Helicobacter Pylori Tipα Protein Induces Cytokines IL-1β And IL-18 Production In THP-1-drived Macrophages Through Activation Of NLRP3 Inflammasome

Objective: Helicobacter pylori infection-induced inflammation in stomach is the main reason of gastric disease. Helicobacter pylori TNF-α-inducing protein(Tipα) is a newly discovered pro-inflammation protein that can induce inflammation in stomach.The NLRP3 inflammasome is a critical regulator of inflammation. The aim of this study was to preliminarily investigate the role of NLRP3 inflammasome in Tipα-induced pro-inflammatory cytokines secretion in PMA-differentiated THP-1 cells.Methods:1. Expression and identification of recombinant Tipα: After identification by PCR, double digestion and gene sequencing, the prokaryotic expression vector p ET30a(+)/Tipα without sequence of signal peptides was transformed into E.coli BL21(DE3) and induced to express Tipα with IPTG. Then the obtained recombinant protein was purified, endotoxin removed and analyzed by Western blot. The concentration of protein was determined by BCA kit.2. Preliminarily explore the proinflammatory effect of Tipα on target cells: The secretion levels of TNF-α, IL-1β and IL-18 in supernatant of culture medium treated with Tipα were detected by ELISA.3. Explore the role of NLRP3/Caspase-1 pathway in Tipα-induced IL-1β and IL-18 secretion in THP-1 cells: We blocked My D88/NF-κB and NLRP3/Caspase-1 pathways by PDTC or the general ROS scavenger, NAC,respectively, and then detected the secretion levels of proinflammatory cytokines and the expression levels of NLRP3 and Caspase-1.Results:1. The prokaryotic expression recombinant vector p ET30a(+)/Tipα was successfully constructed, and the sequence inserted into vector was completely consistent with the Tipα sequence that reported in Genbank. After expression and purification, a soluble 25 k Da-size recombinant protein was attained with above 95% of purity, and the protein could react with anti His-tag monoclonal antibody specifically.2. Tipα protein can significantly induced secretion of TNF-α, IL-1β and IL-18 in THP-1 cells in a time and dose-dependent manner. Levels of TNF-α, IL-1βand IL-18 approached their peaks at 6h post-treament by 40μg/m L of Tipαprotein.3. The results showed that the blockade of NF-κB signaling pathway by PDTC can inhibit the secretion of proinflammatory cytokines and the expression of NLRP3 and Caspase-1.4. When THP-1 cells were pre-treated with ROS scavenger NAC, the Tipα-induced increased IL-1β and IL-18 secretion was obviously eliminated(P < 0.05), while TNF-α level had no significant difference, and the expression levels of Caspase-1 and NLRP3 also have a significant decline.Conclusion:1. Recombinant Tipα protein can promote THP-1-drived macrophages to secrete proinflammatory cytokines TNF-α, IL-1β and IL-18.2. The NLRP3/Caspase-1 pathway may be involved in the Tipα proteininduced IL-1β and IL-18 secretion.