miR-155 Deficiency Ameliorates Autoimmune Inflammation of Systemic Lupus Erythematosus by Targeting S1pr1 in Faslpr/lpr Mice

Qian Xin, Jiangxia Li, Jie Dang, Xianli Bian, Shan Shan, Jupeng Yuan, Yanyan Qian, Zhaojian Liu, Guangyi Liu, Qianqian Yuan, Na Liu, Xiaochun Ma, Fei Gao, Yaoqin Gong and Qiji Liu
J Immunol June 1, 2015, 194 (11) 5437-5445; DOI: https://doi.org/10.4049/jimmunol.1403028

Abstract

MicroRNA-155 (miR-155) was previously found involved in the development of systemic lupus erythematosus (SLE) and other autoimmune diseases and the inflammatory response; however, the detailed mechanism of miR-155 in SLE is not fully understood. To explore the in vivo role of miR-155 in the pathogenesis of SLE, miR-155–deficient Faslpr/lpr (miR-155−/−Faslpr/lpr) mice were obtained by crossing miR-155−/− and Faslpr/lpr mice. Clinical SLE features such as glomerulonephritis, autoantibody levels, and immune system cell populations were compared between miR-155−/−Faslpr/lpr and Faslpr/lpr mice. Microarray analysis, RT-PCR, Western blot, and luciferase reporter gene assay were used to identify the target gene of miR-155. miR-155−/−Faslpr/lpr mice showed milder SLE clinical features than did Faslpr/lprmice. As compared with Faslpr/lpr mice, miR-155−/−Faslpr/lpr mice showed less deposition of total IgA, IgM, and IgG and less infiltration of inflammatory cells in the kidney. Moreover, the serum levels of IL-4 and IL-17a, secreted by Th2 and Th17 cells, were lower in miR-155−/−Faslpr/lpr than Faslpr/lpr mice; the CD4+/CD8+ T cell ratio was restored in miR-155−/−Faslpr/lpr mice as well. Sphingosine-1-phosphate receptor 1 (S1PR1) was found as a new target gene of miR-155 by in vitro and in vivo studies; its expression was decreased in SLE patients and Faslpr/lpr mice. miR-155−/−Faslpr/lpr mice are resistant to the development of SLE by the regulation of the target gene S1pr1. miR-155 might be a new target for therapeutic intervention in SLE.

Introduction

Systemic lupus erythematosus (SLE) (http://www.omim.org/entry/152700) is a typical autoimmune disease characterized by autoantibody production, immune complex deposition, and involvement of multiple organ systems (1). SLE patients show a high serum level of IgM and IgG, and the clinical symptoms are usually accompanied by kidney damage from increased proteinuria level because of the inflammatory cell infiltration.

Immune system dysregulation mediated by immune cells seems to be the main cause of SLE. B cell disorders, including abnormal activation, proliferation, or autoantibody production, are critical reasons for clinical mediation in SLE (2) and have been verified in an SLE mouse model (3). T cells, as well as the proinflammatory cytokines they produce, can promote autoimmune responses in the progression of SLE (4). Recently, many studies showed that Th17 cells expressing IL-17a could mediate the inflammatory response (4). The serum level of IL-17a is increased in SLE patients (5) and the SLE mouse model (6). Although both of these T cell subsets and B cells are regulated by specific microRNAs (miRNAs), the detailed mechanisms of miRNAs controlling the immune cells and whether their targeting can have therapeutic effects are not well known.

miRNAs are a class of endogenous, small noncoding RNAs of 19–22 nt that regulate mRNAs posttranscriptionally (7). By binding to the 3′-untranslated region (UTR) of the target gene mRNA, miRNAs exert their functions by inhibiting elongation, mRNA decay, and mRNA cleavage. Increasing studies have suggested that miRNAs are critical regulators of development and function in the immune system, such as in differentiation of T and B lymphocytes and immune responses (8). Patients and animal models of autoimmune diseases show dysregulated miRNA expression, and functional studies have pinpointed the essential roles of miRNAs in the onset and development of autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and SLE.

miRNA-155 (miR-155) became a focus in several studies because of its essential functions in autoimmune diseases and inflammatory responses such as MS and RA. Several in vivo and in vitro studies reported that silencing miR-155 could ameliorate the disease severity and delay the onset of experimental autoimmune encephalomyelitis (EAE) and RA (9–12). Additionally, miR-155 was found dysregulated in splenocytes from SLE-prone mice as compared with age-matched controls and was closely related to SLE susceptibility (13).

Therefore, miR-155 might be a proinflammatory factor in immune-related disease, and its deprivation might prevent autoimmunity. miR-155 was found overexpressed in the urine of SLE patients but was lower in serum (14, 15). Nonetheless, the study of miR-155 in SLE is still insufficient and little is known about the role of miR-155 in the development of SLE. In the present study, we investigated the role of miR-155 in SLE by generating miR-155−/−Faslpr/lpr mice and found that miR-155 deficiency could ameliorate SLE. Furthermore, we found sphingosine-1-phosphate receptor 1 (S1PR1) as a target of miR-155 by microarray assay and investigated its expression in patient cells.

Materials and Methods

Patient samples

We enrolled 24 patients with SLE and 34 sex- and age-matched healthy controls from Qilu Hospital of Shandong University at Jinan, China. All SLE patients fulfilled the 1997 American College of Rheumatology revised criteria for SLE. Patients with other diseases or concurrent infection were excluded. Blood samples were obtained from all participants after informed consent. The protocol of this study was approved by the Ethics Review Committee for Human Studies at the School of Medicine, Shandong University.

Generation of miR-155–deficient Faslpr/lpr mice and W146 treatment

Wide-type (WT), Faslpr/lpr, and miR-155−/− mice in a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions. The F1 generation was generated by crossing Faslpr/lpr and miR-155−/−mice, then backcrossing them 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. Mice genotypes of lpr and miR-155 mutation were identified by PCR with DNA obtained by tail biopsy. Mice were sacrificed at the end of life, at ∼10 mo.

Fas was genotyped with the following primer sequences: common, 5′-GTAAATAATTGTGCTTCGTCAG-3′, mutant, 5′-TAGAAAGGTGCACGGGTGTG-3′, and WT, 5′-CAAATCTAGGCATTAACAGTG-3′. miR-155 mouse genotyping primers (WT) were: forward, 5′-GTGCTGCAAACCAGGAAGG-3′, reverse, 5′- CTGGTTGAATCATTGAAGATGG-3′, mutant, 5′- CGGCAAACGACTGTCCTGGCCG-3′. W146 (W1020-1MG, Sigma-Aldrich) was dissolved in DMSO. miR-155−/−Faslpr/lpr mice received daily i.p. injections of DMSO or W146 (1 mg/kg) for 3 d and were then sacrificed. All experiments involving animals were approved by the Institutional Animal Care and Use Committee at Shandong University School of Medicine.

Immunostaining of cells and flow cytometry

Abs for mouse CD4-PE, CD8a-PE-Cy7, CD69-FITC, CD45R (B220)-FITC, IgM-PE-Cy7, IgD-PE, CD4-FITC, CD44-PE-Cy7, CXCR5-allophycocyanin, PD-1–PE, and CD138-PE (eBioscience) were used to stain splenocytes or lymph node cells. Suspensions of single splenocytes or lymph node cells were obtained by use of nylon mesh cell strainers (BD Biosciences, C3977-15). Single-cell suspensions were suspended in precooled 1× PBS containing 1% BSA and 0.05% NaN3. A total of 1 × 105 cells was suspended and stained with sets of conjugated Abs for 20 min on ice. Each sample was washed twice and finally resuspended in 500 μl PBS. Flow cytometry (FACSCanto II, BD Biosciences) involved 50,000 events for each test, with analysis by FlowJo v7.6.5.

Mouse urine collection and proteinuria measurement

Urine from mice 30 wk old or 40–50 wk old was collected for analysis by use of metabolic cages (Hatteras Instruments, MMC100). The proteinuria concentration was detected by Coomassie brilliant blue assay at 595 nm absorbance. Urine collection and measurement was repeated three times.

Isolation of PBMCs

PBMCs were isolated from 3-ml heparinized blood samples of patients and controls by Ficoll gradient centrifugation (Lymphoprep, Nycomed Pharma). Cells from the interphase were collected, washed twice with HBSS (HyClone) and stored in 1 ml TRIzol (Invitrogen) at −80°C.

RNA extraction and real-time RT-PCR

Total RNA was extracted from cells with use of TRIzol reagent (Invitrogen, 15596-018) following the manufacturer’s instructions and treated with RNase-free DNase (Promega, M6101). cDNA was prepared with use of Moloney murine leukemia virus reverse transcriptase (Thermo Scientific, EP0352). Reverse transcriptase involved 2 μg RNA, 1 μl random primers at 65°C for 10 min, 2 μl dNTP (10 mM), 4 μl 5× buffer, and 1 μl reverse transcriptase. Diethyl pyrocarbonate H2O was added to 20 μl. Real-time quantitative PCR involved a Roche 480 instrument. Real-time PCR involved 2× FastStart Universal SYBR Green Master mix (Roche, 04913914001), primers (5 μM, 0.5 μl; primer sequences are listed in Supplemental Table I), cDNA (1 μl), and double distilled H2O (3.5 μl). Four replicates were performed for each reaction: 95°C for 10 min, 95°C for 10 s, 60°C for 20 s, and 60°C for 30 s for 40 cycles. All samples were run in duplicate, and mRNA was normalized to GAPDH level. The relative mRNA expression was assessed with the 2−ΔΔCt method.

Protein assays

Mouse spleen cells and human PBMCs were lysed by use of TRIzol. Protein was isolated by the TRIzol method. Protein concentration was quantified by the BCA method (Thermo Scientific, 23225). Equal amounts of protein extracts (50 μg) were loaded and separated by 12% SDS-PAGE, electrotransferred to polyvinylidene difluoride membranes, which were incubated with specific primary Abs overnight at 4°C, and then incubated with HRP-conjugated secondary Abs at room temperature for 1 h and detected by ECL (Thermo Scientific, 32132). Abs for β-actin and S1PR1 were from Epitomics (Burlingame, CA).

Histology, immunohistochemistry, and immunofluorescence

Mice were harvested at age 40 wk. For histology, mouse kidneys were fixed in 4% paraformaldehyde for 24 h at 4°C, then 0.5% paraformaldehyde for 24 h, and then embedded in paraffin and sectioned. Paraffin tissue sections (5 or 7 μm thick) were stained with H&E or periodic acid–Schiff (PAS). For immunohistochemistry, paraffin renal sections were treated with Ag retrieval solution and permeabilized with 0.2% Triton X-100 in PBS, then blocked before incubation with anti-S1pr1 Ab (Abcam, ab23695). Negative controls were substituting the primary Ab with normal serum. For immunofluorescence, kidneys were immediately frozen to −80°C in Tissue-Tek OCT compound (Sakura Finetek, 4583). Frozen sections (5 μm thick) were incubated with blocking buffer and stained with FITC-conjugated anti-mouse C1q, IgG, and IgM Abs. Mouse spleen sections were stained with 100 μg/ml peanut agglutinin (PNA; Molecular Probes, L32458) for 30 min, washed three times for 7–8 min with PBS, and observed under the fluorescence microscope after DAPI staining. Slides were analyzed under a laser-scanning confocal microscope.

Cell transfection and luciferase reporter assay

Human embryonic kidney (HEK)293 cells were cultured in DMEM (Life Technologies, 12100-046) supplemented with 10% FBS (HyClone, SH30396.03), 100 U penicillin, and 100 mg/ml streptomycin (Sangon, BS733). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. miR-155 mimics and inhibitors transfection involved use of transfection reagent in 96-well plates (Thermo Fisher Scientific, R0531) according to the manufacturer’s instructions. HEK293 cells were harvested after 48 h and lysed by passive lysis buffer. Luciferase activity was measured by the Dual-Luciferase reporter assay system.

ELISA of serum levels of Ig Abs and cytokine

Serum titres of IgG, IgM, and IL-21 were detected in mice by use of an ELISA kit (eBioscience, 88-50400-22, 88-50470, and 88-8210). The mouse Th1-, Th2-, and Th17-related cytokines IFN-γ, IL-4, and IL-17a were detected by use of a cytometric bead array mouse kit for Th1/Th2/Th17 (BD Biosciences, 560485).

Microarray analysis

Mouse spleens were frozen in liquid nitrogen immediately after removal. Total RNA was extracted from WT or miR-155−/− mouse spleens. The amount of RNA from each sample was quantified by use of NanoDrop 1000, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA of each sample was used for labeling and array hybridization as follows: 1) reverse transcription by use of an Invitrogen SuperScript double stranded cDNA synthesis kit; 2) double-stranded cDNA labeling with a NimbleGen one-color DNA labeling kit; 3) array hybridization by the NimbleGen hybridization system and washing with the NimbleGen wash buffer kit; and 4) array scanning with the Axon GenePix 4000B microarray scanner (Molecular Devices). Heat maps were drawn from microarray data.

Statistical analysis

Data analysis involved use of GraphPad Prism (GraphPad Software, San Diego, CA). Quantitative data are expressed as mean ± SD or mean ± SEM. Differences between two groups were compared by a Student unpaired two-tailed t test. A p value <0.05 was considered statistically significant.

Results

miR-155−/−Faslpr/lpr mice show mild SLE phenotypes

miR-155 affects immune responses and is involved in the pathology of autoimmune diseases. We found miR-155 overexpressed in the spleen and thymus of Faslpr/lpr mice as compared with WT mice. To investigate whether miR-155 plays a critical role in the development of SLE, miR-155−/−Faslpr/lpr mice and their littermates (Faslpr/lpr mice) were obtained by backcrossing and interbreeding. Mice at 40–50 wk old were surveyed for phenotypes. Splenomegaly is a feature of the SLE mouse model (3). 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 (Fig. 1A). Furthermore, proteinuria level was lower in miR-155−/−Faslpr/lpr than Faslpr/lpr mice, with no significant difference between the two genotypes at 30 wk old (Fig. 1B).

FIGURE 1.
FIGURE 1.

Reduced symptoms of lupus-like disease in 40- to 50-wk-old miR-155-/-Faslpr/lpr mice. (A) Spleens from wild-type (WT), Faslpr/lpr, and miR-155−/−Faslpr/lpr mice were removed and weight was measured. Representative images of spleens show smaller spleen size of miR-155−/−Faslpr/lpr mice than Faslpr/lpr mice (left). Spleen weight and the ratio of spleen mass and body mass from WT (n = 7), Faslpr/lpr (n = 11), and miR-155−/−Faslpr/lpr (n = 7) mice are shown in the histogram (right). (B) Proteinuria level quantitatively measured by Coomassie brilliant blue protein assay (n = 8–10). (C) Paraffin-embedded sections of mouse kidney were stained with H&E and PAS (original magnification ×200). Representative images are shown. Data were evaluated by a two-tailed unpaired t test. Data are mean ± SEM of three independent experiments. *p < 0.05, ***p < 0.001.

To investigate the pathological characteristics in detail, kidney sections of mice were studied by H&E and PAS staining. Although both genotypes showed hyperplasia of the glomerular mesangium, the level was lower for miR-155−/−Faslpr/lpr mice (Fig. 1C). Additionally, glomerular enlargement was ameliorated in miR-155−/−Faslpr/lpr mice.

Reduced total IgM and IgG titres in serum and immune complex deposition in miR-155−/−Faslpr/lpr mouse kidney

Both SLE patients and Faslpr/lpr mice show increased autoantibodies or immune complex deposition in kidneys (16). To further confirm our results, we examined immunofluorescence for IgA, C1q, and IgM in mouse kidney cryosections. Fluorescence intensity was greatly decreased in miR-155−/−Faslpr/lpr mice, which suggests reduced immune complex deposition and kidney inflammation (Fig. 2A). Therefore, miR-155−/−Faslpr/lpr mice showed reduced disease severity as compared with Faslpr/lpr mice.

FIGURE 2.
FIGURE 2.

Knockout of miR-155 in 40-wk-old Faslpr/lpr mice reduces autoantibody titers. (A) Frozen kidney sections from mice glomeruli were stained with FITC-labeled anti-IgM, C1q, or IgA Ab. Confocal microscopy of representative images are from two independent experiments (original magnification ×200). (B) ELISA of titers of total serum IgG and IgM autoantibodies from the indicated mouse strains. (C) Representative images of mouse spleen sections of the three mouse strains stained with PNA (Alexa Fluor 568 conjugate) and DAPI (original magnification ×100). (D) Flow cytometric analysis of Tfh cell proportion in lymph nodes (n = 5–7) and comparison of concentration of IL-21 in sera of the three mouse strains by ELISA (n = 7–17). (E) Quantitative RT-PCR analysis of Bcl6 and IL-21 mRNA levels in mouse spleens. Each dot represents an individual mouse (n = 5–15 for each mouse strain). Data are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Because serum titers of the autoantibodies IgM and IgG are elevated in Faslpr/lpr mice (17), we detected the serum titers of total IgM and IgG induced by B cells. Both IgM and IgG titers were lower in miR-155−/−Faslpr/lpr mice than in Faslpr/lpr mice (Fig. 2B).

Reduced percentage of T follicular helper cells and concentration of IL-21 in the miR-155−/−Faslpr/lpr mouse

Mouse spleen sections stained with PNA confirmed that there were fewer numbers of spontaneous germinal centers in the miR-155−/−Faslpr/lpr spleen compared with the Faslpr/lpr spleen (Fig. 2C). The proportion of Tfh (T follicular helper) cells (CD4+CXCR5+CD44+PD-1+) was significantly reduced in the miR-155−/−Faslpr/lpr mouse lymph nodes. IL-21 is the main cytokine secreted by Tfh cells. ELISA was performed to detect the concentration of IL-21 in mouse serum, and we found that there was a significant reduction of IL-21 in the miR-155−/−Faslpr/lpr mice serum compared with Faslpr/lpr mice (Fig. 2D). Additionally, Bcl6 and IL-21 play a critical role in the regulation of Tfh cell differentiation and can be considered as the marker of Tfh cells. We found that Bcl6 and IL-21 expression was significantly reduced at the mRNA level in miR-155−/−Faslpr/lpr mice spleens (Fig. 2E).

miR-155−/−Faslpr/lpr mice show a decreased CD4+/CD8+ cell proportion and active CD4+ T cell inflammatory cytokines in miR-155−/−Faslpr/lpr serum

Faslpr/lpr mice show abnormalities of T and B cells (18). The SLE mouse model is characterized by increased numbers of activated CD4+ T, B, and plasma cells and an increased ratio of CD4+ to CD8+ T cells as compared with WT mice (3). To investigate the cell levels in detail, we examined spleens and lymph nodes by flow cytometry. The proportion of CD4+CD69+cells (Fig. 3A) was decreased ∼2-fold 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 (Fig. 3B). Compared with Faslpr/lprmice, miR-155−/−Faslpr/lp r mice showed a significantly greater CD4−CD8− T cell population (Fig. 3C). B220+, B220+IgM+, and B220+IgD+ proportions of B cells in spleen did not differ between the genotypes (Fig. 3C).

FIGURE 3.
FIGURE 3.

Quantitative flow cytometry of cell proportions in spleen or lymph nodes from 40-wk-old WT, Faslpr/lpr, and miR-155−/−Faslpr/lpr mice. (A) Proportion of active CD4+ T (CD4+CD69+) cells in spleens and lymph node cells. Splenocytes and lymph node cells from 40- to 50-wk-old mice were stained with the indicated Abs and analyzed by flow cytometry. FACS pseudocolor image shows a representative result from three independent experiments (left). The histograms show mean active CD4+ T proportion of three groups of mouse spleens and lymph nodes (right, n = 7–9). (B) Ratio of CD4+/CD8+ in splenocytes and lymph node cells. A representative result shows the ratio of CD4+ and CD8+ cells from spleens and lymph nodes. CD4+ and CD8+ T cell in spleens and lymph nodes are circled in the FACS pseudocolor image (left).The statistical results of CD4+/CD8+are shown in the histograms (right, n = 7–9) (C) Proportion of CD4−CD8− cells and B cells subsets in mouse spleens. Each symbol represents an individual mouse (left). Splenocytes were labeled with B220, IgM, IgD, and CD138 Abs for subsets of B220+and B220+IgM+, B220+IgD+, and B220+CD138+ cells (right, n = 8–12). (D) Serum levels of T cell–related cytokines IL-4, IL-6, and IFN-γ. Data are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Because aberrant Th cells have been reported in human SLE, we assessed serum levels of the inflammatory cytokines IL-4, IFN-γ, and IL-17a secreted by Th1, Th2, and Th17 cells, respectively, in the mouse. The level of IL-17a and IL-4 was lower in miR-155−/−Faslpr/lpr than Faslpr/lpr mice (Fig. 3D). The level of IFN-γ remained the same in the three mouse groups. The absence of miR-155 in Faslpr/lpr mice may ameliorate the pathological features in part by regulating cell types in SLE.

S1PR1 is negatively regulated by miR-155 in the cell lines and spleen of mice

miRNAs participate in the regulation of mRNA transcription or translation by interacting with the 3′-UTR region of the gene. To determine the target gene of miR-155 in mice, we performed whole-transcriptome analysis of spleens of miR-155−/− and WT mice (accession no. GSE66815, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66815). For genes with >2-fold altered expression, we found 544 upregulated and 508 downregulated genes in miR-155−/− mouse spleens (Fig. 4A). Among the common target genes identified, many are well-established SLE susceptibility genes, such as Malt1 and Pxk. Additionally, genes with altered expression were involved in the immune response and included Cr2, Foxp1, and Zeb2. We confirmed these results by real-time RT-PCR (Fig. 4B).

FIGURE 4.
FIGURE 4.

Microarray assay of gene expression patterns in spleens of WT and miR-155−/− mice and the verification of decreased S1PR1 expression by miR-155 through binding to the 3′-UTR region. (A) Heat map of representative genes with change in expression >2-fold difference at p < 0.05 in spleens. Red or green indicates relative high or low expression levels, respectively (n = 4). (B) Quantitative RT-PCR verification of chip results (n = 8–10 for each mouse strain). Relative fold changes for each gene were set to 1 for WT mice. Data are mean ± SD from three independent biological replicates (experimental error). (C) Venn analysis of chip results (red), predictive human targets (green), and mouse target genes (blue) of miR-155. (D) Schematic view of human and mouse miR-155 binding site of S1PR1. Red shows the seed sequence of miR-155. Vertical lines indicate Watson–Crick and wobble base pairing, respectively. (E) Luciferase reporter assay of HEK293 cells with WT or mutant 3′-UTR vector and contransfected with mimics or inhibitor of miR-155. (F) Western blot analysis of S1PR1 in HEK293 cells transiently transfected with miR-155 mimics or inhibitors for 48 h. NC, normal control. (G) RT-PCR and Western blot analysis and quantification of mRNA and protein levels, respectively, of S1PR1. Band intensity is underneath the gel image. (H) Representative immunohistochemical staining of S1pr1 using hematoxylin in kidneys of Faslpr/lpr and miR-155−/−Faslpr/lpr mice. Data are mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Venn diagrams were created of upregulated genes detected by whole-transcriptome analysis, as well as targets of miR-155 predicted target genes by miRecords in both mice and humans. The union of three sets contains Satb1, H3f3a, Sla, and S1pr1 in the Venn analysis (Fig. 4C). S1PR1, which participates in immune inflammatory responses, was predicted using four commonly used software. As shown in Fig. 4D, the 3′-UTR region of S1PR1 may serve as binding site for miR-155 in humans and mice.

To further confirm that S1PR1 is indeed a target gene of miR-155, we used luciferase assay and transfection experiments. pmirGlo vectors including the S1PR1 3′-UTR sequence and miR-155–specific mimics or inhibitors were cotransfected into HEK293 cells for Dual-Luciferase activity assay. Luciferase activity was decreased as compared with the control, so miR-155 may interact with the S1PR1 3′-UTR and then inhibit gene expression. Luciferase activity was not affected by mutant vector transfection (Fig. 4E). miR-155 inhibitor transfected in HEK293 cells downregulated miR-155 activity and increased the expression of S1PR1. However, when HEK293 cells were transfected with miR-155 mimics, the expression of S1PR1 was decreased (Fig. 4F). In vivo, S1PR1 mRNA and protein levels were upregulated 3-fold in the spleen of miR-155–deficient Faslpr/lpr mice (Fig. 4G). The increased expression of S1PR1 was detected in miR-155−/−Faslpr/lpr mouse kidneys (Fig. 4H). In summary, S1PR1 is a target gene of miR-155, and miR-155 regulates its expression at the posttranscriptional level.

S1PR1-specific antagonist W146 could accelerate inflammatory phenotypes of miR-155−/−Faslpr/lpr mice

We have confirmed that miR-155 could negatively regulate S1PR1 expression in vitro. Next, we explored whether miR-155 is involved in the development of SLE by regulating S1PR1 in vivo. W146 is a specific antagonist S1PR1. miR-155−/−Faslpr/lpr mice were received daily i.p. injections of DMSO or W146 (1 mg/kg). Mice were sacrificed after 3 d and the proportions of cell subtypes were detected. We found that the proportion of activated CD4+ T was increased in the W146-treated mice lymph nodes and spleens (Fig. 5A). Moreover, the ratio of CD4+/CD8+ was also increased (Fig. 5B). Additionally, the proportion of Tfh cell was also increased in lymph nodes of W146-treated mice compared with DMSO-treated mice (Fig. 5C). Overall, miR-155 was involved in development of SLE by regulating the expression of S1pr1 in vivo.

FIGURE 5.
FIGURE 5.

Increased proportion of activated CD4 cells and CD4+/CD8+ T cells in spleens and Tfh cells in the lymph nodes in W146-treated miR-155−/−Faslpr/lpr mice. (A) Proportion of active CD4+ (CD4+CD69+) T cells in splenocytes and lymph node cells from DMSO- and W146-treated miR-155−/−Faslpr/lpr mice. FACS pseudocolor image shows a representative result from three independent experiments (left). The histograms show mean active CD4+ T proportion of two groups of mouse spleens and lymph nodes (right, n = 5–7). (B) Ratio of CD4+/CD8+ in mouse splenocytes and lymph node cells (n = 5–7). (C) Proportion of Tfh cells in lymph node cells (n = 5–7). Data are mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

S1PR1 is downregulated in PBMCs of SLE patients and splenocytes of Faslpr/lpr mice

To confirm whether S1PR1 is involved in the pathogenesis of SLE, we explored S1PR1 expression in human PBMCs from SLE patients and Faslpr/lpr mouse splenocytes. As expected, S1pr1 mRNA and protein expression was decreased in Faslpr/lpr mouse splenocytes and patient PBMCs (Fig. 6).

FIGURE 6.
FIGURE 6.

S1PR1 is downregulated in mice spleen and SLE patient PBMCs. Quantitative RT-PCR analysis of mRNA level (A) and Western blot analysis and quantification of protein level (B) of S1pr1 in spleens of mice. Expression is relative to β-actin level (B). (C and D) Level of S1PR1 in PBMCs from SLE patients and healthy controls (C) and Western blot analysis and quantification of protein level (D). Levels are relative to those of β-actin. Each experiment was repeated at least three times. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In this study, we investigated the role of miR-155 in the pathogenesis of SLE using a knockout mouse model in vivo. miR-155 deficiency could reduce disease severity and ameliorate the lupus-like phenotypes of the Faslpr/lpr mouse. Moreover, total serum levels of IgM and IgG and immune complex deposition in the mouse kidney were decreased in miR-155−/−Faslpr/lpr mice. S1PR1 is a target of miR-155, and its mRNA and protein expression was increased in PBMCs from SLE patients and Faslpr/lpr mouse splenocytes.

miR-155 is located at 21q21.3, the B cell integration cluster, originally considered to be a proto-oncogene associated with lymphoma. miR-155 was implicated in the innate immune function from evidence of dysregulated miR-155 in many autoimmune diseases, which demonstrated that miR-155 was pivotal for the immune and inflammatory response (9, 19–21). miR-155 was found to promote the development of EAE, an established mouse model of MS (9). The expression of miR-155 was elevated in the synovial membrane and synovial fluid macrophages from patients with RA (10). Furthermore, miR-155–deficient mice did not exhibit collagen-induced arthritis (10, 11).

In the EAE model, miR-155–deficient mice demonstrated abnormal proportion and function of immune cells, especially Th1 and Th17 cells (21). Additionally, miR-155–deficient mice showed reduced numbers of T regulatory cells, so miR-155 might promote the development of T regulatory cells (22). Additionally, miR-155 could regulate the germinal center response of B cells, and deprivation of miR-155 in B cells could reduce the secreted IgG1 Ab (23). miR-155–deficient B cells cannot generate reduced extrafollicular and germinal center responses and secrete high-affinity IgG1 Abs (23). Deletion of miR-155 was found to alleviate lupus-like disease in the Faslpr/lpr mouse (24). Therefore, miR-155 plays an important role in the pathogenesis of SLE.

Several studies identified many direct target genes of miR-155. miR-155 could downregulate suppressor of cytokine signaling 1 (25) and src homology 2 domain-containing inositol-5-phosphatase 1 (26) and then play a critical function in the immune response. Additionally, miR-155 promoted Ig class switching in B cells by targeting the transcription factor PU.1 (27). Our in vivo and in vitro studies indicated that S1PR1 is a new target gene of miR-155. S1PR1, a G protein–coupled receptor, can bind to the bioactive signaling molecule S1P for a critical role in the regulation of lymphocyte maturation, migration, and trafficking. Additionally, S1PR1 and S1P signaling is essential for autoimmune diseases such as MS and RA and the inflammatory responses (28, 29).

Although studies of S1PR1 in SLE have been rarely reported, increasing evidence suggests that S1PR1 might be involved in the pathogenesis of SLE. Microarray analysis of Th17 cells from SLE patients showed decreased mRNA level of S1PR1 (30). Fingolimod (FYT720), an analog of sphingosine, which functions as an immunosuppressant, is an effective drug used to treat MS (31). Phosphorylated fingolimod promotes the internalization of S1PR1 and then stops the lymphocytes moving from lymph nodes to peripheral tissues (32). Thus, we next investigated what role, if any, S1PR1 plays in the pathogenesis of SLE. Both the mRNA and protein levels of S1PR1 were decreased more in SLE patients than in healthy controls. Consistent results were obtained in the spleens of Faslpr/lpr mice and littermates. Thus, the expression of S1PR1 might be correlated with the pathogenesis of SLE, and the factor might be a potential therapeutic target for the treatment of SLE. All of these data provide solid evidence for the role of S1PR1 in SLE pathogenesis and development. Further investigations to understand the detailed mechanisms of S1PR1 in SLE are required.

In conclusion, we demonstrated that miR-155–deficient Faslpr/lpr mice are strongly protected against the development of SLE lesions. How this occurs might depend in part on its new target gene S1PR1. miR-155 may be a valid therapeutic target in the treatment of SLE.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Hong Li for help in renal pathology analysis.

Footnotes

  • The gene expression data presented in this article have been submitted to the Gene Expression Omnibus database under accession number GSE66815.

  • This work was supported by Natural Science Foundation of China Grants 81072452, 81273281, and 81471602.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    EAE
    experimental autoimmune encephalomyelitis
    miR-155
    miRNA-155
    miRNA
    microRNA
    MS
    multiple sclerosis
    PAS
    periodic acid–Schiff
    PNA
    peanut agglutinin
    RA
    rheumatoid arthritis
    SLE
    systemic lupus erythematosus
    S1P
    sphingosine 1-phosphate
    S1PR1
    sphingosine-1-phosphate receptor 1
    Tfh
    T follicular helper
    UTR
    untranslated region
    WT
    wild-type.

  • Received December 3, 2014.
  • Accepted March 25, 2015.

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