Macrophage ATP6V0d2 Suppresses Inflammasome Activation And Related Diseases Via Promoting The Autophagosome-lysosome Fusion

Objective:Autophagy-lysosome pathway has a great role in contraining inflammosome activation.Autophagy directly or indirectly regulates the activation of inflammosme in macrophages,but the mechanism of crosstalk between inflammation and autophagy needs to be further developed during inflammatory process;ATP6V0d2 is specificity highly expressed in macrophages and inhibited by LPS,but the mechanism and pathophysiological significance are not yet clear.This article focuses on the functions and mechanisms of ATP6V0d2 in the regulation of autophagy and inflammatory diseases,and the regulation of autophagy during inflammatory process.Method:?1?Genomic data analysis was performed to identify which lysosome-related genes were regulated in macrophages after LPS stimulation and we found Atp6v0d2 that was the most suppressed lysosomal genes.?2?RT-PCR and western blot were used to detect the expression level of Atp6v0d2mRNA and protein of ATP6V0d2 after LPS stimulation in BMDM.?3?The Atp6v0d2 knock out mice were generated.WT and Atp6v0d2^-/-mice were subjected to LPS-induced endotoxic shock.ELISA was used to detect IL-6,TNF-?,and IL-1?in the serum.?4?To investigate potential function of ATP6V0d2 in inflammasome activation,LPS primed WT and Atp6v0d2^-/-BMDMs were stimulated with inflammasome agonists including ATP,SiO2,Alum,MSU.The levels of secreted IL-1?and caspase-1 were determined by western blot.The levels of IL-6,TNF-?,and IL-1?in the supernatants were determined by ELISA.?5?To investigate potential function of ATP6V0d2 in colitis.WT and Atp6v0d2^-/-mice were subjected to DSS-induced colitis.The severity of pathological symptoms of colitis were compared between the WT and Atp6v0d2^-/-mice.DSS model was constructed using Clodronate liposomes to scavenge macrophages or anti-IL-1?to neutralize IL-1?to compare the pathological symptoms and disease severity between wild-type and Atp6v0d2^-/-knockout mice.The mitochondrial ROS levels in Colonic macrophages of wild-type and Atp6v0d2^-/-knockout mice were detected in the DSS model.?6?To investigate whether ATP6V0d2 plays a role in the clearance of damaged organella.Mitochondrial damages were compared in WT and Atp6v0d2^-/-BMDMs upon inflammasome agonists treatment;Mito-SOX staining was used to measure the levels of intracellular mitochondrial ROS;Mito-tracker Green and Mito-tracker DeepRed double staining were used to analyze mitochondrial membrane potential;Mitochondrial morphological damages were also imaged with electron microscopy.Inhibition of mitochondrial ROS using Mtio-TEMPO to detect inflammosome activation.?7?To investigate whether ATP6V0d2 plays a role in the completion of autophagy.The number of autophagosomes in BMDMs was determined by transmission electron microscopy upon inflammatory stimulation.In addition,wild-type and Atp6v0d2^-/-BMDMs were transfected with GFP-RFP-LC3 lentivirus with inflammasome agonists.Subsequently,the GFP and RFP fluorescence puncta were quantified with Confocal microscopy.Wild-type and knock-out BMDMs were treated with rapamycin and the levels of p62 and LC3II were measured.?8?To investigate whether ATP6V0d2 influences the process of autophagy-lysosomes degradation to regulate the activation of inflammosome.In addition to inflammatory stimulation,Rapa and 3MA were respectively used to promote and inhibit autophagy.To compare the activation of inflammasome between WT and Atp6v0d2^-/-BMDMs.Western blot was used to detect the amount of secreted IL-1?and caspase-1 protein in the supernatant.?9?To determine if ATP6V0d2 affects lysosomal pH and function,lysosome pH was determined by Lysotracker and Lysosensor staining and lysosomal hydrolysis of Cathepsin B was determined by western blot and lysosomal acidic degradation was determined by DQ-OVA staining in WT and Atp6v0d2^-/-BMDMs.?10?To determine whether autophagosome-lysosome fusion process is affected by ATP6V0d2,immunofluorescence was used to quantify the co-localization of LAMP1with LC3,STX17,and TOM20 in WT and Atp6v0d2^-/-BMDMs after inflammasome agonist stimulation.?11?Co-immunoprecipitation of ATP6V0d2,STX17 and VAMP8 was used to detect whether ATP6V0d2 binds to the SNARE complex and affects the assembly of the complex.?12?Control retrovirus or retrovirus encoding ATP6V0d2 were transduced into WT and Atp6v0d2^-/-BMDMs.The autophagy flux,mitochondrial damage,and activation of inflammasome were determined.Result:?1?The lysosomal gene,Atp6v0d2 was the most inhibited gene by LPS stimulation in BMDMs by analysis of public available genomic data.?2?LPS inhibited the expression of Atp6v0d2 mRNA and proteint in a time-and dose-dependent manner.?3?LPS induced higher levels of serum IL-1?in Atp6v0d2^-/-mice than WT mice,while the level of IL-6,and TNF-?was comparable.?4?Inflammosome agonists induced higher levels of IL-1?and caspase-1 in Atp6v0d2^-/-BMDMs supernatants than WT BMDMs.?5?The pathology of colitis in Atp6v0d2^-/-mice was more severe than WT mice in DSS-induced colitis model.After removal of macrophages,the pathological symptoms of Atp6v0d2^-/-mice were not significantly different from those of WT mice;The pathological symptoms of Atp6v0d2^-/-mice DSS model after IL-1?neutralization were significantly remission.The mitochondrial ROS in colonic macrophages of Atp6v0d2^-/-mouse was significantly higher than that of wild type.?6?More damaged mitochondria accumulation and higher level of mtioROS in Atp6v0d2^-/-BMDMs than WT BMDMs.After treatment of mito-TEMPO the activtion of inflammosome was contained significantly.?7?More GFP and RFP double fluorescence puncta in Atp6v0d2^-/-BMDMs than WT BMDM after treatment of LPS and ATP,and on the stimulation of rapamycin the levels of p-62 and LC3?were reduced gradually in WT BMDM,while in Atp6v0d2^-/-BMDMs were comparable before and after treatment of rapamycin at different time.?8?In WT BMDM,the activation of inflammosome was decreased in the rapamycin-treated group,and the activation level in the 3MA-treated group was increased,while the effects of activation level of inflammosome in Atp6v0d2^-/-BMDM were more gentle than WT BMDM after treated with rapamycin and 3MA.?9?The lysosomal pH and degradation of DQ-OVA were comparable between WT and Atp6v0d2^-/-BMDMs.?10?LAMP1 co-localized less with LC3,STX17,and TOM20 in Atp6v0d2^-/-BMDM than WT BMDM upon inflammasome agonist treatments.?11?ATP6V0d2 was associated with SNARE complex and promoted the assembly of the complex.?12?Overexpression of ATP6V0d2 significantly promoted autophagy degradation,clearance of damaged mitochondria,and inhibition of inflammasome activation.Conclusion:Although ATP6V0d2 does not affect lysosomal pH and proteinase maturation,it restricts the inflammasome activation in macrophages by mediating the fusion of autophagosomes with lysosomes and subsequent degradation of damaged mitochondria.Atp6v0d2^-/-mice are more susceptible to LPS-induced endotoxic shock and DSS-induced colitis,highlighting the in vivo significance of ATP6V0d2-mediated autophagosome-lysosome fusion and suppression of inflammation.In addition,LPS enhances the activation of inflammosome through down-regulating ATP6V0d2.