Abstract
BackgroundHypoxic-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).MethodsHuman 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.ResultsElevated 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.ConclusionsThis 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.
Highlights
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
Type-1 IFNs activate the Jak-Stat cascade, we investigated the ability of type-1 IFNs to phosphorylate Stat isoforms in M17 human neuroblastoma cells by western blot analysis
IFNα preferentially induces p-Stat-1 opposed to Stat-3, unlike IFNβ that is non-selective in its phosphorylation of the Stat isoforms analysed
Summary
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. Type-1 interferons (IFNs) are a super-family of pleiotropic cytokines that regulate pro-inflammatory gene transcription via the classical Jak/Stat pathway; their role in hypoxia-ischaemia and central nervous system neuro-inflammation remains unknown. Hypoxic-ischaemic brain injury, including stroke and traumatic brain injury (TBI), are the most common acute central nervous system (CNS) neuro-degenerative disorders worldwide. These injuries begin with an initial tissue insult and reduction in oxygen and glucose levels in the surrounding extracellular space, developing a core infarct. Type-1 interferons (IFNs) are key regulators of this cytokine storm [18,19]; their role in CNS neuro-inflammation and hypoxicischaemic injury is still not well understood
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