Fig. 4.

Overexpression of TRAF3 enhances IL-10 and phagocytosis with human macrophages. Human MΦ were transfected with Mock or TRAF3 plasmids. (A) TRAF3 protein expression was determined using flow cytometry with a specific anti-TRAF3 Ab. (Upper) Representative flow cytometry histograms; (Lower) mean ± SEM, n = 4; *P < 0.05. (B) Cytokine levels. MΦ were incubated with STZ with each cys-SPM (10 nM) or vehicle control for 24 h. Results are picograms per milliliter or percent increase of STZ alone; mean ± SEM, n = 3; *P < 0.05. (C and D) Phagocytosis was carried out as in Fig. 3E in the presence of a cys-SPM panel (MCTR3, PCTR3, and RCTR3 1 nM each) or vehicle control. (Left) MFI per cell from one representative experiment; (C, Inset) Kinetics 30 to 60 min (MFI/min). (C, Right) Percent increase of phagocytosis; MFI (TRAF3-OE)/MFI (mock); *P < 0.05, **P < 0.01 vs. time 0. (D, Right) Percent increase of phagocytosis by the cys-SPM panel; mean ± SEM, n = 3; **P < 0.01. For kinetics from three separate donors, see SI Appendix, Fig. S7B. (E and F) Phagocytosis was carried out as in Fig. 3E in the presence of (E) IL-10 (1 pg/ml to 1,000 ng/ml), (F) IL-10 (10 ng/ml) and/or a STAT3 inhibitor (NSC, 100 μM) for 24 h. (E and F, Left) MFI per cell from one representative experiment. (E, Right) Dose–response curve; mean ± SEM, n = 4; P < 0.05. EC₅₀ was estimated using nonlinear regression with log (agonist) vs. response (three parameters). (F, Right) Percent increase of phagocytosis vs. E. coli alone; mean ± SEM, n = 3 or 4; *P < 0.05. Statistical analyses were carried out using (A, B, F) two-tailed Student’s t test, (C and E) one-way ANOVA, or (D) two-way ANOVA. (G) Proposed cys-SPMs/TRAF3/IL-10 axis.