Helicobacter Pylori Induces Cytokines IL-1β And IL-18Production In THP-1-drived Macrophages Through Activation Of NLROP3Inflammasome Via ROS Signaling Pathway

Objectives:NACTH domain-leucine-rich repeat, and PYD-containing protein (NLRP3) can beactivated by diversified activators. Reactive oxygen species (ROS) is one of thesignals of NLRP3signaling pathway. The aim of this study was to investigate theeffects of Helicobacter pylori on NLRP3inflammasomes activation in THP-1(humanmonocytic cell line)-derived macrophages and evaluate the role of ROS.Methods:H. pylori SS1was cocultured with the THP-1-derived macrophages according todifferent MOI (multiplicity of infection) of200:1,100:1,50:1,25:1. Coculturesupernatants and THP-1cells were collected at0h,3h,6h,12h,24h, and IL-1β andIL-18secretion were measured by ELISA. The level of ROS was detected by FCM,and the mRNA expression of NLRP3and caspase-1were quantified usingRealtime-PCR. Western blot was employed to analyze the expression of caspase-1protein. RNA interference technolgy was applied to silence the gene of NLRP3, andthen the effects of NLRP3-siRNA on secretion of IL-1β and IL-18induced by H.pylori were observed. THP-1macrophages were pretreated for30min with NAC (25mM) prior to stimulation with H. pylori SS1at MOI=100, then the contents of IL-1βand IL-18were analyzed by ELISA, and the mRNA expressions of NLRP3andcaspase-1were examined by Realtime-PCR.Results:(1) H. pylori could induce THP-1cells to produce IL-1β and IL-18in a time-anddose-dependent manner. When THP-1macrophages were stimulated with H. pylori at MOI=100:1, IL-1β reached its highest level at6h and IL-18achieved its peak at12h(P<0.05). When THP-1cells were coincubated with H. pylori at MOI=200:1,the levels of IL-1β and IL-18decreased.(2) H. pylori could induce the production of ROS in THP-1cells as determined byFCM. Compared with control cells, the fluorescent intensities of ROS in THP-1cells were markedly increased in a time-dependent manner in response to H.pylori infection, which reached a peak level at6h. Pretreatment with NACsignificantly inhibited the production of ROS induced by H. pylori, the meanfluorescent intensities of ROS decreased by6.79%,33.63%,19.59%and14.68%at3h,6h,12h and24h, respectively.(3) Realtime-PCR results showed that H. pylori could induce the mRNA expressionsof NLRP3and caspase-1in THP-1cells. Compared with the PBS group, bothcaspase-1and NLRP3mRNA levels increased at6h and3h in several andreached their peaks at12h (P<0.01, P<0.05respectively). However, the mRNAexpressions of NLRP3and caspase-1decreased in the NAC inhibition group.(4) Western blot results displayed that the protein expression of caspase-1in H.pylori-stimulated THP-1group increased from3h and achieved peak at12h.Moreover, NAC administration decreased the upregulation of caspase-1proteinlevel induced by H. pylori.(5) When THP-1cells were transfected with NLRP3-siRNA, the production of IL-1βand IL-18decreased.Conclusions:(1) H. pylori could induce THP-1-derived macrophages to produce cytokines IL-1βand IL-18.(2) H. pylori could activate NLRP3inflammasome via ROS signaling pathway.