Exogenous tetracosahexaenoic acid modifies the fatty acid composition of human primary T lymphocytes and Jurkat T cell leukemia cells contingent on cell type

Abstract Tetracosahexaenoic acid (24:6ω‐3) is an intermediate in the conversion of 18:3ω‐3 to 22:6ω‐3 in mammals. There is limited information about whether cells can assimilate and metabolize exogenous 24:6ω‐3. This study compared the effect of incubation with 24:6ω‐3 on the fatty acid composition of two related cell types, primary CD3+ T lymphocytes and Jurkat T cell leukemia, which differ in the integrity of the polyunsaturated fatty acid (PUFA) biosynthesis pathway. 24:6ω‐3 was only detected in either cell type when cells were incubated with 24:6ω‐3. Incubation with 24:6ω‐3 induced similar increments in the amount of 22:6ω‐3 in both cell types and modified the homeoviscous adaptations fatty acid composition induced by activation of T lymphocytes. The effect of incubation with 18:3ω‐3 compared to 24:6ω‐3 on the increment in 22:6ω‐3 was tested in Jurkat cells because primary T cells cannot convert 18:3ω‐3 to 22:6ω‐3. The increment in the 22:6ω‐3 content of Jurkat cells incubated with 24:6ω‐3 was 19.5‐fold greater than that of cells incubated with 18:3ω‐3. Acyl‐coA oxidase siRNA knockdown decreased the amount of 22:6ω‐3 and increased the amount of 24:6ω‐3 in Jurkat cells. These findings show exogenous 24:6ω‐3 can be incorporated into primary human T lymphocytes and Jurkat cells and induces changes in fatty acid composition consistent with its conversion to 22:6ω‐3 via a mechanism involving peroxisomal β‐oxidation that is regulated independently from the integrity of the upstream PUFA synthesis pathway. One further implication is that consuming 24:6ω‐3 may be an effective alternative means of achieving health benefits attributed to 20:5ω‐3 and 22:6ω‐3.


Ethics statement
The study was reviewed and approved by the East of England-Cambridge Central Research Ethics Committee (approval number 19/EE/0096) and all participants gave written informed consent.The purchase and use of primary T cells from StemCell Technologies UK Ltd. was reviewed and approved by the University of Southampton Faculty of Medicine Ethics Review Committee (submission I.D. 49658 and 58050.A1).

Participants and collection of blood samples
The inclusion and exclusion criteria used to select participants in the study were described previously (von Gerichten et al., 2021).Briefly, donors were healthy men and women with an age of 41 (range 21-48) years (n = 10 [4 women]) and median body mass index 25.6 (24.1-26.5)kg/m 2 , blood pressure within age-adjusted normal ranges, nonfasting total cholesterol concentration <7.5 mmol/L, HbA1c concentration <42 mmol/mol, and C-reactive protein concentration <3 mg/L.Participants did not habitually consume fish oil, dietary oil supplements, smoke tobacco, or report any chronic disease.
Volunteers were excluded if they did not meet the inclusion criteria, were pregnant or intending to become pregnant during the study, or were already participating in a clinical study.Nonfasting venous blood samples (100 mL) were collected into tubes containing lithium heparin.
Isolation and culture of CD3 + T cells from whole blood Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood using a histopaque density cushion and centrifugation at 845g for 15 min at room temperature (von Gerichten et al., 2021).PBMCs were collected into RPMI1640 medium containing 10% (vol/vol) heat-inactivated homologous pooled serum (Sigma-Aldrich, Poole, UK; Complete medium; Table 1).CD3 + T cells were isolated by negative selection using the T cell EasySep kit (StemCell Technologies, UK Ltd., Cambridge, UK) according to the manufacturer's instructions.Isolated T cells were washed with 10 mL RPMI1640 and collected by centrifugation at 300g for 10 min at room temperature.CD3 + T lymphocytes were cryopreserved as described (Noakes et al., 2012;Prescott et al., 1999) and stored in liquid nitrogen.Blood donations by participants were suspended during the United Kingdom national restrictions to mitigate the SARS-CoV-2 pandemic.Consequently, to increase the sample number, purified CD3 + T lymphocytes were purchased from StemCell Technologies UK Ltd. (Cambridge, UK; Catalog number 70024.1);these cells were collected from anonymous donors whose characteristics met the inclusion criteria for the study.
In some experiments, Jurkat cells were treated with the carnitine palmitoyl transferase-1 inhibitor Etomoxir (5 μM; Sigma-Aldrich) together with 24:6ω-3 (30 μM) for 48 h.Cells were collected by centrifugation and washed and processed for fatty acid analysis as before.
siRNA knockdown of acyl-CoA oxidase-1 in Jurkat cells and RTPCR analysis Jurkat cells were suspended in serum-free Accell siRNA delivery media (Horizon Discovery Biosciences Ltd., Cambridge, UK) containing glutamine at the density of 1 Â 10 6 cells/mL and treated with either Accell human ACOX1 SMARTPool siRNA (1 μM; Horizon Discovery Biosciences Ltd.) or nontargeted human pool siRNA (1 μM; Horizon Discovery Biosciences Ltd.) and incubated for 72 h at 37 C, in an atmosphere containing 5% (vol/vol) CO 2 .After 72 h, the plates were centrifuged at 300g for 10 min, the supernatant was removed and replaced with RPMI containing 10% human serum and 30 μM of 24:6n-3.The cells were then incubated for a further 96 h.At the end of the incubation, the cells were collected, washed twice in PBS and pelleted for fatty acid composition analysis and to verify ACOX1 knockdown.RNA extraction and qRTPCR were carried out essentially as described (von Gerichten et al., 2021).Briefly, RNA was extracted using the RNeasy Mini kit (Qiagen) with on-column DNAse activity.RNA was eluted in RNase-free water (30 μL).RNA concentration was measured and purity was assessed using a NanoDrop1000 spectrophotometer.cDNA was synthesized by reverse transcription.The level of the acyl-CoA oxidase-1 (ACOX1) transcript was measured by qRTPCR using QuantiTect assay Hs_A-COX1_1_SG (QT00078960) (Qiagen) with QuantiTect Sybr Green PCR kit (Qiagen).Amplified transcripts were quantified using the standard curve method (Cikos et al., 2007) and normalized to the geometric mean of the reference genes 60S ribosomal protein L13-A (RPL13A, Quantitect Primer Assay Hs_RPL13A Primer design reference gene assay [HK-SY-hu]) and succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), Quantitect Primer Assay Hs_SDHA_1_SG (QT00059486).These reference genes have been shown to be stable in CD3 + T lymphocytes and Jurkat cells (Sibbons et al., 2018) by the GeNorm method (Vandesompele et al., 2002).The qRTPCR conditions were those specified by the manufacturer.

Analysis of fatty acid composition by gas chromatography
CD3 + T lymphocytes and Jurkat cells were thawed and suspended in 0.9% (wt/vol) NaCl and the internal standard 17:0 (3 μg) was added.Cell lipids were extracted with chloroform/methanol (2:1, vol/vol; Bligh & Dyer, 1959), dried, dissolved in toluene and converted to fatty acid methyl esters (FAMEs) by incubation with acidified methanol containing 2% (vol/vol) sulfuric acid at 50 C for 2 h (Burdge et al., 2000).The reaction mixture was cooled to room temperature and neutralized with KHCO 3 (0.25 M) and K 2 CO 3 (0.5 M).FAMEs were collected by hexane extraction (Burdge et al., 2000).FAMEs were separated on a BPX-70 fused silica capillary column (30 m Â 0.25 mm Â 25 μm; Trajan, Scientific Europe, Milton Keynes, UK) using an Agilent 6890 gas chromatograph (GC) equipped with flame ionization detection as described (West et al., 2016).Chromatograms were integrated manually by a single operator using OpenLAB CDS ChemStation software (version BC.0301.001;Agilent Technologies, UK).The amounts of individual fatty acids are expressed as mass per million cells at the end of the culture period.Fatty acids were identified by their retention times relative to standards (37 FAMES, Sigma-Aldrich) and confirmed by GC-mass spectrometry (Figure 2) using a 6890 gas chromatograph (Agilent, UK) equipped with an Agilent 5975 mass selective detector set to a mass scan range of m/z 50-550 as described (von Gerichten et al., 2021).

RESULTS
The effect of incubation with 24:6ω-3 on the fatty acid composition of T lymphocytes There was a significant treatment Â activation interaction effect on the amount of 24:0, but not on the  amounts of any of the other SFAs or the MUFAs measured in T lymphocytes (Table 2).
Mitogen stimulation significantly increased the amounts of 18:2ω-6 (2.4-fold) and 20:4ω-6 (1.3-fold) and there was a significant treatment Â activation interaction effect on the amount of 20:4ω-6, but the amounts of the other ω-6 PUFAs that were measured in T cells were not altered (Table 2).Specifically, the mitogeninduced increment in 20:4ω-6 was greater in T lymphocytes incubated with 24:6ω-3 than in untreated cells.The activation-induced change in the amount of 24:1ω-9 was blunted in T cells incubated with 24:6ω-3 compared to untreated T lymphocytes.
The identities of 24:6ω-3 and 22:6ω-3 were confirmed by comparison of the mass spectra of authentic standards with those of the peaks with the corresponding retention times in T lymphocytes (Figure 2).24:6ω-3 was not detected in quiescent or activated CD3 + T cells that were maintained in medium lacking this fatty acid (Table 2, Figure 2), but was significantly incorporated into 24:6ω-3 treated T lymphocytes irrespective of activation status (Table 2).Incubation with 24:6ω-3 increased the amount of 22:6ω-3 in quiescent (fivefold) and activated (sevenfold) T cells compared to T A B L E 3 The effect of incubation with 24:6ω-3 or 18:3ω-3 on the fatty acid composition of Jurkat cells.untreated cells (Table 2).Mitogen stimulation increased the amount of 20:3ω-3 in T cells that were not incubated with 24:6ω-3.However, the magnitude of mitogen-induced change in 20:3ω-3 content was less in cells incubated with 24:6ω-3 than in untreated T cells.There was no single factor effect of 24:6ω-3 on the amounts of 20:5ω-3 or 22:5ω-3, but there was a significant treatment Â activation effect on the amounts of these ω-3 PUFAs (Table 2).

DISCUSSION
Overall, these findings show that although 24:6ω-3 has been regarded as a poor substrate for phospholipid biosynthesis (Voss et al., 1991), both primary CD3+ T lymphocytes and Jurkat cells accumulated 24:6ω-3 when incubated with exogenous 24:6ω-3, which was greater in activated T cells than unstimulated cells, which is consistent with the general increase in the uptake of exogenous fatty acids by mitogen-stimulated T cells (Rode et al., 1982).Exogenous 24:6ω-3 can be incorporated into primary human T lymphocytes and Jurkat cells The cell type-related changes in fatty acid composition induced by treatment with 24:6 ω-3 and acyl-coA oxidase knockdown are which suggests conversion of 24:6ω-3 to 22:6ω-3 via a mechanism involving peroxisomal β-oxidation that is regulated independently from the upstream reactions of the PUFA synthesis pathway (Figure 1).In addition, one possible explanation for increased amounts of saturated or monounsaturated fatty acids are that they represent homeoviscotic adaptations induced by the accumulation of 24:6 ω-3.However, because of such homeoviscotic adaptations and uptake of fatty acids from the culture medium, the changes in T cell fatty acid composition induced by incubation with 24:6ω-3 cannot be assumed to reflect metabolic interconversions alone.Nevertheless, the results of previous studies (Metherel et al., 2019;Metherel & Bazinet, 2019;Moore et al., 1995) support the interpretation of the present findings as showing that both CD3 + T lymphocytes and Jurkat cells can utilize 24:6ω-3 as a substrate for 22:6ω-3 synthesis and that such interconversion can occur irrespective of the integrity of the PUFA synthesis pathway.One interpretation of the similarity between Jurkat cells and primary T lymphocytes in the amount of 22:6ω-3 following incubation with 24:6ω-3 is that the post-endoplasmic reticulum reactions of the PUFA synthesis pathway can be regulated independently from the preceding metabolic steps as suggested previously (Burdge, 2004;Sprecher, 1999).Fibroblasts from patients with Zellweger's disease who lack peroxisomes do not synthesize 22:6ω-3 which supports the conclusion that peroxisomal fatty acid βoxidation is required for 22:6ω-3 synthesis (Moore et al., 1995).However, despite this metabolic block, accumulation of 24:6ω-3 has not been reported in tissues or blood from patients who lack peroxisomes (Martinez, 1995) or from Pex-2/Pex-5 dual knockout mice that do not synthesize peroxisomes or express enzymes involved in peroxisomal fatty acid β-peroxidation (Baes et al., 1997;Faust & Hatten, 1997;Janssen et al., 2000).Moreover, Pex-2/Pex-5 null mice did not differ in liver 22:6ω-3 content from peroxisome replete mice (Janssen et al., 2000).Therefore, the role of peroxisomal fatty acid β-oxidation in 22:6ω-3 biosynthesis remains a matter for debate (Infante & Huszagh, 2001).Direct synthesis of 22:5ω-3 by Δ4 desaturation by the protein product of FADS2 (Park et al., 2015) or by a carnitine plus αtocopherol-dependent mitochondrial pathway (Infante & Huszagh, 2000) have been suggested as alternative mechanisms.Jurkat cells were treated with ACOX1 siRNA in order to investigate whether peroxisomal fatty acid β-oxidation was involved in, at least, some of the changes in fatty acid composition induced by incubation with 24:6ω-3, The present findings show that 64% reduction in ACOX1 mRNA expression by transfection of Jurkat cells with ACOX1 siRNA was accompanied by lower amounts of 22:6ω-3, 22:5ω-3, 20:5ω-3 and 20:3ω-3, and more 24:6ω-3 when cells were incubated with 24:6ω-3 alone.This finding suggests that peroxisomal fatty acid β-oxidation is involved in the synthesis of other ω-3 PUFAs as well as 22:6ω-3, at least in Jurkat leukemia cells, although this interpretation requires more rigorous testing by more direct methods.To the best of our knowledge, the mechanism by which 24:6ω-3 could be converted to 20:5ω-3 has not been described, although it is possible that this may occur via the recycling of carbon atoms from peroxisomal fatty acid βoxidation and utilized in the conversion of 18:3ω-3 to 20:3ω-3, although such recycling of carbon atoms from ω-3 PUFAs has only been reported for labeled 18:3ω-3 which can be utilized in cholesterol synthesis in rodent brain (Cunnane et al., 1994) and whole body SFA and MUFA synthesis in humans (Burdge & Wootton, 2003) and rhesus macaques (Sheaff Greiner et al., 1996).The present findings imply that the suggestion that conversion of 24:6ω-3 is a minor source of ω-3 PUFAs (Metherel & Bazinet, 2019) may depend on cell type.
Treatment of Jurkat cells with Etomoxir differentially decreased the amounts of 24:6ω-3, 22:5ω-3, 22:6ω-3, 18:1ω-7, and 20:4ω-6 in the cells.One possible interpretation is that the synthesis of these unsaturated fatty acids involves mitochondrial β-oxidation, for example by carbon recycling at least in Jurkat cells as occurs in T lymphocytes incubated with [ 13 C-18:3 ω-3] (West et al., 2022).In the absence of findings from experiments using a 24:6ω-3 tracer, it is not possible to draw robust conclusions about the mechanism of 24:6ω-3 metabolism in T lymphocytes or Jurkat cells, which is an important limitation of the present study.
Fatty acid compositions of cell culture media.
T A B L E 1Note: The total fatty acid composition of culture media was determined by gas chromatography as described in the Section 2. 24:6ω-3 was not detected (ND) in media that were not supplemented with this fatty acid.Culture medium: RPMI1640 medium containing 10% (vol/vol) heat-inactivated homologous pooled serum.were made using Student's t test, or the Mann-Whitney U test for data that were not normally distributed.Statistical testing of the interaction between age and T cell activation status (activation state Â life stage) was by 2-way ANOVA with Tukey's post hoc correction for multiple comparisons.A sample size of n = 6 cultures or participants was calculated to provide 87% power to detect a significant difference in the amount of 22:6ω-3 between treatments of 0.4 nmol/10 6 cells with α <0.05.Effect sizes are reported either as Cohen's d, or partial eta squared.
Values are mean ± SEM (n = 6 culture replicates per treatment).All cultures contained 24:6ω-3 (25 μM).Statistical comparisons were done by Student's unpaired t test (equal variances were not assumed).Effect sizes of means that differed significantly (p < 0.05) are reported as Cohen's d, but were not determined (n.d.) for comparisons which failed to meet the threshold for statistical significance.