Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada.
PLoS Pathog. 2022 Mar 18;18(3):e1010415. doi: 10.1371/journal.ppat.1010415. eCollection 2022 Mar.
A hallmark of Entamoeba histolytica (Eh) invasion in the gut is acute inflammation dominated by the secretion of pro-inflammatory cytokines TNF-α and IL-1β. This is initiated when Eh in contact with macrophages in the lamina propria activates caspase-1 by recruiting the NLRP3 inflammasome complex in a Gal-lectin and EhCP-A5-dependent manner resulting in the maturation and secretion of IL-1β and IL-18. Here, we interrogated the requirements and mechanisms for Eh-induced caspase-4/1 activation in the cleavage of gasdermin D (GSDMD) to regulate bioactive IL-1β release in the absence of cell death in human macrophages. Unlike caspase-1, caspase-4 activation occurred as early as 10 min that was dependent on Eh Gal-lectin and EhCP-A5 binding to macrophages. By utilizing CRISPR-Cas9 gene edited CASP4/1, NLRP3 KO and ASC-def cells, caspase-4 activation was found to be independent of the canonical NLRP3 inflammasomes. In CRISPR-Cas9 gene edited CASP1 macrophages, caspase-4 activation was significantly up regulated that enhanced the enzymatic cleavage of GSDMD at the same cleavage site as caspase-1 to induce GSDMD pore formation and sustained bioactive IL-1β secretion. Eh-induced IL-1β secretion was independent of pyroptosis as revealed by pharmacological blockade of GSDMD pore formation and in CRISPR-Cas9 gene edited GSDMD KO macrophages. This was in marked contrast to the potent positive control, lipopolysaccharide + Nigericin that induced high expression of predominantly caspase-1 that efficiently cleaved GSDMD with high IL-1β secretion/release associated with massive cell pyroptosis. These results reveal that Eh triggered "hyperactivated macrophages" allowed caspase-4 dependent cleavage of GSDMD and IL-1β secretion to occur in the absence of pyroptosis that may play an important role in disease pathogenesis.
溶组织内阿米巴（Eh）在肠道中的侵袭标志是急性炎症，主要由促炎细胞因子 TNF-α 和 IL-1β 的分泌所主导。当 Eh 与固有层中的巨噬细胞接触时，通过 Gal-lectin 和 EhCP-A5 依赖性方式招募 NLRP3 炎性小体复合物，激活半胱天冬酶-1（caspase-1），从而导致 IL-1β 和 IL-18 的成熟和分泌。在这里，我们研究了 Eh 诱导半胱天冬酶-4/1 在无细胞死亡的情况下切割 Gasdermin D（GSDMD）以调节人巨噬细胞中生物活性 IL-1β 释放的要求和机制。与 caspase-1 不同，caspase-4 的激活早在 10 分钟时就发生了，这依赖于 Eh Gal-lectin 和 EhCP-A5 与巨噬细胞的结合。通过利用 CRISPR-Cas9 基因编辑的 CASP4/1、NLRP3 KO 和 ASC-def 细胞，发现 caspase-4 的激活独立于经典的 NLRP3 炎性小体。在 CRISPR-Cas9 基因编辑的 CASP1 巨噬细胞中，caspase-4 的激活显著上调，增强了 GSDMD 的酶切，在与 caspase-1 相同的切割位点诱导 GSDMD 孔形成并持续释放生物活性 IL-1β。Eh 诱导的 IL-1β 分泌不依赖于细胞焦亡，这是通过 GSDMD 孔形成的药理学阻断和在 CRISPR-Cas9 基因编辑的 GSDMD KO 巨噬细胞中证实的。这与强力阳性对照物 LPS+ Nigericin 形成鲜明对比，后者诱导主要是 caspase-1 的高表达，其有效地切割 GSDMD，与大量细胞焦亡相关的高 IL-1β 分泌/释放。这些结果表明，Eh 触发的“过度激活的巨噬细胞”允许 caspase-4 依赖性 GSDMD 的切割和 IL-1β 的分泌发生，而没有细胞焦亡，这可能在疾病发病机制中起重要作用。
