Department of Immunobiology, College of Life Science and Technology, Jinan University, Guangzhou, China.
Department of Cell Biology, College of Life Science and Technology, Jinan University, Guangzhou, China.
Apoptosis. 2019 Oct;24(9-10):703-717. doi: 10.1007/s10495-019-01551-x.
ATP acts as a canonical activator to induce NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation in macrophages, leading to caspase-1/gasdermin D (GSDMD)-mediated pyroptosis. It remains unclear whether ATP can induce pyroptosis in macrophages when the NLRP3 pathway is blocked by pathogenic infection. In this study, we used cellular models to mimic such blockade of NLRP3 activation: bone marrow-derived macrophages (BMDMs) treated with NLRP3-specific inhibitor MCC950 and RAW264.7 cells deficient in ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression. The results showed that ATP treatment induced lytic cell death morphologically resembling canonical pyroptosis in both MCC950-treated BMDMs and RAW264.7 cells, but did not cause the activation of caspase-1 (by detecting caspase-1p10 and mature interleukin-1β) and cleavage of GSDMD. Instead, both apoptotic initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspases were evidently activated and gasdermin E (GSDME) was cleaved to generate its N-terminal fragment (GSDME-NT) which executes pyroptosis. The GSDME-NT production and lytic cell death induced by ATP were diminished by caspase-3 inhibitor. In BMDMs without MCC950 treatment, ATP induced the formation of ASC specks which were co-localized with caspase-8; with MCC950 treatment, however, ATP did not induced the formation of ASC specks. In RAW264.7 cells, knockdown of GSDME by small interfering RNA attenuated ATP-induced lytic cell death and HMGB1 release into culture supernatants. Collectively, our results indicate that ATP induces pyroptosis in macrophages through the caspase-3/GSDME axis when the canonical NLRP3 pathway is blocked, suggestive of an alternative mechanism for combating against pathogen evasion.
ATP 作为一种经典的激活剂，可诱导巨噬细胞中 NLRP3（NOD 样受体家族，富含 pyrin 域 3）炎症小体的激活，导致半胱天冬酶-1/gasdermin D（GSDMD）介导的细胞焦亡。目前尚不清楚在 NLRP3 途径被致病性感染阻断的情况下，ATP 是否可以诱导巨噬细胞发生细胞焦亡。在这项研究中，我们使用细胞模型模拟 NLRP3 激活的这种阻断：用 NLRP3 特异性抑制剂 MCC950 处理的骨髓来源巨噬细胞（BMDMs）和缺乏 ASC（含半胱天冬酶募集结构域的凋亡相关斑点样蛋白）表达的 RAW264.7 细胞。结果表明，ATP 处理诱导的裂解细胞死亡形态类似于 MCC950 处理的 BMDMs 和 RAW264.7 细胞中的经典细胞焦亡，但不会导致半胱天冬酶-1 的激活（通过检测半胱天冬酶-1p10 和成熟的白细胞介素-1β）和 GSDMD 的切割。相反，凋亡起始（半胱天冬酶-8 和 -9）和执行（半胱天冬酶-3 和 -7）半胱天冬酶均明显激活，并且 gasdermin E（GSDME）被切割产生其 N 端片段（GSDME-NT），执行细胞焦亡。ATP 诱导的 GSDME-NT 产生和裂解细胞死亡被 caspase-3 抑制剂减弱。在未经 MCC950 处理的 BMDMs 中，ATP 诱导 ASC 斑点的形成，其与半胱天冬酶-8 共定位；然而，在用 MCC950 处理后，ATP 没有诱导 ASC 斑点的形成。在 RAW264.7 细胞中，小干扰 RNA 敲低 GSDME 减弱了 ATP 诱导的裂解细胞死亡和 HMGB1 释放到培养上清液中。总之，我们的结果表明，当经典的 NLRP3 途径被阻断时，ATP 通过 caspase-3/GSDME 轴诱导巨噬细胞发生细胞焦亡，提示针对病原体逃避的替代机制。
