Altered Eicosanoid Production and Phospholipid Remodeling During Cell Culture

The remodeling of polyunsaturated fatty acids (PUFAs) by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48 or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, TxB 2 , PGE 2 and PGD 2 , and the 5-LO pathway, LTB 4 , LTC 4 and 5-HETE which decreased with increasing time in culture. However, the 5-LO metabolites of a 20:3 fatty acid, LTB 3 , all trans-LTB 3 , LTC 3 and 5-HETrE, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace an increasingly scarce arachidonate. In addition, the amount of TNFα induced by LPS stimulation was significantly increased in the cells cultured for 72 h compared to 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species, however without sufficient n-6 fatty acids, resulted in altered lipid mediator biosynthesis and inflammatory response. 5-Lipoxygenase catalyzes oxygenation of arachidonic acid (AA) to 5(S)-hydroperoxy-6- trans -8,11,14-cis-eicosatetraenoic acid (5-HpETE), and further dehydration to the allylic epoxide LTA 4 . LTA 4 is converted by LTA 4 hydrolase to the dihydroxy acid LTB 4 , and by LTC 4 synthase to the glutathione conjugate LTC 4 . LTB 4 is a pro-inflammatory lipid mediator that activates and recruits neutrophils into inflammatory areas whereas cysLTs (LTC 4 and its metabolites LTD 4 , and LTE 4 extravasation, also participate in vascular leakage


Introduction
Almost a century ago, the concept of essential fatty acid efficiency emerged (1) which led to the discovery that animal cells could not make polyunsaturated fatty acids (PUFAs) such as linoleic acid (LA, 18:2, n-6) or α-linolenic acid (ALA, 18:3, n-3). The absence of these fatty acids in the diet caused a diverse number of clinical signs (2) and altered levels of arachidonate (3). Cellular biochemistry responds to a lack of LA and ALA by chain elongation and desaturation of oleoyl-CoA (n-9) to eventually form an unusual PUFA 5,8,11-eicosatrienoic acid (n-9) often called Mead acid after its discoverer (4). This PUFA retains biophysical properties critical for cellular membrane fluidity, but lacks the ability to be transformed into certain lipid mediators. The appearance of Mead acid esterified to cellular phospholipids is a result of the same biochemistry that normally takes place when PUFAs such as ALA and arachidonic acid from the diet are incorporated into complex lipids. Once within the cell these PUFAs are very quickly remodeled into cellular phospholipids. For example, the remodeling of arachidonic acid by what has been termed the Lands cycle has been studied for a number of years (5) and is known to be responsible for the diversity of phospholipid molecular species seen in all cellular membranes. This phospholipid remodeling pathway is a result of CoA esters of PUFAs being synthesized by a family of long chain acyl CoA-synthases (ACSLs) (6) and the action of specific acyltransferases, including the lysophospholipid acyltransferases LPCAT3/MBOAT5 (7) and LPIAT1/MBOAT7 (8), known to incorporate arachidonic acid into phosphatidylcholine and phosphatidylinositol, respectively.
Macrophages, which are a major source of various lipid mediators, respond to a variety of stimuli by activating phospholipase A2 and producing eicosanoids. The RAW 264.7 murine macrophage cell line has been used in numerous studies as a model of primary macrophages (13). RAW cells respond to lipopolysaccharide (LPS) and interferon-γ (IFN-γ) by producing nitric oxide and PGE2 (14). Following calcium ionophore A23187 stimulation, RAW cells also produced modest amounts of leukotrienes and prostaglandins (15). The addition of LPS, phorbol 12-myristate 13-acetate, or A23187 resulted in increase of lysophosphatidylinositol levels (16).
After oxidation of arachidonate and transformation of the free fatty acid into the bioactive chemical structure, the eicosanoid is transported out of the cell so that it can encounter one of many numerous G-protein coupled receptors for prostaglandins (10) and leukotrienes (12,17) found on target cells. However, a large portion of the liberated arachidonate is not by guest, on July 20, 2018 www.jlr.org Downloaded from oxidized, but rather converted back into a CoA ester to complete the remodeling cycle, or to undergo mitochondrial and peroxisomal β-oxidation.
The rapidity of cellular essential fatty acid deficiency, especially for cells carried in tissue culture, has not been widely appreciated. There have been a few studies with either supplementation of arachidonate or other PUFAs and the effect of serum starvation on cellular function (18). In such studies a remarkable effect of depletion of arachidonic acid has been noted (19). Current recommendations for cell culture suggest changing culture media (resupply of PUFAs) only as frequently as 3 days with no suggestion that this period of time could lead to important biochemical changes by depletion of PUFAs (20). There have not been detailed studies of the alteration of the lipidome, specifically those phospholipid molecular species altered as a result of serum starvation or depletion of PUFAs during tissue culture for times as short as 3 days. Reported here is the time-dependent effect of tissue culture on the production of eicosanoids, specifically prostaglandins and leukotrienes, and time-dependent alterations in phospholipids in the membranes of cells carried in culture.

Materials and Methods
Materials. Eicosanoid standards including leukotriene B3 and deuterium-labeled eicosanoids were purchased from Cayman Chemical (Ann Arbor, MI). Phospholipid standards and deuterium-labeled phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). acid. For RP-HPLC-MS/MS, a Shimadzu liquid chromatography system consisting of four LC-20AD pumps, a SIL-20AC autosampler, a CTO-20AC column oven, a FCV-12AH six-port switching valve, and a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (Thermo Fisher Scientific, Waltham, MA) were used (21). An aliquot of each sample (50 µL) was injected into the trap column, an Opti-Guard Mini C18, at a total flow rate of 500 µL/min. Three minutes after sample injection, the valve was switched to introduce the trapped sample to the analytical column, a Capcell Pak C18 MGS3 (Shiseido, Tokyo, Japan). Separation of lipids was achieved by a linear gradient using water and acetonitrile containing 0.1% formic acid. The total flow rate was 120 µL/min, the column temperature was set at 46° C, and the LC column eluent was introduced directly into a TSQ Quantum Ultra. All compounds were analyzed in a negative ion polarity mode. Eicosanoids were quantified by multiple reaction monitoring (MRM

Results
Eicosanoid profiling in A23187-stimulated RAW cells. RAW 264.7 cells (5 × 10 6 cells in 100 mm dish) were cultured for 24, 48 or 72 h without any medium changes. After incubation with or without calcium ionophore A23187, eicosanoids were extracted and quantitated using a targeted RP-LC-MS/MS assay. The stimulated production of arachidonate metabolites derived from the cyclooxygenase pathway, TxB2, PGE2 and PGD2 (Figure 1), was found to decrease for those cells cultured for increasing amounts of time.
In a similar fashion, the absolute quantities of 5-lipoxygenase metabolites LTC4 and LTB4 as well as free arachidonate, generated after calcium ionophore stimulation, were also found to decrease ( each leukotriene species (23). Analysis of the LTA3 nonenzymatic product all-trans-LTB3 was established by HPLC retention time and mass spectrometry and this product was found to be more abundant than LTB3 (Supplemental Figure S2). The structure of the 20:3 fatty acid was determined by the Paternò-Büchi reaction as 5,8,11-eicosatrienoic acid (20:3 n-9) as described elsewhere (24).  Table S1). This negative ion based electrospray ionization assay was based on scheduled ion transitions for molecular ions of  Figure 4). In general, the most significant positive fold abundances in day 1 relative to day 3 corresponded to the arachidonate containing molecular species of PC and PI while the majority of the negative fold abundances were PE and BMP phospholipid species that contained esterified 20:3. As an example, a box plot of the data on PC(16:0e_20:4) revealed a 2-fold decrease from day 1 to 3 ( Figure 4B) while PC(16e:0_20:3) had over a 2-fold increase in abundance from day 1 to 3 ( Figure 4C).
Phosphatidylcholine molecular species PC(18:1e_20:4) and PC(18:0e_20:4) had just the opposite relative abundance changes from Day 1 to Day 3 in culture (Supplemental Figure S3). This pattern of arachidonate-content in these ether PC species was similar to the trend for the stimulated production of LTC4 from these cells at the corresponding day of culture. Overall, these results revealed that the molecular species of arachidonate phospholipids were drastically remodeled during cell culture without medium change and that a new fatty acyl group was being populated into the phospholipid classes to replace an increasingly scarce arachidonate. That new fatty acid was 5,8,11-eicosatrienoic acid also called Mead acid (n-9).
TNF stimulated response of RAW in culture without medium changes. The fatty acid composition of membrane phospholipids in macrophages can influence inflammatory responses (27,28). While lauric acid (C12:0) enhanced the recruitment of Toll-like receptor 4 (TLR4), and its adaptor protein, TIR domain-containing adaptor inducing interferon- (TRIF) and myeloid differentiation factor 88 (MyD88) into lipid rafts and induced TLR4 activation, DHA inhibited the recruitment to lipid rafts and the activation of TLR4 (29). The amount of TNFα induced after 4 hr LPS stimulation was significantly higher in those cells cultured for 72 h compared to 24 h without medium change ( Figure. 5), suggesting that the remodeling of phospholipid molecular species enriched with esterified arachidonate to esterified Mead acid enhanced inflammatory response of macrophage.

Discussion
The ability to carry cells in tissue culture has had an enormous impact on revealing specific biochemical events as well as cell biology. While artifacts that emerge by using such in vitro methods are known, there has been little attention paid to the alterations of lipid composition introduced by such protocols. In addition to this, it has been an established protocol to use serum starvation in many studies, including studies of T and B cell immunology as a practical means to induce specific biochemical events (18,30). This is particularly relevant for the essential biochemical molecules that cannot be synthesized de novo. A striking class of such molecules are the polyunsaturated fatty acids of the n-6 and n-3 families that require exogenous linoleic acid or α-linolenic acid to populate a large family of unsaturated fatty acids found in cellular membranes esterified to phospholipids. This family includes arachidonic acid that is the precursor of a family of bioactive lipid mediators such as prostanoids and leukotrienes. With long term culture without supplementation of these essential fatty acids, the n-6 and n-3 fatty acids containing phospholipids can be depleted. This was readily demonstrated in this study by the depletion of arachidonic acid in phospholipids of all classes, even after just a two or three day continuous culture.
The depletion of arachidonic acid stores is not without consequences as observed for the generation of prostaglandins and leukotrienes following stimulation of cells carried in long term culture. Prostaglandins require a sequence of three double bonds interrupted by methylene groups in a specific structural position along the 20 carbon chain so that a hydrogen atom can be abstracted by PGH synthase at carbon-13 to form an oxygen radical 11-hydrogenperoxy as the initial step of the cyclooxygenase mechanism (31). This 3-double bond structural requirement for cyclooxygenase is observed in arachidonate (5,8,11,14-eicosatetraenoic acid) as well as in dihomoγ-linolenic acid (8,11,14-eicosatrienoic acid), but not in the isomeric Mead acid (5,8,11eicosatetraenoic acid). Thus, the accumulation of Mead acid in cell membrane phospholipids would be expected to have a measurable effect on prostaglandin biosynthesis. This is exactly what was found when RAW 264.7 cells carried in culture for two to three days were stimulated with a calcium ionophore to release arachidonic acid for PGH synthase.
On the other hand, the biosynthesis of leukotrienes involves 5-lipoxygenase abstracting a hydrogen atom from carbon-7 from a fatty acid substrate that also has homo conjugated 3-double bond structure. This structural requirement leads to formation of the 5-hydroperoxy radical that can be further converted by 5-lipoxygenase to form a triene conjugated epoxide. Arachidonate has this structural prerequisite as well as Mead acid, but not dihomo-γ-linolenic acid. In this case of 5lipoxygenase, leukotriene products of the 3 series (LTC3 and LTB3) can be formed but LTA4 hydrolase is not very efficient for the production of LTB3 (32). LTA3 was previously found to inhibit LTB4 production by blocking the binding of LTA4 to LTA4 hydrolase (33). Thus, while the LTC3 time-dependently increased, the all-trans LTB3 and LTB3, which can be also formed from LTA3, were not abundantly found. Nevertheless the all trans-LTB3 product was found to be more abundant consistent with the non-enzymatic hydrolysis of LTA3 derived from Mead acid. These products were defined several years ago (32)(33)(34)(35) and have similar, but somewhat less potent actions through the LTB-R and cysLTR receptors (35).
Cells respond to depletion of essential PUFAs by converting oleic acid (n-9) which can be made from acetate, into a PUFA. These steps involve a Δ 6 -desaturase (FADS1) to form 6,9-octadecadienoic acid. This fatty acid can then be elongated by two carbons (after making the CoA ester) by an elongase such as ELOVL4 to form 8,11-eicosadienoic acid (n-9). Subsequent The use of statistical tools to provide an overview of the significant changes in arachidonoyl and eicosatrienoyl-containing phospholipids readily revealed the significant depletion of arachidonate from most classes of phospholipids. While some molecular species were not significantly altered during the two and three days of unsupplemented culture, as indicated by those unlabeled species in the volcano plots, there were many more phospholipids which did change. In addition, a number of new species became quite apparent, while the most abundant arachidonate species at day one decreased with the appearance of the corresponding 20:3 species of that class. This clearly indicated the rapid operation of the Lands cycle to maintain membrane fluidity by populating PUFA species that were being depleted during the normal remodeling of phospholipids and release of arachidonic acid by various phospholipases. The most abundant phospholipid species containing arachidonic acid including PC and PI in day one, while in day two the PE and PS species that contain 20:3, were becoming much more significant. Even the difference between day three and day two indicated remodeling of phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine molecules species. The overall effect of these changes in PUFA molecular species was revealed by a reduced ability to generate PGE2 and PGD2 as time for these cells in culture progressed as well as the significant diminution of the ability to make LTC4 and LTB4. In part this was made up by an increased production of LTB3 and LTC3 with days in culture as the altered inflammatory cytokine response to LPS.