Fig. 5: Cpt1a genetic knockdown modulates the metabolism of 12-HETE and its metabolites by RAW cells, etomoxir reduces the metabolism of tetranor triene in vivo, and prevents LPS stimulation of RANTES/TNFα generation, oxylipins regulate leukocyte responses, and transcriptomics of human neonatal sepsis proposes multiple genes that support a mitochondrial oxylipin β-oxidation pathway.

a–c Knockdown of Cpt1a alters 12-HETE metabolism. RAW cells were incubated ± LPS (100 ng/ml, for 4 h) with 12-HETE (2.34 µg/ml) added after the first hour (thus added for 3 h). Supernatants were harvested, then lipids were extracted and analyzed for 12-HETE and metabolites using LC/MS/MS as described in Methods (n = 4 (basal nonsil), 5 (basal Cpt1aKD), or 6 (other groups), separate wells of cells, mean ± SEM). d Tetranor triene 12-HETE is increased in vivo during inflammation with CPT1 inhibition. Wild-type mice (female, 7–9 weeks) were injected i.p. with vehicle (PBS), etomoxir (100 μg), and/or LPS (1 μg). After 6 h, lavage was harvested and lipids extracted and analyzed using LC/MS/MS as outlined in Methods (n = 10, individual mice). Outliers are shown as red, or with values. e, f Etomoxir dampens the generation of RANTES and TNF by peritoneal macrophages. Supernatants from macrophages cultured in vitro for 24 h with LPS (100 ng/ml)/etomoxir (25 μM) were tested for RANTES/CCL5 or TNF using ELISAs as described in Methods (n = 3 per group, separate wells of cells, mean ± SEM). a–f *p < 0.05, **p < 0.01, ***p < 0.005 with/without etomoxir, one-way ANOVA with Tukey post hoc test was used. g, h Oxylipin levels detected in vivo promote neutrophil ROS generation. Whole blood was incubated with oxylipin mixtures (Supplementary Table 1) , and then ROS generation in response to stimulation using opsonized S. epidermidis was determined by APF fluorescence as indicated in Methods. Three individual donor samples were separately tested. Monocytes and neutrophils were analyzed using the gating strategy as outlined in Methods. Response to high or low oxylipin doses was expressed either as %APF + cells or % of the vehicle (ethanol) response. Each donor is shown separately. Data were analyzed by repeated-measures two-way ANOVA (g—pairing p < 0.0001, cell type p = 0.018, lipid p = 0.0002, and interaction p = 0.0005; h—pairing p = 0.0008, cell type p = 0.0094, lipid p = 0.0082, and interaction p = 0.0024), Sidak’s post hoc tests are indicated—g Neutrophils ethanol vs low lipid p = 0.0001, neutrophils ethanol vs high lipid p < 0.0001, neutrophils low lipid vs high lipid p = 0.0350, monocytes ethanol vs low lipid p = 0.7020, monocytes ethanol vs high lipid p = 0.8075, monocytes low lipid vs high lipid p = 0.8075—h neutrophils low lipid vs high lipid p = 0.0023, monocytes low lipid vs high lipid p = 0.2363). i Oxylipin levels detected in vivo modulate CD4+ and CD8+ T-cell activation. Whole blood was incubated with oxylipin mixtures (Supplementary Table 1), and then generation of TNF was determined in response to activation using CD3/CD28 beads as indicated in Methods. CD8+ and CD4+ T cells were analyzed using the gating strategy outlined in Methods. Data from individual donors is shown. Two-way ANOVA (repeated measures) was used, pairing p = 0.0039, interaction p = 0.1690, cell type p = 0.0007, effect of lipid p = 0.0253, Sidak’s post hoc tests are indicated—CD4: no lipid vs low lipid p = 0.0461, CD4: no lipid vs high lipid p = 0.7115, CD4: low lipid vs high lipid p = 0.9989, CD8: no lipid vs low lipid p = 0.3742, CD8: no lipid vs high lipid p = 0.0449, CD8: low lipid vs high lipid p = 0.9952). j Human transcriptomics data show significant upregulation of several genes including ACSL and CPT isoforms. Gene expression (log2 expression data) from 35 controls and 26 bacterial confirmed sepsis cases published in⁵⁷ were compared for expression of 32 genes putatively involved in mitochondrial import and β-oxidation of oxylipins, based on known metabolic pathways for long-chain native FA. *p < 0.05, ***p < 0.005, Student’s t-test, two-tailed, then adjusted using Benjamini–Hochberg test.