Elsevier

Brain, Behavior, and Immunity

Volume 80, August 2019, Pages 179-192
Brain, Behavior, and Immunity

Multi-hit early life adversity affects gut microbiota, brain and behavior in a sex-dependent manner

https://doi.org/10.1016/j.bbi.2019.03.006Get rights and content

Highlights

  • Multi-hit early adversity affects sociability in males and anxiety in females.

  • Early adversity differentially alters gut microbiota according to sex.

  • Early adversity elicits sex-specific transcriptome changes in the prefrontal cortex.

  • Behavioral data correlate with prefrontal cortex gene expression and gut microbiota.

Abstract

The accumulation of adverse events in utero and during childhood differentially increases the vulnerability to psychiatric diseases in men and women. Gut microbiota is highly sensitive to the early environment and has been recently hypothesized to affect brain development. However, the impact of early-life adversity on gut microbiota, notably with regards to sex differences, remains to be explored. We examined the effects of multifactorial early-life adversity on behavior and microbiota composition in C3H/HeN mice of both sexes exposed to a combination of maternal immune activation (lipopolysaccharide injection on embryonic day 17, 120 µg/kg, i.p.), maternal separation (3hr per day from postnatal day (PND)2 to PND14) and maternal unpredictable chronic mild stress. At adulthood, offspring exposed to multi-hit early adversity showed sex-specific behavioral phenotypes with males exhibiting deficits in social behavior and females showing increased anxiety in the elevated plus maze and increased compulsive behavior in the marble burying test. Early adversity also differentially regulated gene expression in the medial prefrontal cortex (mPFC) according to sex. Interestingly, several genes such as Arc, Btg2, Fosb, Egr4 or Klf2 were oppositely regulated by early adversity in males versus females. Finally, 16S-based microbiota profiling revealed sex-dependent gut dysbiosis. In males, abundance of taxa belonging to Lachnospiraceae and Porphyromonadaceae families or other unclassified Firmicutes, but also Bacteroides, Lactobacillus and Alloprevotella genera was regulated by early adversity. In females, the effects of early adversity were limited and mainly restricted to Lactobacillus and Mucispirillum genera. Our work reveals marked sex differences in a multifactorial model of early-life adversity, both on emotional behaviors and gut microbiota, suggesting that sex should systematically be considered in preclinical studies both in neurogastroenterology and psychiatric research.

Introduction

The multi-hit hypothesis (or cumulative stress hypothesis) proposes that neuropsychiatric disorders may be precipitated by a combination of two or more major adverse events in particular during development (Maynard et al., 2001, McEwen, 1998, Nederhof and Schmidt, 2012). Subjects exposed to one or more childhood adversities are more likely to become depressed following exposure to stress at adulthood than subjects without early adversity (Chapman et al., 2004, Nemeroff, 2016). Maternal infection and postnatal exposure to psychological stress or traumas have been found to represent important environmental risk factors for the development of psychiatric disorders, including autism, schizophrenia, anxiety-disorders and depression (Brown, 2011, Brown et al., 2014, Nemeroff, 2016). According to the multiple-hit hypothesis, prenatal infection could render the offspring more vulnerable to the deleterious effects of a second postnatal stimulus, such as stress. Previous findings have comforted this hypothesis in animal models combining prenatal inflammation and adolescent or adult stress exposure (Deslauriers et al., 2013, Giovanoli et al., 2013, Monte et al., 2017). However, the impact of prenatal inflammation combined with an early post-natal stress remains underexplored.

The mechanisms underlying the long-term behavioral effects of early adversity are still unclear. A large body of evidence suggests that the medial prefrontal cortex (mPFC), a brain area involved in the regulation of emotional behavior, plays a major role in mediating the effects of early-life stress (Arnsten, 2009, Ulrich-Lai and Herman, 2009). Recent work has demonstrated that the gut microbiota affects gene expression in the mPFC (Gacias et al., 2016, Hoban et al., 2017, Hoban et al., 2016). Gastrointestinal alterations during early-life, notably gut microbiota dysbiosis and loss of barrier function, can disturb brain development and lastingly impair gut-brain communication (Borre et al., 2014, Hsiao et al., 2013, Kim et al., 2017). In particular, maternal immune activation and maternal separation (MS) have been shown to produce intestinal defects such as visceral pain and increased intestinal permeability in association with behavioral outcomes (see Labouesse et al., 2015, O’Mahony et al., 2011 for reviews). Animals exposed to early-life immune or psychological stress also exhibit gut dysbiosis (Amini-Khoei et al., 2019, Bailey and Coe, 1999, Moya-Pérez et al., 2017, Murakami et al., 2017, O’Mahony et al., 2009, Pusceddu et al., 2015). Moreover, numerous studies using maternal immune activation or MS models have shown that microbiota-directed interventions such as probiotic treatments or fecal transplantation modulate brain and behavior, especially stress-related behaviors (De Palma et al., 2015, Giovanoli et al., 2016, Hsiao et al., 2013, Kim et al., 2017, Mattei et al., 2014, Moya-Pérez et al., 2017). Importantly, it has been reported that gut microbiota composition differs according to sex both in animals and humans (Fransen et al., 2017, Hollister et al., 2014, Jašarević et al., 2016, Markle et al., 2013). However, sex differences in gut microbiota in a context of early adversity remain underexplored. Indeed, most of the studies use males and the few studies involving males and females often pool data from both sexes (De Palma et al., 2015, El Aidy et al., 2017, Hsiao et al., 2013, Riba et al., 2018). This issue, which merits further investigation, is of particular importance with respect to the gender differences observed in the prevalence of psychiatric disorders. To support this notion, autism spectrum disorders are more prevalent in men (Werling and Geschwind, 2013), whereas women are more susceptible to anxiety and depression (Steel et al., 2014).

In the present study, we hypothesized that early adversity differentially affects the gut microbiota in males and females and that these differential effects may underlie the sex-related differences observed at the behavioral level. To test this hypothesis, we developed a mouse model of multifactorial early adversity combining prenatal inflammation (lipopolysaccharide (LPS) injection), post-natal MS and unpredictable chronic mild stress (UCMS) in dams and we investigated emotional behavior, mPFC gene expression and gut microbiota composition in male and female offspring (Fig. 1).

Section snippets

Effects of multi-hit early adversity on offspring’s body weight

LPS injection on E17 induced significant hypothermia in dams (t(15) = 4.98, p < 0.001), indicating that bacterial immune activation was effective (Supplementary Fig. S1). There was no significant effect of prenatal LPS on pup body weight on post-natal day (PND)2 (Supplementary Fig. S2A). However, the combination of prenatal LPS and MS (early adversity) significantly decreased the body weight of male pups at PND15 in comparison with control pups (males, t(15) = 2.46, p = 0.003; females, t

Discussion

Several studies have reported sex differences in gut microbiota composition in both humans and animals (Hollister et al., 2014, Markle et al., 2013). There is a growing number of studies investigating the role of the gut-brain axis, especially gut microbiota, in the regulation of stress-related emotional behaviors in animal models of early-life adversity. However, it is not clear whether early stress differentially affects the gut microbiota between males and females (Jašarević et al., 2017,

Animals

Experiments were approved by the Bioethical committee of the University of Bordeaux (N° 50120186-A) according to the European legislation (Directive 2010/63/EU, 22 September 2010). Mice were maintained in a 12-h light/12-h dark cycle (lights on at 0800 h) in a temperature-controlled room (22 °C) with free access to food and water, unless otherwise mentioned. Gestant female C3H/HeNRj mice (n = 30) purchased (Janvier Labs, Le Genest Saint Isle, France) at gestational day 2 were individually

Acknowledgements

This work was supported by the University of Bordeaux, INRA and AVIESAN Immunology, Hematology and Pneumology. M.R. was supported by a stipend of the French Ministry of Research. We warmly thank Céline Monot & Karine Le Roux for technical support and logistics with the microbiological analyses. We are grateful to the INRA MIGALE bioinformatics platform (http://migale.jouy.inra.fr) for providing computational resources. We also thank the Genotoul Get-PlaGe sequencing platform, and Thierry

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