Regulation And Mechanisms Of TAX1BP1 In Monocyte/Macrophage-mediated Pathogenesis Of SLE

ObjectiveSystemic lupus erythematosus(SLE)is an autoimmune disease that is chronic,complex,and publicly classic,in which innate and adaptive immune cells interact to promote its development.TAX1BP1 participates in the regulation of the NF-κB signaling pathway,which is closely related to SLE inflammation,and regulates the cell cycle in T cells and surface Ig M expression in B cells.Our previous study found a mutation(G148D)in TAX1BP1 in SLE families.However,previous studies on the function of TAX1BP1 in T and B cells are insufficient to explain its involvement in SLE,and its function in monocytes/macrophages needs to be investigated.To clarify whether TAX1BP1 is involved in SLE and its mechanism,this study aims to understand the role and biological mechanism of TAX1BP1 in SLE pathogenesis and provide a specific experimental foundation and supporting evidence can aid in the discovery of novel therapeutic targets,through a combination of computer predictions,clinical phenotypes,bioinformatics,and molecular cell biology.Materials and Methods1.Analysis of the structural changes caused by G148 D mutation in TAX1BP1 and prediction of the mutant protein stability and functional changes.(1)TAX1BP1 protein template was obtained from the Uni Prot online database,and the DUET,ELASPIC,I-Mutant Suite,Var Site online tools were used to calculate the energy changes and scores of missense mutations.(2)Alpha Fold2 v2.2.0 was used to predict the mutant structure,and Py Mol and Swiss-PDb Viewer were used for structural diagram and visualization analysis.2.Alteration of TAX1BP1 expression and its correlation with disease in the peripheral blood mononuclear cells(PBMCs)of patients diagnosed with SLE.(1)Samples of plasma,PBMCs,clinical data,and laboratory test results were gathered from healthy controls and patients diagnosed with SLE(n=20).(2)Western blot(WB)analysis was utilized to identify the expression of the TAX1BP1 protein in PBMCs.(3)Plasma from health control and SLE patients was used to stimulate THP-1 cells and identify TAX1BP1 protein expression in the cells.3.Detection of m RNA expression profiles and bioinformatics analysis in monocytes and M1 macrophages after TAX1BP1 knockdown.(1)A TAX1BP1 knockdown lentivirus was constructed and used to infect THP-1cells,which were then stimulated with PMA and inflammatory factors to become M1 macrophages.(2)m RNA transcriptome sequencing was performed on THP-1 cells,TAX1BP1 knockdown THP-1 cells,and M1 macrophages induced from the two types of cells.(3)DESeq2/DEGseq/edge R/Limma/NOIseq were used for differential expression analysis,and the objective was to identify crucial genes,and to achieve this,pathway enrichment analysis was carried out utilizing the GO and KEGG databases.Furthermore,a protein-protein interaction(PPI)network was created through the use of the STRING database and Cytoscape software.4.Detection of phosphorylated protein expression profiles and bioinformatics analysis in monocytes and M1 macrophages after TAX1BP1 knockdown.(1)4DLabel Free mass spectrometry was used to detect phosphorylated proteins in THP-1 cells,TAX1BP1 knockdown THP-1 cells,and M1 macrophages induced from the two types of cells.(2)The t.test function in R was used for differential expression analysis,and The objective was to identify crucial genes,and to achieve this,pathway enrichment analysis was carried out utilizing the GO and KEGG databases.Furthermore,a PPI network was created through the use of the STRING database and Cytoscape software.5.Detection of surface markers and TRAF6 and phosphorylated p65 expression in monocytes and M1 macrophages after TAX1BP1 knockdown.(1)Flow cytometry was used for the identification of surface markers including CD14,CD16,CD80,and CD86.(2)The TAX1BP1 knockdown lentivirus was constructed and used to infect THP-1 cells,which were then stimulated with PMA to become monocyte-derived macrophages.LPS and TNFα were used to stimulate monocyte-derived macrophages,and WB was used to detect TRAF6 and phosphorylated p65 expression.Results1.Multiple databases predicted that the G148 D mutation may result in decreased stability and impaired function of TAX1BP1.The reduction in protein stability may be due to changes in flexibility,hydrogen bonding,and molecular force fields.2.The level of TAX1BP1 expression was notably decreased in the PBMCs of individuals with SLE in comparison to those who were healthy.Additionally,the expression of the TAX1BP1 protein was lower in SLE patients who tested positive for anti-Sm antibodies,anti-ribosomal P protein antibodies,and anti-n RNP/Sm antibodies than in those who tested negative.However,No statistically significant variation in the expression of TAX1BP1 protein was observed between SLE patients who tested positive for other autoantibodies and those who tested negative.There was a negative correlation between disease activity score,SLEDAI-2K,and the he TAX1BP1 protein expression level,whereas there was a positive correlation between complement C3 levels and the TAX1BP1 protein expression level from the PBMCs of SLE patients.The expression of TAX1BP1 protein was higher in SLE patients who were positive for urinary protein than in those who were negative,but there was no statistically significant correlation with other indicators.The TAX1BP1 protein expression level did not show any statistically significant difference between the plasma-stimulated THP-1 cells of SLE patients and those of the normal control group.3.There were significant differences in m RNA expression profiles of THP-1 cells and THP-1-induced M1 macrophages before and after TAX1BP1 knockdown.Specifically,307 differentially expressed genes(103 downregulated and 204 upregulated)were identified in THP-1 cells,while 320 differentially expressed genes(162 downregulated and 158upregulated)were identified in THP-1-induced M1 macrophages after TAX1BP1 knockdown.Enrichment analysis revealed that the genes which were differentially expressed in THP-1 cells showed significant enrichment in the pathways of antigen processing and presentation as well as B cell receptor signaling.,while those in THP-1-induced M1 macrophages were significantly enriched in PI3K-Akt signaling pathway,IL-17 signaling pathway,TNF signaling pathway,Toll-like receptor signaling pathway,chemokine signaling pathway,and systemic lupus erythematosus pathway.Eighteen and 12 key genes were identified in THP-1 cells and THP-1-induced M1 macrophages,respectively,with no overlap between them.4.Knockdown of TAX1BP1 leads to significant differences in phosphorylated protein expression profiles in both THP-1 cells and THP-1-induced M1 macrophages.In THP-1 cells,1327 phosphorylated proteins were downregulated and 845 were upregulated,whereas in THP-1-induced M1 macrophages,141 were downregulated and 1864 were upregulated.Significant enrichment of phosphorylated proteins in the m TOR and PI3 KAkt signaling pathways was observed in THP-1 cells,while significant enrichment of phosphorylated proteins in the B cell receptor signaling pathway,T cell receptor signaling pathway,NF-κB signaling pathway,and NOD-like receptor signaling pathway was observed in THP-1-induced M1 macrophages.Eighteen key genes were identified in THP-1 cells,and several genes with opposite trends in phosphorylated protein expression were identified in THP-1-induced M1 macrophages.5.Knockdown of TAX1BP1 resulted in increased mean fluorescence intensity(MFI)of CD14 in THP-1 cells and decreased MFI of CD16 in THP-1-induced M1 macrophages.Conversely,the MFI of CD80 was increased in both THP-1 cells and THP-1-induced M1 macrophages,while there was no significant change in the MFI of CD86.The expression of phosphorylated p65 and TRAF6 proteins in THP-1-induced monocytes was reduced after LPS stimulation in TAX1BP1-knockdown cells,as was the expression of these proteins after TNFα stimulation.ConclusionThe G148 D mutation may lead to expression or functional abnormalities in TAX1BP1.Reduced expression of TAX1BP1 is involved in the pathogenesis of systemic lupus erythematosus(SLE)and is not induced by cytokines in the blood.Downregulation of TAX1BP1 primarily affects antigen presentation in monocytes and tends to stabilize them in a nonfunctional state,while in M1 macrophages it primarily affects the inflammatory response by regulating the acquired immune-related inflammatory pathways and tending towards functional activation.Both types of cells can participate in SLE through the NF-κB signaling pathway and the NOD-like receptor signaling pathway.Downregulation of TAX1BP1 promotes polarization of monocytes to M1 macrophages,enhances phagocytosis and antigen presentation by monocytes,and increases the proinflammatory effects of M1 macrophages,but this effect is not mediated by the classical NF-κB pathway in monocytes and macrophages.TAX1BP1 participates in SLE disease activity by regulating monocyte antigen presentation,M1 macrophage inflammatory response,suggesting that TAX1BP1 is a potential therapeutic target for SLE.