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Figure 2. Prior chronic OBD supplementation retains a partial proinflammatory memory in the spleen. (A) Experimental schematic of the mouse study to assess immune-lipidome memory in the spleen; (B-I) Flow cytometry plots and quantification of CD11b⁺, CCR2⁺, F4/80⁺/CX3CR1⁺, and MHCII⁺/CX3CR1⁺ cells in CON (10 months of lab chow), OBD (10 months of omega-6-enriched diet), and OBD-R (10 months of OBD diet + 4 months of CON diet) mice; (J) Representative immunofluorescence images showing CCL2 (red) expression; nuclei stained with Hoechst (blue); Magnification; 20×. Scale bar, 50 μm, (K and L) mRNA expression of proinflammatory cytokines TNF-α and CCL2 in CON, OBD, and OBD-R spleens; (M and N) mRNA expression of pro-resolving markers MRC-1 and IFN-γ in CON, OBD, and OBD-R spleens. Data are presented as fold change (FC) relative to CON (baseline FC = 1), normalized to Hprt-1; (O) Pie chart showing the distribution of proinflammatory mediators (PIMs) and specialized proresolving mediators (SPMs) in the spleen; (P) Levels of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in the spleen. Quantification in pg/50 mg tissue; detection limit ≈ 1 pg; (Q) Stacked graphs showing the average concentrations of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the spleen. Quantification values are expressed as pg/50 mg tissue (LV and spleen). Detection limit was ≈1 pg (mass spectrometry); (R) Representative chromatogram for leukotriene B₄ (LTB₄) in the spleen. Statistical comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 vs. CON; #P < 0.05 vs. OBD. Data are presented as mean ± SEM; n = 4/group; ns: non-significant. SPMs: Specialized pro-resolving mediators; LV: left ventricle; CON: control diet; OBD: obesogenic diet; OBD-R: obesogenic diet reversal; SEM: standard error of the mean.