Abstract
Background In this study we evaluated the molecular mechanism by which pyroptosis is induced in mononuclear macrophages isolated from Saba pigs following infection with pathogenic E. coli high pathogenicity island (HPI). Mononuclear macrophages were divided into four treatment groups: control, Lipopolysaccharide (LPS) + adenosine triphosphate (ATP), HPI positive (+) strain and HPI negative (-) strain. The mononuclear macrophages and their culture supernatants were collected at 0.5, 3, 6, 9, 12 and 24 h after infection. DNA changes were detected by TUNEL staining and the integrity of the cell membrane was evaluated by propidium iodide (PI) staining. Changes in mRNA expression levels of NLRP3, caspase-1, IL-1β, and IL-18 gene in mononuclear macrophages were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and caspase-1 protein expression was detected by indirect immunofluorescence. IL-1β and IL-18 concentration in the mononuclear macrophage culture supernatant were measured by ELISA. Results Compared with the control group, TUNEL and PI staining of mononuclear macrophages was significantly increased following infection with the HPI + /HPI - strains ( P < 0.01 or P < 0.05), with significantly higher levels detected in the HPI + group compared with those in the HPI - group ( P < 0.01 and P < 0.05). Compared with the control group, the expression levels of NLRP3, caspase-1, IL-1β, and IL-18 in the HPI groups were upregulated after pathogenic E. coli infection, with significantly higher levels detected in the HPI + group compared with those in the HPI - group ( P < 0.01 or P < 0.05). Conclusions These findings showed that pathogenic E. coli HPI infection of Saba pigs results induced pyroptosis of mononuclear macrophages characterized by increased expression of NLRP3, caspase-1, IL-1β and IL-18 mRNA in mononuclear macrophages, the induction of cell membrane pore formation, nuclear DNA damage, and the secretion of IL-1β and IL-18 to enhance the inflammatory response.
Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain