Abstract
BackgroundSuperparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS).MethodsPrimary murine microglial cells were pretreated with IONPs (1–50 μg Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α was quantified by ELISA. The activity of IL-1β converting enzyme (ICE) and TNF-α converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively.ResultsConfocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1β, but not TNF-α. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE.ConclusionsOur results demonstrated a contrasting effect of IONPs on the production of IL-1β and TNF-α by LPS-stimulated microglia, in which the attenuation of IL-1β by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing.
Highlights
Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity
Primary microglial cells were pretreated with IONPs (1–50 μg Fe/mL), and stimulated with LPS (100 ng/mL) for 24 h
Confocal microscopy was used to monitor the uptake of IONPs, and the images showed the accumulation of dark brown dots in the cytoplasm of cells exposed to IONPs (Figure 1A)
Summary
Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. Activated microglia produce pro-inflammatory cytokines and cytotoxic factors, including interleukin (IL)-1β, nitric oxide (NO), tumor necrosis factor (TNF)-α and reactive oxygen species (ROS) [13,14]. These mediators play a key role in the prevention of brain cells from further damage and to promote the repair of the damaged tissue. IONPs were found to be engulfed by BV2 cells, which induced a large number of cellular vesicles, swelling of endoplasmic reticulum and morphological alterations of mitochondrial cristae [22] These results indicate that the functionality and morphology of resting microglia are altered in response to nanoparticle exposure. We reported that IONPs suppressed the production of IL-1β by activated microglia via the secretory lysosomal pathway of cytokine processing
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