Figure 5. Oleic acid (OA) reverses palmitic acid (PA)-dependent hyper-inflammation in response to lipopolysaccharide (LPS) in vitro, and PA-dependent enhanced endotoxemia disease severity in vivo.

Primary bone marrow-derived macrophages (BMDMs) were isolated from age-matched (6–8 weeks) C57BL/6 female and male mice. BMDMs were plated at 1×10⁶ cells/mL and treated with either media (Ctrl), LPS (10 ng/mL) for 24 hr, PA (PA stock diluted in 0.83% EtOH; 0.5 mM PA conjugated to 2% bovine serum albumin) for 12 hr, or OA (200 μM; diluted in endotoxin-free water). Controls for all treatments are shown next to experimental groups treated additionally with LPS (10 ng/mL) for 24 hr. Supernatants were assessed via ELISA for (A) TNF, (B) IL-6, and (C) IL-1β secretion. Age-matched (6–8 weeks) female BALB/c mice were fed standard chow (SC) or ketonic diet (KD) for 2 weeks and injected intraperitoneal with 7 mg/kg LPS. (D) Temperature loss and (E) survival were monitored every 2 hr. For (A–C), experiments were run three times and data are representative of (A) two experiments and (B, C) one experiment. For all plates, all treatments were performed in triplicate, and a student’s t-test was used for statistical significance. For (D), a Mann Whitney test was used for pairwise comparisons. For (E), a log-rank Mantel-Cox test was used for survival curve comparison. For (D, E), experiments were run three times, and data are representative of one experiment, n=5 mice/group. β symbols indicate KD + Veh vs KD + OA significance, and ∞ symbols indicate KD + Veh vs. SC + Veh. For all panels, *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Error bars shown mean ± SD.