MiR-155 Deficiency Ameliorates Autoimmune Inflammation Of Systemic Lupus Erythematosus By Targeting SlPr1 In Fas^lpr/lpr Mice

Systemic lupus erythematosus (SLE) (OMIM 152700) is a typical systemic autoimmune disease, characterized by a large number of pathogenic autoantibodies, and immune complex deposition in the joints, liver, renal, skin and other tissues, which cause multi-system and multiple organ injuries in SLE patients. Clinical manifestations of SLE are diverse and complicated. The incidence of SLE was significantly higher in female than that of male. Etiology of SLE is complex, and it can be caused by both environmental and genetic factors. So far, the specific pathogenesis of SLE has not been fully elucidated.Immune system disorders is present in SLE patients, with a large number of autoantibody production and immune complex accumulated in the kidneys, eventually resulting in the kidney failure. About one third SLE patients are afflicted with abnormity of immune cells including the abnormal activation of T cells and B cells as well as the unbalanced ratio of CD4+ and CD8+ T cells.MicroRNA (miRNA) was found as a class of endogenous, important regulators of gene expression in the past 20 years and involved in the regulation of many biological processes such as cell proliferation, apoptosis and differentiation. By binding to the 3’-UTR of the target gene mRNA, miRNAs exert their functions by inhibiting elongation, decay and cleavage of mRNA. Increasing studies have suggested that miRNAs are critical regulators of development and function in the immune system, which include miR-181a, miR-146a and miR-155. MiR-155 plays a role in various physiological and pathological processes. MiR-155 is located at human chromosome 21q21.3, which was initially identified as a gene that was transcriptionally activated by promoter insertion at a common retroviral integration site in B-cell lymphomas and was formerly called BIC (B-cell integration cluster). Abnormal expression of miR-155 plays important role in development of tumor and autoimmune diseases. Studies have demonstrated that overexpression of miR-155 or BIC gene can cause diffuse large B-cell lymphoma or Hodgkin’s lymphomas. It has been demonstrated miR-155 may participate the development of B cells, T helper (Th) cells, regulatory T (Treg) cells, dendritic (DC) cells, T follicular helper (Tfh) cells and other immune response process. Additionally abnormal expression of miR-155 also takes part in a variety of autoimmune disease pathogenesis, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. MiR-155 and related inflammatory factors are elevated in rheumatoid arthritis (RA) patients’macrophages isolated from synovium and synovial fluid, besides, miR-155-deficient mice were resistant to collagen-induced arthritis. MiR-155-/- mice were significantly resisted the occurrence of experimental autoimmune encephalomyelitis (EAE), and the proportion of Thl and Thl7 cells was significantly reduced in miR-155-/- EAE mice’s spleens and lymph nodes. Moreover miR-155 was elevated in various organs and tissues of SLE patients as well as animal models of SLE.In summary, miR-155 may play a proinflammatory role in autoimmune diseases. Although the relationship between miR-155 and SLE is well documented in several studies, the detailed molecular mechanism is still unclear. In order to clarify the role of miR-155 in lupus mice and whether the absence of miR-155 can ameliorate autoimmune inflammation of SLE, we cross the miR-155 deficient mouse into the SLE background and analyze whether depletion miR-155 may release the SLE affection phenotype. Furthermore we conducted a thorough inquiry of miR-155 targeted genes. The results acquired from this study are shown as follow:PartⅠConstruction and phenotype analysis of miR-155-/-Faslpr/lprmiceMRL/lpr mice, with the elevated expression of miR-155 in their spleens and thymuses, are recognized as the most favorable model of SLE. In order to explore the in vivo role of miR-155 during the pathogenesis of SLE, we cross the miR-155 deficient mouse into the Faslpr/lpr background. Faslpr/lpr and miR-155-/- mice in a C57BL/6 background were purchased from Jackson Laboratory. The F1 generation was generated by crossing Faslpr/lpr and miR-155-/- mice, then backcrossed with Faslpr/lpr mice. Mice homozygous for the lpr mutation and heterozygous for the miR-155 mutation were interbred to maintain the miR-155-/-Faslpr/lpr and Faslpr/lpr strains.Wild type mice, Faslpr/lpr mice and miR-155-/-Faslpr/lpr mice at 40-50 weeks old were sacrificed and surveyed for phenotypes. Spleen weight and spleen weight/body weight ratio are important indicators of SLE severities, so firstly we measured spleen weight and spleen weight/body weight ratio of these mice. Although both miR-155-/-Faslpr/lpr mice and Faslpr/lpr mice showed splenomegaly, miR-155-/-Faslpr/lpr spleens were significantly smaller than Faslpr/lpr spleens. Moreover, the ratio between spleen weight and body weight was decreased 1.4 fold. Because autoantibody and immune complex are usually deposited in kidneys of lupus mice, next we detected the extent of kidney damage of these mice. Mice urine for 24 h was collected by metabolic cages and the proteinuria concentration was detected by coomassie brilliant blue (CBB) assay. We found that proteinuria level was lower in miR-155-/-Faslpr/lpr than Faslpr/lpr mice, with no significant difference between the two genotypes at 30 weeks old. These results suggest that depletion miR-155 in FasIpr/lpr mice can significantly relieve lupus nephritis. Indeed, histopathological analysis showed increased kidney tissue structural disorders in Faslpr/lpr mice, including glomerular enlargement, glomerular basement membrane thickening and significant inflammatory cell infiltration, while there was only mild inflammatory cell infiltration in glomerular and reduced mesangial proliferation in miR-155-/-Faslpr/lpr mice. In addition, immunofluorescence experiments demonstrated that the deposition of immune complex IgM, IgA and C1q was significantly reduced in miR-155-/-Faslpr/lpr mice renal, and ELISA results showed that both IgM and IgG antibody titers decreased in the serum.In addition, we also detected T and B lymphocyte subtypes’proportion by flow cytometry. The proportion of CD4+CD69+ T cells was about a two-fold decrease in the miR-155-/-Faslpr/lpr mouse. MiR-155 deficiency could rescue the increased ratio of CD4+ to CD8+ T cells in both spleens and lymph nodes. Cytometric beads array demonstrated that the level of IL-17a and IL-4 was lower in miR-155-/-Faslpr/lpr than Faslpr/lpr mice The level of IFN-y remained same in the three groups. PNA staining showed that there was spontaneous formation of germinal center in Faslpr/lpr mice spleen. However, the number of germinal center was reduced in the miR-155-/-Faslpr/lpr mice spleens. Studies have shown that T follicular helper (Tfh) cells involved in the development of SLE, so the proportion of Tfh cells and secreted cytokine levels were detected. Results showed that compared with Faslpr/lpr mice, the proportion of Tfh cells was significant decreased in the miR-155-/-Faslpr/lpr mice lymph nodes. ELISA results showed that the concentration of IL-21, follicular helper type T (Tfh) related cytokines, was reduced in the miR-155-/-Faslpr/lpr mice serum, and expression of 11-21 and Bcl6 mRNA levels decreased in the mice spleen. B220+, B220+IgM+ and B220+IgD+ proportions of B cells did not differ between the genotypes. The above results indicated that the absence of miR-155 in Faslpr/lpr mice may ameliorate the pathological features in part by regulating cell types in SLE.In summary, the absence of miR-155 in Faslpr/lpr mice may ameliorate the pathological features of SLE.Part Ⅱ MiR-155 plays a critical role in the pathogenesis of SLE by regulating S1pr1MiRNAs participate in the regulation of translation by binding to the gene’s mRNA 3’-UTR region. To determine the target genes of miR-155 in mice, we performed whole-transcriptome analysis of spleens of miR-155-/- and WT mice. For genes with more than two-fold altered expression, we found 544 upregulated and 508 downregulated genes in miR-155-/- mouse spleens.Venn diagrams were created using upregulated genes detected by whole-transcriptome analysis, as well as miR-155 predicted target genes by miRecords in both the mouse and human. The union of three sets contains Satbl, H3f3a, Sla and Slprl in the Venny analysis.S1PR1 (Sphingosine-1-phosphate receptor 1), also known as endothelial differentiation gene 1 (EDG1), is a protein encoded by the S1PR1 gene in human. S1PR1 can exert its function by inactivating JAK-STAT pathway. It has been confirmed that S1PR1 participate in the development of various autoimmune diseases, and our study also found that S1pr1 is abundantly expressed in blood, spleens and lymph nodes of mice. In addition, we also found S1PR1 is decreased in Faslpr/lpr lupus mice spleens and SLE patients’peripheral blood lymphocytes compared with the controls, suggesting that it may be involved in the development of SLE.In order to verify whether S1PR1 is a target gene of miR-155, we construct pmirGLO-S1PR1-3’-UTR vector. Next we co-transfected mimics or inhibitor of miR-155 as well as the constructed plasmid into HEK 293 cells and found that miR-155 mimics can decrease luciferase activities while miR-155 inhibitor can significantly increase the luciferase activities. However, when the miR-155 binding site is mutated, this phenomena immediately disappear. We further proved that S1PR1 can be elevated by miR-155 inhibitor and decreased by miR-155 mimics. Taken together, these results suggest that S1PR1 is a direct target gene of miR-155.After confirming that miR-155 could negatively regulate S1PR1 expression in vitro, we then verified this results in vivo. We found that both protein and mRNA levels of Slprl were increased in miR-155-/-Faslpr/lpr spleens of mice compared with these of Faslpr/lpr mice, and elevated expression of S lprl was also detected in the kidney of miR-155 deficient mice by immunohistochemistry experiments.In order to verify whether miR-155 participate in the development of SLE by regulating expression of Slprl,we injected DMSO or W146, the S1PR1 specific antagonist, by intraperitoneal to miR-155-/-Faslpr/lpr mice. And we found that, W146 treated mice demonstrated increased CD4+/CD8+ T ratio in spleens and lymph nodes, besides the percentage of CD4+CD69+ T cells as well as Tfh cells were also increased compared with the control mice. These results indicate that miR-155 plays an important role in the development of SLE by regulating Slprl in mice.The results of this section showed that miR-155 took part in the development of SLE via regulating Slprl.Part Ⅲ Characterization of the transcirptional regulation mechanism of human S1PR1 geneWe have proved that miR-155 could participate in the development of SLE by regulating expression of Slprl, and this fact indicated that S1PR1 may be a new therapeutic target. In addition, we collected 24 patients with SLE and 34 healthy subjects’peripheral blood from Shandong University Qilu Hospital during 2010 and 2012. Real-time quantitative PCR and Western blot results showed that both mRNA levels and protein levels of S1PR1 were decreased in peripheral blood cells of patients with SLE compared with healthy controls. Furthermore, the results were also confirmed in normal mouse with Faslpr/lpr lupus mices plenocytes. This indicated that S1PR1 may be associated with pathogenesis of SLE. In fact, previous studies have demonstrated S1PR1 was involved in the pathogenesis of other autoimmune diseases. FTY720 (Fingolimod), a well-known drug for the treatment of autoimmune disease multiple sclerosis (MS), can inhibit S1PR1 function. All these results indicate that S1PR1 exert its important roles during the development of tumor and autoimmune diseases, so it’s necessary and important to identify in which way S1PR1 is regulated. So we investigated the transcriptional regulation mechanism of S1PR1 in this section.Promoter of S1PR1, range from transcription start site to upstream-1 kb region, were cloned into pGL3-Basic vector, and a lot of truncated plasmids were also constructed. All these plasmids were transfected and followed by the detection of dual luciferase reporter gene activity. When the sequence was truncated from -29 to-12 bp, the dual luciferase activity was significantly decreased, and this result suggested that potential transcription factors may bind to this sites. And when this region was mutated or truncated, the luciferase activity was significantly decreased comparing with the wild type vector. Mobility shift assay (EMSA) also showed that this region possessed the transcription factor binding site. Software prediction result showed that transcription factors such as ETS1, FLI1, ELK1, STAT1, STAT3, STAT4, STAT6 and C-MYC may bind to this region. Supershift results confirmed that p-STAT1 could be the key transcriptional factor of S1PR1.We also found that the level of S1PR1 was significantly reduced in STAT1-knockdown cells. Consistent with the reduction of S1PR1 caused by depletion of STAT1, overexpression of STAT1 resulted in up-regulation of S1PR1. And after luciferase plasmids transfected into STAT1-knockdown or overexpression cells, we draw our conclusion that overexpression of STAT1 can stimulate S1PR1 transcription while knockdown STAT1 decrease S1PR1 transcription, besides this process can be blocked by mutation or deletion of the-29 to-12 bp region.To verify the expression of STAT1 involved in the regulation S1PR1, we designed a series of experiments, and the results showed that S1PR1 was increased both in mRNA and protein levels along with the activation of STAT1 after incubated with IFN-y, and decreased after treatment with STAT1 specific inhibitor fludarabine. In addition, ChIP assays were performed and the results also confirmed that the transcription factor STAT1 can be combined directly with S1PR1 promoter region under physiological conditions.In order to explore the relationship between STAT1 and S1PR1 on the transcriptional level, we extract RNA of 41 healthy individuals’peripheral blood cells and detect the expression of STAT1 as well as S1PR1. And we found STAT1 expression levels were significantly positively correlated with S1PR1 expression. (R2= 0.4439, P 0.01).The experimental results of this part show that transcription factor STAT1 could bind to upstream region of -29- -12 bp in S1PR1 gene and positively regulate its expression.