Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain
Introduction
The brain is highly enriched in long chain polyunsaturated fatty acids (LC-PUFAs) that are essential bioactive compounds with a large range of physiological roles such as cerebral plasticity, cell survival and neuroinflammation (Bazinet and Laye, 2014, Laye et al., 2018). Docosahexaenoic acid (DHA, 22:6 n-3) and arachidonic acid (AA, 20:4 n-6) are respectively the major n-3 and n-6 PUFAs in the brain. Despite the fact that DHA is poorly synthesized from its dietary precursor α-linolenic acid (ALA) (<1%) (Plourde and Cunnane, 2007), ALA-derived DHA has been recently reported to be sufficient for brain DHA supply (Domenichiello et al., 2014). However, preformed DHA can be provided through the diet to increase DHA levels in the brain (Orr et al., 2013, Joffre et al., 2016). N-3 LC-PUFA supplementation is the most efficient way to increase DHA levels and decrease AA levels in the brain (Murthy et al., 2002, Hiratsuka et al., 2009, Orr et al., 2010, Moranis et al., 2012, Fan et al., 2016, Lacombe et al., 2017).
N-3 LC-PUFAs exert protective actions against inflammation in the brain (Lonergan et al., 2004, Li et al., 2015, Shi et al., 2016, Dong et al., 2017, Fourrier et al., 2017). In vivo, fish oil supplementation providing n-3 LC-PUFAs, including DHA and eicosapentaenoic acid (EPA), is associated with a decreased cerebral expression of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β, induced by pro-inflammatory stimuli such as lipopolysaccharide (LPS, the gram-negative bacteria endotoxin), IL-1β (Dong et al., 2017) or aging (Labrousse et al., 2012, Orr et al., 2013, Dehkordi et al., 2015, Shi et al., 2016). In vitro data suggests that the anti-inflammatory activity of DHA in the brain is mediated through microglia, the brain’s innate immune cells (De Smedt-Peyrusse et al., 2008, Antonietta Ajmone-Cat et al., 2012, Pettit et al., 2013, Chang et al., 2015, Fourrier et al., 2017). Recent studies indicate that the mechanisms underlying the anti-inflammatory effect of DHA and EPA in the brain involve n-3 LC-PUFA derived bioactive mediators, called oxylipins, which promote the resolution of inflammation (Serhan et al., 2000, Levy et al., 2001, Hong et al., 2003, Serhan et al., 2015, Rey et al., 2016).
LC-PUFAs are converted by cyclooxygenase (COX), lipoxygenases (LOX) and cytochrome P450 (CYP) into oxylipins (Fig. 1). Several studies have reported their pro or anti-inflammatory activities in the brain (Orr et al., 2013, Rey et al., 2016, Laye et al., 2018). Oxylipins derived from DHA and EPA include anti-inflammatory and pro-resolving metabolites such as eicosanoids, resolvins, protectin and maresin (Bazan, 2009, Serhan et al., 2011). Oxylipins derived from AA and its precursor linoleic acid (LA) are mostly pro-inflammatory compounds and include prostaglandins (PG), leukotrienes (LT) or thromboxanes (Tx) and hydroxyoctadecadienoic acids (HODE) (Fig. 1) (Calder, 2006). Oxylipin levels are tightly regulated by inflammatory stimuli or insults in peripheral tissues and immune cells (Yang et al., 2009, Balvers et al., 2012a, Willenberg et al., 2016). In the brain, n-3 and n-6 derived oxylipins are produced during injury or ischemic conditions, including from microglia (Farias et al., 2008). The expression of oxylipin’s synthesizing enzymes is also tightly regulated by inflammatory stimuli (Rosenberger et al., 2004, Birnie et al., 2013, Taha et al., 2017), including in the brain (Laye et al., 2018). Administration of pro-inflammatory cytokines or LPS induces COX-2 expression in neuronal and endothelial cells (Cao et al., 1996, Nadjar et al., 2005, Rummel et al., 2006). COX-2, 5-LOX, 15-LOX and several isoforms of CYP expression are also induced in the hippocampus and cortex of rodents after traumatic brain injury (Birnie et al., 2013). A PUFA dietary intervention modulates both cellular levels of PUFAs and oxylipins. As a result, n-3 LC-PUFA supplementation increases oxylipins derived from EPA and DHA and decreases compounds derived from AA (Balvers et al., 2012b, Hashimoto et al., 2015). Conversely, dietary n-6 PUFA supplementation increases AA-derived oxylipins and decreases oxylipins derived from EPA (Taha et al., 2016). Changes of oxylipins levels triggered by dietary n-6 and n-3 PUFA supply could be linked to the regulation of the enzymes COX-2 and 15-LOX (Rao et al., 2007, Kim et al., 2011, Taha et al., 2017). However, the influence of dietary n-3 LC-PUFAs on the conversion enzymes and oxylipin profiles in specific brain regions has been poorly addressed. N-3 LC-PUFAs are promising molecules due to their modulation of brain inflammation (Lonergan et al., 2004, Li et al., 2015, Shi et al., 2016, Dong et al., 2017, Fourrier et al., 2017). Thus, we explored whether n-3 LC-PUFA dietary supplementation provides protection in an LPS model of inflammation through differential production of oxylipins.
We hypothesized that inflammatory stimuli can differently regulate n-3 LC-PUFA derived-oxylipins production in the brain, dependent on the dietary n-3 LC-PUFA supply. To address this, we compared the oxylipin profile in the hippocampus of LPS treated mice fed with isocaloric diets with identical ALA content but supplemented or deficient in n-3 LC-PUFAs.
Section snippets
Animals and treatment
All experiments were performed on male C57Bl6/J mice obtained from Janvier Labs at post-natal day 21 (Le Genest-Saint-Isle, France). Mice were maintained under standard housing conditions on corncob litter in a temperature (23 ± 1 °C) and humidity (40%) controlled animal room with a 12 h light/dark cycle (07–19 h), with ad libitum access to food and water. A total of 88 mice were used in this study. Half of the mice were fed with a diet enriched with n-3 LC-PUFA and the other half with a diet
Dietary n-3 LC-PUFA supplementation modifies hippocampal fatty acid composition
We first evaluated the impact of either diet on the hippocampal fatty acid composition (Table 4). Total n-3 PUFA and n-6 PUFA levels were significantly different in the hippocampus of the n-3 LC-PUFA supplemented group as compared to the n-3 LC-PUFA deficient group (DHA: F(1,12) = 25.22, p < 0.001, EPA: F(1,12) = 305.3, p < 0.0001, n-3 DPA: F(1,12) = 18.27, p < 0.001, AA: (F(1,12) = 211.1, p < 0.0001, n-6 docosapentaenoic acid (DPA): F(1,12) = 192.6, p < 0.0001, adrenic acid: F(1,12) = 174.4,
Discussion
In this work, we provide evidence that dietary n-3 LC-PUFA supplementation modulates hippocampal oxylipins and alters LPS-induced pro-inflammatory cytokine expression in the hippocampus, suggesting that n-3 LC-PUFAs could promote the resolution of hippocampal neuroinflammation through the release of oxylipins.
In the n-3 LC-PUFA supplemented diet, the amount of EPA and DHA corresponds to a daily intake of 40 mg/day/mouse (26.5 mg EPA and 14 mg DHA per mouse). To extrapolate to humans (Nair and
Acknowledgement
We thank C.Tridon, S. Delbary and B. Péré for taking care of the mice and Genotoul Plateforme for oxylipin analyses.
Funding source
This work was supported by INRA, ANRT, ACTIA (Association Nationale de la Recherche et de la Technologie), Idex Bordeaux University, ANR MoodFood, Fondation pour la Recherche Médicale (FRM) and Société Française de Nutrition (SFN). J.C. Delpech is the recipient of a post-doctoral fellow from Région Aquitaine. A.D. Greenhalgh is the recipient of the Agreenskills post-doctoral grant support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
Declaration of interest
None
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