Sex and estradiol influence glial pro-inflammatory responses to lipopolysaccharide in rats

Summary

There is a greater prevalence of neuroinflammatory diseases in females than males. Microglia, the major immunocompetent cells of the central nervous system, play a key role in neuroinflammation. We aimed to determine if inherent differences in toll-like receptor 4 mediated pro-inflammatory response in glia could possibly contribute to the skewed female prevalence of neuroinflammatory disorders. In addition, in order to identify if estradiol (E2), the major female sex steroid contributes to a heightened pro-inflammatory response, estradiol was added both in vivo and in vitro. Microglia and astrocytes were isolated from neonatal pups and stimulated with lipopolysaccharide (LPS) in the presence and absence of E2. Hippocampal microglia were isolated from adult male and female rats and stimulated ex vivo with LPS. Male neonatal microglia and astrocytes produced greater IL-1β mRNA than females. However, when co-incubated with varying doses of estradiol (E2), the E2 produced anti-inflammatory effects in the male microglia but a pro-inflammatory effect in female microglia. LPS-induced IL-1β mRNA was attenuated by E2 in female but not male adult hippocampal microglia. However, females supplemented with E2 in vivo produced a potentiated IL-1β mRNA response. TLR4 mRNA was decreased by LPS in both microglia and astrocytes but was not affected by sex or E2. CD14 mRNA was increased by LPS and may be elevated more in females than males in microglia but not astrocytes. Therefore, sexual dimorphic differences do occur in both neonatal and adult microglia though maturity of the microglia at the time of isolation influences the pro-inflammatory response.

Introduction

Microglia are the predominant immunocompetent cells within the central nervous system. They release pro-inflammatory mediators in response to injury or inflammation. Microglia have been implicated in many neurodegenerative and neuroinflammatory diseases including multiple sclerosis, Alzheimer's disease and chronic pain (Watkins et al., 2007, Perry et al., 2010, Streit, 2010). Females have a greater prevalence than males in all of these pathologies, (Streit, 2005, Streit et al., 2005, Achiron and Gurevich, 2009, Fillingim et al., 2009, Amor et al., 2010, Streit, 2010). Therefore, the question arises as to whether microglia, which contribute to neuroinflammation, also contribute to the difference in pro-inflammatory response between males and females.

There is recent evidence to suggest that morphological and phenotypic differences exist between male and female microglia, but that the differences change with age. For example, neonatal male microglia display an amoeboid morphology and have a more classically activated phenotype compared to neonatal female microglia (Schwarz et al., 2012). However, unstimulated microglia isolated from naive female 60-day-old rats present with a more classically activated state with higher levels of IL-1 expression and lower IL-10 expression (Schwarz et al., 2012). Purine receptor expression on microglia, known to be involved in microglial activation, also appears to vary between sex and age (Crain et al., 2009). However, no studies have investigated whether these sexually dimorphic differences remain when the microglia are stimulated with an immune challenge.

It is becoming increasingly recognized that toll-like receptors (TLR) are involved not only in the recognition of pathogens but also in the recognition of endogenous danger signals (Erridge, 2010). TLR4 is a pattern recognition receptor that has been implicated, within the central nervous system (CNS), as importantly contributing to the pathological processes underlying neuropathic pain, multiple sclerosis, and Alzheimer's disease through the release of neuroinflammatory and neuroexcitatory substances as a consequence of TLR4 activation (Marta, 2009, Morales et al., 2010, Nicotra et al., 2012). Therefore, in this series of studies we investigated whether there is a potential sex difference in changes in mRNA expression in neonatal microglia following an immune challenge using lipopolysaccharide, the classic TLR4 ligand. CD14 and MD2 are co-receptors for the TLR4 complex and are shown to be involved in neuroinflammatory conditions (Nadeau and Rivest, 2002, Cao et al., 2009, Loram et al., 2011). Therefore, the subsequent changes to TLR4 and its co-receptors will be investigated.

The second aim of this study was to identify whether this difference in phenotype of the microglia between neonates and adults arises from changes in estrogen levels. Estrogens are not only critically involved in reproduction, but can also modulate neuronal function, acting as a neurosteroid, and can attenuate or potentiate inflammatory responses in immune cells both in vivo and in vitro (Compagnone and Mellon, 2000, Vegeto et al., 2006, Calippe et al., 2010). Neonatal cells are not yet exposed to the cyclical fluctuations of estrus seen in adults. Therefore, microglia from both P0/1 pups and microglia isolated from adult rats were investigated to compare these two states. We have selected to test the influence of 17β-estradiol (E2), as it is the predominant circulating estrogen. The effect of E2 on glial cells was tested both in vitro and in vivo to identify if differences exist between the two conditions.

While microglia are the major immunocompetent cell within the CNS, astrocytes also have the capacity to produce pro-inflammatory mediators and are now implicated in a number of neuroinflammatory diseases (Pineau et al., 2010). Astrocytes have been identified to have sexually dimorphic differences in vivo (Suarez et al., 1991, Johnson et al., 2008). However, only one study has investigated the immune responses of isolated astrocytes in vitro (Santos-Galindo et al., 2011). Therefore, we investigated whether astrocytes from male and female neonatal pups responded differently to LPS in vitro in the presence and absence of E2.