| Research background:Systemic lupus erythematosus(SLE)is an autoimmune disease,and its pathogenesis has not been clarified.Macrophages(M0)participate in body immunity.M0 can differentiate into pro-inflammatory M1 macrophages under the stimulation of lipopolysaccharide.M1 macrophages secrete Tumor necrosis factor-α.TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and other landmark inflammatory cytokines.TNF-α,IL-1β and IL-6 can be used as marker genes of M1 macrophages,and their expression levels are positively correlated with SLE activity.DNA methylation is a form of chemical modification of DNA.DNA methylation can alter genetic expression without altering the sequence of nucleic acids.Abnormal genome-wide DNA methylation levels are associated with SLE.However,It has not been reported whether the M1 macrophage marker genes are regulated by DNA methylation.Human Apolipoprotein B m RNA Editing Enzyme Catalytic Subunit3A(APOBEC3A)is an innate host limiting factor.It has the activity of inhibiting retrovirus and endogenous retrofactor.APOBEC3 A is mainly expressed in monocytes and macrophages,It can assist in active DNA demethylation.Studies have shown that APOBEC3 A can affect interferon-γ RNA methylation,and it regulates the expression of interferon-γ in peripheral blood mononuclear cells.APOBECA affects inflammation in SLE organs and tissues.Therefore,we speculated whether APOBEC3 A could be involved in regulating DNA methylation in promoter regions of M1 macrophage marker genes(TNF-α,IL-6 and IL-1β)in SLE patients.Our aim is to provide new evidence for further exploration of SLE pathogenesis and therapeutic targets.Research objective:1.We identified M1 macrophage marker genes TNF,IL6,and IL1βwhether the expression of is regulated by DNA methylation.2.We overexpressed APOBEC3 A and explored the effects of APOBEC3 A on TNF,IL6 and IL1β effect of DNA methylation level in gene promoter regionResearch method:1.We isolated peripheral blood monocytes from 6 normal persons and 6 SLE patients by immunomagnetic bead separation.We induced colony factors into M0 macrophages by stimulating them with macrophages.Then,Lipopolysaccharide(LPS)was used to induce M1 macrophages.The expression levels of TNF-α,IL-6 and IL-1β and promoter methylation in M0 macrophages and M1 macrophages were detected by real-time quantitative PCR and bisulfite sequencing.Meanwhile,we detected the expression level of APOBEC3 A gene.2.We induced M0 macrophages from 5 mice and induced them into M1 macrophages using LPS in vitro.Then we amplified APOBEC3 A by lentivirus transfection.We detect TNF-α,IL-6,IL-1β expression and promoter region methylation level by real-time quantitative PCR and bisulfite sequencing method.Research results:1.The expression levels of TNF-α(P=0.2485)and IL-6(P=0.1732)in normal human M1 macrophages were increased.The expression level of IL1β(P=0.0296)was significantly increased.TNF(P=0.0006)and IL6(P=0.0351)promoter methylation levels were significantly decreased,while IL-1β promoter methylation levels were not significantly changed.2.The expression levels of TNF-α(P=0.0380),IL-6(P=0.0006)and IL-1β(P=0.0034)in M1 macrophages of SLE patients were significantly increased.The SLE patients with M1 macrophages TNF(P=0.0003),IL6(P=0.0008),IL1β(P=0.0453)promoter region show low levels of methylation status.3.APOBEC3 A expression level in M1 macrophages of SLE patients was significantly increased(P=0.0395).Pearson correlation analysis showed that APOBEC3 A was positively correlated with m RNA of TNF-α(P=0.0032,r=0.9535),IL-6(P=0.0018,r=0.9647)and IL-1β(P=0.0485,r=0.8143).4.We overexpressed APOBEC3 A in mouse M1 macrophages and found that the expression levels of IL-6(P=0.0377)and IL-1β(P=0.0444)were significantly increased.Meanwhile,the methylation levels of IL6promoter-1807(P=0.0219)and IL1β promoter-352(P=0.0462)were significantly decreased.There was no significant change in methylation level of TNF promoter region.Research conclusion:1.We compared the expression levels of TNF-α,IL-6,IL-1β and promoter DNA methylation levels in M0 and M1 macrophages from healthy persons and M1 macrophages from SLE patients and healthy persons.Then we determined whether the expression of M1 macrophage marker genes TNF,IL6 and IL1β was regulated by DNA methylation.2.APOBEC3 A expression was significantly increased in M1 macrophages of SLE patients.APOBEC3 A may regulate the methylation level of IL6 and IL1β promoter region of M1 macrophages and then affect the expression level of IL-6 and IL-1β. |
