Anti-Inflammatory Effects of Progesterone in Lipopolysaccharide-Stimulated BV-2 Microglia

Beilei Lei,Daniel T Laskowitz,Hana N Dawson,David S Warner,Brian Mace,Michael L James,Karin E Peterson

doi:10.1371/journal.pone.0103969

Abstract

Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E2 (PGE2), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone’s effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.

Highlights

Numerous studies indicate that progesterone regulates multiple non-reproductive functions in the brain including cognition, memory, and neurogenesis [1,2,3,4]
Localization of progesterone receptors in BV-2 cells To study the effects of progesterone in microglia, the expression of progesterone receptors was first examined
Utilizing immunofluorescence staining with antibody recognizing both PR-A and PR-B isoforms, under complete medium condition, co-localization of progesterone receptors with nucleus (DAPI, blue) was observed, suggesting nuclear localization of progesterone receptors in BV-2 cells (Figure 1)

Summary

Introduction

Numerous studies indicate that progesterone regulates multiple non-reproductive functions in the brain including cognition, memory, and neurogenesis [1,2,3,4]. Progesterone elicits its effects via progesterone receptors (PRs), which include classical nuclear PRs (two major isoforms PR-A and PR-B) and recently recognized membrane PRs [3,5,6,7]. The classical mechanism of progesterone action is mediated by nuclear PRs, which function as transcription factors by binding to specific progesterone response elements within the promoter region of target genes to modulate transcription and genomic networks [3,4,8]. Non-classical mechanisms have been recently suggested to involve membrane PRs and cytoplasmic kinase activation and signal cascades [3,4,8].

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Conclusion