Entamoeba histolytica ‐Induced Activation of Caspase‐4 Regulates Gasdermin D Cleavage to Mediate IL‐1β Secretion in Macrophages

Abstract

A hallmark of Entamoeba histolytica (Eh) invasion in the gut is acute intestinal inflammation dominated by the secretion of pro-inflammatory cytokines. Eh in contact with macrophages activates caspase-1 by the recruitment of the NLRP3 inflammasome complex in a Gal-lectin and Eh cysteine proteases 5 (EhCP-A5)-dependent manner, resulting in the maturation and secretion of interleukin (IL)-1β and IL-18. Although it is well-defined that caspase-1 activation is important in mediating the secretion of IL-1β and IL-18, the role caspase-4 play in the overall pro-inflammatory response is less identified. We have recently shown that Eh activates caspase-1/4 in macrophage that cleaved gasdermin D (GSDMD) leading to the insertion of N-terminal effector domain into the cell membrane, driving both pore formation and IL-1β secretion without causing significant cell death. In this study, we interrogated the requirements and activation mechanism for Eh-induced caspase-4 activation in cleaving GSDMD and in mediating bioactive IL-1β release. Similar to caspase-1, caspase-4 activation was dependent on Eh Gal-lectin and EhCP-A5 binding to macrophages that triggered ATP release that acted as a second signal. To determine if Eh-induced activation of caspase-4 interacted with the canonical NLRP3 inflammasome to regulate GSDMD-mediated pro-inflammatory cytokine release, CRISPR-Cas9 gene editing of caspase-1, 4, ASC, and NLRP3 THP-1 cells were utilized. Unlike caspase-1, caspase-4 activation in response to Eh was triggered within 10 min and was independent of ASC, NLRP3, and caspase-1. In the absence of caspase-1, caspase-4 activation was significantly up regulated that enhanced the cleavage of GSDMD to induce robust IL-1β secretion. Eh-induced caspase-4 played a major role in triggering GSDMD pore formation and bioactive IL-1β release as quantified by HEK-Blue™ reporter cell IL-1β assay independent of pyroptosis. This was in marked contrast to the positive control, LPS + Nigericin that induced high expression of caspase-1, enhanced GSDMD cleavage, IL-1β secretion, and massive pyroptosis. These results suggest that Eh induced “hyperactivated macrophages” led to caspase-4 dependent GSDMD cleavage and IL-1β secretion in the absence of pyroptosis important in disease pathogenesis.