| IntroductionSystemic lupus erythematosus is an autoimmune disease associated with a variety of the immune mechanisms. Its features include the production of autoantibodies, formation of persistent chronic inflammation and damage of multiple target organs. The pathogenesis of SLE is not totally clear yet. Recently,more and more research focuses on the role of innate immunity in the pathogenesis of SLE. Toll-like receptors can promote and exacerbate SLE by identifying self-nucleic acid and activating NF-κB and other inflammatory signaling pathways. Many regulated or mediated inflammation factors are involved in Toll-like receptor signaling pathway, including CCAAT/enhancer binding protein β(C/EBPβ), tumor necrosis factor alpha-induced protein 3(TNFAIP3) and TNFAIP3 interacting protein1(TNIP1),etc.C/EBPβ is a transcription factor of eukaryotic cell and plays an important role in a variety of physiological and pathological processes of cell differentiation, tumorigenesis and immune response, etc. TNFAIP3 and TNIP1 have been identified as SLE susceptibility genes by genome-wide association study(GWAS). And many single nucleotide polymorphisms(SNPs) of TNFAIP3 and TNIP1 are associated with SLE genetic susceptibility significantly in multiple ethnic populations. Meanwhile,C/EBPβ could be combined with the promoter of TNFAIP3 gene in macrophages and promote TNFAIP3 m RNA expression. In TNIP1 gene knockout mice model, C/EBPβ LAP and products of its target gene(Csf3, Nos2 and S100a8) were significantly increased, and lupus-like performances appeared, but classic inflammatory NF-κB signaling pathway and MAPK pathway unaffected.Therefore, C/EBPβ may be involved in the pathogenesis of SLE, and C/EBPβ, TNFAIP3 and TNIP1 interrelate in SLE. But the C/EBPβ expression and function in SLE patients, and interreaction of TNFAIP3, TNIP1 and C/EBPβ related to SLE pathogenesis are not clear.ObjectiveThe aim of our project is to investigate the expression of C/EBPβ, and TNIP1 TNFAIP3 in PBMC of patients with SLE and their relationship with disease activity, and to evaluate the impact of SLE patients plasma treatment on C/EBPβ expression in monocyte-macrophages, as well as the regulation of C/EBPβ expression by TNFAIP3 and TNIP1 in monocytes.MethodsThe m RNA and protein expression of C/EBPβ, TNFAIP3 and TNIP1 in PBMC of SLE patients and normal controls were detected by quantitative PCR(Q-PCR) and western blotting(WB), and the correlation bewteen C/EBPβ m RNA expression with disease activity score and with TNFAIP3 / TNIP1 m RNA expression was analysed. THP-1 cells were stimulated by plasma from SLE patients and normal control, C/EBPβ m RNA and protein expression in these THP-1 cell drived monocyte-macrophages were detected by Q-PCR and WB. At last the protein expression of C/EBPβ, TNFAIP3 and TNIP1 in THP-1 cell after TNFAIP3 small interfering RNA(si RNA) transfection or TNIP1 interference slow virus infected were also detected by Q-PCR and WB. Data were expressed as mean ± SEM, The unpaired t-test or Mann–Whitney U test was performed to compare difference between experiment and control group, and for correlation analysis, Spearman correlation analysis was applied.(Graph Pad Prism 5.0 for windows). For all statistical analyses, significance levels were set at P < 0.05, and markly significance levels were set at P < 0.01.Results1. The m RNA expression of C/EBPβ in PBMC of SLE patients was higher than normal control group(p<0.05).2. The m RNA expression of C/EBPβ increased in PBMC of SLE patients high ANA titer(≥1: 160) compared with the patients with low ANA titers(≤1: 80)(p<0.05); The m RNA expression of C/EBPβ increased in PBMC of patients anti-Sm antibody positive and anti-n RNP antibody positive SLE patients(p< 0.01). The m RNA expression of C/EBPβ in PBMC from other autoantibody-positive SLE patients and in patients with negative showed no significant difference(p<0.05).3. The m RNA expression of C/EBPβ in PBMC of SLE patients was positively correlated with disease activity SLEDAI scores(p<0.05,r>0); and negatively correlated with complement components C3, C4(p<0.05,r<0). The rest showed no statistically significant correlation.4. The m RNA expression of C/EBPβ in PBMC of SLE patients was positively correlated with TNFAIP3 and TNIP1 m RNA expression(p<0.05,r>0).5. C/EBPβ LAP expression in PBMC of SLE patients was significantly higher than that in normal controls(p<0.01); C/EBPβ LIP, TNIP1 and TNFAIP3 protein expression in PBMC of SLE patients were lower than normal controls(p<0.05).6. C/EBPβ LAP expression in THP-1 cells induced monocyte-macrophages which stimulated by plasma from SLE patients increased compared with normal control(p<0.05),and C/EBPβ LIP expression is not changed.7. C/EBPβ LAP increased in THP-1 cell after TNFAIP3 small interfering RNA(si RNA) transfection or TNIP1 interference slow virus(p<0.05).Conclusion1. C/EBPβ m RNA expression in PBMC of patients with SLE elevated and especially in PBMC of patients with the characteristic antibodies, and positively correlated with disease activity score, and negatively correlated with C3, C4; in PBMC of patients with SLE C/EBPβ LAP expression increased, and LIP reduced. The prompt C/EBPβ expression contribute to the pathogenesis of SLE, and is associated with disease activity.2. C/EBPβ m RNA expression was positively correlated with TNIP1 / TNFAIP3 m RNA expression in PBMC of patients with SLE. TNIP1 and TNFAIP3 protein decreased in PBMC of patients with SLE. It suggests that C/EBPβ could enhance TNIP1 / TNFAIP3 transcripts but downregulate of TNIP1 and TNFAIP3 protein expression in PBMC of patients with SLE,which may be due to abnormal translation.3. In THP-1 cells induced monocyte-macrophages which stimulated by plasma from SLE patients, C/EBPβ m RNA expression significantly increased, C/EBPβ LAP expression increased, while C/EBPβ LIP expression is not affected. That demonstrates SLE patients plasma can induce monocyte-macrophage C/EBPβ expression and functional.4. C/EBPβ LAP increased in THP-1 cell after TNFAIP3 si RNA transfection or TNIP1 interference slow virus, indicating that the inhibition of TNIP1 and TNFAIP3 protein can induce C/EBPβ functional protein expression in monocytes. |
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