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Figure 5. Diet switch to control diet activates macrophage memory and function at hematopoietic and local levels. (A) Experimental schematic of in vitro LPS-induced immune memory. BMDMs, splenic MΦ, and peritoneum MΦ isolated from 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, with or without LPS (100 ng/mL, 4 h), to examine MΦ memory; BMDM mRNA expression of (B) CCL2, (C) TNF-α, (D) MRC-1, and (E) ARG-1 in the presence or absence of LPS from CON, OBD, and OBD-R mice. Splenic MΦ mRNA expression of (F) CCL2, (G) TNF-α, (H) MRC-1, and (I) ARG-1; Peritoneum MΦ mRNA expression of (J) CCL2, (K) TNF-α, (L) MRC-1, and (M) ARG-1. Representative immunofluorescence images showing MRC-1 (purple), CCL2 (red), F4/80 (green), and nuclei (Hoechst, blue) in (N) BMDMs, (O) splenic MΦs, and (P) peritoneal MΦs with or without LPS from CON, OBD, and OBD-R mice. Magnification, 100×. Scale bar, 10 μm. Phagocytosis index quantified by the percentage of pHrodo-Red-positive macrophages at 0, 15, 30, and 60 min in (Q) BMDM, (R) splenic MΦs, (S) peritoneal MΦs; (T) Experimental schematic of the in vitro 12-HETE and LPS-induced immune memory model; (U) Representative immunofluorescence images showing expression of MRC-1 (green), CCL2 (red), and nuclei (Hoechst, blue) depicting 12-HETE-induced immune memory in BMDMs isolated from CON, OBD, and OBD-R mice. Magnification, 40×. Scale bar, 50 μm. Comparisons between groups were analyzed using two-way ANOVA with Sidak’s multiple comparisons test for -LPS and +LPS samples. *P < 0.05 vs. CON
