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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Pyroptosis of Macrophages Induced by Clostridium perfringens Beta-1 Toxin
Siyu Zhang1, Lingling Ma1, Fuyang Song1
1Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western China, College of Life Science, Ningxia University, Yinchuan 750021, China.
Abstract:
Clostridium perfringens beta-1 toxin (CPB1) is responsible for necrotizing enteritis and enterotoxemia. However, whether the release of host inflammatory factors caused by CPB1 is related to pyroptosis, an inflammatory form of programmed cell death, has not been reported. A construct expressing recombinant Clostridium perfringens beta-1 toxin (rCPB1) was created, and the cytotoxic activity of the purified rCPB1 toxin was assessed via CCK-8 assay. The rCPB1-induced macrophage pyroptosis by assessing changes to the expression of pyroptosis-related signal molecules and the pyroptosis pathway of macrophages using quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopic assays. The results showed that the intact rCPB1 protein was purified from an E. coli expression system, which exhibited moderate cytotoxicity on mouse mononuclear macrophage leukemia cells (RAW264.7), normal colon mucosal epithelial cells (NCM460), and human umbilical vein endothelial cells (HUVEC). rCPB1 could induce pyroptosis in macrophages and HUVEC cells, in part through the Caspase-1-dependent pathway. The rCPB1-induced pyroptosis of RAW264.7 cells could be blocked by inflammasome inhibitor MCC950. These results demonstrated that rCPB1 treatment of macrophages promoted the assembly of NLRP3 inflammasomes and activated Caspase 1; the activated Caspase 1 caused gasdermin D to form plasma membrane pores, leading to the release of inflammatory factors IL-18 and IL-1β, resulting in macrophage pyroptosis. NLRP3 may be a potential therapeutic target for Clostridium perfringes disease. This study provided a novel insight into the pathogenesis of CPB1.
Insights
Clostridium perfringens beta-1 toxin (CPB1) triggers pyroptosis, a programmed cell death, in macrophages and endothelial cells. This inflammatory response involves the NLRP3 inflammasome and Caspase-1, offering potential therapeutic targets for C. perfringens infections.
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Clostridium perfringens beta-1 toxin (CPB1) is a key virulence factor in necrotizing enteritis and enterotoxemia.
- The role of CPB1 in inducing pyroptosis, an inflammatory form of programmed cell death, remained uninvestigated.
Purpose of the Study:
- To investigate whether CPB1 induces pyroptosis in host cells.
- To elucidate the molecular mechanisms underlying CPB1-induced pyroptosis.
Main Methods:
- Recombinant CPB1 (rCPB1) was expressed and purified.
- Cytotoxicity was assessed using CCK-8 assays.
- Pyroptosis was analyzed via quantitative real-time PCR, immunoblotting, ELISA, immunofluorescence, and electron microscopy.
- Inflammasome activation was studied using the inhibitor MCC950.
Main Results:
- Purified rCPB1 exhibited moderate cytotoxicity in RAW264.7, NCM460, and HUVEC cells.
- rCPB1 induced pyroptosis in macrophages (RAW264.7) and HUVEC cells via a Caspase-1-dependent pathway.
- rCPB1 treatment promoted NLRP3 inflammasome assembly and Caspase-1 activation.
- Activated Caspase-1 led to Gasdermin D cleavage, pore formation, and release of IL-18 and IL-1β, causing pyroptosis.
Conclusions:
- CPB1 induces pyroptosis in macrophages and HUVEC cells through the NLRP3 inflammasome and Caspase-1 pathway.
- NLRP3 inflammasome activation is a critical mechanism in CPB1-mediated cell death.
- Targeting the NLRP3 inflammasome may represent a novel therapeutic strategy for Clostridium perfringens diseases.
