Study On The Activation Mechanism Of NLRP3 Inflammasome In Inflammatory Responses Initiated By Leptospira Infection

Leptospirosis is an important global zoonotic disease.After infection with leptospires,animals such as rats usually have no clinical symptoms and shed spirochetes from their urine for a long period of time,whereas all human individuals fall ill.Leptospira causes multi-organs(such as kidney,liver and lung)damages in severe cases.Previous studies showed that different inflammatory responses initiated by Leptospira were associated with tissue damages and pathogen clearance in human or animals,but the mechanism remains unknown.Recent reports found that NLRP3 inflammasome is closely related to the inflammatory response induced by pathogens in human or animals.However,whether NLRP3 inflammaosome participates in mediating inflammotory response remains unclear.In this research,THP-1 and mouse macrophages were infected with L.interrogans to establish infection model and the activation mechanism of NLRP3 inflammasome was subsequently demonstrated.The results of this study contributed to elucidate pathogenic mechanism of Leptospira as well as provide novel ideas for the prevention and therapy of leptospirosis and refers to original findings with high academic value.1.Activation detection of NLRP3 inflammasome in macrophages initiated by Leptospira infection and its effect on the release of inflammatory cytokines.(1)Establishment of macrophage infection model: THP-1 and MPM cells were infected with L.interrgans strain Lai at MOI of 100(leptospire:cell=100:1)and co-cultured at at 37?5% CO2 for 1 h,2 h,4 h,12 h,and 24 h.For inhibitory tests,THP-1 and MPM cells were transfected with NLPR3 gene silencing sequence or pretreated with NLRP3 inhibitor glybenclamide or Caspase-1 inhibitor Z-YVAD-FMK for 30 min and subsequentlyinfected with Leptospira interrogans for 1 h,2 h,4 h,12 h and24 h.(2)NLRP3 mRNA levels detection:GAPDH and ?-actin were used asinternal reference for THP-1 and MPM cells,the total mRNA was extractedusing TRIzol method and then transcribed into c DNA and was subsequently quantifiedusing real-time fluorescent quantitative real time PCR.The results showed that the expression of NLRP3 mRNA level in L.interrogans-infected THP-1 cells increased by 4.05,0.34,0.33,0.06 and 1.66 times after infctedfor 1 h,2 h,4 h,12 h and 24 h,respectively(P<0.05),while that in L.interrogans-infected MPM cells was increased by9.42,17.08,28.12,4.56 and 0.17 time,s respectively(P<0.05).(3)NLPR3 protein levels determination: Flow cytometry determination showed that the expression rates of NLRP3 protein in infected THP-1 cells increased from 9.26% of normal cells to 94.01%,89.24%,31.80%,19.74%,11.28% at the time point(P<0.05).The expression rates of NLRP3 protein in infected MPM cells were increased from 18.71% of normal cells to 58.78%,43.64%,36.42%,76.46%,85.21% at the time points of 1 h,2 h,4 h,12 h and 24 h afte infection(P<0.05).(4)Effect of NLRP3 inhibitor on the inflammatory cytokines secretion in Leptospiras-infected macrophage: The NLRP3-mediated secretion of IL-1?,IL-18 and IL-33 was detected by ELISA combined with the NLRP3 inhibitory test.The results showed that the IL-1? of THP-1 cells pretreated with NLRP3 inhibitor decreased to 0.004,0.01,0.006,0.02,and 0.007 times of unpretreated cells infected for the same time duration(P<0.05);the IL-1? of MPM cells pretreated with NLRP3 inhibitor decrased to 0.14,0.12,0.007,0.22 and 0.009 times of unpretreated cells infected for the same time duration(P<0.05).The IL-18 of THP-1 cells pretreated with NLRP3 inhibitor fell to 0.07,0.06,0.06,0.37 and 0.39 times of unpretreated cells infected for the same time duration(P<0.05);the IL-18 of MPM cells pretreated with NLRP3 inhibitor fell to 0.30,0.11,0.19,0.28 and 0.40 times of unpretreated cells infected for the same time duration(P<0.05).IL-33 levelof THP-1 and MPM cells rose after 12 h of infection with L.interrogans,IL-33 level of of THP-1 cells fell to 0.59 and 0.60 times of unpretreated cells infected for the same time duration(P<0.05);the IL-33 level of MPM cells fell to the 0.30 and 0.28 times of unpretreated cells infected for the same time duration(P<0.05).The results suggested that the L.interrogans infection caused IL-1?,IL-18 and IL-33 increase in both THP-1 and MPM cells,which was inhibited by NLRP3 specific inhibitor(P<0.05);(5)Effect of NLRP3 gene silencing on the inflammatory cytokines secretion in Leptospira-infected macrophage:The NLRP3-mediated secretion of IL-1?,IL-18 and IL-33 was detectedusing ELISA combined with the NLRP3 gene silencing.The results showed that the IL-1? of THP-1 cells with NLRP3-si RNA decreased to 0.03,0.02,0.04,0.02 and 0.02 times of cells without gene scilence at the same time duration of infection(P<0.05);the IL-1? of MPM cells with NLRP3-si RNA fell to 0.12,0.17,0.11,0.98 and 0.23 times of cells without gene scilence at the same time duration of infection(P<0.05).The IL-18 of THP-1 cells with NLRP3-si RNA fell to0.60,0.31,0.56,0.22 and 0.68 times of of cells without gene scilence at the same time duration of infection(P<0.05);the IL-18 of MPM cells with NLRP3-si RNA decreased to0.21,0.17,0.18,0.13 and 0.12 times of of cells without gene scilence at the same time duration of infection(P<0.05).IL-33 of THP-1 and MPM cells rose after 12 h of infection.the IL-33 of THP-1 cells decreased to 0.42 and 0.42 times of cells without gene scilence at the same time duration of infection(P<0.05);the IL-33 of MPM cells decreased to 0.19 and 0.22 times of cells without gene scilence at the same time duration of infection(P<0.05).The NLRP3 gene silence significantly inhibited IL-1?,IL-18 and IL-33 secrtion in Leptospira-induced THP-1 and MPM cells.(6)Effect of caspase-1 inhibitor on the nflammatory cytokines secretionin Leptospira-infected macrophage:The Z-WEHD-FMK mediated secretion of IL-1?,IL-18 and IL-33 was detected by ELISA combined with the Caspase-1inhibitory test.The results showed that the IL-1? of THP-1 cells pretreated with caspase-1inhibitor fell to 0.51,0.38,0.37,0.43 and 0.59 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-1? of MPM cells pre-treated with caspase-1 inhibitor fell to 0.17,0.14,0.07,0.26 and 0.12 times of un-pretreated cells at the same infection time duration(P<0.05).The IL-18 of THP-1 cells pretreated with caspase-1 inhibitor fell to 0.70,1.29,0.57,0.71 and 0.89 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-18 of MPM cells pretreated with Caspase-1 inhibitor dropped to 0.30,0.42,0.78,0.28 and 0.60 times of un-pretreated cells at the same infection time duration(P<0.05).IL-33 of THP-1 and MPM cells rose after 2 h of infection.the IL-33 of THP-1 cells fell to 0.70 and0.68 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-33 MPM cells dropped to 0.35 and 0.47 times of un-pretreated cells at the same infection time duration(P<0.05).Caspase-1 inhibitor Z-WEHD-FMK effectively inhibited thesecreation of inflammatory cytokine IL-1?,IL-18 and IL-33 in Leptospira-infected THP-1 and MPM cells(P<0.05).2.Effect of ROS and lysosomal pathway on the NLRP3 inflammasome activation in Leptospira infected macrophages(1)Establishment of macrophage infection model: THP-1 and MPM cells were infected with L.interrgans strain Lai at MOI of 100(leptospire:cell=100:1)and co-cultured at at 37?5% CO2 for 1 h,2 h,4 h,12 h,24 h.For the inhibitory tests,THP-1 and MPM cells were pretreated with NADPH inhibitor apocynin,ROS inhibitor NAC and cathepsin B inhibitor CA-074 me for 30 min andthen infected with L.interrgans for 1 h,2 h,4 h,12 h,24 h.(2)Detection of ROS levels in macrophages infected with L.interrogans: The ROS specific fluorescent probe DCFH-DA marker was used to stain the cells and the fluorescence were detected using fluorescence microscope.The results showed that the ROS specific fluorescence intensity increased in infected THP-1 and MPM cells,with brighter fluorescence in MPM cell.The fulorescence intensity became weaker in NADPH or ROS inhibitor pretreated MPM cells,with less degree of changes in ROS inhibitor pretreated THP-1cells.(3)Effect of ROS inhibitor on the inflammatory cytokines secretion in Leptospira-infected macrophage: The secretion of IL-1?,IL-18 and IL-33 was detected by ELISA combined with the ROS inhibitory test.The results showed that the IL-1? of THP-1 cells pretreated with ROS inhibitor dropped to 0.03,0.03,0.07,0.07 and 0.02 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-1? of MPM cells pretreated with ROS inhibitor dropped to.12,0.11,0.02,0.02 and 0.03 times of of un-pretreated cells at the same infection time duration(P<0.05).The IL-18 of THP-1 cells pre-treatedwith ROS inhibitor dropped to 0.33,0.42,0.03,0.36 and 0.47 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-18 of MPM cells pretreated with ROS inhibitor fell to 0.24,0.24,0.10,0.18 and 0.14 times of un-pretreated cells at the same infection time duration(P<0.05).IL-33 in THP-1 and MPM cells rose after 12 h of infection,the IL-33 of THP-1cellspretreated with inhibitor fell to 0.81 and 0.71 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-33 of MPM cells pre-treated with ROS inhibitor fell to 0.39 and 0.47 times of un-pretreated cells at the same infection time duration(P<0.05).ROS inhibitor NAC effectively inhibited the expression of inflammatory cytokines IL-1?,IL-18 and IL-33(P<0.05).(4)Effect of cathepsin B inhibitor on the inflammatory cytokines secretion in Leptospire-infected macrophages: The CA-074 me mediated secretion of IL-1?,IL-18 and IL-33 was detected by ELISA combined with the cathepsin B inhibitory test to confirm or exclude cathepsin B participating inthe activation of the NLRP3 inflammatoryinflammasome.The results showed that the IL-1?of THP-1 cells pretratedwith cathepsin B inhibitor fell to 0.15,0.19,0.25,0.17 and 0.16 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-1? of MPM cells pretreated with cathepsin B inhibitor fell to 0.06,0.01,0.02,0.06 and 0.03 times of un-pretreated cells at the same infection time duration(P<0.05).The IL-18 of THP-1 cells pretreatedwith cathepsin B inhibitor fell 0.20,0.26,0.35,0.33 and 0.42 times of un-pretreated cells at the same infection time duration(P<0.05);the IL-18 of MPM cells pretreated with cathepsin B inhibitor fell to0.27,0.22,0.20,0.18 and 0.14 times of un-pretreated cells at the same infection time duration(P<0.05).IL-33 in THP-1 and MPM cells increased after 12 h of infection,the IL-33 of THP-1 cells preteated with cathepsin B inhibitorfell to 0.84 and 0.85 times(P<0.05);the IL-33 of MPM cells pre-treated with cathepsin B inhibitor fell to 0.21 and 0.22 times of un-pretreated cells at the same infection time duration(P<0.05).Cathepsin B inhibitor CA-074 me effectively inhibited the secretion of inflammatory cytokines IL-1?,IL-18 and IL-33(P<0.05).Conclusion: 1.NLRP3 mRNA protein expression levels were significantly up-regulated in THP-1 and MPM cells infected with L.interrogan.The secretion level of NLRP3 inflammasome charercteristic inflammatory cutokine IL-1?,IL-18 and IL-33 increased in both Leptospira-infected THP-1 and MPM cells,which issignificantly reduced by NLRP3 or caspase-1 inhibitor.It suggested that L.interrogans infection initiated the NLRP3 inflammasome activation in both human and mouse macrophages.Further more,L.interrogans induced earlier and higher level of IL-1? secretion in human macrophages than in mouse macrophages,while the secretion levels of IL-18 and IL-33 in both human and mouse macrophages were lower.The obove results suggest that NLRP3 inflammasome madiated different level of inflamatory cytokines in human and mouse macropages which is of high innovation and important academic value.2.The ROS levels were significantly up-regulated in L.interrogans infected THP-1 and MPM cells,with higher upregulation in MPM cells.The upregulationof inflammatory cytokine IL-1,IL-18 and IL-33 levels were significantly reduced in both Leptospira-infected THP-1 and MPM cells by the ROS and cathepsin B inhibitor,The results suggests that both ROS pathway and lysosomal pathway participated in the activation of NLRP3 inflammasome in L.interrogans infected human and mouse macrophages,which provides scientific basis to reveal the activation mechanism of NLRP3 inflammasome initiated by L.interrogans infection in macrophage of differen hosts.