The Pathogenic Roles Of IFI44L In Systemic Lupus Erythematosus And Its Molecular Mechanisms

Systemic lupus erythematosus(SLE)is a chronic,relapsing autoimmunedisease resulting in multi-organ dysfunction which is a severe threat to human health.Interferon which is a kind of cytokines regulating immune participated in onset and development of SLE.Interferons not only function themselves,they can also activate some downstream genes which called interferon-inducible genes including Interferon induced protein 44like(IFI44L).Some researchs of genes profile difference have found IFI44L upregulated in SLE serum and synovium.Genome-wide association study showed IFI44L significantly associated with SLE.In addition,genome-wide methylation of SLE CD4~+T cell identified different loci in IFI44L.Therefore,we speculated abnormal expression of IFI44L may play important role in pathogenesis of SLE.In this study,we designed to explore the pathogenic functions of IFI44L in SLE and its molecular mechanisms by following three parts.PartⅠThe expression of IFI44L in PBMCObjective:To detect the expression of IFI44L in PBMC from SLEpatients and in health PBMC stimulated by IFN-αor SLE serum.Methods:PBMCs of SLE patients and health controls were isolated.IFN-αor SLE serum were added into treatment group and cells were collected after 3h,6h,12h,24h and 48h.Real-time PCR and Western blot was performed to detect the m RNA and protein of IFI44L.Results:Both m RNA and protein of IFI44L was significantly upregulated in 20 SLE patients’PBMC compared with 20 health controls.There was significant difference between two groups by t-test(p<0.01).Both m RNA and protein of IFI44L were significantly upregulated treated by IFN-αor SLE serum after 3h,6h,12h,24h and 48h.There was significant difference compared to 0h group by t-test(p<0.01).Conclusion:IFI44L was significantly upregulated in SLE PBMC.PartⅡThe epigenetic mechanisms of IFI44L over-expression in SLE PBMCObjective:To investigate the methylation level of IFI44L promoter inSLE patients’PBMC and PBMC treated by IFN-αor SLE serum.Methods:PBMCs from SLE patients and health controls were isolated by density gradient centrifugation.IFN-αor SLE serum were added into treatment group and cells were collected after 3h,6h,12h,24h and 48h.Total DNA was isolated and treated by Sodium bisulfite.Methylation level was detected by pyrosequencing.Results:Compared with 20 healthy controls,the methylation level of IFI44L promoter in 20 SLE patients’PBMC was downregulated.There was significant difference between two groups by t-test(p<0.01).The methylation level of IFI44L promoter in 6 health patients’PBMC didn’t change treated by IFN-αor SLE serum.There was no significant difference compared to 0h group by t-test(p<0.05).Conclusion:Over-expression of IFI44L in SLE PBMC might relate with the hypomethylation of IFI44L promoter.PartⅢPossible mechanisms of IFI44L in pathogenesis of SLEObjective:To explore the effect of ERK pathway inhibitor on expression of IFI44L and the effect of IFI44L expression alteration on TBK1/IRF3 pathway and downstream related cytokines.Methods:PBMC from SLE patients and health controls were isolated by density gradient centrifugation in sterile procedure.SLE patients’PBMC was treated with U0126 or PD98059 and collected after 3h,6h,12h,24h and 48h.Health PBMC which was treated with U0126 or PD98059 were treated by IFN-αor SLE and collected after 12h and 24h.Real-time PCR was performed to detect the m RNA levels of IFI44L,TBK1,IRF3,and STING.Western blot was used to detect the protein level of IFI44L.Cell culture supernatant was collected and TNF-α,CCL5,IFN-βwere detected by ELISA.Results:IFI44L m RNA or protein in 6 SLE patients’PBMC treated by U0126 or PD98059 was upregulated.IFI44L m RNA or protein in 6 normal PBMC which was treated with U0126 or PD98059 was upregulated after treated by IFN-αor SLE serum.There was significant difference compared to 0h group or compared with solvent groups by t-test(p<0.05).TBK1,IRF3 and STING m RNA in health PBMCs and TNF-α,CCL5,IFN-βin cell culture supernatant were upregulated transfected with IFI44L overexpressed plasmid and were downregulated transfected with IFI44L si RNA.There was significant difference compared to control group(p<0.05).Conclusion:Inhibition of MEK/ERK pathway contributed to high-level IFI44L,which promoted TBK1/IRF3 pathway and downstream inflammatory cytokines contributing to occurrence and development of SLE.