Soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro.

BACKGROUND: Neuro-inflammation has long been implicated as a contributor to the progression of Alzheimer's disease in both humans and animal models. Type-1 interferons (IFNs) are pleiotropic cytokines critical in mediating the innate immune pro-inflammatory response. The production of type-1 IFNs following pathogen detection is, in part, through the activation of the toll-like receptors (TLRs) and subsequent signalling through myeloid differentiation factor-88 (Myd88) and interferon regulatory factors (IRFs). We have previously identified that neuronal type-1 IFN signalling, through the type-1 interferon alpha receptor-1 (IFNAR1), is detrimental in models of AD. Using an in vitro approach, this study investigated the TLR network as a potential production pathway for neuronal type-1 IFNs in response to Aß. METHODS: Wildtype and Myd88(-/-) primary cultured cortical and hippocampal neurons were treated with 2.5 µM Aß1-42 for 24 to 72 h or 1 to 10 µM Aß1-42 for 72 h. Human BE(2)M17 neuroblastoma cells stably expressing an IRF7 small hairpin RNA (shRNA) or negative control shRNA construct were subjected to 7.5 µM Aß1-42/Aß42-1 for 24 to 96 h, 2.5 to 15 µM Aß1-42 for 96 h or 100 ng/ml LPS for 0.5 to 24 h. Q-PCR was used to analyse IFNα, IFNß, IL-1ß, IL-6 and TNFα mRNA transcript levels. Phosphorylation of STAT-3 was detected by Western blot analysis, and cell viability was assessed by MTS assay. RESULTS: Reduced IFNα, IFNß, IL-1ß, IL-6 and TNFα expression was detected in Aß1-42-treated Myd88(-/-) neurons compared to wildtype cells. This correlated with reduced phosphorylation of STAT-3, a downstream type-1 IFN signalling mediator. Significantly, Myd88(-/-) neuronal cultures were protected against Aß1-42-induced neurotoxicity compared to wildtype as determined by MTS assay. Knockdown of IRF7 in M17 cells was sufficient in blocking IFNα, IFNß and p-STAT-3 induction to both Aß1-42 and the TLR4 agonist LPS. M17 IRF7 KD cells were also protected against Aß1-42-induced cytotoxicity. CONCLUSIONS: This study confirms that the neuronal type-1 IFN response to soluble amyloid is mediated primarily through TLRs. This production is dependent upon Myd88 and IRF7 signalling. This study suggests that targeting this pathway to modulate neuronal type-1 IFN levels may be beneficial in controlling Aß-induced neurotoxicity.

Type-1 interferons contribute to oxygen glucose deprivation induced neuro-inflammation in BE(2)M17 human neuroblastoma cells.

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, IFNß, IL-1ß, 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 IFNß 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, IFNß, 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.