Elsevier

Brain, Behavior, and Immunity

Volume 41, October 2014, Pages 22-31
Brain, Behavior, and Immunity

Named Series: Diet, Inflammation and the Brain
Nutritional n-3 PUFAs deficiency during perinatal periods alters brain innate immune system and neuronal plasticity-associated genes

https://doi.org/10.1016/j.bbi.2014.03.021Get rights and content

Abstract

Low dietary intake of the n-3 polyunsaturated fatty acids (PUFAs) is a causative factor of neurodevelopmental disorders. However the mechanisms linking n-3 PUFAs low dietary intake and neurodevelopmental disorders are poorly understood. Microglia, known mainly for their immune function in the injured or infected brain, have recently been demonstrated to play a pivotal role in regulating maturation of neuronal circuits during normal brain development. Disruption of this role during the perinatal period therefore could significantly contribute to psychopathologies with a neurodevelopmental neurodevelopmental component. N-3 PUFAs, essential lipids and key structural components of neuronal membrane phospholipids, are highly incorporated in cell membranes during the gestation and lactation phase. We previously showed that in a context of perinatal n-3 PUFAs deficiency, accretion of these latter is decreased and this is correlated to an alteration of endotoxin-induced inflammatory response. We thus postulated that dietary n-3 PUFAs imbalance alters the activity of microglia in the developing brain, leading to abnormal formation of neuronal networks. We first confirmed that mice fed with a n-3 PUFAs deficient diet displayed decreased n-3 PUFAs levels in the brain at post-natal days (PND)0 and PND21. We then demonstrated that n-3 PUFAs deficiency altered microglia phenotype and motility in the post-natal developing brain. This was paralleled by an increase in pro-inflammatory cytokines expression at PND21 and to modification of neuronal plasticity-related genes expression. Overall, our findings show for the first time that a dietary n-3 PUFAs deficiency from the first day of gestation leads to the development of a pro-inflammatory condition in the central nervous system that may contribute to neurodevelopmental alterations.

Introduction

Long chain PUFAs, acid (DHA; 22:6n-3) and arachidonic acid (AA; 20:4n-6), are essential lipids and key structural components of neuronal membrane phospholipids. They are metabolized from precursors alpha-linolenic acid (ALA; 18:n-3) for n-3 PUFAs and linoleic acid (LA; 18:2n-6) for n-6 PUFAs which cannot be synthesized de novo by mammals and have to be provided through the diet (Sinclair, 1975). DHA and AA accumulate rapidly in the brain during the later stages of gestation and early postnatal life via placenta and maternal milk or infant formula (Clandinin et al., 1980, Martinez, 1992, Green and Yavin, 1996, Wainwright, 2002). They are highly concentrated in the central nervous system (CNS) and are necessary for normal brain development and function (Xiao et al., 2005, Labrousse et al., 2012, Larrieu et al., 2012, Moranis et al., 2012, Luchtman and Song, 2013). A dietary supply of n-3 PUFAs confers advantages to cognitive development while n-3 PUFAs deficits impair it (Simopoulos, 1991, Innis, 2000, Salem et al., 2001, Wainwright, 2002, Hoffman et al., 2009, Lafourcade et al., 2011, Labrousse et al., 2012, Larrieu et al., 2012). More specifically, we previously demonstrated that spatial memory is impaired in adult mice exposed to n-3 PUFA deficiency from the first day of gestation (Moranis et al., 2012). These animals also display higher depressive-like symptoms in the Porsolt forced swimming test (FST) as well as an impairment of exploratory behaviors with emotional load (Lafourcade et al., 2011, Larrieu et al., 2012).

In a typical western diet, the estimated ratio of n-6/n-3 PUFAs is around 20/1 in marked contrast with the optimal 4/1 ratio (Simopoulos, 2000, Simopoulos et al., 2000) leading to long chain n-3 PUFA replacement by long chain n-6 PUFAs in membranes. The relative excess of dietary n-6 PUFAs promotes the formation of AA, the fatty acid precursor of inflammatory molecules such as prostaglandins, thus promoting neuroinflammatory processes and the subsequent development of inflammatory-based CNS disorders (Calder, 2008, Laye, 2010). Moreover, n-3 PUFAs, such as DHA and their derivatives (resolvins, neuroprotectins, maresins) exert anti-inflammatory effects and inhibit the production of pro-inflammatory cytokines (Calder, 2013). We and others previously demonstrated in vitro that DHA prevents bacterial endotoxin lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in microglial cells (De Smedt-Peyrusse et al., 2008, Antonietta Ajmone-Cat et al., 2012). In vivo, we found that mice exposed throughout life to an n-3 PUFA deficient diet displayed lower brain DHA level (cortex and hippocampus being more susceptible) and a higher production of interleukin (IL)-6 in plasma and hippocampus in response to LPS (Mingam et al., 2008). In addition, dietary supplementation with long-chain n-3 PUFAs such as DHA protects from LPS or IL-1-induced (Kavanagh et al., 2004, Song et al., 2008) and aging-associated (Labrousse et al., 2012) brain cytokine production. Altogether, PUFAs are potent immunomodulators and modifying their brain content may disrupt neuroimmune interactions.

Microglia are the main cellular component of the brain innate immune system and are key players in the regulation and maintenance of CNS homeostasis (Hanisch and Kettenmann, 2007, Prinz et al., 2011). Microglia respond to injury and inflammation with production of pro- or anti-inflammatory factors, including prostaglandins, nitric oxide, cytokines, and chemokines (Gehrmann et al., 1995). In addition to this classical role a number of recent studies revealed a major role for microglia in normal brain development, especially in neural circuit formation (Paolicelli et al., 2011, Schafer et al., 2012). Microglia participate in synapse elimination via the activation of the complement system leading to maturation of CNS networks (Wake et al., 2009, Tremblay et al., 2010, Paolicelli et al., 2011, Schafer et al., 2012). In addition, microglial cells are able to shift their functional phenotypes once stimulated by inflammatory factors (Hanisch, 2013). By analogy with peripheral macrophages, microglia phenotypes have thus been characterized by some authors into four main states (M1, M2a, M2b and M2c), depending on their inflammatory and phagocytic properties (Ransohoff and Perry, 2009, Perry et al., 2010, Chhor et al., 2013). However, little is known about microglia phenotypes during the perinatal period, a key period for long chain PUFAs accumulation in the developing brain.

Thus, we hypothesized that the long term effect of n-3 PUFA deficiency on behavior could be linked to its effect on microglia during the perinatal period. So the aim of this study was to assess the effect of n-3 PUFA deficiency on microglia during the early post-natal stage of brain development. We therefore investigated the effect of an adequate or deficient dietary supply of ALA to the mother and the consequences on brain PUFA levels and microglia functionality of the pups at birth (postnatal day, PND0) and at weaning (PND21) in the hippocampus of mice, a structure that is crucial for cognitive performances development.

Section snippets

Animals

Studies were carried out according to the Quality Reference System of INRA (http://www.international.inra.fr/content/download/947/11111/file/requirements) and approved by the national ethical committee for care and use of animals (A5012093). The mice were housed in groups of 4–5 per cage and maintained in a temperature and humidity controlled facility on a 12 h light–dark cycle with food and water ad libitum.

Diets

After mating, C57BL6/J and CX3CR1-eGFP females were fed with a diet containing 6% fat in

N-3 PUFA deficiency affects brain fatty acid levels in the developing brain

To assess the impact of n-3 PUFAs deficiency on brain lipid composition, we evaluated the fatty acid content of the cerebral cortex at PND0 and PND21 in mice. The n-3 PUFA deficiency induced a significant decrease in n-3 PUFAs and specifically of DHA (22:6 n-3) (diet: F(1,18) = 235.5, p < 0.0001) associated with a sharp increase in n-6 PUFA such as docosapentaenoic acid (or DPA, 22:5 n-6) (diet: F(1,18) = 2628.1, p < 0.0001) (Table 4). These changes were significantly higher at PND21 (for DHA from

Discussion

In this work, we showed that an n-3 PUFA deficiency given over the gestation and lactation phases altered microglia phenotype and motility in the post-natal brain. This was paralleled by an increase in pro-inflammatory factors expression at PND21. Such impairment could be involved in behavioral alterations observed in the brain of rodents exposed to n-3 deficient diet.

PUFAs represent potent immunomodulatory agents. In accordance with previous reports, DHA levels were decreased in the brain of

Acknowledgments

The microscopy was conducted in the Bordeaux Imaging Center of the University of Bordeaux Segalen (Labex BRAIN (ANR 10 LABX 43). The help of Philippe Legros, Christel Poujol, Sébastien Marais, Magali Mondin is acknowledged. Thanks are due to J.C. Helbling, C. Tridon, P. Birac, and M. Cadet for excellent technical support. This research was financially supported by Institut National de la Recherche Agronomique (INRA, France), Fondation pour la Recherche Médicale (FRM, France), Agence Nationale

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