The essential dietary fatty acid α-linolenic acid (ALA) can be oxidised into 18 carbon oxylipins or metabolised into longer chain n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Oxylipins from n-3 PUFAs are generally acknowledged as less pro-inflammatory or capable of resolving inflammation compared to n-6-derived oxylipins(Reference Gabbs, Leng and Devassy1). The regulation of the partitioning of ALA between PUFA synthesis and oxidation to oxylipins is not yet understood and might be impaired in human T cells by age-related immune dysregulation; namely immunosenescence.
To address this, peripheral blood CD3+ T cells from healthy younger adult volunteers (18–30 years; n = 10) and older adult volunteers (58–74 years; n = 6) were cultured for 48 h, with or without concanavalin A (10μg/mL) in 10% (v/v) pooled donor plasma with an 8:1 or 5:1 linoleic acid (LA) to ALA ratio (n-6/n-3 ratio). ALA metabolites were detected either by gas chromatography-mass spectrometry for cellular PUFA or by LC-MS/MS for oxylipins in cell culture supernatants. Metabolite to ALA ratios were calculated for the primary PUFA synthesised from ALA in T cells, eicosatrienoic acid (20:3n-3 / ALA), and the most abundantly oxidised metabolites of ALA, 9- and 13-hydroxyoctatrienoic acid (HOTrE / ALA). Multiple t-test (unpaired, two-tailed) with Holm-Sidak correction was performed for statistical analysis (GraphPad Prism 8.4.3). Results for stimulated and unstimulated cells were similar; only results for stimulated cells are reported here.
In younger adult T cells, HOTrE / ALA was significantly higher than 20:3n-3 / ALA for 5:1 n-6/n-3 (0.44±0.09 vs 0.11±0.01, P = 0.005) and 8:1 n-6/n-3 (0.20±0.02 vs 0.13±0.02, P = 0.019). Further, there was significantly higher HOTrE / ALA in cells treated with 5:1 n-6/n-3 compared to 8:1 n-6/n-3 (0.44±0.09 vs 0.20±0.02, P = 0.041), but 20:3n3 / ALA did not differ between n-6/n-3 ratios.
Likewise, HOTrE / ALA from T cells treated with a 5:1 n-6/n-3 were significantly higher compared to 20:3n-3 / ALA in both younger (0.49±0.09 vs 0.11±0.01, P < 0.001) and older (0.45±0.05 vs 0.28±0.02, P = 0.018) adults. The ratio of 20:3n-3 / ALA was significantly higher in older adults compared to younger adults (0.28±0.02 vs 0.11±0001, P < 0.001), but there was no difference in HOTrE / ALA comparing older and younger adults.
These findings show that ALA is used preferentially by mitogen-stimulated T cells for constitutive production of anti-inflammatory lipid mediators rather than synthesis of longer chain PUFA in both younger and older adults. The lipid mediator production in younger adults is greater with a lower (5:1) n-6/n-3 ratio. Further, elongation of ALA is higher in older than in younger adults. This has implications for understanding the effects of dietary PUFA on immune function and healthy aging.
Acknowledgments
The authors wish to acknowledge Dr Debra Skene and the Metabolomics Core Facility, School of Bioscience and Medicine, University of Surrey, for providing support and resources for the LC-MS analysis of oxylipins.
