Fig. 3
Enhanced immune effector cells and function in old mice are normalized upon challenge. a–c Synoviocytes from the small joints were extracted, and their composition was analyzed by flow cytometry. Gating strategy for myeloid subsets is shown in a. Pie charts show the frequencies of Ly6G+ neutrophils (n=6–12 mice), SiglecF+ eosinophils (n=2–8 mice/group), F4/80+CD11b- (n=2–5 mice/group), F4/80+CD11b+ (n=2–5 mice/group), F4/80-CD11b+ (n=2–5 mice/group), and Ly6G-SiglecF-F4/80-CD11b- (other; n=2–5 mice/group) subsets among synoviocytes of young (average age of 14 weeks) and old (average age of 85 weeks) mice before (b), or of young (average age of 22 weeks) and old (max. n = 8 mice, average age of 97 weeks) mice 56 days after (c) immunization with G6PI. d, e Synoviocytes from the small joints were extracted and directly stimulated with 2.5 μM of the Ca2+ ionophore A23187 or vehicle (unstim) for 3 h. Subsequently, lipid mediators were quantified by UPLC-MS-MS. Amounts of lipid mediators were related to the amounts of free fatty acids, and frequencies of metabolites among all analyzed mediators are depicted in the pie charts representing synovial cells of young (average age of 9 weeks) and old (average age of 102 weeks) mice before (d, n=3 mice/group); or of young (average age of 29 weeks) and old (average age of 102 weeks) mice 8 weeks after G6PI immunization (e, n=6 mice/group). f, g Peritoneal lavage cells from young or old mice were analyzed by flow cytometry. Representative plots are shown in (f). Pie charts (g) show summarized lavage compositions from young (left, n=11 mice, average age of 10 weeks) or old (right, n=11 mice, average age of 106 weeks). h Peritoneal lavage cells from young (n=7 mice in 4 analyses, average age of 16 weeks) and old mice (n=9 mice in 4 analyses, average age of 86 weeks) were cultured overnight to recover peritoneal macrophages. Subsequently, adherent cells were stimulated with 1% SACM or vehicle (unstim) for 3 h or left untreated. Lipid mediators were quantified by UPLC-MS-MS. Amounts of lipid mediators were related to the amounts of free fatty acids and frequencies of metabolites among all analyzed mediators are depicted in the pie charts (n=4 mice/group). All pie charts show average values. Statistical testing was performed as described in the methods section