The Role Of The NLRP3 Inflammasome In Radiation-Induced Pyroptosis In Macrophages

Background As one of the important treatments,radiotherapy was used mainly for killing variety of malignant tumor cell.But it inevitably kill some normal cells the same time which is the most important limit of its clinical application.More and more studies have found that radiation,in addition to killing rapidly dividing cells,can also lead to a significant reduction in immune cells and release of proinflammatory cytokines,thus affecting the repair of damaged cells and tissue,inhibiting immunity or even lead to systemic response syndrome.Macrophages are widely distributed in various tissues and organs of the body and play an important role in regulating cell function,promoting inflammation subsided and repairing damaged tissue.Act as the earliest cell type that respond to radiation damage in the innate immune system,macrophages was found to recruit,activate,transform and promote the repair of damaged tissue in early and late stage of radiation.Damaging macrophages will seriously hinder the regression of inflammation and tissue repair after exposed to radiation.Studies have found that NLRP3 inflammasome mediated cell pyroptosis is an important model of cell death in macrophage under stimulation such as virus infection,heat stress injury and flagellin.And NLRP3 inflammasome activation was found to mediated radiation induced damage of lung tissue and oral mucosa.Therefore,to explore the role of NLRP3 inflammasome activation-mediated pyroptosis in radiation-induced macrophage death may provide a new strategy for reducing the side effects of radiotherapy and improving the therapeutic effect.Objective Our group intends to study the effect of radiation on macrophage NLRP3 inflammasome and cell pyroptosis in vivo and in vitro,and to elucidate the mechanism of NLRP3 inflammasome in radiation-induced macrophage pyroptosis using Nlrp3 knockout mice.Methods Primarily cultured BMDM were exposed to different doses of radiation and the cell viability,the proportion of double positive cells(pyroptotic cells)and the secretion of proinflammatory cytokines were test using CCK-8 kit,Pyroptosis test kit,LDH CytotoxicityAssay Kit and ELISA kit(TNF-α,IL-1β,IL-18,IFN-γ,IL-6,MCP-1 and IL-12p40).The effect of radiation on macrophage NLRP3 inflammasome was analyzed by RT-PCR and Western Blot.Primarily cultured WT and Nlrp3 knockout BMDM were exposed to different doses of radiation and the cell viability,the proportion of double positive cells(pyroptotic cells)and the expression level of NLRP3 inflammasome activation-related protein were test to determine the role of NLRP3 inflammasome in radiation induced macrophage pyroptosis.In vivo experiments,immunofluorescence and Western Blot were used to analyze the expression of NLRP3 inflammasome activation-related protein in the spleen of WT and Nlrp3 knockout mice,and to verify the effect of radiation on NLRP3 inflammasome.The morphology of radiated spleen macrophages in the two genotypes of mice was observed by transmission electron microscopy to determine the damage manner of macrophages induced by radiation.The holistic protective effect of Nlrp3 knockout on radiation-induced death of mice was studied by survival curves of the two genotypes of mice.Results 1.Radiation induce inflammasome activation and pyroptosis in BMDM.CCK-8 test showed that the survival rate of BMDM decreased after radiation in a doseand time-dependent manner.FAM-YVAD-FMK and PI-labeled radiation-induced BMDM showed an increase of double positive cell proportion and LDH activity in a dose-dependent manner.ELISA test showed that M1-related cytokines(TNF-α,IFN-γ and IL-12),inflammasome activation related cytokines(IL-1β and IL-18)and other inflammatory cytokines(IL-1α and MCP-1)increased in 10 Gy and 20 Gy radiation group.5 Gy radiation group showed an increase of IL-1β,IL-18 and IFN-γ,while the contents of TNF-α,IL-12p40,IL-1α and MCP-1 did not change significantly.In addition,cleaved-Caspase-1(p10)in BMDM was increased after exposed to high dose radiation(10 Gy and 20 Gy),while cleaved-Caspase-1(p10)in low dose radiation(5 Gy)group was not significantly changed compare to control group.2.Nlrp3 knockout significantly inhibited radiation-induced inflammasome activation and pyroptosis in BMDM.The percentage of 10 Gy radiation-induced BMDM cell death decreased from 25.98%to 5.45% and the formation of cleaved-Caspase-1(p10)and IL-1β induced by 10 Gy radiation in BMDM was significantly decreased after Nlrp3 knockout.In addition,the proportion of FAM-YVAD-FMK and PI double-positive BMDM and LDH activity induced by 10 Gy radiation were significantly decreased after Nlrp3 knockout.3.Nlrp3 knockout has a protective effect on radiation-induced injury The cleaved-Caspase-1(p10)and the level of serum IL-1β increased after 9.5 Gy radiation.The level of cleaved-Caspase-1(p10)and serum IL-1β in spleen of Nlrp3 knockout radiation(9.5 Gy)group were not significantly higher than those in control group.Immunofluorescence staining showed that the cleaved-Caspase-1(p10)in the spleen of mice exposed to 9.5 Gy was increased and the increased cleaved-Caspase-1(p10)mainly expressed in the marginal zone(rich in macrophages).The cleaved-Caspase-1(p10)in splenic marginal zone in radiated Nlrp3 knockout group was not significantly different from the control group.Electron microscopic results showed that,after 9.5 Gy radiation,splenic macrophages of WT mice showed pyroptosis-like changes(cell swelling,small vacuoles format on cell surface and fall into the cell spase,without obvious nuclear shrinkage,rupture or dissolved),while the splenic macrophages of Nlrp3 knockout radiation(9.5 Gy)group mice showed no significant change.In addition,survival analysis showed that the survival rate and survival time of Nlrp3 knockout radiation(9.5 Gy)mice were significantly higher than WT radiated mice.Conclusions Low dose radiation induce macrophages to secrete a variety of inflammatory cytokines,high dose radiation not only promote secretion of inflammatory factors,but also induce NLRP3 inflammasome activation-mediated macrophage pyroptosis.Targeted inhibition of NLRP3 inflammasome activation and secondary macrophage pyroptosis can be used as an important strategy to limit radiation-induced macrophage injury,proinflammatory response and related tissue damage.