背景:炎症小体激活和随后释放的促炎细胞因子,包括白细胞介素1β(IL-1β)已被广泛报道有助于视网膜变性的进展,包括年龄相关性黄斑变性(AMD),是西方世界失明的主要原因。GasderminD(GSDMD)的作用,炎性体激活后的焦亡的关键执行者,然而,不太成熟。在这项研究中,我们旨在描述GSDMD在健康和退化视网膜中的作用,并揭示其作为IL-1β释放管道的作用,包括通过细胞外囊泡(EV)介导的释放。
方法:GSDMD突变体和基因敲除小鼠,利用体外炎症模型和建立良好的体内视网膜变性(光氧化损伤;PD)模型来探索GSDMD在调节IL-1β释放和传播视网膜炎症中的作用和病理贡献。整个视网膜的RNA测序用于研究变性期间GSDMD介导的炎症。使用纳米颗粒跟踪分析研究了EV在GSDMD介导的IL-1β释放中的作用,ELISA和EV抑制范例。最后,使用GSDMD特异性siRNA检查靶向GSDMD的治疗功效.
结果:在这项工作中,我们发现GSDMD缺陷小鼠的视网膜功能保存得更好,增加光感受器的生存能力和减少炎症。RNA-Seq分析揭示GSDMD可通过NF-κB信号传导级联和促炎细胞因子的释放在视网膜中传播炎症。我们还显示IL-1β以GSDMD依赖性方式包装并通过EV释放。最后,我们证明,使用RNAi损害GSDMD功能或阻断EV释放能够降低无细胞上清液和EV中的IL-1β含量。
结论:综合来看,这些结果表明,致裂孔形成蛋白GSDMD在视网膜炎症的传播中起关键作用,特别是通过释放EV封装的IL-1β。使用遗传或药理学抑制剂靶向GSDMD可以提供抑制炎症级联反应和延迟视网膜变性进展的治疗机会。
BACKGROUND: Inflammasome activation and the subsequent release of pro-inflammatory cytokines including Interleukin 1β (IL-1β) have been widely reported to contribute to the progression of retinal degenerations, including age-related macular degeneration (AMD), the leading cause of blindness in the Western World. The role of Gasdermin D (GSDMD), a key executioner of pyroptosis following inflammasome activation, however, is less well-established. In this study we aimed to characterise the role of GSDMD in the healthy and degenerating retina, and uncover its role as a conduit for IL-1β release, including via extracellular vesicle (EV)-mediated release.
METHODS: GSDMD mutant and knockout mice, in vitro models of inflammation and a well-established in vivo model of retinal degeneration (photo-oxidative damage; PD) were utilised to explore the role and pathological contribution of GSDMD in regulating IL-1β release and propagating retinal inflammation. RNA sequencing of whole retinas was used to investigate GSDMD-mediated inflammation during degeneration. The role of EVs in GSDMD-mediated IL-1β release was investigated using nanoparticle tracking analysis, ELISA and EV inhibition paradigms. Finally, the therapeutic efficacy of targeting GSDMD was examined using GSDMD-specific siRNA.
RESULTS: We identified in this work that mice deficient in GSDMD had better-preserved retinal function, increased photoreceptor survivability and reduced inflammation. RNA-Seq analysis revealed that GSDMD may propagate inflammation in the retina via NF-κB signalling cascades and release of pro-inflammatory cytokines. We also showed that IL-1β was packaged and released via EV in a GSDMD-dependent manner. Finally, we demonstrated that impairing GSDMD function using RNAi or blocking EV release was able to reduce IL-1β content in cell-free supernatant and EV.
CONCLUSIONS: Taken together, these results suggest that pyroptotic pore-forming protein GSDMD plays a key role in the propagation of retinal inflammation, in particular via the release of EV-encapsulated IL-1β. Targeting GSDMD using genetic or pharmacological inhibitors may pose a therapeutic opportunity to dampen inflammatory cascades and delay the progression of retinal degeneration.