co-cultured in transwell or in direct contact. A. The release of IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9 was analyzed on activated-M1-like, ASC alone and on activated-M1-like co-cultured with ASC in transwell (A-M1 + ASC Indirect) or in contact (A-M1 + ASC Direct) (N = 10). Data were expressed as median with 95% confidence interval. B. The release of IL10, CD163 was analyzed on activated-M1-like, ASC alone and on activated-M1-like co-cultured with ASC in transwell (A-M1 + ASC Indirect) or in contact (A-M1 + ASC Direct) (N = 10). Data were expressed as median with 95% confidence interval. Statistical analysis was performed with the Kruskal–Wallis & Dunn's post hoc for unpaired data, ∗P < 0.005 indicates if the presence of ASC influenced the effect of M1-like by comparing the M1-like alone group with the ASC alone group and M1-like treated with ASC group; oP < 0.004 indicates significant effects of culture conditions (transwell and cell-contact) by comparing M1-like group with M1-like treated with ASC in transwell group and M1-like treated with ASC in contact group. ND = not detected. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Fig. 5 Effects of PGE2 released by ASC as a mediator of M2-like macrophage polarization. A. the release of PGE2 was analyzed on activated-M1-like macrophages, ASC alone and on activated-M1-like co-cultured with ASC in transwell (A-M1 + ASC Indirect) or in contact (A-M1 + ASC Direct) (N = 8). Data were expressed as median with 95% confidence interval. Statistical analysis was performed with the Kruskal–Wallis & Dunn's post hoc for unpaired data, ∗P < 0.002 indicates if the presence of ASC influenced the effect of M1-like by comparing the M1-like alone group with the ASC alone group and M1-like treated with ASC group; oP < 0.001 indicates significant effects of culture conditions (transwell and cell-contact) by comparing M1-like group with M1-like treated with ASC in transwell group and M1-like treated with ASC in contact group. B. TNFα, IL6, IL10, CD163 and CD206 expression was analyzed on activated-M1-like, treated with PGE2 (400 ng/ml) with or without PGE2 receptor antagonist (EP4) (N = 6). Data were expressed as median with 95% confidence interval. Statistical analysis was performed, ∗P < 0.01 indicates the differences between groups using the non-parametric Mann–Whitney test with Bonferroni correction for four comparisons. C. TNFα, IL6 and IL10 release was analyzed on activated-M1-like, treated with PGE2 (400 ng/ml) with or without PGE2 receptor antagonist (EP4) (N = 6). Data were expressed as median with 95% confidence interval. Statistical analysis was performed, ∗P < 0.01 indicates the differences between groups using the non-parametric Mann–Whitney test with Bonferroni correction for four comparisons. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Fig. 6 ASC modulation of PGE2 and COX2 on CD14+ synovial macrophages. The release of PGE2 (pg/ml) and COX2 (% GAPDH) expression were evaluated on CD14+ synovial macrophages obtained from OA patients, ASC alone and CD14+ synovial macrophages co-cultured with ASC (N = 4). Data were expressed as median with 95% confidence interval. ∗P < 0.05 was considered statistically significant and indicates the differences between CD14+ synovial macrophages alone and co-cultured with ASC groups using Friedman ANOVA & Dunn's post hoc for paired data. Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10.1016/j.joca.2017.01.011) Copyright © 2017 The Authors Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10. 1016/j. joca Copyright © 2017 The Authors Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10. 1016/j. joca Copyright © 2017 The Authors Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1161-1171DOI: (10. 1016/j. joca Copyright © 2017 The Authors Terms and Conditions