The Role And Mechanism Of Activated NLRP3 Inflammasome Of Aged Macrophages In Non-exudative Age-related Macular Degeneration

Background:Age-related macular degeneration（AMD）is a main disease causing irreversible visual impairment in patients over 60 years old.Clinically,AMD is divided into dry AMD（non-exudative type）and wet AMD（exudative type）.Early dry AMD is usually asymptomatic,or only companied with mild visual distortion or reduced visual ability in darkness in some cases.Advanced dry AMD usually progresses in a rapid speed,eventually resulting in severe vision loss,permanent visual impairment or even blindness,which has a great impact on the quality of life and becomes a serious public health issue.In recent years,although more attention has been paid to dry AMD,the pathogenesis is still unclear.Effective methods for early diagnosis and treatment of dry AMD have not yet been found.Therefore,it is of great importance to investigate the mechanism of the development of dry AMD.Studies have found that age-related macular degeneration（AMD）is now regarded as an intraocular"inflammmaging"disease,in which the inflammatory response plays a pivotal role.NLRP3 inflammasome and inflammatory factors（IL-18,IL-1b,IL-6,TNF-a,etc.）accumulate in the retina and choroid of dry AMD patients.Increasing inflammatory factors can damage the cells in retina and promote inflammatory responses in RPE cells,which further promotes the development of dry AMD.Aged macrophages are closely related to inflammatory response.The expression of NLRP3 inflammasome in aged macrophages is up-regulated which can promote the release of various cytokines and aggravate the inflammatory response.Some studies have found the accumulation of aged macrophages close to the drusen in dry AMD,and the spatial distribution and phenotype of aged macrophages in the eyes of AMD patients are different from those of normal eyes.As AMD progressess,the number of aged macrophages in retina and choroid significantly increases.Therefore,it is suggested that aged macrophages and NLRP3 inflammasome are involved in the occurrence and development of early dry AMD.Purpose:To verify that hydrogen peroxide（H₂O₂）can induce an aged macrophage model;to explore whether the NF-κb pathway is involved in the aging of macrophages;to study the effect of NLRP3 activation in aged macrophages on the inflammatory state of retinal pigment epithelial cells.In vivo experiments were conducted to confirm that an early AMD c57 mice model can be established by blue light;to observe the effect of eradicating aged macrophages on the occurrence and development of AMD;and to investigate whether aging macrophages affect the development of AMD through the NLRP3 inflammasome activation.This study aimed to provide basis and new targets for the treatment and further research of dry age-related macular degeneration.Methods:（1）Establishment of H₂O₂-induced aged macrophage model:Macrophages were treated with 1000μM,900μM,800μM,700μM,600μM,500μM,400μM,300μM,200μM and 100μM H₂O₂ for 1h,1.5h and 2h,respectively.The proliferation of macrophages treated with different concentrations of H₂O₂ for different time was detected by CCK8 to determine the medium lethal concentration and treatment time of H₂O₂.Macrophages were treated with 400μM H₂O₂ for 2h to induce aged macrophage model.Immunofluorescence staining,Western Blot（WB）and q PCR were used to detect the expression of aging marker p16^INK4a.The changes of cell function（proliferation,phagocytosis,migration ability）were detected by CCK8method,neutral red staining and scratch assay.The expression of NLRP3 and its downstream inflammatory factors（Caspase-1,IL-18,IL-1b）were also detected;（2）Verifying that the NF-κb signaling pathway WAS involved in the aging of macrophages:The macrophages were treated with 10μM BAY 11-7082（NF-κb signaling pathway inhibitor）for 2h to inhibit the NF-κb signaling pathway;WB and q PCR were used to detect the aging marker,p16INK4a.Changes in cell function（proliferation,phagocytosis,migration ability）were detected.The expression of NLRP3 inflammasome and its downstream inflammatory factors（Caspase-1,IL-18,IL-1b）was measured;（3）NLRP3 knockdown:Macrophages were transfected with NLRP3 si RNA to obtain NLRP3-knockdown macrophages.The knockdown effect was detected by immunofluorescence staining,WB and q RT-PCR methods;（4）Co-culture system:Macrophages（lower chamber）and ARPE-19 cells（upper chamber）were co-cultured for 3d in Transwell chambers.The expression of NLRP3inflammasome and its downstream inflammatory factors（Caspase-1,IL-18,IL-1b）in ARPE-19 cells was detected by WB method;（5）In vivo experiment:Clodronate liposomes（0.5μl,5mg/ml）were intravitreally injected to remove macrophages in the eyes of aged healthy c57 mice（18 months old）.Early AMD model of aged mice was established by blue light（2000lux）exposure.HE staining was used to detect retinal histopathological changes.ERG was used to measure the amplitude of a-wave and b-wave.WB was used to measure the expression of NLRP3 inflammasome and its downstream inflammatory factors（Caspase-1,IL-18,IL-1b）.The number of aged macrophages in mice choroid was observed by immunofluorescence staining.After eradicating macrophages,NLRP3-knockdown macrophages were injected into the vitreous cavity.The expression inflammatory factors in the retina and choroid was detected by WB method to verify the role of aged macrophages and NLRP3 inflammasome in early AMD.Results:（1）In vitro experiments:1)Aged macrophage model was successfully constructed:After the macrophges were treated by 400μM H₂O₂ for 2h,the aged macrophage model was successfully established.The expression of aging marker p16^INK4a was significantly up-regulated;the proliferation,phagocytosis and migration abilities of aged macrophages were significantly decreased;The expression levels of NLRP3inflammasome and its downstream inflammatory factors,Caspase-1,IL-18 and IL-1b,in aged macrophages were significantly increased（p<0.05）;2)NF-κb pathway was involved in H₂O₂-induced macrophage aging:The NF-κb pathway in macrophages was successfully inhibited after macrophages were treated with 10μM BAY 11-7082 for 2h.Inhibition of NF-κb pathway could down-regulate the expression of p16INK4a,improve the ability of cell proliferation,phagocytosis and migration,and effectively alleviate the aging of macrophages induced by H₂O₂.3)Aged macrophages promoted the up-regulation of inflammatory responses in ARPE-19 cells through the activation of the NLRP3 inflammasome:NLRP3 gene in macrophages was successfully knocked down by small interfering RNA（si RNA）technique.Compared with aged macrophages,after NLRP3-knockdown macrophages were treated by H₂O₂,the expression of NLRP3inflammasome and its downstream inflammatory factors was significantly decreased（p<0.05）.As shown by the results of co-culture,the expression of NLRP3 inflammasome and downstream inflammatory factors in ARPE-19 cells were upregulated in aged macrophages.In contrast,in the co-culture system of H₂O₂-treated NLRP3-knockdown macrophages+ARPE-19 cells,the expression of the above inflammatory factors in ARPE-19 cells was significantly decreased（p<0.05）.（2）In vivo experiments:1)The effect of scavenging intraocular macrophages on retinal histopathology:In normal aged c57 mice,the thickness of the retinal ONL layer became thinner and the cells were disordered after blue light exposure;2)Effects of scavenging macrophages on the inflammatory state in the retina:The expression of NLRP3,IL-18 and IL-1b in normal aged c57 mice was significantly up-regulated after exposure to blue light（p<0.05）.The expression of the above inflammatoy factors was not significantly up-regulated after scavenging intraocular macrophages;3)The effects of intravitreal injection of NLRP3-knockdown macrophages on retinal histopathology and the expression of inflammatory factors:Compared with the aged c57 mice exposed to blue light,the thickness of the retinal ONL layer was similar to that of the normal eyes and the cells were neatly arranged,the amplitudes of a-wave and b-wave were higher,and NLRP3,Caspase-1,IL-18 and IL-1b expression levels were not significantly up-regulated in the aged mice injected with NLRP3-knockdown macrophages after blue light exposureConclusion:1.400μM hydrogen peroxide treatment for 2h can successfully establish aged macrophage model;2.NF-κb signaling pathway is involved in hydrogen peroxide-induced macrophage aging,and blocking NF-κb signaling pathway can reduce the degree of macrophage aging;3.Aged macrophages can promote the up-regulation of inflammatory factors in ARPE-19 cells through the activation of NLRP3 inflammasome and the release of inflammatory factors;4.Blue light exposure can be used to establish an early AMD model in aged mice,and intraocular aged macrophages can promote the development of early AMD through NLRP3 activation.