Amelioration Of Amyloid Β Induced Retinal Inflammatory Responses By A LXR Agonist TO901317 Is Associated With Inactivation Of The NF-ΚB Signaling And NLRP3 Inflammasome

BackgroudAge-related macular degeneration(AMD) is one main disease which cause irreversible blindness among the elderly in the developed countries. Both eyes are affected simultaneously or successively. The vision is progressively impaired. AMD is divided into two types: dry AMD and wet AMD. Dry AMD is considered early AMD and is the most common clinical type of AMD, which accounted for 80%- 85% of all AMD. So far the pathogenesis of dry AMD has not been fully elucidated, and there is no effective therapeutic methods. The inflammatory response, retinal pigment epithelium(RPE) cell aging, metabolic disturbance are generally believed to facilitate the pathogenesis of dry AMD.Drusen, an extracellular material deposited between the RPE and the Bruch’s membrane, is a hallmark of early AMD and has been suggested as a high-risk factor for the development of chronic inflammation. Amyloid β(Aβ) is the main component of drusen and is the pathogenic peptide associated with neurodegenerative diseases such as Alzheimer’s disease(AD) and AMD. It induces neuroinflammation and the formation of the Nod-like receptor protein-3(NLRP3) inflammasome.By using a retinal inflammation mouse model induced by Aβ, we explored potential mechanisms of inhibition of retinal inflammation by activation of LXRs and one of their target genes, ABCA1 with a LXR agonist TO901317(TO90). The study provides scientific and theoretical basis for developing the new drugs to treat retinal inflammation.Part I A mouse model of retinal inflammation induced by the intravitreal injection of Aβ1-40PurposeTo establish a mouse model of retinal inflammation induced by the intravitreal injection of amyloid β(Aβ), a major component in drusen.MethodsThis study is to determine the optimal concentration of Aβ1-40 in inducing retinal inflammatory responses in C57BL/6J mice. The m RNA expressions of inflammatory cytokines interleukin-6(IL-6), the tumor necrosis factor-α(TNF-α) and nucleotide-binding oligomerization domain leucine-rich repeats containing pyrin domain 3(NLRP3) inflammasome were examined at 24 hours after the intravitreal injection of 2μl of PBS with different concentrations(0.5, 1.0, 1.5, 2.0 μg/μl) of Aβ1-40. After determination of the concentration, mice were injected with optimal concentrations of Aβ1-40 or Aβ40-1 and the eyeballs were enucleated at days 1, 4 and 14 postinjection. The expression of inflammatory cytokine and the activation of NLRP3 inflammasome in the neuroretina and the RPE/choroid complex were analyzed by real-time PCR. The severity of retinal damage was evaluated with HE staining at day 4 postinjection, and retinal functions were evaluated by ERG at days 4 and 14. Differences between groups were analyzed using ANOVA followed by a post hoc Bonferroni correction.ResultsThe optimal concentration of Aβ1-40 that induced retinal inflammation was 1.0 μg/μl. Compared with Aβ40-1, Aβ1-40 significantly upregulated the expressions of IL-6, TNF-α and NLRP3 inflammasome genes in the neuroretina and the RPE/choroid complex at days 1 and 4 postinjection. The differences in inflammatory cytokine gene expression between these groups were not significant at day 14, except for the TNF-α in the neuroretina group. No structural disorganization or inflammatory cell infiltration was observed in the retina in each group under light microscopy at day 4. At days 4 and 14 after intravitreal injection, the amplitudes of ERG a- and b-waves significantly decreased in the Aβ1-40 injected mice compared to the untreated and the Aβ40-1 injected mice. No significant differences were observed between the untreated and Aβ40-1 groups at day 14, except for the amplitudes of the dark-adapted ERG b-wave recorded with light intensities of 1.0 and 10.0 cd·s/m2.ConclusionOur data show that Aβ1-40 induces a transient proinflammatory response in the neuroretina and the RPE/choroid complex, and causes retinal functional impairment. These responses mimic those observed in the early stages of age-related macular degeneration(AMD) patients. We find inflammasome is activated in this process. This novel model is useful for investigating the pathogeneses of early AMD. Part II Amelioration of Amyloid β induced retinal inflammatory responses by a LXR agonist TO901317 is associated with inactivation of the NF-κB signaling and NLRP3 InflammasomePurposeRetinal chronic inflammation is a key pathogenic process in age-related macular degeneration(AMD). Amyloid β(Aβ) peptide, a major component of drusen which is the hallmark of AMD, is known to facilitate inflammatory responses in vivo. The purpose of this study was to investigate whether activation of liver X receptors(LXRs) ameliorates retinal inflammatory responses induced by Aβ1-40 and to explore the underlying mechanism.MethodsRetinal inflammatory responses were induced with intravitreal injection of Aβ1-40 peptide in C57BL/6J mice. A synthetic LXR ligand TO901317(TO90, 50 mg/kg/d) or vehicle was intragastrically administrated from 3 days before to 4 days after Aβ1-40 injection. The expressions of pro-inflammatory genes TNF-α and IL-6 were examined by real-time PCR. The levels of LXRα, LXRβ and their target gene ABCA1, as well as NLRP3, caspase-1 and IL-1β in the neuroretina and the RPE/choroid complex were detected with real-time PCR and western blotting. The changes of phosphorylated transcription inhibition factor-κBα(p-IκBα) in the neuroretina and the RPE/choroid complex were detected with western blotting. Retinal function was assessed with electroretinogram(ERG).ResultsThe m RNA expressions of LXRα and LXRβ in the neuroretina of the Aβ1-40-injected mice decreased. No significant difference was found on the protein expressions of LXRs and ABCA1 in both neuroretina and RPE/choroid complex between the Aβ1-40-injected group and the untreated group. TO90 enhanced the expressions of LXRα and ABCA1 at both m RNA and protein levels in the Aβ1-40-injected mice, while the LXRβ expression was unchanged. TO90 preserved ERG a- and b-wave amplitudes in the Aβ1-40-treated mice. Meanwhile, compared with the Aβ1-40 plus vehicle-treated group, the m RNA levels of the pro-inflammatory cytokines TNF-α and IL-6 were significantly decreased in the Aβ1-40 plus TO90-treated group. Furthermore, TO90 downregulated the phosphorylation of IκBα as well as the expressions of NLRP3, caspase-1 and IL-1β in the neuroretina and the RPE/choroid complex in the Aβ1-40-injected animals.ConclusionActivation of LXRα and ABCA1 with TO90 inhibits retinal inflammatory responses induced by Aβ1-40 in mice. It appears that the anti-inflammatory effect is mediated mainly by LXRα. Furthermore, the beneficial effect is associated with inhibition of the NF-κB signaling pathway and the NLRP3/caspase-1/IL-1β axis. LXR agonist may become a new class of anti-inflammatory agent for AMD.