Role Of Icaritin In Regulating Foxp3/IL17a Balance In Systemic Lupus Erythematosus And Its Effects On The Treatment Of MRL/lpr Mice

Systemic lupus erythematosus (SLE) is a female predominant autoimmune disease characterized by multi-organ dysfunction. The pathogenesis of SLE is complex. Several studies have revealed that CD4+T cells are critical in the initiation and maintenance of autoimmune responses in SLE. Regulatory T cells and Th17cells are subsets of effector CD4+T cells. A fine balance between Treg cells and Th17cells regulates immune homeostasis. However, this balance is destroyed in autoimmune diseases such as SLE.The treatment of SLE includes hydroxychloroquine, corticosteroids, immunosuppressive drugs and so on. However, these drugs are only partially effective. Sometimes, these indiscriminate suppressors of immune-mediated inflammatory cure SLE at the cost of considerable adverse effects. Undoubtedly there is a need for safer and more effective treatments for SLE.Icaritin (ICT) is an active ingredient extracted from Chinese herbals Epimedium genus. It has been used as an aphrodisiac, tonic and antirheumatic in traditional medicine in China. However, recent studies have shown that ICT has a wide range of pharmacological and biological activities. Zhang et al have found that ICT can down-regulate ER expression. Li has indicated that ICT inhibits T cell activation and prolongs skin allograft survival in mice [4]. The polymorphisms of oestrogen receptor alpha and antoreactivity of CD4+T cells are known to be associated with the onset and development of SLE [5]. Together, these data suggest that ICT may have therapeutic effects on SLE.In this study, we found that ICT can regulate Foxp3/IL17a balance, enhance Treg cells suppressive activities, and inhibit over-activation of CD4+T cells from SLE. We aslo observed that ICT regulated Foxp3/IL17a balance by increasing STAT5b expression and histone methylation modification. Further experiments confirmed that ICT-treated mice exhibited amelioration of renal disease and ICT might be a potential new drug for the treatment of SLE. Part Ⅰ ICT regulates Foxp3/IL17a balance Section I Effect of ICT on the expression level of Foxp3and IL17a geneObjective:To explore the effect of ICT on the expression level of Foxp3and IL17a gene.Methods:CD4+T cells were isolated from SLE patients by positive selection using magnetic beads. CD4+T cells were treated with40uM/L ICT for72h. Foxp3and IL17a mRNA levels were determined by real-time RT-PCR. Foxp3protein level was examined by western blotting. Detection of IL17a level was performed by ELISA.Results:Compared to control group, Foxp3mRNA level (Control group vs ICT group:1±0.0vs2.288±0.3962, P=0.0019) and protein level (Control group vs ICT group:0.6167±0.07638vs1.46±0.2254, p=0.0036) were significantly increased in ICT-treated group, while IL17a mRNA level (Control group vs ICT group:1±0.0vs0.4077±0.1839, P=0.002) and protein level (Control group vs ICT group:41.86±12.82vs30.34±0.7566, P=0.0344) were significantly decreased in ICT-treated group.Conclusion:ICT can increase the expression level of Foxp3while decrease IL17a gene expression. Section Ⅱ Modulation of Treg Cell function and CD4+T cell autoreactivity by ICTObjective:To explore the effect of ICT on the suppressive capacity of Treg cells and autoreactivity of CD4+T cells in vitro.Methods:CD4+CD25+CD127-T cells, CD4+CD25-T cells, CD4+T cells and CD19+B cells were isolated from SLE patients using magnetic beads. CD4+CD25+CD127-T cells were treated with40uM/L ICT for72h. CD4+CD25+CD127-T cells and CD4+CD25" T cells were cocultured at the ratio of1:1for5days. Proliferation of CD4+CD25-T cells was determined by measuring CFSE dilution with the FACS CANTO (BD Biosciences). Detection of IL10and TGF-β levels were performed by ELISA. CD4+T cells were treated with40uM/L ICT for72h. CD4+T cells and autologus CD19+B cells were cocultured at the ratio of1:4for8days. Detection of IgG, IFN-γ, IL-6and TNF-α levels were performed by ELISA. CD40L and CD70expression level were detected by flow cytometry.Results:Compared to control group, the suppressive capacity of Treg cell in ICT-treated group were significantly enhanced (Control group vs ICT group:33.67±10.97vs63.1±2.265, p=0.0104). Importantly, ICT had no influence on the overall proliferation of Treg cells. The level of IL10from supernatant from the coculture cells in ICT-treated group was siganificantly increased compared with control group (8.19±35.01vs196.2±38.21, p=0.0039). There was no significant difference in TGF-β levels (110.2±3.942vs114.4±5.568, p=0.1626). Expression of CD40L,CD70and production of IgG were significantly decreased in ICT-treated group(Contol group vs ICT group:2±0.1vs1±0.2, P=0.029；3.6±0.26vs1.46±0.25,P=0.00378；43.14±12.52vs6.476±3.364,p=0.0013). Production of IFN-γ,IL-6,TNF-αwere also significantly decreased in ICT-treated group(Control group vs ICT group:330.6±17.23vs194.56±6.762,P=0.0252；521.6±61.2vs251.1±58.9,P=0.0337；750.2±32.753vs420.6±23.341,p=0.0045, respectively)Conclusion:ICT can enhance the suppressive capacity of Treg Cell and inhibit the autoreactivity of CD4+T cell in vitro. Part Ⅱ The molecular mechanisms of ICT in modulating Foxp3/IL17a balanceSection Ⅰ Effect of ICT on histone methylation status at Foxp3and IL17a promoterObjective:To explore the effect of ICT on histone methylation status at Foxp3and IL17a promoter region.Methods:Used the cells from section I of part I. Amounts of H3K4me3、 H3K9me3and H4acethlation within the foxp3and IL17a promoter were analyzed by chromatin immunoprecipitation (ChIP) and real-time PCR. Results:Compared to control group, H3K4me3enrichment at the Foxp3promoter was significantly increased in ICT-treated group (Control group vs ICT group:1±0.0vs11.1±3.21, P=0.0081); H3K9me3enrichment at the IL17a promoter was significantly increased in ICT-treated group (Control group vs ICT group:1±0.0vs4.367±1.106, P=0.0341) There was no significant difference in H4acethlation level at Foxp3and IL17a promoter region.Conclusion:ICT can increase H3K4me3enrichment at the foxp3promoter and H3K9me3enrichment at the IL17a promoter. Section Ⅱ Genome-wide gene expression profiling in CD4+T cells stimulated with ICTObjective:To detect whole genome gene expression profiling in ICT-treated CD4+T cells.Methods:CD4+T cells were isolated from SLE patients by positive selection using magnetic beads. CD4+T cells were treated with40uM/L ICT for72h. Total RNA was extracted and Affymetrix microarray was performed. Four differentially expressed genes were further verified by Real-time PCR.Results:Compared to control group, ICT-treated group resulted in a total of1007differentially expressed genes with at least3fold changes,395over-expressed (accounting for60%of differentially expressed genes) and612under-expressed (accounting for40%of differentially expressed genes) respectively. The Gene Ontology (GO) analysis and pathway analysis results showed that most of the genes regulated by ICT are mainly associated with immune response process. We selected four differentially expressed genes (IRF-4, STAT5a, STAT5b, HIF-1) for validation. These4genes are transcription factors (TF), which have been reported to regulate both Foxp3and IL17a expression. The change trend was consistent with microarray results. Compared to controls, the expression of STAT5b is obviously increased (Control group vs ICT group:1±0vs2.467±0.1508, p=0.0339).Conclusion: The differentially expressed genes of ICT-treated group are mainly associated with immune response process. STAT5b may be the checkpoint for regulation of Foxp3/IL17a expression by ICT. Section Ⅲ Effect of ICT on foxp3/IL17a expression after down-regulating STAT5b expressionObjective:To explore the effect of ICT on Foxp3/IL17a expression after down-regulating STAT5b expression.Methods:CD4+T cells were isolated from SLE patients by positive selection using magnetic beads. STAT5b-siRNA and control-siRNA was transfected into CD4+T cells by transient electroporation. Then CD4+T cells were treated with40uM/L ICT for24h. STAT5b, Foxp3and IL17a mRNA were evaluated by real-time PCR. STAT5b protein levels were examined by western blotting.Results:Compared to control-siRNA group, STAT5b mRNA level and protein level were significantly decreased. After down-regulating STAT5b expression, Foxp3and IL17a had no significant changes in ICT-treated group (Control group vs ICT group:1±0vs1.073±0.07959, p=0.17;1±0vs1.293±0.01839, p=0.18, respectively)Conclusion:Down-regulating STAT5b in CD4+T cells can inhibit the effect of ICT on the modulation of Foxp3/IL17a balance. Part Ⅲ The therapeutic effect of ICT on the MRL/lpr miceObjective:To explore the therapeutic effect of ICT on the MRL/lpr mice.Methods:Twenty14-week-old female MRL/lpr mice were randomized into two group. Ten of them were treated with ICT, which was dissolved in Carboxymethyl Cellulose (CMC) by oral gavage daily for four weeks, another10mice for control group received the same volume of CMC. The level of serum anti-dsDNA, IL-10, TGF-β and IFN-γ were assayed by ELISA. Urinary albumin was determined by albustix paper. The sediment of IgG immune complex and C3in kidney was determined by direct immunofluorescenee, and pathologieal damage of kidney was determined by HE staining method.Results:1. Compared to control group, the ICT-treated mice have lower level of dsDNA and IL-10(Control group vs ICT group:67140±4836vs55080±9596, p=0.0371;6441±479.9vs5627±486.6, p=0.0116, respectively). There are no significant difference in both of TGF-β and IFN-γ (110.2±3.942vs114.4±5.568, p=0.1626;493.9±25.94vs497±41.58, p=0.8675, respectively)2. ICT-treatment decreased urinary albumin after four weeks of treatment (Control group vs ICT group:300±73.02967vs84.2857±38.72105, P=0.0198)3. Slighter deposition of IgG and C3to the mesangium and capillary loop of glomeruli in ICT-treated MRL/lpr mice was observed.4. In accordance with the histopathologic changes of kidney, ICT-treated MRL/lpr mice had much milder GN and TIN damage score (Control group vs ICT group:8.36±1.067vs6.72±0.7935, p=0.0129;8.16±1.328vs6.04±1.054, p=0.012, respectively)Conclusion:ICT exerts an ameliorative effect on renal damage in MRL/lpr mice.