Figure 1.

12,13-diHOME promotes inflammatory macrophage polarization and reduces phagocytic capacity, (a) Representative flow cytograms of IL-1β (CD68⁺ IL-1β^Hi; upper panel) and CD206 (CD68⁺CD206^Hi; lower panel) THP-1 macrophages following treatment with 37.5 or 75 μM 12,13-diHOME for 24 or 48 hr (gating strategy provided in Supplementary figure 1). As controls, cells were stimulated 48 hr with 20 ng ml⁻¹ IFNγ and 100 ng ml⁻¹ LPS together to promote an IL-1β phenotype or 20 ng ml⁻¹ IL-4 to induce a CD206 phenotype. (b) IL-1β⁺/CD206⁺ THP-1 macrophage ratio following treatment with 37.5 or 75 μM 12,13-diHOME or vehicle (DMSO; control) for 24 or 48 hr (Brown–Forsythe and Welch ANOVA test). (c) Temporal gene expression (1, 2, 4, 8, 12, 24, and 48 hr) of macrophage cytokines associated with inflammatory functional states (IL-1β, TNFα, IL-6, and NFκB (RELA)) and anti-inflammatory functional states (IL-10, TGFβ) following exposure of primary human macrophages to 12,13-diHOME. Mean gene expression of triplicate reactions quantitated by qRT-PCR followed with 2^−ΔΔCT calculation; each follow-up timepoint was compared to baseline. Significant (p < 0.05) findings are indicated with an asterisk (linear mixed model, Supplementary table 2). Dose-dependent reduction in phagocytosis of fluorescently labeled E. coli following exposure to 12,13-diHOME (50 or 100 μM) exposure is observed in (d) Raw264.7 (blue) and THP-1 (red) macrophages and (e) confirmed using a gentamicin protection assay (Kruskal–Wallis test). All box plots indicate the interquartile range (IQR) and median.