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. 2021 Jun 1;12:701626. doi: 10.3389/fimmu.2021.701626

Figure 3.

Figure 3

PA reverts the deleterious effect of a LA-rich diet by increased IL-10 production and enhanced suppression capacity of Treg cells. Mice were fed a LA-rich diet or control-diet 4 weeks prior to immunization and were additionally treated with PA (LA+PA) or water (control; LA) by daily oral gavage starting from day 0. (A, B) Ex vivo proliferation assay of CD4+ T cells isolated from the spleen on day 10 of EAE. Isolated cells were re-stimulated with MOG35-55 in vitro and proliferation was assessed by flow cytometry 72h later. (A) Quantification of proliferating cells from control, LA or LA+PA treated mice (n=6 per group). (B) Representative histograms of proliferating CD4+ T cells. (C, D) Splenic cells were isolated on day 10 of EAE and re-stimulated with MOG35-55 in vitro for 48h. Quantification of (C) IL-17A and (D) IL-10 in cell culture supernatants analyzed by ELISA (n=6 per group). (E, F) Treg suppression assay of ex vivo obtained CD4+CD25+ splenic T cells isolated from control, LA or LA+PA treated mice on day 10 of EAE. CD4+CD25+ T cells were co-cultured with e450 labeled CD4+CD25- cells for 72h (n=5 per group). (F) Representative histograms of proliferating CD4+ cells co-cultured with or without Treg cells analyzed by flow cytometry. (G–I) Splenic naïve CD4+ T cells were treated with PBS (control), LA (250µM) or LA+PA (PA 150µM) under Treg polarizing conditions. Flow cytometry analysis of CD25+FoxP3+ cells (G) and IL10+FoxP3+ cells (H) in viable CD4+ cells was performed 96h later (n=6 from 2 independent experiments). (I) Quantification of IL-10 in cell culture supernatants of Treg differentiation assays analyzed by ELISA. (J, K) IL-10 signaling was blocked by the addition of an IL-10 receptor blocker (1µg/ml) to CD4+ T cell differentiation assay under Treg polarizing conditions (n=6 from two independent experiments). Flow cytometry analysis of CD25+FoxP3+ cells (J) and IL10+FoxP3+ cells (K) in viable CD4+ cells was performed 96h later (n=6 from 2 independent experiments). ns, not significant. *p<0.05, **p<0.01, ***p<0.001 using one-way ANOVA and Tukey’s posthoc test.