| Mycobacterium neoaurum(M.neoaurum)is a rapidly growing Nontuberculous mycobacteria(NTM)with smooth colonies and golden yellow color.M.neoaurum is widely distributed in air,soil,water,animal saliva and body surface,which is easy to cause infection of the injured site and soft tissue.Bacillus Calmette-Guérin(BCG),a live attenuated strain of Mycobacterium bovis,is a vaccine that naturally causes tuberculosis in cattle and other animals,including humans.Inflammasome is a multi-protein complex assembled in the cytoplasm,which is composed of pattern recognition receptor,adapter protein ASC and cysteine protease caspase-1.It can be activated by microbial infection,endogenous danger signals and environmental stimuli.Inflammasome complex can activate caspase-1,promote the release of inflammatory cytokines IL-1βand IL-18,and then regulate the occurrence of pyroptosis.However,the association between M.neoaurum and BCG infection and IL-1βand IL-18 release,inflammasome assembly,and pyroptosis remains unclear.Therefore,this paper mainly studied the mechanism of action between M.neoaurum and BCG infection and inflammasome.Firstly,to investigate the secretion mechanism of IL-1βand IL-18 induced by M.neoaurum and BCG in mouse macrophages,we infected mouse macrophages with M.neoaurum and BCG.The results showed that IL-1βand IL-18 were significantly expressed,and their secretion reached the maximum at 15 h after infection.Therefore,15h was selected as the best infection time(15 h was the infection time in subsequent experiments).We further detected the maturation of IL-1βand IL-18 by RT-PCR and Western blot.The m RNA expression levels and protein expression levels of IL-1βand IL-18 were significantly increased,indicating that the infection of M.neoaurum and BCG induced the maturation and secretion of IL-1βand IL-18.RT-PCR and Western blot were used to detect whether M.neoaurum and BCG infection induced caspase-1 activation.The results showed that after M.neoaurum and BCG infected mouse macrophages,caspase-1 gene expression and pro-caspase-1 protein cleavage were induced,and active caspase-1 was produced.Next,mouse macrophages were treated with different concentrations of caspase-1 inhibitor(Ac-y VAD-CHO),and then infected with M.neoaurum and BCG.The results showed that the secretion of IL-1βand IL-18 in mouse macrophages decreased in a dose-dependent manner.These results indicated that the secretion of IL-1βand IL-18 in mouse macrophages induced by M.neoaurum and BCG depended on the activation of caspase-1.Secondly,we studied that M.neoaurum and BCG induced secretion of IL-1βand IL-18 in mouse macrophages depend on which inflammasome.First,we knocked down the function of NLRP3 and AIM2 genes in mouse macrophages by small interference RNA transfection technology,and then infected them with M.neoaurum and BCG.The results showed that the secretion of IL-1βand IL-18 in mouse macrophages was significantly decreased,suggesting that the secretion of IL-1βand IL-18 in mouse macrophages induced by M.neoaurum and BCG was related to NLRP3 and AIM2.Further,we detected whether the infection of M.neoaurum and BCG induced the assembly of NLRP3 and AIM2 inflammasome.Using RT-PCR and western blot,we found that the infection of M.neoaurum and BCG induced the gene expression and protein expression of NLRP3,AIM2,ASC,caspase-1,indicating that M.neoaurum and BCG induced the assembly of NLRP3 and AIM2 inflammasomes in mouse macrophages.Since K~+effluence is associated with almost all activation pathways,macrophages were treated with different concentrations of Glibenclamide,an ATP-dependent selective K~+channel inhibitor,and then infected with M.neoaurum and BCG to detect IL-1βand IL-18 secretion in the cell supernatant.It was found that inhibition of ATP-dependent K~+channel activation significantly decreased the secretion of IL-1βand IL-18 in macrophages induced by M.neoaurum and BCG infection.On this basis,we also identified the relationship between K~+efflux and the assembly of NLRP3 and AIM2 inflammasome,and found that inhibition of K~+efflux inhibited the assembly of NLRP3 and AIM2 inflammasome induced by M.neoaurum and BCG infected macrophages.Finally,study on pyroptosis induced by M.neoaurum and BCG infection of mouse macrophages.In order to detect the membrane damage of mouse macrophages induced by M.neoaurum and BCG infection,we infected mouse macrophages with M.neoaurum and BCG,and used cytotoxicity kit to detect the membrane damage.The results showed that M.neoaurum and BCG infection caused an increase in the proportion of cells with cell membrane damage.After further using different concentrations of caspase-1 inhibitor to treat mouse macrophages,it was found that cell membrane damage caused by M.neoaurum and BCG was dependent on caspase-1 activation.Further,we detected the expression and cleavage of GSDMD in M.neoaurum and BCG infected mouse macrophages.Found that M.neoaurum and BCG infection induced the cleavage of GSDMD and produced active GSDMD-NT,indicating that M.neoaurum and BCG infection induced pyroptosis of mouse macrophages.In conclusion,M.neoaurum and BCG infection can promote the maturation and secretion of IL-1βand IL-18,induce the activation of caspase-1 and the assembly of NLRP3 and AIM2inflammasome,and this pathway is regulated by K~+channel.At the same time,M.neoaurum and BCG could cause pyroptosis of macrophages.The study indicates that NLRP3 and AIM2inflammasome are an immune defense mechanism against the invasion of M.neoaurum and BCG,and pyroptosis is involved in the infection process of M.neoaurum and BCG.This study provides a new research direction for further exploring the immunopathogenic mechanism of NTM and Mycobacterium tuberculosis. |
