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.

Three cases of large retinal capillary hemangiomas treated with verteporfin and photodynamic therapy.

OBJECTIVE: To investigate the efficacy of verteporfin and photodynamic therapy in the treatment of large retinal capillary hemangiomas. METHODS: Case reports of 3 patients with large retinal capillary hemangiomas treated with photodynamic therapy using verteporfin. Standard verteporfin dosages (6 mg/m(2) of body surface area) were given. Both standard and modified photodynamic protocols were followed. Modified protocols included shorter verteporfin infusion times and longer light exposure times. RESULTS: Pretreatment best-corrected Snellen visual acuity of the 3 affected eyes were 20/100, 20/50, and 2/200, respectively. All cases had associated exudative retinal detachments involving the macula. Cases 1 and 2 were classic endophytic retinal capillary hemangiomas. Case 3 was a reactive retinal capillary hemangioma. Case 1 had 2 photodynamic therapy treatments, and after 8 months, visual acuity improved to 20/40. Two years after initiating photodynamic therapy, the visual acuity was 20/30 and there was no reperfusion of the hemangioma. Case 2 had 3 photodynamic therapy treatments. The hemangioma was fibrotic, and 20 months after initiating photodynamic therapy visual acuity improved to 20/30. Case 3 had 1 treatment, 11 weeks later and visual acuity improved to 20/400. Four months after treatment, visual acuity returned to counting fingers because of tractional elevation of the macula as the capillary hemangioma fibrosed. Vitrectomy surgery was performed, and choroidal and retinal neovascularization was discovered. Three months after vitrectomy visual acuity was 20/400. In cases 1 and 2, the capillary hemangioma ultimately regressed, and the exudative detachment resolved. CONCLUSIONS: Verteporfin and photodynamic therapy were effective in achieving closure of large retinal capillary hemangiomas. In all cases, the hemangioma underwent fibrosis with consequent macular puckering due to retinal traction. In all cases, the visual acuity improved.