Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids

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Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids Brian S. Finlin, Beibei Zhu, Catherine P. Starnes, Robert E. McGehee, Charlotte A. Peterson, Philip A. Kern Journal of Nutritional Biochemistry Volume 24, Issue 9, Pages 1571-1579 (September 2013) DOI: 10.1016/j.jnutbio.2013.01.007 Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 1 Effect of adipocyte coculture on the expression of differentiation markers in polarized macrophages. Polarized macrophages (M1, M2a, or M2c) and differentiated adipocytes were prepared as described under “Methods” and cultured alone or with adipocytes as indicated. Total RNA was prepared from the macrophages and subjected to gene expression analysis by real-time reverse transcriptase (RT-)PCR. The data are represented as means±S.E. (n=3). (A) Mannose receptor (MR) expression in macrophages. *Compare to M1 macrophages cultured alone (P<.05). †Compared M2a macrophages cocultured with adipocytes to M2a macrophages cultured alone (P<.05). (B) IL-10 expression in macrophages. *Compare to M1 or M2c macrophages cultured alone (P<.05). †Compare M2c macrophages cocultured with adipocytes to M2c macrophages cultured alone (P<.05). (C) CD163 expression in macrophages. *Compare to M1 or M2a macrophages cultured alone (P<.05). †Compare M2c macrophages cocultured with adipocytes to M2c macrophages cultured alone (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 2 Effect of adipocyte coculture on the expression of TSP-1, CTGF and collagen I in macrophages. Total RNA was prepared from the macrophages from the experiment in Fig. 1 and subjected to gene expression analysis by real-time RT-PCR. The data are represented as means±S.E. (n=3). (A) TSP-1 mRNA expression in macrophages. *Compare M1 macrophages cocultured with adipocytes to M1 macrophages cultured alone (P<.05). **Compare M2c macrophages cocultured with adipocytes to M2c macrophages cultured alone (P<.05). (B) CTGF expression. *The effect of adipocyte coculture on macrophage CTGF expression (P<.05). (C) Collagen I mRNA expression in macrophages. *Compare M1 macrophages cocultured with adipocytes to M1 macrophages cultured alone (P<.05). **Compare M2c macrophages cocultured with adipocytes to M2c macrophages cultured alone (P<.05). (D) TSP-1 mRNA expression in adipocytes. *Compare adipocytes cocultured with M1, M2a or M2c macrophages to adipocytes cultured alone (P<.05). (E) CTGF mRNA expression in adipocytes. *Compare adipocytes cocultured with M1or M2c macrophages to adipocytes cultured alone (P<.05). (F) Collagen I mRNA expression in adipocytes. (G) Inhibition of CTGF induction by inhibiting TGF-β. *Compare 1D11 to control antibody (P<.05). **Compare SB505124 to DMSO control (P=.056). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 3 Effect of adipocyte coculture on the expression of TSP-1 protein in the medium. The medium from the coculture experiment in Fig. 1 was collected and analyzed for TSP-1 protein expression by ELISA. The data are represented as means±S.E. (n=3). †Compare M1 macrophages cocultured with adipocytes to M1 macrophages cultured alone (P<.05). ††Compare M2c macrophages cocultured with adipocytes to M2c macrophages cultured alone (P<.05). *Effect of coculture (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 4 Effect of treatment with recombinant IL-10 or TGF-β on IL-10 and TSP-1 in M2c macrophages. M2c macrophages were serum starved for 12 h and then were treated for 6 h with vehicle control, IL-10 (20 ng/ml) or TGF-β (5 ng/ml) as indicated. Total RNA was prepared and subjected to gene expression analysis by real-time RT-PCR. The data are represented as means±S.E. (n=3). (A) IL-10 expression. *Compare IL-10 expression in cells treated with recombinant IL-10 to control treated M2c macrophages. (B) TSP-1 expression. *Compare TSP-1 expression in cells treated with recombinant IL-10 to control treated M2c macrophages (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 5 Effect of siRNA-mediated reduction of IL-10 on IL-10 and TSP-1 in M2c macrophages cocultured with adipocytes. M2c macrophages were treated with the indicated siRNA and then cocultured with adipocytes as indicated. Total RNA was prepared and subjected to gene expression analysis by real-time RT-PCR. The data are represented as means±S.E. (n=3). (A) IL-10 expression. *Compare IL-10 expression in M2c macrophages cocultured with adipocytes and treated with IL-10 siRNA to control siRNA treated M2c macrophages (P<.05). (B) TSP-1 expression. *Compare TSP-1 expression in M2c macrophages cocultured with adipocytes and treated with IL-10 siRNA to control siRNA treated M2c macrophages (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 6 Effect of FFAs on IL-10 and TSP-1 in M2c macrophages cocultured with adipocytes. M2c macrophages were cultured alone or cocultured with adipocytes in the presence of the indicated FFAs (C, control; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Lin, linoleic acid; O, oleic acid; P, palmitic acid) at a concentration of 100 μM. Total RNA was prepared and subjected to gene expression analysis by real-time RT-PCR. The data are represented as means±S.E. (n=3). (A) TSP-1 expression. *,**Compare TSP-1 expression in cells treated with the indicated FFA to control-treated M2c macrophages (P<.05). (B) *,**Compare IL-10 expression in cells treated with the indicated FFA to control-treated M2c macrophages (P<.05). (C) The medium from the coculture experiment done in the absence and presence of 100 μM DHA was collected and analyzed for TSP-1 protein expression by ELISA. The data are represented as means±S.E. (n=3). *Effect of DHA treatment on TSP-1 in adipocyte–M2c macrophage cocultures (P<.05). (D) The coculture experiment was repeated in the presence of 20 ng/ ml IL-10 as indicated; open columns indicate TSP-1 expression; shaded columns indicate IL-10 expression. The data are represented as means±S.E. (n=3). *Compare TSP-1 expression to control cells; **compare TSP-1 expression to DHA-treated cells (P<.05). #Compare IL-10 expression to control cells; ##compare IL-10 expression to DHA-treated cells (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Fig. 7 Effect of using adipocyte conditioned medium versus coculture on the ability of FFAs to inhibit TSP-1 expression. M2c macrophages were treated with adipocyte conditioned medium in the presence of the indicated FFA or cocultured with adipocytes in the presence of the indicated FFA as indicated (C, control; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Lin, linoleic acid; O, oleic acid). Total RNA was prepared and subjected to gene expression analysis by real-time RT-PCR. The data are represented as means±S.E. (n=3) TSP-1 expression. #Compare TSP-1 expression in cells treated with conditioned media or cocultured with adipocytes (P<.05). *Compare TSP-1 expression in M2c macrophages treated with the indicated FFA to control-treated M2c macrophages (P<.05). Journal of Nutritional Biochemistry 2013 24, 1571-1579DOI: (10.1016/j.jnutbio.2013.01.007) Copyright © 2013 Elsevier Inc. Terms and Conditions