NLRP3 Inflammasome Plays Carcinogenic Role In AML Through Caspase-1/IL-1β Pathway

BackgroundAcute myeloid leukaemia(AML)is a clonal hematologic malignancy characterized by clonal and abnormally differentiated hematopoietic progenitors that proliferate and accumulate in bone marrow,peripheral blood and other tissues[1,2].Though cytogenetic heterogeneity and enormous molecular heterogeneity of AML have become increasingly apparent and the therapeutic strategy has made great progress,the overall prognosis of AML patients remains poor.The search for more effective agents urgently need to be addressed.Abnormalities of immune system have long been recognized in AML,but the detailed immunological mechanism of AML remains unclear.Inflammasome is one of the major complexes involved in innate immune response,and NLRP3 represents the most extensively investigated inflammasome sensors[3,4].NLRP3 inflammasome is an intracellular multi-protein complex containing NLRP3,ASC,and pro-caspase-1[5].The exact mechanism of NLRP3 inflammasome activation is still under investigation,and a spatially and temporally separated two-step process including priming and activation is generally accepted.Signal one upregulate transcription of pro-IL-1β,pro-IL-18 and NLRP3 via NF-κB activation by Toll-like receptor(TLR)signaling,and signal two in inflammasome activation involves the sensing of PAMPs and DAMPs by NLRs.NLRP3 activators are structurally and chemically diverse and the mechanism of inflammasomes activation is not fully understood[5-8].Interestingly,Hornung and colleagues reported a new pathway "alternative inflammasome".Unlike canonical NLRP3 activation signaling,lipopolysaccharide(LPS)triggers the NLRP3 inflammasome activation in human monocytes via TLR4-TRIF-RIPK1-FADD-CASP8 independently from an observed signal two[9].When stimulated,the NLRP3 inflammasome promotes caspase-1 activation and subsequently converting pro-IL-1β and pro-IL-18 into their mature bioactive forms.Mature IL-1β and IL-18 is then released to extracellular space[6].In addition,IL-1β itself induces synthesis of pro-IL-1βthrough IL-1RI-MyD88-NF-κB pathway,resulting in a positive feedback loop[10].Increasing evidence emphasizes that inflammasomes promote inflammation,and chronic inflammatory response triggered by a variety of immune cells is crucial for cancer establishment and progression[4,11-13].As effector of NLRP3 inflammasome,IL-1β is a pluripotent pro-inflammatory cytokine involved in cell proliferation,differentiation and considered to play a pivotal role in tumor pathogenesis.Recent evidence suggests that high serum concentrations of IL-1β and IL-18 are strictly correlated to poor prognosis of cancer patients[14].Despite mounting clinical and experimental evidence demonstrated NLRP3 inflammasome’s carcinogenic activities,its role in cancer is diverse and sometimes contrasting[10,13,14].Whether NLRP3 inflammasome has a beneficial or detrimental effect on the development of cancer draw more and more attention.Recent studies have identified that NLRP3 inflammasome activation and IL-18 signaling pathway induce tumoricidal activity of NK cells and regulate gut microbiota,leading to antitumorigenic effects on colitis-associated colorectal cancer.However,multiple studies have shown that NLRP3-IL-1β-IL-1R signaling axis conducted carcinogenic properties in the growth and metastasis of gastric carcinoma,melanoma,mammary tumors and fibrosarcoma in a context-dependent manner[11,15].Moreover,the activation of inflammasome enhances migration and metastasis of prostate cancer,head and neck squamous cell carcinoma,lung cancer,hepatocellular carcinoma and skin cancer.As a result,several small molecules,antagonists,and monoclonal antibodies against components of the inflammasome are developed to control cancer[11,16-19].It was reported that NLRP3 inflammasome plays a carcinogenic role in lymphoma through IL-18.Our previous research has shown that the polymorphisms of IL-1β(rs 16944)and IL-18(rs 1946518)may contribute to poorer survival of AML patients[20].However,the role of NLRP3 inflamasome in the development and treatment of AML remains poorly understood.In this study,we investigated the functions and potential underlying mechanisms of NLRP3 inflammasome in AML.Altogether,the present study demonstrates for the first time that NLRP3 inflammasome increases drug-resistance and plays carcinogenic role in AML Caspase-1/IL-1β pathway.ObjectiveThis study aimed to demonstrate NLRP3 inflammasome was activated in bone marrow of ND AML and search for critical molecule involved in AML through bayesian network.Furthermore,we intended to explore the role of NLRP3 activation and inactivation in biological effects of AML cells.Additionally,we intended to investigate the role of NLRP3 inflammasome in resistance of leukemia cells of ND AML patients to chemotherapy drug.Materials and MethodsPatients and SpecimensBone marrow(BM)aspirates included in this study contained 84 newly-diagnosed(ND)AML patients and 16 healthy controls.The sera of BM samples were collected from EDTA stabilized bone marrow and stored at-80℃ for determination of cytokines.Bone marrow mononuclear cells(BMMCs)were isolated by density-gradient centrifugation with Ficoll-Hypaque.A total of 2x106BMMCs were stabilized in TRIZOL and stored at-80℃ for RNA extraction.The other leukemia cells of ND AML patients were used for cell culture experiment.1.Detect the mRNA expression level of NLRP3 inflammasome associated molecules and IL-1β concentration in bone marrow of ND AML and normal controls1.1 Real-time PCR:A total of 63 newly diagnosed AML patients and 16 controls were chosed for determining the expression level of NLRP3 inflammasome components,NF-κB and IL-1R.Total RNA was extracted from BMMCs using TRIZOL.The mRNA expression level of NLRP3,ASC,Caspase-1,IL-1β,IL-18,NF-κB and IL-1R were determine by RT-PCR.1.2 Analyze bayesian network based on mRNA expression level of NLRP3 inflammasome components,NF-κB and IL-1R.1.3 ELISA:A total of 70 newly diagnosed AML patients and 15 controls were chosed to detect level of IL-1β and IL-18 in BM supernatant.The concentration of IL-1β was detected by human ELISA kits from R&D Systems.2.Explore the biological role of NLRP3 inflammasome after further activation in leukemia cells of ND AML patients.2.1 Leukemia cells of ND AML patients(1x106 cells/ml)were cultured in RPMI1640 culture medium supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin and incubated at 37℃ in humidified atmosphere with 5%CO2.To further activate NLRP3 inflammasome,leukemia cells of ND AML patients were stimulated with LPS(1 μg/ml).2.2 After incubated for 24h,48h and 72h,cells were seeded in 96-well plates respectively for cell proliferation analysis using CCK8 method.2.3 In addition,after cultured for 48h,cells were also harvested for apoptosis analysis by Flow cytometry and RNA extraction for RT-PCR,while supernatants were collected for ELISA analysis.3.NLRP3 activated THP-1 cells cocultured with leukemia cells of ND AML patients3.1 THP-1 cells were cultured in RPMI 1640 culture medium supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin and incubated at 37℃ in humidified atmosphere with 5%CO2.3.2 Activated THP-1 cells by LPS:THP-1 cells were stimulated with LPS.After being cultured for 48h,cells were harvested for RNA extraction and supernatants were collected for ELISA analysis.3.3 Activated THP-1 cells by LPS+ATP:THP-1 cells(1×106 cells/ml)were stimulated with LPS(1μg/ml)for 6h followed by ATP(5mmol/L)for 1h,then washed with normal saline for three times.100ul THP-1 cells(5×104 cells/ml)in the upper chamber cocultured with 600μl leukemia cells of ND AML patients(1 × 106 cells/ml)in the bottom chamber using Transwell chamber.Anti-IL-1β and anti-IL-18 were added for neutralization.After being incubated for 48h,leukemia cells in the bottom chamber were harvested for apoptosis assay.In addition,we cultured LPS+ATP activated THP-1 cells for 48h,then supernatants were collected for ELISA analysis.4.Treatment with IL-1β,IL-18 for leukemia cells of ND AML patientsTo investigate the role of NLRP3 inflamasome effectors in AML,IL-1β or/and IL-18 were added into leukemia cells of ND AML patients.After incubated for 24h,48h,72h and 96h,cell proliferation analysis was performed.Besides,after cultured for 48h,apoptosis analysis was performed.5.Inactivation of NLRP3 with Caspasel inhibitorTo inhibit the activation of caspase-I,leukemia cells of ND AML patients were pretreated with Caspase-1 inhibitor Z-YVAD-FMK for 1h prior to LPS stimulation.Cell proliferation analysis was performed at 24h,48h and 72h.Besides,supernatants were collected for ELISA analysis and cells were harvested for apoptosis analysis after cultured for 48h.6.Inactivation of NLRP3 with NF-κB inhibitorFor inhibition of NLRP3 inflammasome activation via NF-κB signaling pathway,NF-κB inhibitor Bay11-7082 was used to treat leukemia cells of ND AML patients for 1h prior to LPS stimulation.Cell proliferation analysis was performed at 24h,48h and 72h.Besides,supernatants were collected for ELISA analysis and cells were harvested for apoptosis analysis after being cultured for 48h.7.Activation and inactivation of NLRP3 inflammasome on protein levelLeukemia cells of ND AML patients were treated with LPS,LPS+ATP,LPS+ATP+Z-YVAD-FMK and LPS+ATP+Bay11-7082.The cells were pretreated with Z-YVAD-FMK and Bay11-7082 for lh and then stimulated with LPS for 6h followed by ATP for 1h.Then cells were harvested to detect the protein expression of pNF-KB(65KD),pro-caspase-1(48KD),pro-IL-1β(31KD),active caspase-1(20KD)and active IL-1β(17KD)by western blot analysis.8.Explore the role of NLRP3 inflammasome in antitumor effect of chemotherapy drug.To determine the role of IL-1β and IL-18 in antitumor activity of chemotherapy drug,ADR,DNR and Ara-C were added into leukemia cells of ND AML patients with or without NLRP3 inflammasome effectors.Furthermore,to explore the role of NLRP3 activation in antitumor effect of chemotherapy drug,ADR,DNR and Ara-C were added into leukemia cells of ND AML patients with or without LPS.cell proliferation and apoptosis analysis were performed.Result1.NLRP3 inflammasome was activated in bone marrow of ND AML patients1.1 The mRNA expression levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18,NF-κB and IL-1R in bone marrow of ND AML patients and normal controls:RT-PCR results showed that the mRNA expression levels of NLRP3,IL-1β,NF-κB and IL-1R were significantly higher in the patient group than in the control group.We also found upregulation of the mRNA levels of ASC,Caspase-1 and IL-18,but did not reach significance.1.2 The bayesian network results suggest that NLRP3 inflammasome act through NF-κB and IL-1R in AML.1.3 The ELISA results confirmed that the concentration of IL-1β was significantly elevated in BM supernatant of ND AML patients compared with controls.2.Further activatation of NLRP3 inflammasome by LPS promoted proliferation and inhibited apoptosis in leukemia cells of ND AML patients2.1 NLRP3 inflammasome in leukemia cells of ND AML patients was further activated by LPS:LPS stimulation significantly increased the mRNA expression of Caspase-1 and IL-1β in leukemia cells of ND AML patients.NLRP3 and NF-κB mRNA expression level also elevated in LPS group,but no significant difference was found.Moreover,ELISA results showed that LPS obviously increased cytokines IL-1β and IL-18 secretion.2.2 After the treatment of LPS,the CCK8 results indicated significant increase in OD value,and apoptosis analysis by Flow cytometry showed significant decrease in apoptosis rate.The results indicated that NLRP3 inflammasome activation in leukemia cells of ND AML patients promoted proliferation and inhibited apoptosis significantly.3.Activated THP-1 cells inhibit apoptosis of leukemia cells of ND AML patients3.1 The RT-PCR results showed that the mRNA expressions of Caspase-1 and IL-1β were statistically increased by LPS in THP-1 cells,Though NLRP3 and NF-κB mRNA expression levels have the same trend,no significant difference was found.Besides,ELISA results showed that LPS and LPS+ATP significantly increased the concentration of cytokines IL-1β in culture supernatants.These findings confirmed that LPS and LPS+ATP activated NLRP3 inflammasome in THP-1 cells.3.2 Apoptosis analysis by Flow cytometry showed NLRP3 inflammasome activated THP-1 cells inhibited apoptosis of lukemia cells of ND AML patients,and the inhibiting effect can be reversed by the use of neutralizing antibody anti-IL-1β.However,anti-IL-18 did not function in this way.4.IL-1β not IL-18 promoted proliferation and inhibited apoptosis of leukemia cells of ND AML patientsCCK8 results showed that IL-1β and IL-1β+IL-18 had remarkable promoting effect on leukemia cells proliferation,but IL-18 alone had no significant effect.In addition,Apoptosis analysis by Flow cytometry showed IL-1β and IL-1β+IL-18 significantly decreased the apoptosis rate of leukemia cells.Similarly,IL-18 alone had no obvious effect.The data verified that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in AML.5.Inactivation of NLRP3 inflammasome by Caspasel inhibitor can inhibit proliferation and induce apoptosisThe ELISA results showed that Caspasel inhibitor Z-YVAD-FMK downregulated secretion of IL-1β and IL-18 in leukemia cells of ND AML patients.CCK8 showed Z-YVAD-FMK suppressed the proliferation of leukemia cells of ND AML patients.And apoptosis analysis by Flow cytometry showed Z-YVAD-FMK increased apoptosis rate of leukemia cells.6.Inactivation of NLRP3 by NF-κB inhibitor can inhibit proliferation and induce apoptosisELISA results showed that NF-κB inhibitor Bay11-7082 obviously decreased cytokines IL-1β and IL-18 secretion.Bay11-7082 inhibited the proliferation and promoted apoptosis rate of leukemia cells of ND AML patients.7.Activation and inactivation of NLRP3 inflammasome on protein levelsWestern blot showed that LPS and LPS+ATP up-regulated the protein expression of pNF-KB(65KD),pro-caspase-1(48KD),pro-IL-1β(31KD),active caspase-1(20KD)and active IL-1β(17KD).And protein expressions were decreased by Z-YVAD-FMK or Bay11-7082.8.NLRP3 inflammasome increased resistance of leukemia cells of ND AML to ADR and DNR8.1 The influence of NLRP3 inflammasome effectors on efficacy of chemotherapy drug:ADR,DNR and Ara-c induced apoptosis of leukemia cells,IL-1βsignificantly attenuated the antitumor effect of ADR and DNR,and apoptotic cell significantly reduced.However,the efficacy of Ara-c was not affected.Moreover,IL-18 showed little effect on antitumor effect of chemotherapy drug.8.2 NLRP3 inflammasome activation enhanced the resistance of leukemia cells to ADR and DNR:LPS activated NLRP3 inflammasome reduced the antitumor effect of ADR and DNR,the cell proliferation was significantly increased and cell apoptosis was significantly decreased.Interestingly,NLRP3 inflammasome activation had no obvious influence on antitumor effect of Ara-c.Conclusion1.NLRP3 inflammasome was activated in bone marrow of ND AML patients and NLRP3 inflammasome may acted through NF-κB and IL-1R in AML.2.Further activation of NLRP3 inflammasome by LPS promoted proliferation and inhibited apoptosis of leukemia cells of ND AML patients.NLRP3 inflammasome activated THP-1 cells inhibited apoptosis of leukemia cells of ND AML patients.Inactivation of NLRP3 inflammasome can inhibit proliferation and induce apoptosis.3.NLRP3 inflammasome plays carcinogenic role in AML through Caspase-1/IL-1β pathway.4.NLRP3 inflammasome increased resistance of leukemia cells of ND AML to ADR and DNR.5.Regulating NLRP3 inflammasome activity may provide a novel approach for AML therapy.