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. 2019 Nov 5;33(12):14784–14797. doi: 10.1096/fj.201901872R

Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice

Joan P Graves 1, J Alyce Bradbury 1, Artiom Gruzdev 1, Hong Li 1, Caroline Duval 1, Fred B Lih 1, Matthew L Edin 1, Darryl C Zeldin 1,1
PMCID: PMC6894073  PMID: 31690125

Abstract

Inflammatory stimuli, such as bacterial LPS, alter the expression of many cytochromes P450. CYP2C and CYP2J subfamily members actively metabolize fatty acids to bioactive eicosanoids, which exhibit potent anti-inflammatory effects. Herein, we examined mRNA levels of the 15 mouse Cyp2c and 7 mouse Cyp2j isoforms in liver, kidney, duodenum, and brain over a 96-h time course of LPS-induced inflammation and resolution. Plasma and liver eicosanoid levels were also measured by liquid chromatography with tandem mass spectrometry. Expression changes in Cyp2c and Cyp2j isoforms were both isoform and tissue specific. Total liver Cyp2c and Cyp2j mRNA content was reduced by 80% 24 h after LPS but recovered to baseline levels by 96 h. Total Cyp2c and Cyp2j mRNA in kidney (−19%) and duodenum (−64%) were reduced 24 h after LPS but recovered above baseline by 72 h. Total Cyp2c and Cyp2j mRNA content in brain was elevated at all time points after LPS dosing. Plasma eicosanoids transiently increased 3–6 h after administration of LPS. In liver, esterified oxylipin levels decreased during acute inflammation and before recovering. The biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.—Graves, J. P., Bradbury, J. A., Gruzdev, A., Li, H., Duval, C., Lih, F. B., Edin, M. L., Zeldin, D. C. Expression of Cyp2c/Cyp2j subfamily members and oxylipin levels during LPS-induced inflammation and resolution in mice.

Keywords: cytochromes P450, ipopolysaccharide, gene expression, fatty acids, eicosanoids

Members of the cytochromes P450 gene superfamily are known to metabolize both endogenous compounds and xenobiotics (1–3). Cytochrome P450 isoforms from the CYP2C and CYP2J subfamilies are believed to be the major isoforms involved in the epoxygenation of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) and/or hydroxylation of AA to hydroxyeicosatrienoic acids (1, 2, 4). The EETs and hydroxyeicosatrienoic acids regulate many biologic processes, including vasodilation, angiogenesis, inflammation, and nociception (5). Both microsomal epoxide hydrolase (EPHX)1 gene) and soluble epoxide hydrolase (sEH, EPHX2 gene) hydrolyze EETs to biologically less-active dihydroxyeicosatrienoic acids (DHETs) (6). sEH inhibitors, which potentiate EET effects by reducing EET hydrolysis, have progressed to clinical trials for the treatment of hypertension and neuropathic pain.

The regulation of cytochrome P450 epoxygenases and EETs during inflammation is complex. LPS binds the transmembrane TLR4/myeloid differentiation factor 2 dimer, initiating a complex signaling cascade that activates the transcription factor NF-κB and results in the release of proinflammatory cytokines including TNF-α, IL-1, and IL-6 (7–9). LPS has been shown to suppress transcript and enzyme levels of hepatic and extrahepatic cytochromes P450 in rats (10). A similar study in mice found decreased hepatic expression of Cyp2c29, Cyp2c44, and Cyp2j5 and renal expression of Cyp2c44 and Cyp2j5 24 h after LPS administration (11). Conversely, EETs have been shown to regulate the inflammatory effect of LPS. In vitro, EETs attenuate LPS-induced NF-κB activation and cellular adhesion molecule expression (12). In vivo, mice with transgenic endothelial overexpression of either CYP2J2 or CYP2C8, or genetic disruption of sEH, have increased EETs and decreased proinflammatory cytokine and cell adhesion molecule expression (13). Taken together, these data suggest a model in which LPS suppresses P450 epoxygenase expression and EET production in the acute phase of inflammation. These effects would dampen the anti-inflammatory effects of EETs to potentiate proinflammatory pathways. Eventual recovery of epoxygenase expression and EET production may aid in the resolution of inflammation (14). Importantly, however, a comprehensive analysis of mouse Cyp2c and Cyp2j expression and EET levels in vivo during LPS-induced acute inflammation and inflammatory resolution has never been performed.

In this study, we used 15 mouse Cyp2c- and 7 mouse Cyp2j-specific Sybr Green primers sets (15, 16) to examine mRNA levels of these 22 P450s in liver, kidney, duodenum, and brain over a 96-h time course after LPS dosing. These tissues are known to express CYP2C and CYP2J isoforms and are involved in inflammatory disease models that are responsive to EETs (13, 15, 16–18). We found that total Cyp2c and Cyp2j isoform expression was significantly reduced in mouse liver, kidney, and duodenum at 24 h and then recovered to baseline levels. Conversely, LPS increased total Cyp2c and Cyp2j mRNA levels in brain throughout the 96-h time course. In contrast, plasma P450-derived oxylipins transiently increased during the first 6 h after LPS dosing and then returned to baseline levels.

MATERIALS AND METHODS

Reagents and supplies

Sybr Green oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA). MicroAmp Optical 384-Well Reaction Plates, an ABI ViiA 7 Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA), and Power Sybr Green PCR Master Mix (Thermo Fisher Scientific) were used for the quantitative PCR (qPCR) experiments.

Animal treatment

All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and were approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee. Mice were maintained on standard NIH31 chow (Research Diets, New Brunswick, NJ, USA).

Wild-type male C57BL/6 mice were given a single dose of 1 mg/kg LPS (purified Escherichia coli 0111: B4; MilliporeSigma, Burlington, MA, USA) in PBS, or PBS vehicle alone as control, by intraperitoneal injection. Mice were euthanized, and tissues were collected at 3, 6, 24, 48, 72, and 96 h after LPS administration. The PBS control mice were euthanized, and tissues were collected at the 96-h time point. Five mice per group were assessed at each time point.

RNA and cDNA preparation

Total RNA was isolated from the collected tissues using RNeasy Midi Kits from Qiagen (Germantown, MD, USA) according to the manufacturer’s instructions. One microgram of total RNA was treated with 1 U of DNase I (Thermo Fisher Scientific) for 15 min. The reaction was inactivated by addition of 1 μl of 25 mM EDTA and heat treatment at 65°C for 10 min. RNase-free water was added to a total volume of 50 μl. A High-Capacity cDNA Reverse-Transcription Kit from Thermo Fisher Scientific was used according to the manufacturer’s protocol to produce cDNA from the DNase I–treated total RNA. The final concentration of the cDNA was 10 ng/μl, which was diluted to a working concentration of 1 ng/μl with RNase-Free Water (Qiagen) for qPCR experiments.

Sybr Green qPCR

Specific primer sets of oligonucleotides for all the mouse Cyp2c and Cyp2j subfamily members were developed and their qPCR conditions optimized as previously reported (15, 16). Ptgs1 (Mm00477214_m1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) (Mm00478374_m1) expression was measured using commercially available TaqMan reagents (Thermo Fisher Scientific) according to the manufacturer’s protocols. qPCR data were analyzed with the 2−ΔΔCt method (19). Graphs were generated using the Prism 6.0C program (GraphPad Software, La Jolla, CA, USA). Samples below the limit of detection (Ct >35) are noted as not detected.

There were significant differences in the relative expression of the glyceraldehyde 3-phosphate dehydrogenase, 18s rRNA, β-actin, β-2 microglobulin, and peptidylprolyl isomerase (cyclophilin)-like (Ppil) housekeeping genes at various time points post-LPS treatment in mouse liver, duodenum, and brain compared with the 0-h control (Supplemental Fig. S1). In contrast, no significant differences in the relative expression of Ppil over the same LPS time course were observed in the kidney using the forward 5′-CAGACGCCACTGTCGCTTT-3′ and reverse 5′-TGTCTTTGGAACTTTGTCTGCAA-3′ primers (20). Detection of renal Cyp2c and Cyp2j transcripts without and with normalization to Ppil (Supplemental Fig. S2) revealed similar expression patterns for the 96-h LPS time course. Therefore, for all tissues, the Ct values were averaged and used to calculate individual sample ΔCt. The mean ΔCt values of the control group were then used to calculate the ΔΔCt values for individual samples.

Outliers were detected and removed according to the Grubbs outlier test (21). The significance between treated and control samples was calculated by the Student’s t test.

PCR efficiency of the mouse Cyp2c and Cyp2j Sybr Green primer sets

To standardize the copy number of the various Cyp2c and Cyp2j transcripts, the absolute expression level was determined according to previously published methods (22, 23). Using individually cloned cDNAs for all the subfamily members, an absolute standard curve was generated for the Sybr Green primer sets listed in Supplemental Table S1. The absolute standard curve was generated in triplicate with equimolar template in the biologically relevant range of 0.00457–0.71 fmol/μl. All values used to establish the individual standard curves were in the linear logarithmic exponential phase of the PCR. Following absolute quantification, the transcript levels of the Cyp2c and Cyp2j subfamily members could be empirically compared with each other, which then allowed for quantitative determination of the relative transcript copy number and the gene expression profile (Supplemental Table S2).

Liquid chromatography with tandem mass spectrometry analysis

In total, 200 μl of plasma from LPS-treated or control mice was mixed with 200 μl of 0.1% acetic acid in 5% methanol and 10 μl of internal standard. Eicosanoids were isolated by liquid-liquid extraction with ethyl acetate, passed over phospholipid removal columns, and dried in a vacuum centrifuge. Concentrations are reported as picograms per milliliter. Levels of both free and esterified oxylipins and fatty acids were determined in liver tissue obtained at each time point. Liver tissue was homogenized in 0.1% acetic acid in 5% methanol (9 μl/mg tissue) containing 10 μM trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid kindly provided by Bruce Hammock (University of California–Davis, Davis, CA, USA). In total, 20 mg of liver lysate (200 μl) was extracted as above to determine free, nonesterified levels of oxylipins. A second 200-μl aliquot of liver lysate was incubated for 15 min at 37°C with 2 μl (3.6 U) of porcine pancreas phospholipase A2 (PLA2; MilliporeSigma) in 200 μl 2× PLA2 buffer {0.4 M Tris pH 8.0, 0.4 M NaCl, 0.6 mM CaCl2, 10 μM trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}.

RESULTS

Expression of the mouse Cyp2c and Cyp2j subfamily members in livers of LPS-treated mice

Mice were dosed with intraperitoneal injection of LPS and humanely killed at 0, 3, 6, 24, 48, 72, and 96 h after treatment. These time points were chosen to examine Cyp expression and eicosanoid levels at the peak of inflammation (3 and 6 h after LPS), at the end of clinical signs (24 h after LPS), and during resolution of inflammation (48–96 h after LPS) (24). In this study, Ptgs2 expression was only detectable in liver during acute inflammation at the 3- and 6-h time points (Fig. 1A). All of the Cyp2c and Cyp2j isoforms except Cyp2c65, Cyp2j12, and Cyp2j13 were detectable in liver. mRNA expression of most isoforms was unchanged at 3 and 6 h after LPS treatment; however, Cyp2c66, Cyp2c67, Cyp2c70, and Cyp2j8 were significantly induced early after LPS administration. In contrast, Cyp2j9 was significantly reduced as early as 3 h after LPS. Liver expression of 13 isoforms (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69, and Cyp2c70) was significantly reduced at 24 h after LPS administration (Fig. 1A and Supplemental Fig. S3A, C). Cyp2c39 expression was significantly increased at the 24-h time point but returned to baseline levels 96 h after LPS dosing. Of the 13 Cyp2c isoforms that were significantly reduced at the 24-h time point, 3 remained below baseline at 48 h, but 7 rebounded above baseline and were significantly increased at the 48- and/or 72-h time points. Thus, whereas expression of the liver P450s were generally reduced 24 h after LPS treatment, the Cyp2c and Cyp2j genes exhibited isoform-specific responses to LPS administration.

Figure 1.

Figure 1

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Expression of the mouse Cyp2c and Cyp2j mRNAs in liver during LPS-induced inflammation and resolution. Mouse livers were collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of bacterial LPS. A) RT-PCR quantification of mRNA expression for each of the Cyp2c and Cyp2j isoforms. Baseline (time 0) values were set to 1 for each isoform. B) Adjustment for primer efficiency reveals relative mRNA expression of each of the Cyp2c and Cyp2j isoforms (arbitrary units). Blank cells indicate values that were below the limit of detection (Ct >35). Colored bars to the right of each chart denote the scale of induction or suppression (A) or relative mRNA quantification (B) for each detectable isoform. Data shown are means ± se; n = 5/ time point. *P < 0.05.

Adjusting for the efficiency of the Sybr Green primers allows for an accurate comparison of relative P450 mRNA levels. Examination of adjusted mRNA levels revealed that the majority of Cyp2c/Cyp2j mRNA content in liver is from 3 isoforms; at baseline, Cyp2c29 makes up 70% of total Cyp2c/Cyp2j mRNA content in the liver, whereas Cyp2c70 and Cyp2j5 make up 10 and 11%, respectively (Figs. 1B and 2, and Supplemental Fig. S3B, D). Cyp2c29 is the most predominant Cyp2c/Cyp2j transcript for all time points, except at 24 h after the LPS administration, when Cyp2j5 was expressed at slightly higher levels (31%) than Cyp2c29 (29%). Because all other Cyp2c and Cyp2j isoforms together make up a small fraction of total Cyp2c/Cyp2j mRNA content (2–8%), the responses of Cyp2c29, Cyp2c70, and Cyp2j5 dominate total hepatic Cyp2c/Cyp2j mRNA content over the entire LPS time course. LPS treatment reduced total hepatic Cyp2c and Cyp2j mRNA content by 81% at 24 h (Fig. 2; P < 0.05). Hepatic Cyp2c and Cyp2j mRNA content rebounded to levels that were 40% higher than baseline at 72 h (P < 0.05) before returning to basal levels at the 96-h time point (Fig. 2).

Figure 2.

Figure 2

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Mouse Cyp2c and Cyp2j mRNAs in liver over the 96-h LPS time course. Pie charts represent adjusted expression of hepatic Cyp2c and Cyp2j isoforms for the 0-, 3-, 6-, 24-, 48-, 72-, and 96-h time points after LPS injection. The size of each pie chart is proportional to the combined Cyp2c and Cyp2j mRNA levels at each time point. The percentage of the total mRNA at each time point is indicated for each isoform that was expressed at >1% total mRNA. The bar graph shows summation of the Cyp2c and Cyp2j mRNAs for each time point (arbitrary units).

Expression of the mouse Cyp2c and Cyp2j subfamily members in kidneys of LPS-treated mice

LPS-induced changes in renal P450 expression was less than that observed in the liver. Four of the Cyp2c isoforms were not detectable in kidney (Cyp2c37, Cyp2c40, Cyp2c50, and Cyp2c65). Moreover, kidney levels of 7 of the detectable Cyp2c isoforms (Cyp2c29, Cyp2c38, Cyp2c39, Cyp2c54, Cyp2c66, Cyp2c67, and Cyp2c70) were not significantly changed at any time over the entire 96-h LPS time course (Fig. 3A and Supplemental Fig. S4A, C). LPS suppressed Cyp2c44 mRNA levels 3, 6, and 24 h after LPS dosing and reduced Cyp2c69 transcript levels at 3–48 h. Expression levels for Cyp2c44 and Cyp2c69 returned to baseline levels by the 48- and 72-h time points, respectively. Cyp2j5 and Cyp2j11 transcript levels were significantly decreased only at 24 h after LPS administration. LPS induced a transient 2–3-fold increase in Cyp2j8 and Cyp2j9 expression 6 h after LPS dosing; however, expression of both genes returned to baseline levels by the 24-h time point.

Figure 3.

Figure 3

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Expression of the mouse Cyp2c and Cyp2j mRNAs in kidney during LPS-induced inflammation and resolution. Mouse kidneys were collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of bacterial LPS. A) RT-PCR quantification of mRNA expression for each of the Cyp2c and Cyp2j isoforms. Baseline (time 0) values were set to 1 for each isoform. B) Adjustment for primer efficiency reveals relative mRNA expression of each of the Cyp2c and Cyp2j isoforms arbitrary units). Blank cells indicate values that were below the limit of detection (Ct >35). Colored bars to the right of each chart denote the scale of induction or suppression (A) or relative mRNA quantification (B) for each detectable isoform. Data shown are means ± se; n = 5/time point. *P < 0.05.

After adjustment for mouse Cyp2c and Cyp2j Sybr Green primer pair efficiency, Cyp2j5 was the most abundantly expressed Cyp2c/Cyp2j isoform in the kidney at all time points (Figs. 3B and 4, and Supplemental Fig. S4B, D). Cyp2j5 accounted for 87–91% of total Cyp2c/Cyp2j mRNA content in the kidney. Cyp2c44, Cyp2c54, Cyp2j6, and Cyp2j11 were expressed at much lower levels throughout the LPS time course (2–5% of total mRNA content). Total Cyp2c and Cyp2j mRNA content was reduced by 16% 24 h after LPS administration (Fig. 4; P < 0.05) but was not significantly different from vehicle-treated mice at any other time point.

Figure 4.

Figure 4

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Mouse Cyp2c and Cyp2j mRNAs in kidney over the 96-h LPS time course. Pie charts represent adjusted expression of renal Cyp2c and Cyp2j isoforms for the 0-, 3-, 6-, 24-, 48-, 72-, and 96-h time points after LPS injection. The size of each pie chart is proportional to the combined Cyp2c and Cyp2j mRNA levels at each time point. The percentage of the total mRNA at each time point is indicated for each isoform that was expressed at >1% total mRNA. The bar graph shows summation of the Cyp2c and Cyp2j mRNAs for each time point (in arbitrary units).

Expression of the mouse Cyp2c and Cyp2j subfamily members in duodenums of LPS-treated mice

Changes in expression of the Cyp2c and Cyp2j isoforms in the duodenum were similar to those observed in the liver. Only 12 of the 22 genes were detectable in vehicle-treated tissues. Expression of Cyp2c38, Cyp2c44, and Cyp2c66 was significantly reduced 24 h after LPS administration (Fig. 5A and Supplemental Fig. S5A, C). Cyp2c29 expression tended to be reduced at the 24- and 48-h time points (P = 0.05, P = 0.07, respectively). Expression of the Cyp2c and Cyp2j isoforms returned to baseline levels or higher by the 72-h time point. At 96 h post-LPS dosing, 7 of the 12 detectable isoforms were expressed at levels significantly higher than baseline.

Figure 5.

Figure 5

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Expression of the mouse Cyp2cs and Cyp2j mRNAs in duodenum during LPS-induced inflammation and resolution. Mouse duodenums were collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of bacterial LPS. A) RT-PCR quantification of mRNA expression for each of the Cyp2c and Cyp2j isoforms. Baseline (time 0) values were set to 1 for each isoform. B) Adjustment for primer efficiency reveals relative mRNA expression of each of the Cyp2c and Cyp2j isoforms (in arbitrary units). Blank cells indicate values that were below the limit of detection (Ct >35). Colored bars to the right of each chart denote the scale of induction or suppression (A) or relative mRNA quantification (B) for each detectable isoform. Data shown are means ± se; n = 5/ time point. *P < 0.05.

After adjusting for the efficiency of the primer sets used, Cyp2c29 and Cyp2c65 were the most abundantly expressed of the Cyp2c/Cyp2j isoforms; together, they accounted for ∼70% of total duodenal Cyp2c/Cyp2j mRNA content (Figs. 5B and 6 and Supplemental Fig. S5B, D). Cyp2c55, Cyp2c66, Cyp2c68, and Cyp2j6 each accounted for 5–10% of total duodenal Cyp2c/Cyp2j mRNA content. Total Cyp2c and Cyp2j mRNA content trended toward lower expression 24 and 48 h after LPS dosing (P = 0.06, P = 0.14, respectively). Total duodenal Cyp2c/Cyp2j mRNA content rebounded to levels that were 3-fold higher than baseline at the 96-h time point (Fig. 6, P < 0.05).

Figure 6.

Figure 6

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Mouse Cyp2c and Cyp2j mRNAs in duodenum over the 96-h LPS time course. Pie charts represent adjusted expression of duodenal Cyp2c and Cyp2j isoforms for the 0-, 3-, 6-, 24-, 48-, 72-, and 96-h time points after LPS injection. The size of each pie chart is proportional to the combined Cyp2c and Cyp2j mRNA levels at each time point. The percentage of the total mRNA at each time point is indicated for each isoform that was expressed at >1% total mRNA. The bar graph shows summation of the Cyp2c and Cyp2j mRNAs for each time point (arbitrary units).

Expression of the mouse Cyp2c and Cyp2j subfamily members in brains of LPS-treated mice

The expression pattern of the Cyp2c and Cyp2j mRNAs in brain was different from that in other tissues. None of the Cyp2c and Cyp2j mRNAs were significantly reduced 24 h after LPS administration (Fig. 7A and Supplemental Fig. S6A, C). Cyp2c39 expression was elevated at the 24-h time point, and expression of 4 of the isoforms (Cyp2c39, Cyp2j6, Cyp2j9, and Cyp2j12) was significantly increased from 48 to 96 h after LPS dosing. Expression of 8 of the 13 detectable brain Cyp2c and Cyp2j isoforms remained unchanged from basal levels at all time points.

Figure 7.

Figure 7

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Expression of the mouse Cyp2cs and Cyp2j mRNAs in brain during LPS-induced inflammation and resolution. Mouse brain was collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of bacterial LPS. A) RT-PCR quantification of mRNA expression for each of the Cyp2c and Cyp2j isoforms. Baseline (time 0) values were set to 1 for each isoform. B) Adjustment for primer efficiency reveals relative mRNA expression of each of the Cyp2c and Cyp2j isoforms (arbitrary units). Blank cells indicate values that were below the limit of detection (Ct >35). Colored bars to the right of each chart denote the scale of induction or suppression (A) or relative mRNA quantification (B) for each detectable isoform. Data shown are means ± se; n = 5/time point. *P < 0.05.

Adjustment for the Sybr Green primer set efficiencies revealed that Cyp2c54 (43% of total Cyp2c/Cyp2j content), Cyp2j9 (32%), and Cyp2j6 (16%) were the most abundant of the brain isoforms at baseline. Expression of Cyp2c54 was relatively constant over the 96-h time course; however, Cyp2j6 and Cyp2j9 mRNA levels increased 3–4-fold from 24 to 96 h after LPS dosing. Thus, whereas brain had an equal ratio of Cyp2c and Cyp2j mRNAs at baseline, Cyp2j mRNA content was greater than Cyp2c content by 76–24%, respectively, at 96 h (Figs. 7B and 8 and Supplemental Fig. S6B, D). Total Cyp2c and Cyp2j mRNA content was unchanged at 3–6 h after LPS dosing but approximately doubled at the 24-, 48-, 72-, and 96-h time points (P = 0.06, 0.003, 0.02, and 0.004, respectively).

Figure 8.

Figure 8

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Mouse Cyp2c and Cyp2j mRNAs in brain over the 96-h LPS time course. Pie charts represent adjusted expression of Cyp2c and Cyp2j isoforms in brain for the 0-, 3-, 6-, 24-, 48-, 72-, and 96-h time points after LPS injection. The size of each pie chart is proportional to the combined Cyp2c and Cyp2j mRNA levels at each time point. The percentage of the total mRNA at each time point is indicated for each isoform that was expressed at >1% total mRNA. The bar graph shows a summation of the Cyp2c and Cyp2j mRNAs for each time point (arbitrary units).

Plasma fatty acid and oxylipin levels in LPS-treated mice

Plasma was collected at each time point, and levels of AA, linoleic acid (LA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and their oxylipin metabolites were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The fatty acids and oxylipin metabolites are reported as a percentage of baseline levels (Fig. 9). At 3 and 6 h after LPS injection, plasma levels of AA (203, 218%) and LA (249, 301%) were significantly increased and returned to near-baseline levels by the 24-h time point (Fig. 9 and Supplemental Fig. S7A, B). The ω-3 fatty acids EPA and DHA followed a similar pattern; plasma levels significantly increased (268 and 251%) 6 h after LPS dosing and then returned to near-baseline levels within 24 h of LPS dosing (Fig. 9 and Supplemental Fig. S7C). These findings are consistent with the release of free fatty acids from membrane stores following transient activation of PLA2 by LPS.

Figure 9.

Figure 9

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Effect of LPS-induced inflammation on plasma levels of AA, LA, EPA, DHA, and their oxylipin metabolites. Plasma fatty acid and oxylipin metabolite levels were determined by LC-MS/MS in samples collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of LPS. Corresponding epoxide and diol concentrations were combined to estimate changes in total epoxygenase metabolite levels for each regioisomer. Data shown are reported as percentages of baseline. Colored bars to the right of each chart denote the scale of increase or decrease in oxylipin levels relative to baseline; n = 5/time point. *P < 0.05.

Plasma oxylipin levels closely mirrored those of the parental fatty acids. Of the AA epoxides, only 14,15-EET levels were consistently detectable. Relative levels for each are reported as percentage of baseline, wherein the 0-h PBS control was set to 100%. Plasma levels of 14,15-EET + DHET; 11,12-EET + DHET; 8,9-EET + DHET; and 5,6-EET + DHET were significantly increased at 3 h (252, 268, 228, and 195% of baseline, respectively) and 6 h (297, 377, 217, and 189% of baseline, respectively) after LPS administration and then decreased toward baseline levels by the 24-h time point (Fig. 9 and Supplemental Fig. S8). Plasma levels of prostaglandin D₂ (PGD2) and prostaglandin E₂ (PGE2) peaked 3 h after LPS administration and remained elevated at 6 and 24 h after LPS compared with baseline levels (Fig. 9). Plasma levels of the LA-derived 9,10- epoxy-12Z-octadecenoic acid (9,10-EpOME) + 9,10-dihydroxy-12-octadecenoic acid (DiHOME) and 12,13-EpOME + DiHOME increased by 219 and 298%, respectively, at 3 h, increased by 280 and 410%, respectively, at 6 h, and then decreased toward baseline levels by the 24-h time point (Fig. 9 and Supplemental Fig. S9A, B). Similarly, plasma levels of EPA-derived 17,18-epoxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (EpETE) + dehydroepiandrosterone (DHEQ) and docosahexaenoic acid (DHA)-derived 19,20-4Z,7Z,10Z,13Z,16Z (EpDPE) + dihydroxy-docosapentaenoic acid (DiHDPA) increased to 240 and 268% of baseline, respectively, at 3 h and increased to 339 and 489% of baseline, respectively, at 6 h post-LPS dosing and then returned toward baseline levels at the 24-h time point (Fig. 9 and Supplemental Fig. S9C, D).

Effect of LPS on levels of free and esterified fatty acids and oxylipins in liver

Levels of free (nonesterified) and esterified fatty acids and their oxylipin metabolites in liver were also measured by LC-MS/MS. Relative levels for each are reported as a percentage of baseline, wherein the 0-h PBS control was set to 100% (Fig. 10). Hepatic levels of free AA, EPA, and DHA remained largely unchanged from baseline, except for significant increases in EPA (220%) at the 24-h time point and in DHA (129%) at the 48-h time point. Esterified AA, EPA, and DHA levels in liver also remained relatively constant except for significant increases in AA (125%) and DHA (123%) at the 48-h time point and a significant decrease in EPA (53%) at the 24-h time point. In contrast, free LA levels in liver significantly decreased 6–96 h after LPS dosing, whereas membrane-bound LA levels were significantly increased for the same time points.

Figure 10.

Figure 10

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Effect of LPS-induced inflammation on levels of free and membrane-bound hepatic fatty acids and their oxylipin metabolites. Free and esterified fatty acids and oxylipin metabolite levels were determined by LC-MS/MS in liver samples collected at 0, 3, 6, 24, 48, 72, and 96 h after 1-mg/kg injection of LPS. Corresponding epoxide and diol concentrations were combined to estimate changes in total epoxygenase metabolite levels for each regioisomer. Data shown are reported as percentages of baseline. Colored bars to the right of each chart denote the scale of increase or decrease in oxylipin levels relative to baseline; n = 5/time point. *P < 0.05.

Free and esterified oxylipin levels were minimally or inconsistently altered throughout the 96-h time course after LPS treatment, with a few notable exceptions. Compared with baseline concentrations, levels of free 9,10-EpOME + DiHOME and free 19,20-EpDPE + DiHDPA were significantly elevated at multiple time points from 3 to 96 h after LPS dosing. Levels of esterified 8,9-EET + DHET (at the 24-, 48-, 72-, and 96-h time points) and 14,15-EET + DHET (at the 6-, 24-, 72-, and 96-h time points) were significantly decreased relative to baseline, whereas levels of esterified 5,6-EET + DHET (at the 48-, 72-, and 96-h time points) and 11,12-EET + DHET (at the 48-h time point) were significantly increased.

The level of the free 9,10-EpOME + DiHOME was significantly increased in liver at 3, 24, 48, and 96 h after LPS dosing. By contrast, the level of esterified 9,10-EpOME + DiHOME was decreased significantly at the 6-, 48-, 72-, and 96-h time points. Levels of free 12,13-EpOME + DiHOME remained constant throughout the LPS time course. Levels of esterified 12,13-EpOME + DiHOME levels were also largely unchanged after LPS treatment except for a small (27%) but significant increase at the 72-h time point. Thus, for these LA-derived oxylipins, changes in free and esterified forms appeared to mirror each other. In contrast, the level of the free 17,18-EpETE + DHEQ remained relatively constant in liver throughout the LPS time course, whereas the esterified level initially decreased significantly for the 3-, 6-, and 24-h time points and then significantly increased by the 72- and 96-h time points. The level of the free 19,20-EpDPE + DiHDPA was significantly increased throughout the entire LPS treatment time course, whereas the esterified levels were unchanged from baseline at all time points. Thus, for the EPA- and DHA-derived oxylipins, changes in free and esterified forms did not mirror each other.

DISCUSSION

Inflammation is known to alter the expression and function of many hepatic P450 enzymes (11, 25). Although P450 expression is usually suppressed in inflammatory models, expression may also be induced or unaffected (26). A comprehensive analysis of inflammation-induced changes in the Cyp2c and Cyp2j isoforms and their bioactive oxylipin metabolites has never been performed. The current study revealed several important findings: 1) regulation of Cyp2c and Cyp2j expression during a 96-h time course after LPS-induced inflammation and resolution is isoform and tissue specific; 2) PCR primer efficiency adjustments permit absolute quantitation of relative expression of the 15 mouse Cyp2c and 7 Cyp2j isoforms; 3) LPS induced a pattern of expression of Cyp2c/Cyp2j isoforms in liver and duodenum that was dissimilar to that observed in kidney and brain; 4) plasma levels of AA, LA, EPA, and DHA, and their bioactive epoxide/diol metabolites, increase at 3–6 h after LPS treatment, consistent with early transient activation of PLA2; and 5) the LPS-induced decrease in levels of esterified oxylipins in liver suggest that membrane-bound oxylipins may be released early during inflammation.

Previous reports have assessed LPS-induced expression changes of a small subset of P450 genes in select tissues using assays that we have recently shown to be nonspecific for their intended target (15, 16). Expression of murine Cyp2c29, Cyp2c44, Cyp2c55, and Cyp2j5 isoforms was previously shown to be down-regulated after LPS administration (11, 27). Expression of renal Cyp2c44 and Cyp2j5; pulmonary Cyp2c44, Cyp2j5, and Cyp2j9; and myocardial Cyp2c44 was also reduced during a time course of LPS treatment (11). The data in the current manuscript suggest that the effect of LPS-induced inflammation is both isoform and tissue specific. Expression of Cyp2c29, one of the most abundantly expressed isoforms in liver and duodenum at baseline, is reduced by >90% in both tissues at 24 h. In contrast, expression of the other major liver Cyp2c/Cyp2j isoforms, Cyp2c70 and Cyp2j5, are suppressed by only 44–66%. Similarly, in duodenum, Cyp2c29 expression is reduced by 96% at 24 h, whereas Cyp2c65 and Cyp2j6 are reduced by <30%. In both tissues, Cyp2c29 is the most abundantly expressed isoform at baseline, but it represents a more minor constituent of the P450 pie after 24 h of LPS-induced inflammation.

We determined primer PCR efficiency against standard curves for each cDNA and adjusted RT-PCR values to allow for direct comparisons of the abundance of each Cyp2c and Cyp2j isoform at each time point. This adjustment step is critical to understanding the functional role of LPS in suppression of each isoform. For example, whereas LPS strongly suppresses expression of many isoforms in the liver, including Cyp2c38, Cyp2c40, Cyp2c50, Cyp2c54, and Cyp2c55, such suppression is likely of minimal consequence because these isoforms combine to make up <1% of all Cyp2c/Cyp2j mRNAs in the liver. Absolute mRNA quantification of each isoform reveals the importance of LPS on expression of Cyp2c29, Cyp2c70, and Cyp2j5 in liver; Cyp2j5 in kidney; Cyp2c29 and Cyp2c65 in duodenum; and Cyp2j54, Cyp2j6 and Cyp2j9 in brain. It is primarily the regulation of these key isoforms that contributes to the overall Cyp2c/Cyp2j expression after LPS-induced inflammation.

The overall pattern of LPS-induced changes in Cyp2c/Cyp2j isoform expression was different in each of the 4 tissues examined. Total hepatic Cyp2c/Cyp2j expression was unchanged during early inflammation, decreased substantially at 24 h post-LPS, and then rebounded to higher-than-baseline levels at 72 h post-LPS before returning to basal levels at the 96-h time point. Total Cyp2c/Cyp2j expression in duodenum was qualitatively similar to that in liver, except that the decrease in Cyp2c/Cyp2j expression at 24 h was followed by a delayed recovery and above-basal expression at the 96-h time point. In contrast, LPS induced minimal changes in total renal Cyp2c/Cyp2j expression, resulting in a small but significant 16% reduction in Cyp2c/2j mRNA at the 24-h time point. Surprisingly, LPS did not suppress total Cyp2c/Cyp2j expression in brain at any time point. Instead, total brain Cyp2c/Cyp2j mRNA levels steadily increased throughout the time course of LPS-induced inflammation and reached a statistically significant 2-fold induction at 48-, 72-, and 96-h time points.

Other studies also suggest that the mechanisms through which LPS regulates P450 expression are complex and may involve various transcription factors that are both P450 isoform and tissue specific (11, 26, 28). Our findings of changes in hepatic Cyp2c and Cyp2j transcript levels over the 96-h LPS time course, as well as time-dependent changes in fatty acid metabolism, are consistent with LPS acting via a pretranslational mechanism, as proposed by others in refs. 11 and 26–28. The observed isoform-, time-, and tissue-dependent responses of P450 genes to LPS suggest that the mechanism for each Cyp2c and Cyp2j isoform may involve a diverse set of intracellular signaling molecules and transcription factors that depend on the specific isoform and tissue. Induction of P450 epoxygenase gene expression and anti-inflammatory eicosanoid production in brain may protect against neurodegenerative inflammation and might limit Parkinson or Alzheimer disease progression (29–32).

The effect of LPS on P450 expression appeared stronger in the Cyp2c subfamily as compared with the Cyp2j subfamily. Cyp2c isoforms make up 89% of liver Cyp2c/Cyp2j mRNA and 95% of duodenal Cyp2c/Cyp2j mRNA expression. Most of the Cyp2c isoforms in these tissues were repressed by more than 80% 24 h after LPS. In contrast, Cyp2j mRNA in these tissues was more modestly suppressed by LPS. Total Cyp2c/Cyp2j mRNA in kidney, which is dominated by Cyp2j5 expression (93% of total), was minimally altered by LPS (16% reduction at 24 h post-LPS). In addition, brain Cyp2c/Cyp2j mRNA is evenly split between Cyp2c and Cyp2j mRNA at baseline; however, LPS strongly induces the abundant cerebral Cyp2j isoforms (∼3-fold by 96 h post-LPS). Although the diminished suppression and/or induction of Cyp2j isoforms goes against the prevailing paradigm of inflammation-induced Cyp suppression, other studies have reported Cyp2j induction after LPS treatment (33).

A limitation of the current study was that we quantified mRNA levels rather than protein expression or activity. In general, CYP protein levels and CYP enzymatic activity track well (34, 35), although there are examples in which the CYP protein is unstable (e.g., CYP2J6) and activity is far lower than would be predicted based on protein expression (36). Unfortunately, there are very few isoform-selective antibodies that can distinguish between the various murine CYP2C and CYP2J proteins, which are highly homologous.

LPS treatment promotes a robust inflammatory response via a complex signaling cascade, resulting in the secretion of proinflammatory cytokines (7, 9). P450-derived EETs have potent anti-inflammatory effects, which occur through the suppression of NF-κB signaling and inhibition of IκB kinase (12). Several additional studies support the role of CYP-derived oxylipins as anti-inflammatory mediators in vivo (13, 37). Together, these data suggest that the suppression of P450 epoxygenase activity and epoxy fatty acid (EpFA) production may serve to potentiate the acute proinflammatory effects of LPS.

LPS induced a transient increase in circulating fatty acids and oxylipins. Plasma AA levels increased 2-fold over baseline at 3 and 6 h after LPS treatment. Similar 2–3-fold transient increases were observed for LA, EPA, and DHA. This suggests transient activation of PLA2 activity during the first several hours of inflammation, as reported by others in refs. 38 and 39. Plasma oxylipin levels also transiently increased during the first several hours after LPS dosing; however, the oxylipin increases were severalfold higher than their corresponding parental fatty acids. For instance, 14,15-EET + DHET increased 2.5-fold over baseline, and EpOMEs + DiHOMEs increased 2–4-fold over baseline. Cyp2c/Cyp2j mRNA levels were unchanged during early inflammation, which suggests that P450 epoxygenase levels were also likely similar to baseline levels during acute LPS-induced inflammation. Together, these data suggest that transient release of fatty acids increases substrate concentrations, which results in enhanced EpFA biosynthesis and release. The concentration of most analytes remained elevated, if not significantly so, at the 24- and 48-h time points, at a time when multiple tissues showed significant reduction in Cyp2c/Cyp2j mRNA levels. Thus, our data suggest that PLA2-induced release of substrate, rather than P450 expression, is a major factor in determining EpFA levels in plasma after LPS.

Human CYP2C and CYP2J isoforms are known to metabolize EPA and DHA to fatty acid epoxides (40, 41); however, kinetic data on the corresponding mouse isoforms is lacking. We suspect that in vitro kinetic assays using mouse CYPs would be similar to those observed for human CYPs. Interestingly, although the levels of EPA and DHA are similar to those of AA, the amounts of EPA and DHA metabolites are far more abundant than those of AA. The precise mechanisms of EpFA production and disposal are poorly understood. In vivo, steady-state levels of AA, EPA, and DHA metabolites result from a combination of formation, release, membrane reesterification, hydrolysis, and ultimately, excretion or catabolism. The abundance of LA-, EPA-, and DHA-derived EpFAs over EETs is consistent with previously published reports (6, 42); however, the regulation of specific EpFA levels by PLA2, CYP, EPHX, and acyl-CoA synthetase long-chain (ACSL) enzymes is complex and poorly studied (6, 43, 44). Like EETs, EPA and DHA epoxides have anti-inflammatory effects; however, they appear to activate alternative receptor pathways and also have distinct effects, including antiarrhythmic properties (45, 46).

Liver, as a large organ that abundantly expresses P450s, may represent a significant source of EpFA production. We measured both free and phospholipid-bound (esterified) levels of fatty acids and oxylipin metabolites in liver at each point in the 96-h time course. Interestingly, free levels of AA and 14,15-EET + DHET were relatively stable and did not follow the transient increase and decrease that was observed for these analytes in plasma. Thus, whereas esterified AA levels were consistent or mildly increasing over the 96-h time course, esterified 14,15-EET + DHET dropped to levels that were significantly lower than baseline at 6–96 h after LPS treatment. At 24 h, liver tissue had lost 35% of its esterified 14,15-EET + DHET content, which represented a loss of nearly 100 ng of 14,15-EET from liver membranes. Together, these data suggest the possibility that, in addition to de novo formation of EETs from liberated AA, direct release of EETs from phospholipids may account, at least in part, for a significant portion of circulating EETs following LPS treatment.

In summary, we demonstrate that the expression of the 15 mouse Cyp2cs and 7 mouse Cyp2js is altered over a 96-h time course of LPS inflammation and resolution in an isoform- and tissue-specific manner. LPS induced strong suppression of total Cyp2c/Cyp2j content in liver and duodenum that rebounded above baseline during resolution of inflammation. In contrast, Cyp2c/Cyp2j expression in kidney is only modestly regulated by LPS, whereas Cyp2c/2j expression in brain is increased by LPS. The acute rise and decline of plasma fatty acid, epoxide, and diol metabolites suggests that LPS-induced PLA2 activation, rather than P450 expression, regulates circulating EpFA levels. Our findings, together with the known anti-inflammatory effects of EETs, suggest that the biphasic suppression and recovery of mouse Cyp2c and Cyp2j isoforms and associated changes in eicosanoid levels during LPS-induced inflammation and resolution may have important physiologic consequences.

ACKNOWLEDGMENTS

The authors thank Lois Wyrick [National Institute of Environmental Health Sciences (NIEHS), U.S. National Institutes of Health (NIH); contractor] for graphic arts assistance. This work was supported by the Division of Intramural Research, NIEHS/NIH, (Z01 ES025034 to D.C.Z.). The authors declare no conflicts of interest.

Glossary

AA

arachidonic acid

DHA

docsahexaenoic acid

DHEQ

dehydroepiandrosterone

DHET

dihydroxyeicosatrienoic acid

DiHDPA

dihydroxy-docosapentaenoic acid

DiHOME

dihydroxy-12-octadecenoic acid (DiHOME

EET

epoxyeicosatrienoic acid

EPA

eicosapentaenoic acid

EpDPE

4Z,7Z,10Z,13Z,16Z

EpETE

epoxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid

EpFA

epoxy fatty acid

EPHX

epoxide hydrolase

EpOME

epoxy-12Z-octadecenoic acid

LA

linoleic acid

LC-MS/MS

liquid chromatography with tandem mass spectrometry

PLA2

phospholipase A2

Ppil

peptidylprolyl isomerase (cyclophilin)-like

Ptgs2

prostaglandin-endoperoxide synthase 2

qPCR

quantitative PCR

sEH

soluble epoxide hydrolase

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

AUTHOR CONTRIBUTIONS

J. P. Graves, J. A. Bradbury, A. Gruzdev, M. L. Edin, and D. C. Zeldin designed the research; J. P. Graves, J. A. Bradbury, F. B. Lih, and M. L. Edin performed research; A. Gruzdev and C. Duval contributed new reagents or analytical tools; J. P. Graves, A. Gruzdev, M. L. Edin, and D. C. Zeldin analyzed data; and J. P. Graves, A. Gruzdev, C. Duval, M. L. Edin, and D. C. Zeldin wrote the manuscript.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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Associated Data

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Supplementary Materials

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