Autophagic regulation of inflammasome-mediated hyperactive state in living macrophages

摘要 核苷酸结合结构域（NBD）和富含亮氨酸重复序列（LRR）的蛋白质（NLR）组装成 功能性超分子组织中心称为炎性小体，其作为宿主的界面 防御病原体或其他类型的危险。典型炎性小体激活caspase-1， 炎性蛋白酶在激活时触发两种重要生物反应：1）细胞因子加工 白细胞介素-1b（IL-1b），和2）质膜Gasdermin D孔形成以增强炎性细胞 死亡（焦亡）。细胞因子分泌和焦亡在稳态条件下偶联，如GSDMD 渗透IL-1b的质膜上的孔也可损害细胞膜的完整性 导致溶解性细胞死亡。有趣的是，在炎性小体激活后的过度活跃状态下，细胞显示出 持续的细胞因子分泌，但保持活力，即使这一过程依赖于GSDMD孔形成 在质膜上。重新校准GSDMD以仅维持GSDMD的释放的潜在机制 对白细胞介素，而不是焦亡，知之甚少。 自噬被认为是炎症的重要调节因子。在我们最近的研究中，我们发现， 炎性小体由一种促进炎性小体组装的攻击小体样机制调节（Yang） 而且还诱导自噬以抑制胱天蛋白酶-1加工和IL-β分泌（Yin）（Magupalli等人，科学， 2020年）。我们的研究与之前的报道一致，即缺乏关键自噬基因的小鼠， 造血细胞中的Atg 16 L1对葡聚糖硫酸钠诱导的急性结肠炎高度敏感， 分泌大量IL-1b 和IL-18细胞因子。另外这些 炎症表型是 通过在小鼠中注射抗IL-1b和IL-18抗体减轻。总体而言，我们和其他公布的结果 证明自噬直接调节炎性小体激活下游信号的程度。 然而，目前尚不清楚自噬是否可以调节细胞过度活跃状态和细胞自燃状态之间的转换， 巨噬细胞 在本申请中，我们将研究自噬和巨噬细胞之间的潜在联系 使用炎性小体测定和细胞成像来检测超活化。

Abstract Nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing proteins (NLRs) assemble into functional supramolecular organizing centers called the inflammasomes, which serve as an interface of host defense against pathogens or other types of danger. Canonical inflammasomes activate caspase-1, an inflammatory protease that upon activation, triggers two important biological responses: 1) processing of cytokine interleukin-1b (IL-1b), and 2) formation of plasma membrane gasdermin D pores to potentiate inflammatory cell death (pyroptosis). Cytokine secretion and pyroptosis are coupled under a steady-state condition, as the GSDMD pores on the plasma membrane, which permeates IL-1b, can also compromise the cell membrane integrity leading to the lytic cell death. Interestingly, in a hyperactive state following inflammasome activation, cells show sustained cytokine secretion but retain viability, even though this process is dependent on GSDMD pores formed on the plasma membrane. The underlying mechanisms which recalibrate GSDMD to only sustain the release of the interleukin, but not pyroptosis, are poorly understood. Autophagy is regarded as an important regulator of inflammation. In our recent studies, we found that inflammasomes are regulated by an aggresome-like mechanism that promotes inflammasome assembly (Yang) but also induces autophagy to dampen caspase-1 processing and IL-b secretion (Yin) (Magupalli et al., Science, 2020). Our studies are consistent with previous reports showing that mice lacking the key autophagic gene, Atg16L1, in hematopoietic cells, were highly susceptible to dextran sulphate sodium-induced acute colitis, with the secretion of a high amount of IL-1b and IL-18 cytokines. In addition, these inflammatory phenotypes were alleviated by injection of anti-IL-1b and IL-18 antibodies in mice. Collectively, our and other published results demonstrate that autophagy directly regulates the extent to which inflammasomes activate downstream signals. However, it is unclear if autophagy can regulate the switch between a hyperactive state and a pyroptotic state of macrophages. In this application, we will investigate the potential link between autophagy and macrophage hyperactivation using inflammasome assays and cellular imaging.