Elsevier

Brain, Behavior, and Immunity

Volume 51, January 2016, Pages 212-222
Brain, Behavior, and Immunity

Estrogen protects the blood–brain barrier from inflammation-induced disruption and increased lymphocyte trafficking

https://doi.org/10.1016/j.bbi.2015.08.020Get rights and content

Highlights

  • We report a novel protective action of estrogen on the BBB.

  • Estrogen enhances inter-endothelial cell tight junction function.

  • Estrogen’s actions are mediated by GPR30 signaling and phosphorylation of annexin A1.

  • Estrogen limits lymphocyte extravasation after challenge with inflammatory cytokines.

  • Estrogen limits inflammation-induced immune cell trafficking through ERβ/annexin A1 pathways.

Abstract

Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood–brain barrier (BBB) and it is recognized as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesize that estrogen can limit BBB breakdown following the onset of peripheral inflammation.

Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modeled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammation-induced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models.

Together, our data provide novel mechanisms for the protective effects of estrogen, and enhance our understanding of the beneficial role it plays in neurovascular/neuroimmune disease.

Introduction

Several lines of evidence suggest that the sex differences seen in vascular and neural diseases may be at least partly linked to differing sex hormone complements. In particular, a number of beneficial effects have been attributed to the principal female hormone estrogen, including in conditions as diverse as Parkinson’s disease, Alzheimer’s disease, head injury and multiple sclerosis (Gillies and McArthur, 2010). One important working hypothesis is that the neuroprotective effects of estrogen may be related to its known anti-inflammatory and immunomodulatory actions (Czlonkowska et al., 2005, Nadkarni and McArthur, 2013).

The endothelium of the blood–brain barrier (BBB) is at the forefront of the defensive features of the central nervous system, regulating its interactions with the immune system. In particular, there is accumulating evidence that BBB function is compromised during peripheral inflammation, leading to inappropriate passage of cells and molecules into the brain parenchyma (Carvey et al., 2009). Estrogen has been shown to exert a variety of anti-inflammatory effects, including reducing iNOS activity (Cignarella et al., 2009), directly regulating the cytokine milieu (Gameiro et al., 2010) and altering expression of vascular and leukocyte adhesion molecules (Dietrich, 2004). Whilst initial studies have identified estrogen as being able to modulate BBB tight junction proteins such as claudin 5 (Burek et al., 2014), full characterization of the effects of estrogen upon the BBB is lacking, and represents an underexplored aspect of hormonal protection.

We have previously studied the importance of the anti-inflammatory protein annexin A1 (ANXA1) in the BBB, where it plays a major role in the regulation of tight junction expression, contributing to limited barrier permeability (Cristante et al., 2013). Although ANXA1 was originally described as a glucocorticoid second messenger (Flower and Blackwell, 1979), we and others have since reported that it can also be modulated by estrogen (Solito et al., 2003, Davies et al., 2007, Nadkarni et al., 2011), leading us to hypothesize that estrogen may exert protective effects upon the BBB in inflammation through the regulation of this protein.

Using a combined in vivo/in vitro approach, we examined the response of the BBB to peripheral inflammatory challenge and the ability of the principal estrogen: estradiol (E2) to restore homeostasis in this system. We report here the presence of sexual dimorphism in the response of the BBB to peripheral pro-inflammatory challenge in vivo, and a dual protective role for estradiol upon the inflamed cerebral endothelium in vitro, mediated through the regulation of ANXA1 and ICAM-1 expression. Together these actions control movement of both small molecules and immune cells across the cerebral endothelium.

Section snippets

Reagents

All reagents are from Sigma–Aldrich (Poole, UK) unless otherwise stated. Cell culture medium and solutions were purchased from Lonza (Basel, Switzerland). The ERα agonist PPT (4,4′,4″-(4-propyl-(5)-pyrazole-1,3,5-triyl)trisphenol), the ERβ agonist DPN (2,3-bis(4-Hydroxyphenyl)-propionitrile), the GPR30 agonist G-1 ((±)-1-[(3aR,4S,9bS)-4-(6-bromo-1,-3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone), the ERβ antagonist PHTPP

Estrogen-dependent sex differences in the BBB response to inflammatory challenge

We initially characterized the response of the murine BBB to a simple systemic inflammatory model, the administration of bacterial lipopolysaccharide (LPS; 3 mg/kg i.p.), with assessment of barrier permeability through extravasation of intravenous Evan’s blue dye in young adult (2 month) male and female C57Bl/6 mice. A marked sex difference was notable in male animals showing a clear and reproducible, but transient, increase in BBB permeability 4 h post-LPS administration, whilst such change was

Discussion

Steroid hormones have long been known to possess anti-inflammatory properties, indeed synthetic glucocorticoid receptor agonists are amongst the most clinically potent anti-inflammatory drugs available, but it is only relatively recently that the anti-inflammatory potential of estrogen has begun to be closely studied (Nadkarni and McArthur, 2013). The primary interface between the CNS and the inflammatory response, the BBB, has been examined as a target for estrogen action (Oztas et al., 2001,

Conclusions

The prevalence of inflammatory cerebrovascular diseases shows a distinct male predominance, with numerous studies showing women to be relatively protected. Understanding why this is the case remains a challenge. Here, we identify a powerful protective action of the female sex steroid estrogen, showing that this hormone can both enhance inter-endothelial cell tight junction function and limit lymphocyte extravasation following challenge with inflammatory cytokines. Moreover, we identify these

Author contributions

Elisa Maggioli: Performed in vivo and in vitro experiments, data analysis.

Simon McArthur: Performed in vivo work, the immunocytochemical and confocal microscopic analysis, qPCR, data analysis, contributed to writing and discussion of the manuscript.

Claudio Mauro: Performed shRNA lentivirus production and contributed to discussion of the manuscript.

Julius Kieswich: Animal handling and support in vivo experiments.

Dennis Kusters: Performed ANXA1 expression and purification.

Chris Reutelingsperger:

Financial interest

The authors declare no conflict of interests.

Acknowledgments

This work was supported by ARUKPPG2013B-2 and FISM-3/15/F14 to ES, Barts and the London Trust to MY, and British Heart Foundation Fellowship FS/12/38/29640 to CM.

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    EM and SMcA contributed equally to this work.

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