title: Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells.
abstract: BACKGROUND
  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. 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).
  
  
  METHODS
  Primary 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.
  
  
  RESULTS
  Confocal 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, No,1, Sec, 4, Roosevelt Road, Taipei 10617, Taiwan. jenliang@mail.ypu.edu.tw.
authors.forename: Hsin-Ying
authors.initials: HY
authors.lastname: Wu
authors.forename: Min-Chun
authors.initials: MC
authors.lastname: Chung
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Wang
authors.forename: Chung-Hsiung
authors.initials: CH
authors.lastname: Huang
authors.forename: Hong-Jen
authors.initials: HJ
authors.lastname: Liang
authors.forename: Tong-Rong
authors.initials: TR
authors.lastname: Jan
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003911:Dextrans / Q000494:pharmacology*; D005434:Flow Cytometry; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D058185:Magnetite Nanoparticles; D051379:Mice; D008807:Mice, Inbred BALB C; D017628:Microglia / Q000187:drug effects* / Q000276:immunology; D018613:Microscopy, Confocal; D053758:Nanoparticles; D061251:Primary Cell Culture; D055571:Secretory Pathway / Q000187:drug effects* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D003911:Dextrans; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D058185:Magnetite Nanoparticles; D014409:Tumor Necrosis Factor-alpha; C482199:lipopolysaccharide, E coli O55-B5; D002401:Cathepsin B; C533915:Ctsb protein, mouse; C065507:ferumoxides
references.citation: Nat Rev Mol Cell Biol. 2009 Feb;10(2):148-55
references.pmid: 19122676
references.citation: Biomaterials. 2011 Jan;32(2):547-55
references.pmid: 20880574
references.citation: Brain Res Brain Res Rev. 1995 Mar;20(3):269-87
references.pmid: 7550361
references.citation: Glia. 2002 Nov;40(2):140-55
references.pmid: 12379902
references.citation: Int J Biochem Cell Biol. 1997 May;29(5):715-20
references.pmid: 9251238
references.citation: Angew Chem Int Ed Engl. 2008;47(29):5362-5
references.pmid: 18551493
references.citation: Int J Nanomedicine. 2011;6:2791-8
references.pmid: 22114506
references.citation: Contrast Media Mol Imaging. 2009 Nov-Dec;4(6):305-11
references.pmid: 19941323
references.citation: PLoS One. 2011;6(9):e25524
references.pmid: 21991395
references.citation: ACS Nano. 2008 May;2(5):889-96
references.pmid: 19206485
references.citation: ACS Nano. 2010 May 25;4(5):2595-606
references.pmid: 20329742
references.citation: Eur J Neurosci. 2001 Aug;14(4):609-17
references.pmid: 11556886
references.citation: Nat Rev Immunol. 2003 Jun;3(6):472-82
references.pmid: 12776207
references.citation: Brain Res Bull. 2012 Jan 4;87(1):10-20
references.pmid: 22024597
references.citation: Biomaterials. 2010 Dec;31(34):9015-22
references.pmid: 20739059
references.citation: Int J Nanomedicine. 2011;6:1779-86
references.pmid: 21980240
references.citation: Science. 1985 Apr 26;228(4698):497-9
references.pmid: 3872478
references.citation: Curr Med Chem. 2009;16(10):1278-94
references.pmid: 19355885
references.citation: Eur J Biochem. 2003 May;270(10):2109-19
references.pmid: 12752430
references.citation: Environ Health Perspect. 2010 May;118(5):589-95
references.pmid: 20439179
references.citation: Nat Cell Biol. 2010 Jan;12(1):19-30; sup pp 1-13
references.pmid: 19966785
references.citation: Blood. 2011 Jul 7;118(1):9-18
references.pmid: 21562044
references.citation: Semin Cell Dev Biol. 2002 Aug;13(4):279-84
references.pmid: 12243727
references.citation: Immunobiology. 2009;214(7):601-12
references.pmid: 19268389
references.citation: Immunobiology. 2009;214(7):543-53
references.pmid: 19250700
references.citation: Science. 2003 Feb 28;299(5611):1400-3
references.pmid: 12610307
references.citation: Glia. 2010 Jan 1;58(1):114-24
references.pmid: 19544382
references.citation: J Microencapsul. 1996 May-Jun;13(3):245-55
references.pmid: 8860681
references.citation: Trends Neurosci. 1996 Aug;19(8):312-8
references.pmid: 8843599
references.citation: Toxicol Lett. 2011 Aug 10;205(1):26-37
references.pmid: 21596115
references.citation: ACS Nano. 2011 Nov 22;5(11):8629-39
references.pmid: 21974862
references.citation: Nanotechnology. 2010 Feb 19;21(7):75102
references.pmid: 20090199
references.citation: Biomaterials. 2010 Jul;31(19):5063-71
references.pmid: 20381862
references.citation: Indian J Biochem Biophys. 2008 Apr;45(2):75-90
references.pmid: 21086720
references.citation: Nat Rev Mol Cell Biol. 2002 Feb;3(2):122-31
references.pmid: 11836514
references.citation: Acta Biomater. 2013 Sep;9(9):8454-65
references.pmid: 23727247
references.citation: NMR Biomed. 2008 Oct;21(8):820-9
references.pmid: 18470957
references.citation: J Neurosci. 2001 Sep 15;21(18):7046-52
references.pmid: 11549714
references.citation: J Neurosci Res. 1998 Oct 1;54(1):68-78
references.pmid: 9778151
references.citation: J Cereb Blood Flow Metab. 2010 Jan;30(1):15-35
references.pmid: 19756021
references.citation: Brain Behav Immun. 1995 Dec;9(4):345-54
references.pmid: 8903851
references.citation: Blood. 2011 Aug 11;118(6):1570-8
references.pmid: 21693760
references.citation: Int J Nanomedicine. 2011;6:1229-35
references.pmid: 21753874
references.citation: Biochem Pharmacol. 2009 Jan 1;77(1):86-100
references.pmid: 18848818
references.citation: Toxicol Appl Pharmacol. 2009 Aug 15;239(1):106-15
references.pmid: 19520096
references.citation: Int J Biochem Cell Biol. 2002 Jan;34(1):1-5
references.pmid: 11733179
references.citation: Int J Nanomedicine. 2010 Sep 07;5:593-9
references.pmid: 20856834
references.citation: Glia. 2001 Nov;36(2):165-79
references.pmid: 11596125
references.citation: Environ Health Perspect. 2007 Nov;115(11):1631-7
references.pmid: 18007996
references.citation: Mol Imaging. 2011 Jun;10(3):206-14
references.pmid: 21496449