Chadee, KrisWang, Shanshan2022-05-022022-05-022022-04Wang, S. (2022). Entamoeba histolytica-induced activation of caspase-4 regulates Gasdermin D cleavage to mediate IL-1β secretion in macrophages (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/114594The parasite Entamoeba histolytica (Eh) is named for its capability to lyse healthy host tissues. A hallmark of Eh invasion in the gut is acute intestinal inflammation dominated by the secretion of pro-inflammatory cytokines. Eh in direct contact with macrophages activates caspase-1 by the assembly of 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. Pyroptosis, an inflammatory mode of lytic programmed cell death, is essential for host defence and other danger signals. Eh activates caspase-4/1 in macrophage that cleaved GSDMD, the executioner of cell pyroptosis, 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, I interrogated the requirements and molecular mechanism for Eh-induced caspase-4 activation in cleaving GSDMD and in initiating a robust pro-inflammatory response. The noncanonical caspase-4 is considered inextricably connected to the NLRP3 inflammasome, however, the concise mechanism of this interaction remains unclear. 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 CASP4/1, ASC, NLRP3, GSDMD THP-1 cells were utilized. In the absence of caspase-1, caspase-4 activation was significantly upregulated and enhanced GSDMD pore formation to trigger robust IL-1β secretion as quantified by HEK-BlueTM reporter cell. The fundamental function of pyroptosis is to trigger vigorous inflammatory responses to defend against pathogens. However, excessive and prolonged pyroptosis can lead to severe inflammatory diseases or immunological conditions, including sepsis and autoimmune disorders. The most outstanding finding of this work revealed that Eh did not trigger extensive cell pyroptosis for robust IL-1β secretion, rather Eh induced “hyperactivated macrophages” that led to caspase-4 dependent GSDMD cleavage and IL-1β release. 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 pyroptotic cell death. Edman degradation and in vitro caspase cleavage assays showed that caspase-4 cleaved GSDMD at the same position as caspase-1 (human) to form pores in the plasma membrane. This work sheds new light on the functional role of caspase-4 in regulating GSDMD pore forming activity in innate host defenses against Eh.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.GasdermincaspaseparasitemacropahgeEntamoba histolyticainnate immunityinflammationamebiasisEducation--HealthEducation--SciencesMicrobiologyParasitologyHealth SciencesImmunologymaster thesis10.11575/PRISM/39714