title: Type-1 interferons contribute to oxygen glucose deprivation induced neuro-inflammation in BE(2)M17 human neuroblastoma cells.
abstract: BACKGROUND
  Hypoxic-ischaemic injuries such as stroke and traumatic brain injury exhibit features of a distinct neuro-inflammatory response in the hours and days post-injury. Microglial activation, elevated pro-inflammatory cytokines and macrophage infiltration contribute to core tissue damage and contribute to secondary injury within a region termed the penumbra. Type-1 interferons (IFNs) are a super-family of pleiotropic cytokines that regulate pro-inflammatory gene transcription via the classical Jak/Stat pathway; however their role in hypoxia-ischaemia and central nervous system neuro-inflammation remains unknown. Using an in vitro approach, this study investigated the role of type-1 IFN signalling in an inflammatory setting induced by oxygen glucose deprivation (OGD).
  
  
  METHODS
  Human BE(2)M17 neuroblastoma cells or cells expressing a type-1 interferon-α receptor 1 (IFNAR1) shRNA or negative control shRNA knockdown construct were subjected to 4.5 h OGD and a time-course reperfusion period (0 to 24 h). Q-PCR was used to evaluate IFNα, IFNβ, IL-1β, IL-6 and TNF-α cytokine expression levels. Phosphorylation of signal transducers and activators of transcription (STAT)-1, STAT-3 and cleavage of caspase-3 was detected by western blot analysis. Post-OGD cellular viability was measured using a MTT assay.
  
  
  RESULTS
  Elevated IFNα and IFNβ expression was detected during reperfusion post-OGD in parental M17 cells. This correlated with enhanced phosphorylation of STAT-1, a downstream type-1 IFN signalling mediator. Significantly, ablation of type-1 IFN signalling, through IFNAR1 knockdown, reduced IFNα, IFNβ, IL-6 and TNF-α expression in response to OGD. In addition, MTT assay confirmed the IFNAR1 knockdown cells were protected against OGD compared to negative control cells with reduced pro-apoptotic cleaved caspase-3 levels.
  
  
  CONCLUSIONS
  This study confirms a role for type-1 IFN signalling in the neuro-inflammatory response following OGD in vitro and suggests its modulation through therapeutic blockade of IFNAR1 may be beneficial in reducing hypoxia-induced neuro-inflammation.
authors.forename: Myles Robert
authors.initials: MR
authors.lastname: Minter
authors.forename: Moses
authors.initials: M
authors.lastname: Zhang
authors.forename: Robert Charles
authors.initials: RC
authors.lastname: Ates
authors.forename: Juliet Marie
authors.initials: JM
authors.lastname: Taylor
authors.affiliation: Department of Pharmacology, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville 3010, VIC, Australia. pcrack@unimelb.edu.au.
authors.forename: Peter John
authors.initials: PJ
authors.lastname: Crack
mesh_terms: D000704:Analysis of Variance; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D005947:Glucose / Q000172:deficiency*; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D007249:Inflammation / Q000378:metabolism*; D009447:Neuroblastoma / Q000473:pathology; D010766:Phosphorylation; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D053633:Receptor, Interferon alpha-beta / Q000235:genetics / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014162:Transfection
chemical_list: D016207:Cytokines; C508032:Ifnar1 protein, mouse; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D053633:Receptor, Interferon alpha-beta; D005947:Glucose