Abstract
BackgroundLuteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization.MethodsResting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium.ResultsLuteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures.ConclusionsOur findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.
Highlights
Luteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species
Effects of luteolin on selected pro-inflammatory markers As a basis to study the genome-wide transcriptional effects of luteolin on activated microglia and to validate our cell culture system, we initially performed a dose-response curve for luteolin
To correlate the microscopic findings with functional data, we studied the influence of microglia-secreted products on caspase-related apoptotic signaling in the neuronal cell model. 661W cells cultured in the presence of conditioned medium (CM) from LPS-stimulated BV-2 cells displayed a strong induction of caspase 3/7 activity (Fig. 7I)
Summary
A plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization. The resident macrophages of the nervous system, have important roles in immune regulation [1,2] and neuronal homeostasis [3,4]. Ramified microglia perform a very active and continous surveillance function with their long protrusions [6,7]. They receive permanent tonic inhibitory inputs from neurons to prevent microglial neurotoxicity [8,9]. Activated microglia in the vicinity of degenerating neurons have been identified in a broad spectrum of neurodegenerative disorders including Alzheimer’s disease [13], Parkinson’s disease [14], amyotrophic lateral sclerosis [15], multiple sclerosis [16], and inherited photoreceptor dystrophies [17,18]
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