Elsevier

Placenta

Volume 36, Issue 4, April 2015, Pages 454-462
Placenta

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators

https://doi.org/10.1016/j.placenta.2015.01.007Get rights and content

Highlights

  • Term labour is associated with decreased AMPK activity in fetal membranes.

  • With no labour, AMPK activity is decreased in preterm fetal membranes with PROM.

  • AMPK activators decrease infection-induced pro-inflammatory cytokines.

  • AMPK activators decrease IL-1β-induced MMP-9 expression.

Abstract

Introduction

In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour.

Methods

Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1β-stimulated MMP-9 expression.

Results

AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1β-induced MMP-9 expression.

Discussion

The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research.

Introduction

Of the 3.1 million worldwide annual neonatal deaths, around 35% are due to complications of preterm birth [1]. There is increased incidence of cerebral palsy, learning difficulties and respiratory illnesses in children born premature [2]; the morbidities associated with preterm birth can extend to adulthood and are inversely related to gestational age. As such, the impact of preterm birth affects not only the child and family; data from the USA suggest that the annual cost of preterm birth has surpassed US$26 billion [3]. These emotional and societal costs arise due to a lack of effective therapeutics that can stop preterm labour.

Spontaneous term and preterm labour share a common pathway, comprising of increased uterine contractions, cervical ripening and rupture of fetal membranes [4]. While term labour is due to normal physiological activation of this pathway, preterm labour is due to pathological activation such as infection or inflammation. Fetal membranes can also rupture in the absence of labour, i.e. pre-labour rupture of membranes (PROM). Preterm PROM (PPROM) occurs in 30–40% of all spontaneous preterm births and is associated with higher rates of neonatal mortality and morbidity [7]. Infection is considered the biggest aetiological factor involved in PPROM [7], [8]. Pathogenic microorganisms can trigger the inflammatory response via activation of Toll-like receptors (TLRs) [9], which are highly expressed at the maternal-fetal interface. Such pathogenic organisms include lipopolysaccharide (LPS; ligand to TLR4), flagellin (TLR5) and the viral dsRNA analogue poly(I:C) (TLR3), which are known to stimulate the production of cytokines and metalloproteinase (MMP)-9 in fetal membranes in the context of infection-mediated preterm labour [10].

Recent evidence implicates adenosine monophosphate (AMP)-activated protein kinase (AMPK) in modulating acute inflammatory reactions. AMPK is a serine/threonine protein kinase that consists of three heterogenic subunits, a catalytic (α) subunit and two regulatory subunits (β and γ); all of which exist in at least 2 isoforms. Initially AMPK was shown to be involved in regulation of fatty acid and cholesterol synthesis [11]. However, data indicate that activation of AMPK might be a useful strategy for beneficial regulation of the inflammatory response. For example, in vitro, activation of AMPK is associated with inhibition of LPS- or IL-1β-induced cytokine production [12], [13], [14]. In vivo, activation of AMPK has been shown to inhibit the production of pro-inflammatory mediators in serum and their expression in the central nervous system of rats injected with a sublethal dose of LPS [13]. Enhanced activation of AMPK also resulted in diminished severity of LPS-induced acute lung injury in mice [12].

AMPK has been detected in human placenta, where its expression is decreased in the obese placenta [15]. To our knowledge, however, the expression or the role of AMPK has not been investigated in human fetal membranes. Therefore, the aims of this study were (i) to establish the effect of human spontaneous term and preterm labour on AMPK activity in human fetal membranes; (ii) to compare AMPK activity in the absence of labour in fetal membranes obtained at preterm gestation from women with PROM or with intact membranes at delivery; and (iii) to determine the effect of activators of AMPK on infection- or inflammation-induced expression of pro-inflammatory and pro-labour mediators in human fetal membranes and primary amnion cells. Tissues were treated in the presence of TLR ligands LPS, flagellin and poly(I:C), and primary amnion cells with the pro-inflammatory cytokine IL-1β.

Section snippets

Tissue collection

The Research Ethics Committee of Mercy Hospital for Women approved this study. Written, informed consent was obtained from all participating women. All tissues were obtained from women who delivered healthy, singleton infants. All tissues were brought to the research laboratory and processed within 15 min of the Caesarean delivery. Women with any underlying medical conditions such as diabetes, asthma, polycystic ovarian syndrome, preeclampsia and macrovascular complications were excluded.

Localisation of AMPKα in fetal membranes

The first aim of this study was to determine the localisation of total AMPKα and phosphorylated Thr172 AMPKα (p-AMPKα) in human fetal membranes. For these IHC studies, samples were obtained from women at the time Caesarean section. Representative images from 1 patient are shown in Fig. 1. In fetal membranes, AMPKα and p-AMPKα was present in amnion epithelium, chorionic trophoblasts and decidua. Staining for AMPKα and p-AMPKα was both nuclear and cytoplasmic. There was additional staining of

Discussion

There is increasing evidence that the activation of AMPK can inhibit inflammatory responses induced by various stimulants, and accordingly, AMPK activity is suppressed with increased inflammation [38]. AMPK has not previously been described in human fetal membranes and the inflammatory responses that lead to labour. This study describes a novel role for AMPK in the regulation of pro-labour mediators in human fetal membranes. AMPK activity in fetal membranes was decreased after term labour, as

Funding

Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Additional funding was provided by the Medical Research Foundation for Women and Babies and the Mercy Research Foundation.

Disclosure summary

The authors have nothing to declare.

Conflict of interest

The authors have nothing to declare.

Acknowledgements

The following are gratefully acknowledged: Clinical Research Midwives Genevieve Christophers, Gabrielle Pell, and Rachel Murdoch for sample collection; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation.

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