Oxylipin metabolism is controlled by mitochondrial β-oxidation during bacterial inflammation

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

Oxygenated polyunsaturated fatty acids (PUFA) (oxylipins) are essential bioactive lipid mediators generated during inflammation/infection. They are generated by cyclooxygenases (COX), lipoxygenases (LOX), or cytochrome P450s (CYP), expressed in a variety of cells and tissues1,2,3,4,5,6,7,8,9,10. They signal through activation of G protein-coupled receptors (GPCRs) at sub nM concentrations1,5,6,7,8,9,10,11,12,13,14,15,16. Oxylipin signaling requires deactivation; however, our understanding of how this happens during infection is poor. Systemic pathways for individual oxylipins, including thromboxane, prostacyclin and hydroxyeicosatetraenoic acids (HETEs) were uncovered in healthy humans decades ago17,18,19,20. There, infusion of exogenous labeled lipids enabled determination of half-life and metabolites, some of which appear immediately, slowly disappearing over several minutes21. Urinary metabolite analysis became the gold standard for whole body oxylipin analysis. Separately, peroxisomal β-oxidation of individual oxylipins was explored using liver microsomes. There, partial β-oxidation revealed truncated products termed dinors (minus 2 carbons) and tetranors (minus 4 carbons). These were identified as stable intermediates in tissue, plasma and urine21.

Aside from peroxisomes, mitochondria contain fully competent β-oxidation machinery, used for the first steps of FA-dependent energy metabolism, FA oxidation (FAO)22,23. Here, FA are converted to acetyl-CoA which enters the tricarboxylic cycle (TCA) providing substrates for oxidative phosphorylation (OxPhos)24. Although mediated by distinct enzymes to peroxisomes, mitochondrial β-oxidation also involves sequential removal of 2-carbon fragments from the carboxyl terminus. More recently, there has been significant interest in how mitochondrial FAO supports innate and adaptive immunity in T cells and macrophages25,26. Saturated FA such as palmitate or stearate are considered the main β-oxidation substrates; however, oxylipins generated abundantly during inflammation have not been considered19. We recently showed that endogenously generated platelet oxylipins are removed by carnitine palmitoyltransferase-1 (CPT1), the mitochondrial import protein for FAs27. However, it is not known whether mitochondria also remove oxylipins in other leukocytes, nor how this might be regulated during inflammation/infection, if this impacts lipid signaling, and if it supplies acetyl-CoA to OxPhos. These are important questions since large amounts of diverse oxylipins are generated during inflammation/infection, with downstream autocrine and paracrine signaling being a major contributor to the overall inflammatory response. Currently, there is significant interest in how mitochondria contribute to inflammation, and their role in immunometabolism26,28,29. One view is that in lipopolysaccharide (LPS)-treated macrophages (e.g., M1 phenotype) mitochondrial OxPhos is shut down in favor of increased glycolysis, along with the increased synthesis of FA30. How this process impacts the bioavailability of oxylipins and their inflammatory bioactivity is unknown.

To address these questions, we examine the role of mitochondria in removing oxylipins in macrophages during inflammatory activation in vitro and in vivo, and how modulation of physiological oxylipin levels impacts leukocyte signaling relevant to an acute inflammatory challenge (macrophages, neutrophils, T cells). Genetic and lipidomic approaches characterize a metabolic β-oxidation network for oxylipins and their metabolites, during inflammation. The study identifies a mechanism for dampening oxylipin signaling that could be more broadly relevant in inflammation and infection.

Inhibition of CPT1 prevents secretion of diverse peritoneal oxylipins in vivo

Mitochondrial β-oxidation of native long-chain FA requires uptake of their FA-CoA derivatives via the outer membrane transporter, CPT1, of which there are three isoforms, a,b,c. However, whether oxylipin species can be imported via CPT1 in macrophages is unknown. To test this in vivo, the pan CPT1 inhibitor etomoxir was injected intraperitoneally (i.p.) into wild-type C57BL/6 mice alone or with the bacterial lipid LPS (1 μg), then peritoneal lavage harvested after 6 h, and analyzed using LC/MS/MS. A large number of oxylipins were detected in cell-free supernatant with several showing expected elevations in response to LPS (Fig. 1a, b and Supplementary Fig. 1a). These are broadly categorized into lipids generated by 12/15-LOX, COX-1/2, or CYP450, although some are from more than one pathway (Supplementary Data File 1). Many specialist pro-resolving mediators (SPM) including resolvins or protectins were not conclusively detected. For maresin1 (Mar1, 7R,14S-diHDOHE), very small peaks were seen that fell below our limit of quantitation (LOQ) for the assay. When injecting larger amounts of sample and broadening the retention time window, two peaks were seen with one having the same retention time as the authentic standard (Supplementary Fig. 1b). It was not possible to generate a convincing MS/MS spectrum from tissues that matched the standard due to the low levels present. The detection of two isomers suggests diastereomers of 7,14-di-HDOHE, and the peaks may also contain co-eluting stereoisomers. This is suggestive of non-enzymatic origin and this peak is more appropriately named 7,14-diHDOHE.

a, b Plots for individual lipids showing that inhibition of CPT1 enhances oxylipin levels, with a bigger impact during inflammatory challenge with LPS. Wild-type mice (female, 7–9 weeks) were injected i.p. with vehicle (PBS), etomoxir (100 μg), or LPS (1 μg). After 6 h, lavage was harvested and lipids extracted using SPE then analyzed using LC/MS/MS as outlined in Methods. A series of example lipids from 12/15-LOX, COX, and CYP450 are shown (n = 10). c, d Plots for individual lipids showing the impact of etomoxir ± LPS on oxylipin release by peritoneal macrophages in vitro. Peritoneal macrophages were isolated as described in Methods, then cultured in serum-free medium in the presence of etomoxir (25 μM) with/without LPS (100 ng/ml). After 24 h, the supernatant was harvested and lipids extracted using SPE, then analyzed using LC/MS/MS. A series of lipids based on enzymatic origin are shown, n = 3 per sample. For all panels, data are mean ± SEM, one-way ANOVA with Tukey post hoc test, stats are shown for the effect of etomoxir only, where significant. Where no bar is shown no significant difference was seen.

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Etomoxir stimulated small increases in several oxylipins, however when included with LPS, a far stronger impact was seen, and levels of many lipids were effectively doubled (Fig. 1a, b). This suggests that CPT1 in peritoneal mitochondria actively transports bioactive oxylipins during health, but in infection, its role is significantly enhanced.

CPT1 regulates the metabolism of endogenously generated oxylipins by peritoneal macrophages

In vivo, peritoneal oxylipins could be metabolized by mitochondria in resident macrophages, B cells and/or peritoneal membrane (mesothelium). Naive resident macrophages, which express COX-1 and 12/15-LOX (Alox15), are likely to be the primary basal source31. Thus, naive peritoneal macrophages from wild-type mice were assessed for mitochondrial removal of endogenous oxylipins. We focused on extracellular forms since most are secreted to activate GPCRs extracellularly. Serum-free media was used to prevent contamination with blood-derived oxylipins. LPS (24 h) stimulated prostaglandin (PG) secretion by naive resident peritoneal macrophages, consistent with COX-2 induction (Fig. 1c and Supplementary Fig. 1c). Although 12/15-LOX is highly expressed in naive peritoneal macrophages, its loss in culture leads to the relatively low generation of monohydroxy FA (12-HETE and 12/15-HEPEs) (Fig. 1d and Supplementary Fig. 1d)32. Overall, oxylipin species generated by peritoneal macrophages were similar to those in lavage, although the total number/diversity was less with fewer CYP-derived species and no SPMs detected (Supplementary Fig. 1c).

Etomoxir significantly increased detected levels of several monohydroxy lipids and COX-2 products (Fig. 1c, d). This indicates that both their formation and removal is increased by inflammation, similar to in vivo (Fig. 1a, b). 12-HETE, 12-HEPE and 15-HEPE increased with etomoxir but were suppressed by LPS, likely due to inflammation-associated loss of 12/15-LOX (Fig. 1d and Supplementary Fig. 1d). Importantly, in vivo and in vitro, COX-2 and 12/15-LOX products were consistently increased by CPT1 inhibition.

Several mono- and di-hydroxy oxylipins were only detected when CPT1 was blocked, indicating that their mitochondrial metabolism exceeds their generation (Fig. 1d and Supplementary Fig. 1d). Thus, CPT1 prevents their secretion. In contrast, during LPS challenge, the eicosapentaenoic acid product 17,18-diHETE, generated by soluble epoxide hydrolase oxidation of CYP-derived 17,18-EET, was suppressed by etomoxir (Supplementary Fig. 1e). Overall, these data indicate that peritoneal macrophage oxylipin secretion (12/15-LOX or COX-2 derived) is counterbalanced by CPT1-mediated uptake into mitochondria. However, CYP-derived lipids show the opposite, where CPT1 inhibition dampens levels.

Bone marrow-derived macrophages (BMDM) consume diverse oxylipins from serum

Macrophages are exposed to oxylipins from other immune or stromal cells during inflammation. Serum contains numerous oxylipins from 12-LOX and COX-1, primarily monohydroxy isoforms and thromboxane, generated by white cells and platelets, but few PGs. Serum forms during innate immune responses, thus oxylipin metabolism may occur in local or systemic sites. To examine this, we tested primary murine bone marrow-derived cells differentiated to macrophages, then treated with MCSF, alone or along with LPS/IFN-γ or IL-4 (henceforth referred to as M0, M1, and M2) respectively as in vitro of models for macrophage inflammation33. All macrophage phenotypes consumed significant amounts of exogenous monohydroxy oxylipins from 10% serum-containing medium, including 12-HETE/12-HEPE (12-LOX, generated by platelets), 5-HETE/5-HEPE (5-LOX, generated by neutrophils), and 9-/13-HODEs (Supplementary Fig. 2a). 11-HETE/11-HEPE and 15-HETE/15-HEPE were removed by M0(MCSF) and M2(IL-4) cells; however, they appeared higher or unchanged for M1(LPS/IFN-γ) since they were simultaneously generated by COX-2, induced by LPS/IFN-γ (Supplementary Fig. 2a). Furthermore, M1(LPS/IFN-γ) cells secreted large amounts of PGs and thromboxane due to COX-2, causing a net increase in their extracellular levels. (Supplementary Fig. 2a). Thus, the overall pattern was consumption of serum oxylipins by all macrophage populations, coupled with simultaneous generation of COX-2 PGs by M1(LPS/IFN-γ). Last, low dose etomoxir (25 μM) significantly increased PGs released by M1(LPS/IFN-γ) cells (Supplementary Fig. 2b). Overall, this demonstrates that mitochondrial uptake of PGs suppresses their secretion from classic inflammatory BMDM cells (M1(LPS/IFN-γ)), in the same manner as seen with naive peritoneal macrophages and in vivo with LPS peritonitis, while all BMDM cell types consume significant amounts of HETEs and HEPEs.

Dynamic control of oxylipins by CPT1 during the inflammatory challenge in RAW macrophages

Next, we used RAW cells, a model for macrophage inflammatory responses in vitro, amenable to genetic modification. These don’t express 12/15-LOX, therefore, to examine the impact of CPT1 on its products, cells stably overexpressing Alox15 (RAWAlox15) were generated. Basally, neither RAWmock nor RAWAlox15 cells secreted many oxylipins, with only 9/-HODE, two PGs, and three di-hydroxy products of CYP released in low amounts (Supplementary Fig. 3a). LPS (24 h, 100 ng/ml) stimulated robust secretion of many oxylipins, including several PGs (COX-2) 9/13-HODEs, and 11- and 15-HETEs, HETrEs and HEPEs (likely from COX-2) (Fig. 2a and Supplementary Figs. 3a, b and 4a–c). The primary 12/15-LOX products 12-HETE and 14-HDOHE were only detected in RAWAlox15 cells, and their generation was increased by LPS (Fig. 2a).

a Blockade of CPT1 leads to a significant elevation of 12/15-LOX-derived oxylipins in RAW cells. RAWmock and RAWAlox15 cells were cultured with etomoxir (25 μM) and/or LPS (100 ng/ml) for 24 h before harvest and SPE extraction of supernatant for oxylipin analysis using LC/MS/MS. Bar charts showing how oxylipins from 12/15-LOX are impacted by etomoxir (n = 6, separate wells of cells, mean ± SEM). b CPT1 inhibition prevents the metabolism of 12(S) or 12(R)HETE and their tetranor triene and diene metabolites. RAW cells were supplemented with 1.5 μg 12(S) or 12(R)-HETE/106cells for 3 h with/without etomoxir (25 μM), then supernatants were analyzed for levels of 12-HETE and its triene and diene tetranor products using LC/MS/MS (n = 3 (LPS alone) or 4 (all other samples), mean ± SEM, separate wells of cells). For all panels, comparisons are with/without etomoxir, one-way ANOVA with Tukey post hoc test, stats are shown for the effect of etomoxir only, where significant.

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CPT1 inhibition had little impact on basal oxylipin secretion (Supplementary Fig. 3a). However, it significantly elevated the LPS-dependent generation of monohydroxy FA (HETEs, HDOHEs, HETrEs, and HODEs) from RAWAlox15 cells (Fig. 2a). These data indicate that secreted oxylipins normally represent only a fraction of the overall amounts made by 12/15-LOX. Thus, LPS increases CPT1-mediated removal of 12/15-LOX products, similar to in vivo and peritoneal macrophages. Many primary 12/15-LOX products (12-HETE, 14-HDOHE) were seen, while others (9-HOTrE, 9-HODE) may be byproducts of secondary propagation. In etomoxir/LPS-treated RAWAlox15 (but not RAW cells), a lipid suggestive of resolvinD5 (RvD5, 7S,17S-diHDOHE) was detected, however of the two peaks, only one matched the retention time of the authentic standard (Supplementary Fig. 4a, b). The lipid was very low in abundance, and it was not possible to generate a reliable MS/MS spectrum from cell supernatants. As there were two peaks, they likely represent diastereomers of 7,17-di-HDOHE, and the individual peaks may also contain co-eluting stereoisomers. Thus, we labeled this 7,17-diHDOHE and suggest it arises from non-enzymatic oxidation. No SPMs were detected in any other samples from RAW or RAWAlox15 cells.

Several COX-2 derived PGs and 14,15-diHETE (both CYP) were significantly decreased by etomoxir in RAW cells (Supplementary Fig. 4c). This mirrored the impact of CPT1 inhibition on peritoneal macrophage 17,18-diHETE. In summary, while LOX products were consistently elevated by etomoxir in RAW or all primary macrophages, for RAW cells alone, both COX and CYP-derived lipids were suppressed.

Exogenous 12-HETE is metabolized to diene and triene tetranor metabolites via mitochondrial and non-mitochondrial β-oxidation in RAW cells

To determine the fate of oxylipins removed by CPT1, we examined the metabolism of exogenous 12-HETE by RAW macrophages. 12(S)-HETE was rapidly removed following LPS stimulation, coinciding with the formation and subsequent metabolism of two tetranor 12-HETEs (Supplementary Fig. 4d–f). These were confirmed by MS/MS as a triene, 8-hydroxy-4Z,6E,10Z-hexadecatrienoic acid (comparison with an authentic standard), and a diene, proposed as 8-hydroxy-6,10-hexadecadienoic acid (through comparison with34) (Supplementary Fig. 5a–c). Both will be 8(S) since they originate from 12(S)-HETE. As confirmation, 12(S)-HETE-d8 was added to RAW cells (3 h), and a deuterated diene was detected (Supplementary Fig. 5d, e). The MS/MS fragmentation of these lipids is shown (Supplementary Figs. 12–14). For the tetranor diene, fragmentation matched the expected gas-phase chemistry of hydroxylated FAs, where cleavage occurs next to the hydroxyl group (Supplementary Schemes 13 and 14). For the triene, a daughter ion at m/z 165.1 was seen, with high-resolution MS/MS of the standard confirming this as C11H17O (Supplementary Fig. 6a and Supplementary Scheme 12). NMR for the triene is shown in Supplementary Fig. 7. Almost all triene and diene were detected extracellularly, and over 4–8 h, both disappeared from supernatant and cell pellets (Supplementary Fig. 4e, f). This suggests that macrophages generate and secrete primary and secondary metabolites of HETEs, but then re-internalize them for further metabolism. We next incubated triene tetranor 12(S)-HETE with RAW macrophages, and after 3 h, diene was detected extracellularly (Supplementary Fig. 6b). This indicates that diene forms via saturation of the triene, confirming the structure as 8(S)-hydroxy-6E,10Z-hexadecadienoic acid.

Next, the impact of CPT1 inhibition on the metabolism of exogenous 12(S)-HETE or 12(R)HETE was tested. Both HETEs were rapidly removed by LPS-stimulated RAW cells, with around 0.15% or 0.4% remaining after 3 h, for the S and R forms, respectively (Fig. 2b, left panel). Cellular and supernatant 12(S)-HETE and 12(R)HETE were both increased by CPT1 inhibition, with 12(R)HETE most strongly impacted. Based on the impact of etomoxir, the primary removal appeared to be non-mitochondrial (Fig. 2b). 12-HETE was converted to tetranor trienes and dienes, which were detected mainly outside the cells (Fig. 2b and Supplementary Fig. 8). These were around 5–8-fold increased by CPT1 blockade, confirming that they are also dynamically metabolized by mitochondria in LPS-stimulated macrophages (Fig. 2b and Supplementary Fig. 8). In the case of the diene, both S and R enantiomers were similarly elevated by etomoxir treatment, while for the triene, the 12(R)HETE was most strongly impacted.

Since most removals appeared to be non-mitochondrial, we analyzed for esterification of 12(S)-HETE into phospholipid pools via Land’s cycle, as previously described35. First, to determine which molecular species were formed, a precursor scanning LC/MS/MS analysis was undertaken, scanning for negative ion precursors of m/z 319.2, the carboxylate anion of HETEs. Cells were supplemented with 12(S)-HETE for 3 h, then harvested and lipid extracts were analyzed. Several precursor ions were found, between m/z 738–810 that were absent in RAW cells not supplemented with 12-HETE (Fig. 3a, b). Their structures were subsequently confirmed using MS/MS, showing they originate from a series of expected HETE-containing PE or PC lipids using MS/MS scans. Next, these lipids were quantified using LC/MS/MS. Substantial amounts of 12-HETE-PE/PC were generated during the 3 h incubation with 12(S)-HETE (1.4 μg/106 cells), totaling ca. 88% of the added oxylipin. When cells were concurrently stimulated with LPS, the proportion of HETE incorporated reduced to ca. 73%, with significant reductions in levels of three lipids and trends toward a reduction in all (Fig. 3c). This identifies esterification of 12-HETE into PLs to be the dominant route of removal in RAW cells, accounting for considerably more than β-oxidation and is in line with previous work by Pawlowski et al.35. The partial dampening of this phenomenon by LPS may be a result of phospholipase activities being upregulated during inflammation. Further studies are needed to determine the impact of inflammation on all competing pathways of oxylipin removal in order to build up a more comprehensive picture of which are the dominant events under defined conditions relevant to specific situations such as inflammation, wounding, coagulation, etc.

a–c Cells (106) were incubated for 3 h with 12(S)-HETE ± LPS before lipids were extracted and analyzed as in Methods. Precursor LC/MS/MS was undertaken as described in Methods, scanning for ions that fragment to generate HETE. a Chromatogram showing elution of precursors that generate product ions with m/z 319.2. b MS spectrum from 10–15 min showing PE and PC species that contain 12-HETE. c Quantification of 12-HETE PE and PC species that are formed following incubation of RAW cells with 12(S)-HETE (n = 3, mean ± SEM, separate wells of cells) compared with/without LPS, Student’s t-test, two-tailed. Where no bar is shown no significant difference was seen. d Cpt1a knockdown dampens cellular levels of PGs in RAW cells. RAW cells expressing either the non-silencing (RAWnonsil) or Cpt1a knockdown siRNA (RAWCpt1aKD) were treated with LPS (100 ng/ml) for 24 h and cell levels of PGs measured using LC/MS/MS as in Methods (n = 6, mean ± SEM, separate wells of cells). Student’s t-test, two-tailed. Where no bar is shown no significant difference was seen.

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CPT1a gene silencing confirms dysregulation of oxylipin secretion in RAW cells

Considerably higher amounts of etomoxir than we used (~10-fold higher) can have off-target effects due to an impact on adenine nucleotide translocase36,37. To rule this out, we targeted CPT1a using a lentiviral approach. Silencing to around 14% control levels was achieved in RAW264.7 macrophages, stably expressing shRNA that targets Cpt1a (RAWCpt1aKD), but not those expressing a non-silencing control (RAWnonsil) (Supplementary Fig. 9a–c). CPT1 maintains mitochondrial health since it imports FAs to maintain cardiolipin and phospholipid pools inside mitochondria38. Thus, CPT1-deficient cells do not proliferate normally and may alter their bioenergetic and lipid metabolism profiles. The balance between mitochondrial and peroxisomal β-oxidation and their ability to support elongase activities required to generate PUFA for oxylipin synthesis may have adapted. Similar to low dose etomoxir, the overall impact on endogenous LPS-stimulated PG synthesis in RAW cells was the suppression of supernatant and cell levels (Figs. 3d and 4). Furthermore, the CYP450 product (17,18-diHETE) was suppressed, as also seen for etomoxir with naive peritoneal macrophages and RAW cells. Endogenous generation of several monohydroxy eicosanoids was suppressed by silencing of Cpt1a (Fig. 4). These data further confirm the dual role of CPT1 in both generation and removal of oxylipins depending on their enzymatic source and cell type.

RAW cells expressing either the non-silencing (RAWnonsil) or Cpt1a knockdown siRNA (RAWCpt1aKD) were treated with LPS (100 ng/ml) for 24 h and secretion of PGs, 17,18-diHETE, or monohydroxy oxylipins were measured using LC/MS/MS as in Methods (n = 6, mean ± SEM, separate wells of cells). Student’s t-test, two-tailed. Where no bar is shown no significant difference was seen.

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Since RAW cells do not generate 12-HETE, this metabolite was exogenously added to cultured cells to evaluate mitochondrial oxylipin removal. In basal RAW cells, Cpt1knockdown did not prevent the metabolism of 12-HETE; instead, there was a small increase, perhaps by esterification or peroxisomal metabolism (Fig. 5a). In contrast, 12-HETE removal by LPS-stimulated RAW cells was partially inhibited by Cpt1 knockdown (Fig. 5a). Furthermore, the triene and diene tetranor metabolites were significantly reduced by Cpt1knockdown in LPS-treated but not basal RAW cells (Fig. 5b, c). This indicated that following Cpt1 knockdown, RAW cells showed reduced mitochondrial metabolism of 12-HETE coupled with the lower formation of its tetranor products post LPS stimulation.

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.

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Inflammation accelerates 12-HETE conversion to triene tetranor 12-HETE via mitochondrial β-oxidation in vivo

In vivo inhibition of CPT1 significantly elevated many oxylipins in the peritoneal cavity following the LPS challenge. To examine β-oxidation of oxylipins via mitochondria in vivo, the formation of tetranor metabolites was next measured. The diene metabolite was absent; however, tetranor 12-HETE triene was significantly elevated on CPT1 blockade during inflammatory stimulation (Fig. 5d). This confirms that inflammation in vivo not only increases the formation and mitochondrial uptake of oxylipins (shown in Fig. 1), but also their metabolism to tetranors.

Oxylipin metabolism by mitochondria is not sustaining oxidative phosphorylation (OxPhos)

Mitochondrial β-oxidation of FA is directly linked with OxPhos. It forms acetyl-CoA, a substrate for the TCA cycle as well as NADH and FADH2, substrates for complexes I and II, and also transfers electrons to flavoprotein-ubiquinone oxidoreductase (ETF-QO)39. Thus, oxylipins could contribute to OxPhos. However, LPS suppresses mitochondrial respiration, through multiple mechanisms including aconitase inhibition and nitric oxide binding to complex IV, making this unlikely28. Here, LPS treatment profoundly decreased cellular respiration consistent with previous data (Supplementary Fig. 9d)40,41. Importantly, LPS did not impact mitochondrial DNA (mtDNA), or cause organelle damage as measured by DNA lesion frequency (Supplementary Fig. 9e, f). Thus, mitochondria are still present in LPS-treated cells, although β-oxidation of FAs is largely uncoupled from OxPhos.

Blocking CPT1 dampens cytokine/chemokine generation by peritoneal macrophages

CPT1 inhibition doubled the levels of PGs generated by inflammatory activated peritoneal macrophages, in particular the abundant prostacyclin (PGI2) metabolite 6-keto-PGF1α and PGE2 (Fig. 1c). Thus, we next sought to examine whether this impacted PG-mediated autocrine signaling. Several studies have reported that oxylipins such as monohydroxy FAs, PGE2 or iloprost (PGI2 analog) can suppress cytokine and chemokine generation in monocytes or macrophages, including by EP receptor signaling, or PPAR-γ activation42,43,44,45,46, thus we tested the impact of etomoxir on generation of TNF or RANTES in LPS-stimulated peritoneal macrophages. For both proteins, there was a significant decrease in the generation when etomoxir was present (Fig. 5e, f). This supports the idea that removing oxylipins can dampen their signaling in macrophages.

Oxylipin levels detected in vivo regulate leukocyte responses in a concentration-dependent manner

Oxylipins are primarily secreted to act on other cell types in a paracrine manner. Next, to address whether macrophage mitochondrial β-oxidation impacts wider oxylipin signaling between other cell types, human phagocyte and T-cell responses to these lipids were determined. Neutrophils were chosen since they are known to be sensitive to PGE2, via EP receptor signaling47,48, while T cells can be regulated by PPARγ, a transcription factor that is regulated by many LOX-derived oxylipins49,50,51,52,53,54,55. Here, reactive oxygen species (ROS) production elicited by Staphylococcus epidermidis, a common pathogen in human peritonitis was measured by aminophenyl fluorescein (APF) fluorescence of whole blood leukocytes56. Two concentrations were used that represented amounts of 52 individual oxylipins detected in vivo, in the presence or absence of etomoxir (with LPS) (Supplementary Table 1). Here, ROS generation by neutrophils was consistently enhanced by higher or lower oxylipin levels. However, there was a significant concentration-dependent impact in three individual donors (Fig. 5g, h). Thus, the higher amount, consistent with levels detected during CPT1 inhibition, was significantly more potent at activating neutrophils. Monocyte ROS generation was unaffected at either dose (Fig. 5g, h). Next, T-cell responses in peripheral blood mononuclear cell (PBMC) were determined by measuring TNF generation following TCR-specific stimulation of CD4+ and CD8+ T-cell populations. Here, we found that in general oxylipins had a statistically significant suppressive effect on TNF production in both subsets. Post-test analysis revealed that the lower level of oxylipins alone significantly suppressed TNF in CD4+ cells. In the case of CD8+ cells, only the high level showed significance, while there was a trend at both doses (Fig. 5i). Taken together, these pilot data indicate that modulation of oxylipin levels, within physiological amounts detected in vivo can regulate immune responses of neutrophils and T cells in vitro, supporting the concept that oxylipin removal by mitochondria during inflammation could regulate broader cellular innate immune responses.

Transcriptional analysis of human neonatal bacterial sepsis identifies gene candidates responsible for mitochondrial oxylipin metabolism

The enzymes that import oxylipins into mitochondria are not conclusively known, but it is very likely that those importing long-chain FA are involved. These include five acyl-CoA synthetase long-chain family members (ACSL1,3-6), as well as CPT1a, 1b, 1c, and CPT2. The gene products mediate the formation of −CoA and then -carnitine derivatives that are required for long-chain FA uptake across mitochondrial membranes (Supplementary Fig. 10and Supplementary Table 2). This is followed by mitochondrial β-oxidation, which catalyzes sequential removal of 2-carbon fragments to generate acetyl-CoA, and chain shortened metabolites. Genes that encode proteins that metabolize long-chain PUFA include HADHA, HADHB, ECI1, DECR1, and ACADVL (Supplementary Fig. 15 and Supplementary Table 2)22. A set of 33 genes were compiled including also isoforms with a preference for medium or shorter chain FA. Their expression during human infection in vivo was tested using a microarray from human neonatal whole blood cells (35 cases, 26 controls). Here, infants suspected of infection had blood cultures conducted, and a diagnosis of bacterial sepsis was confirmed57. Out of the genes tested, 22 were significantly different with 9 up- and 13 downregulated in sepsis. Notably, several upregulated genes encode proteins involved in the generation of long-chain FA-CoAs and their interconversion to acyl-carnitines in mitochondria (Figs. 5j and 6a). For the most relevant, seven were significantly upregulated (ACSL1,3,4, CPT1b, CPT2, ACADVL, HADHB) while three were downregulated (ACSL5, ECI1, HADHA) (Figs. 5j and 6a). This indicates a high degree of regulation during complex bacterial infection in seriously ill humans, suggesting the specific isoforms that may be involved in oxylipin removal. We also interrogated transcriptomic data from stromal tissues extracted from mice challenged (i.p.) with a cell-free supernatant from a clinical isolate of S. epidermidis (SES)58. Here, there was overall upregulation, showing a similar trend to the human bacterial dataset (Fig. 6b, c). Although most individual genes were not statistically significant, Cpt1a showed around 2-fold induction, which was significant 6 h post infection.

a Plots for the eight significantly upregulated genes in the human neonatal dataset are shown. n = 35 and 26 for infection and controls, respectively. Student’s t-test, two-tailed, then adjusted using Benjamini–Hochberg test. b, c Transcriptomics of mouse peritonitis shows significant upregulation of Cpt1a at 6 h post SES. Gene expression data from peritoneal membranes harvested post SES challenge were analyzed for expression of 32 genes (n = 3 per group) *p < 0.05, Student’s t-test, two-tailed, adjusted using Benjamini–Hochberg test. c The plot for Cpt1a expression. d, eTranscriptomics reveals Ascl1 and Eci1 to be highly upregulated in response to LPS/IFN-γ in murine BMDM. Transcriptomics data obtained from GEO database were analyzed as outlined in Methods for expression of 34 genes selected for potential or known involvement in mitochondrial β-oxidation. Samples were BMDM treated with either LPS/IFN-γ or IFNγ alone as indicated. For all genes, the log2fold change was calculated and plotted using Pheatmap in R (d). e Box and whisker plots for normalized expression (using Limma and Oligo Bioconductor packages) of Eci1 in mouse datasets with n = 3 for all groups, adjusted using Benjamini–Hochberg test (n = 3 for all groups except for GSE53053 M0 (n = 2)). Box shows median, and interquartile ranges (IQR). The ends of the whisker are at 1.5 × IQR above the third quartile (Q3) and 1.5 × IQR below the first quartile (Q1). If minimum or maximum values are outside this range, then they are shown as outliers.

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Consistent upregulation of ACSL isoforms is revealed across multiple human and murine macrophage datasets, and inhibition of ACSLs dampens 12-HETE metabolism by β-oxidation in RAW cells

Next, data from three mouse BMDM or three human PBMCs studies were downloaded from GEO59,60,61,62,63,64. Several isoforms of ACSL were found to be upregulated, including ACSL1,3,4 (human study) or Acsl1,5 (SES peritonitis and in vitro BMDM experiments). Notably, ACSL1 expression was upregulated in all studies (Figs. 6d, e and 7a, b). For BMDM, Acsl1 was consistently significantly induced on stimulation using either LPS/IFN-γ or IFN-γ alone (Figs. 6d and 7a). Two of the mouse BMDM datasets also indicated significant increases in Eci1, encoding 3,2-enoyl-CoA isomerase (Supplementary Fig. 15 and Fig. 6d, e). In human studies, ACSL1 was also induced by LPS/IFN-γ or IFN-γ (Fig. 7b). To confirm the functional relevance of ACSL upregulation, the impact of the pharmacological inhibitor Triascin C on 12-HETE metabolism was tested. 12-HETE removal was strongly inhibited while the formation of tetranors was dampened between 60 and 95% (Fig. 7c). This is consistent with the requirement for CoA-esters for 12-HETE β-oxidation (mitochondrial or peroxisomal), and confirms the involvement of ACSLs. We note that 12-HETE uptake into phospholipid pools via Land’s cycle esterification also requires ACSL activity. Last, we saw a small non-significant suppression of 12-HETE removal by LPS in RAW cells (Fig. 7c). This is consistent with the process being mainly via Land’s cycle esterification, which was in turn slightly suppressed by LPS (Fig. 3a–c).

a Plots for normalized expression (using Limma and Oligo Bioconductor packages) of Acsl1 in the mouse datasets, with n = 3 for all groups except for GSE53053 M0 (n = 2) adjusted using Benjamini–Hochberg test. b ACSL1 is strongly induced in human M1 macrophages. Human transcriptomics data for ACSL1 expression were downloaded from GEO and normalized expression level plotted. GSE46903 (n = 3, 10 for M0, M1 respectively), GSE35499 (n = 7), GSE5099 (n = 3). Data were normalized using Limma and Oligo Bioconductor packages as outlined in Methods, then adjusted using the Benjamini–Hochberg test. Box shows median, and interquartile ranges (IQR). The ends of the whisker are at 1.5 × IQR above the third quartile (Q3) and 1.5 × IQR below the first quartile (Q1). If minimum or maximum values are outside this range, then they are shown as outliers. c Triascin C alters metabolism of 12-HETE. RAW cells were cultured for 3 h in serum/phenol red-free medium with 12(S)-HETE (1.4 μg/106 cells), with/without 100 ng/ml LPS with/without 7 μM Triascin C. Cells and supernatant were harvested and 12-HETE and its tetranor metabolites measured using LC/MS/MS (n = 3, mean ± SEM, separate wells of cells). Significance was tested using ANOVA with Tukey post hoc test. The impact of LPS (either with or without Triascin C) was not significant for any conditions or lipids, except for diene. d Etomoxir modulates levels of AA in RAW cells. RAW cells were cultured for 3 h in serum/phenol red-free medium with 12(S)-HETE (1.4 μg/106 cells), with/without 100 ng/ml LPS with/without 25 μM etomoxir. Cells and supernatant were harvested and 16:0, 18:0, and 20:4 measured using LC/MS/MS (n = 3, mean ± SEM, separate wells of cells). Significance was tested using ANOVA with Tukey post hoc test. For 16:0 and 18:0, there were no significant differences found.

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Arachidonate (20:4) levels increase in response to inflammation and are dampened by CPT1 activity

Pharmacological or genetic inhibition of CPT1/Cpt1a dampened PG generation in RAW cells (Figs. 3d and 4a and Supplementary Fig. 4c). As a potential explanation, we considered whether LPS-driven inflammation leads to elevated PUFA biosynthesis, which is then dampened by CPT1 inhibition. Specifically, β-oxidation generates acetyl-CoA, which is converted to malonyl-CoA, used to chain elongate and desaturate essential FA to form PUFA via the gene products of ELOVL and FADS. To examine this, the impact of LPS/etomoxir on saturated FA (e.g., 16:0, 18:0) and PUFA (e.g., 20:4) levels was tested. No significant effect was seen for saturated FA, although etomoxir tended to decrease them following LPS treatment (Fig. 7d). In contrast, LPS stimulated significant increases in 20:4 levels. Etomoxir had opposing effects on this, dampening AA in resting cells, while enhancing its levels during LPS stimulation (Fig. 7d). Here, LPS most likely increases 20:4 by promoting its phospholipaseA2 (PLA2)-dependent release from PL membranes. Some 20:4 is then dynamically removed via CPT1, and thus is sensitive to etomoxir. In contrast, the negative impact of etomoxir in resting cells may relate to the reduction in the supply of malonyl-CoA for elongation. Importantly, the impact of etomoxir on 20:4 was only seen during LPS stimulation, consistent with upregulation of ACSLs and accelerated oxylipin consumption we observed during inflammation. Last, the impact of inflammatory activation on FADS or ELOVL expression was examined, using the same GEO datasets tested above. However, no consistent trends were seen for any isoforms (data not shown). This indicates that the elongation pathway is not upregulated at the gene expression level by inflammation. In summary, the elevation of 20:4 following inflammatory activation most likely arises from activities of phospholipases, and CPT1 then plays a role in its subsequent removal. However, we were unable to elucidate the reason for the suppression of PG biosynthesis by inhibition of CPT1, and further studies are required.

Oxylipins are soluble mediators generated during inflammation and infection that link lipid metabolism with immunity. Dynamically controlling their removal is essential to dampen inappropriate inflammation. Here we show that blocking the mitochondrial enzyme CPT1, effectively doubles secreted levels of endogenously generated oxylipins in vivo during the inflammatory challenge. Using exogenous 12-HETE, we propose a paradigm in which macrophage removal of oxylipins and their metabolites involves cycles of secretion and re-internalization of partially oxidized tetranor intermediates, followed by further mitochondrial metabolism. Extending previous studies, we found high levels of secreted free acid tetranors in vitro and in vivo, thus regardless of whether formed in peroxisomes or mitochondria, these lipids appear to undergo repeated cycles of acylation/de-acylation (to CoA and carnitine forms, via ACSLs and CPT) facilitating their rapid secretion, re-internalization and further β-oxidation34,65. This demonstrates a complex cellular metabolism for tetranors, and raises questions as to why they are secreted, instead of being simply metabolized to terminal end products. Tetranor compounds can only be formed via β-oxidation (either peroxisomal or mitochondrial). For this to occur, the lipids must be taken up by the cells. Transport of oxylipins into cells is long known to be mediated by a combination of diffusion, and in the case of PGs, prostaglandin transporters such as the protein encoded by SLCO2A166. These proteins have for decades been considered important for supporting the cellular inactivation of individual PG species, and their role in tetranor metabolism could be explored.

Our finding that oxylipin metabolism is strongly stimulated by LPS has implications for the rare genetic disorder, CPT1 deficiency, where symptomatic episodes and death are triggered by acute infection67. Also, rates of hospitalization and deaths in infants with the CPT1avariant p.P479L are higher for homozygotes with respiratory, dental, and aural infections68,69. This variant reduces CPT1a activity by around 50%68,69. The observation that infection causes severe symptoms indicates that mitochondrial β-oxidation is a protective response to pathogen challenge. This is in line with our findings that CPT1-dependent oxylipin removal is elevated by the bacterial product LPS, and suggests that this may be an important part of the physiological response to infection, functioning as an “off-switch” for pathologic signaling. Whether oxylipin levels are elevated in patients with CPT1a deficiency is not known, and future studies to examine this are warranted.

Mitochondrial uptake of short-/medium-chain native FAs is independent of CPT1; however, long-chain native FA require first activation to acyl-CoA derivatives by acyl-CoA synthetases (ACSL), then conversion to acyl-carnitines, via CPT1 (Supplementary Fig. 10). Following import into mitochondria via an inner membrane transporter, FA are re-converted to acyl-CoAs by CPT2. We note that so far, evidence for CPT1 being able to import oxylipins relies on the use of etomoxir and Cpt1a silencing. Due to the lack of availability of oxylipin-CoAs, we are as yet unable to directly test for enzyme activity with isolated mitochondria. Acyl-CoAs are used for mitochondrial β-oxidation, with sequential removal of two C fragments, and generation of acetyl-CoA. Using a human sepsis cohort and multiple human and murine in vitro models, we propose a biochemical pathway for oxylipin uptake and mitochondrial metabolism during infection (Supplementary Fig. 15). A consistent pattern across all studies was upregulation of ACSL1/Acsl1, while we also saw upregulation of ACSL3,4 (human) and Acsl5 (mice) (Figs. 5j, 6a–e, and 7a, b). Consistent with this, the pan-ACSL inhibitor, Triascin C was highly effective at preventing 12-HETE metabolism to the diene and triene tetranor metabolites in RAW cells (Fig. 7c). ACSL1 is already known to shuttle FAs to mitochondria for β-oxidation and lipid synthetic pathways although others may also be involved70,71,72,73,74,75,76. As yet, the specific ACSLs that acylate oxylipins are not known, although Klett found that all six isoforms are active in vitro77. Given our findings, characterizing which specific ACSLs support the import of oxylipins into mitochondria warrants study. ACSL1 is known to be critical for shuttling FA into mitochondria for β-oxidation in the heart, skeletal muscle, and adipose tissues70,71,72,73,74. Furthermore, in the liver, ACSL1 and CPT1 were shown to be physically associated75,76. Thus, ACSL/CPT1 may be a critical checkpoint enabling higher rates of oxylipin metabolism during infection. Indeed, a critical role for ACSL1 in sepsis outcome has been proposed78. Regulating import of long-chain FAs into the mitochondrial matrix, we found increased CPT1a or b, dependent on the model (Fig. 6a–c), along with CPT2 induction in human sepsis (Fig. 6a). Murine BMDM stimulated with IFN-γ also upregulated Eci1 (Fig. 6d, e). This encodes DCI, which converts 3-cis or trans-enoyl-CoA to 2-trans-enoyl-CoA during mitochondrial β-oxidation and was previously shown to be induced during Hepatitis C virus infection and required for virus replication79,80. Last, upregulation of the β subunit of the mitochondrial trifunctional protein (encoded by HADHB) and very long-chain acyl-CoA dehydrogenase (encoded by ACADVL) are also fully consistent with their proposed role in oxylipin β-oxidation (Fig. 6aand Supplementary Fig. 15).

Macrophages rely primarily on glycolysis for energy when stimulated with LPS/IFN-γ, and conversely use FAO to supply OxPhos with substrates when exposed to immune-modulatory stimuli such as IL-426. However, recent studies have uncovered a significant role for lipid metabolism in “M1” cells even in the absence of OxPhos, with FA synthetic pathways (FAS) being upregulated by LPS26. Our observation that etomoxir partially suppressed 18:0 and 16:0 levels in LPS-stimulated RAW cells is consistent with this (Fig. 7d). This anabolic event is proposed to supply inflammatory activated cells with lipids for processes such as proliferation (sphingolipids, glycerolipids, phospholipids, etc.), and generation of oxylipins26. However, in the case of PUFA substrates for oxylipin synthesis, FA elongation/desaturation of the essential FA, linoleic and α-linolenic acid by FADS and ELOVLgene products is the key event required. Although we found significant elevations in 20:4 (Fig. 7d), this was most likely mediated via PLA2 activities since there was no impact of etomoxir and FADS and ELOVL genes were not upregulated by LPS. Here, we focused down on inflammatory activated macrophages that generate oxylipins, where metabolism will favor glycolysis and FAS, and reduced OxPhos. We show for the first time that CPT1 mediates exogenous and endogenously generated oxylipin β-oxidation by macrophages, which is elevated during inflammation. The impact of blocking CPT1 varied by sub-family of oxylipins, based on either:

1. (i)

   Cell type: etomoxir consistently elevated LOX-derived oxylipins and various other monohydroxy-oxylipins, while suppressing CYP/sEH metabolites in all macrophages. However, COX-derived PGs increased in vivo and in primary macrophages, but were suppressed in RAW cells.

   
   

2. (ii)

   How CPT1 was targeted: in RAW cells, etomoxir or gene silencing suppressed COX-derived PGs and CYP/sEH oxylipins. In contrast, monohydroxy oxylipins were increased by etomoxir but suppressed by CPT1 knockdown in RAW cells. Etomoxir was used at 25 μM, well below concentrations found to induce off-target effects on adenine nucleotide translocase36,37. Indeed, previous studies using high levels of etomoxir led to FAO being incorrectly proposed as required for alternative activation of M2 macrophages (100–200 μM)81. Nowadays, lower concentrations are recommended that block ~90% of β-oxidation without side-effects (e.g., 10–25 μM)38. As a second approach, shRNA knockdown stably reduced expression of CPT1a. However, this causes adaptive changes to the metabolic status of macrophage mitochondria, beyond acute inhibition of CPT1. Recent publications implicate CPT1 in maintaining mitochondrial health due to its requirement for importing FAs for maintaining mitochondrial cardiolipin and phospholipid pools38. Thus, cells lacking CPT1 do not proliferate normally and may have adapted to a deficiency in OxPhos through alterations in other lipid and energy metabolism pathways. Thus, constitutive knockdown may directly impact the balance between mitochondrial and peroxisomal β-oxidation and the ability to support elongase activities in the cells. Nevertheless, both inhibitory approaches caused significant changes to oxylipin secretion, both for PGs, 12-HETE and its two tetranor metabolites, supporting the hypothesis that mitochondria can be a significant site of oxylipin regulation in macrophages.

   
   

Our data reveal complex modulation of oxylipin removal and formation by CPT1 as follows:

1. (i)

   Removal: oxylipins and their metabolites can be degraded by two separate β-oxidation pathways, with only mitochondrial enzymes relying on CPT1. However, peroxisomes and mitochondria display metabolic interplay where FA degradation intermediates are transferred for metabolism between the organelles82. Thus, stable knockdown of CPT1 may require compensatory increases in peroxisomal β-oxidation. It has been proposed that truncated unsaturated FA metabolites may need to transfer to mitochondria for complete metabolism82. Indeed, 12-HETE-triene was suggested as the terminal peroxisomal metabolite for 12-HETE, with further metabolism proposed to require mitochondria18. Our data provide supportive evidence for this interplay between organelles since blockade of CPT1 had a far greater impact on the metabolism of tetranor metabolites than exogenous 12-HETE in LPS-stimulated cells. Thus, 12-HETE can be metabolized in either organelle, but peroxisomal-generated tetranors are then shuttled to mitochondria, with their oxidation significantly stimulated by inflammation.

   
   

2. (ii)

   Generation: oxylipins are generated from PUFA, which are themselves synthesized via chain elongation and desaturation of linoleic and α-linolenic acids. This process requires malonyl-CoA, which is formed from acetyl-CoA, a product of which themselves depend at least in part on CPT1 for formation. Thus, while both synthesis and degradation of oxylipins simultaneously occur in macrophages, steady-state levels will depend on the cells’ metabolic status, and whether CPT1 is mainly supporting degradation (by β-oxidation) or synthesis (by supplying TCA intermediates) of individual oxylipins. Thus, CPT1 could either increase or decrease levels of oxylipins and their metabolites concurrently, as seen in our study. Detailed flux analysis of this phenomenon is required to further our understanding since a role for regulation of elongation/desaturation was not clearly seen in our study.

   
   

Here, we show that mitochondrial oxylipin removal takes place during inflammation on a background of reduced OxPhos, a well-known response to LPS stimulation by macrophages28. However, β-oxidation requires a basal level of OxPhos both to regenerate NAD+ and oxidized flavin cofactors in the electron transferring flavoprotein complex, which reduces ubiquinone. While OxPhos was suppressed, there was a low residual activity remaining in both RAW and peritoneal cells that appeared sufficient to sustain β-oxidation, observed by the formation/metabolism of oxylipins and HETE tetranors. Our experiments using exogenous 12-HETE suggest that non-mitochondrial pathways such as peroxisomal oxidation and also esterification is probably responsible for removing most. This agrees with previous studies where exogenous 12-HETE added to mammalian cells was removed by peroxisomes forming a series of metabolites, down to C12:118,34,83,84, and a significant amount is converted to esterified pools35. However, these studies did not consider the impact of inflammation and the focus was exclusively on exogenous 12-HETE, not endogenous oxylipins generated by the cells. Here, we extend this range of oxylipins generated by macrophages, and reveal that during LPS inflammation, mitochondria (via CPT1) effectively halve secreted amounts of oxylipins en masse, and are a key site for removing peroxisomal metabolites of 12-HETE. We demonstrate that this alters the outcome of leukocyte activation, suggesting that mitochondrial β-oxidation may act as a brake for dampening host inflammatory responses in vivo during infection. Further studies are required to test this idea in vivo, including characterization of the ability of other leukocytes such as neutrophils and T cells to remove oxylipins via β-oxidation The potential implications for humans with CPT1a deficiency, relating to severely augmented inflammation during infection should be considered and studies initiated to test this idea. Also, whether this phenomenon contributes to infection-associated host inflammation in general, in cases where mitochondrial health is compromised, should be considered.