Open Access

The MicroRNA miR-22 Represses Th17 Cell Pathogenicity by Targeting PTEN-Regulated Pathways

Li Wang, Rong Qiu, Zhaoyang Zhang, Zhijun Han, Chao Yao, Guojun Hou, Dai Dai, Wenfei Jin, Yuanjia Tang, Xiang Yu and Nan Shen
ImmunoHorizons June 1, 2020, 4 (6) 308-318; DOI: https://doi.org/10.4049/immunohorizons.2000008

Abstract

Multiple sclerosis is a chronic autoimmune disease driven by pathogenic Th17 cells. In this study, we dissected the role of miR-22 in pathogenic Th17 cells by autoantigen-specific disease models. We first showed that miR-22 was upregulated in peripheral lymphoid organs and spinal cords of mice developed autoimmune encephalomyelitis. Although miR-22 was upregulated in multiple Th cell subsets, it was dispensable for Th cell differentiation in vitro. Whereas miR-22−/− mice exhibited milder symptoms of disease in an active experimental autoimmune encephalomyelitis model, adoptive transfer of miR-22−/− 2D2 Th17 cells into naive recipient mice promoted higher disease incidence and severity compared with mice transferred with control 2D2 Th17 cells. Global transcriptional analysis of miR-22–deficient pathogenic Th17 cells revealed upregulated genes in phosphatase and tensin homologue (PTEN)–mediated pathways, and Pten was further identified as one of its potential targets. Therefore, we identified that Th17 cell–intrinsic miR-22 could protect mice from autoimmunity by targeting PTEN-regulated pathways.

Introduction

Multiple sclerosis (MS) is widely regarded as a classical autoimmune disease (1). Although considerable effects has been made into understanding the pathogenesis of MS, effective treatment strategy for this disease is not available (2). Thus far, accumulating evidences have proven that myelin reactive lymphocytes cells have been involved in the pathogenesis of MS (3). More specifically, whereas decreased regulatory T cell (Treg) function and increased frequencies of Th17 and Th1 cells have been reported in MS (4, 5), IL-17A upregulation and pathogenic Th17 cells were reported to be associated with the pathogenesis of MS (6, 7). Murine experimental autoimmune encephalomyelitis (EAE) model helps to understand the pathogenesis of MS and might eventually lead to the development of effective therapeutics for human MS (8).

IL-17–producing Th17 cells display a high grade of plasticity (9, 10). They are abundant in the lamina propria of small intestine, and exhibit critical roles for mucosal defense (11, 12). In addition, proinflammatory Th17 cells accumulate in inflammation sites and contribute to multiple inflammatory disorders (13). Th17 cells can also be generated with a group of cytokine combinations, which include TGF-β1, IL-6 or IL-6, IL-1β, and IL-23 in vitro (14, 15).

MicroRNAs (miRNAs) regulate gene expression at the posttranscription level (16). Our group and other groups have shown that miRNAs were important factors in the modulation of autoimmunity (17, 18). In our previous work, we reported that miR-21 was upregulated in lupus patients’ CD4+ T cells and highly expressed in spinal cords of EAE mice (19). miR-183-96-182 cluster was shown to promote Th17 cell pathogenicity (17), and miR-155 was identified to target Jarid2 to promote Th17 cell effector function (20). miR-22, a highly conserved miRNA, which has been shown to control the function of several types of immune cells (21). Aberrant expression of miR-22 has been observed in MS patients and other autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus (22, 23). A recent report found that miR-22 could control the activation of dendritic cell through activating AP-1 transcription factor complexes and histone deacetylase HDAC4 (21). Furthermore, it has been reported that miR-22 was highly expressed in the Th17 cells differentiated in vitro compared with naive CD4+ T cells (17). Interestingly, it has been shown that anti–miR-22 locked nucleic acid treatment could reduce the activity of Th17 cells and reverses mouse emphysema by repressing dendritic cell function (21). However, intrinsic role of miR-22 in pathogenic Th17 cells is still unknown.

In this study, we found that expression of miR-22 was increased in spleen and spinal cords of mice developed EAE, and it was highly upregulated in inflammatory Th17 cells derived in vitro. Whereas global deletion of miR-22 displayed ameliorated disease symptoms without disrupted Th17 cell differentiation, Th17 cell–intrinsic loss of miR-22 induced higher morbidity and mortality of EAE disease. Phosphatase and tensin homologue (PTEN), which is a tumor suppressor and plays an important role in Th17 cell differentiation (24), was shown as a potential target of miR-22 in pathogenic Th17 cells. Collectively, these data demonstrated that Th17-intrinsic miR-22 acts as a suppressor of pathogenic Th17 cell–mediated autoimmune disease by targeting PTEN-regulated pathway.

Materials and Methods

Mice

miR-22+/− mice were from the Nanjing Biomedical Research Institution of Nanjing University (Nanjing, China). miR-22−/−, littermate control, 2D2, and 2D2 miR-22−/− mice on the C57BL/6 background were bred and maintained under specific pathogen-free conditions. All animals were raised in microisolator cages with sterile water, autoclaved diet, and filtered air. Six- to eight-week-old mice were employed for all experiments. All animal experiments were performed in compliance with the guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Renji Hospital of Shanghai Jiao Tong University School of Medicine.

T cell differentiation in vitro

Naive CD4+ T cells were isolated from spleen of 6- to 8-wk-old mice using a CD4+ CD62L+ T Cell Isolation Kit II (catalog no. 130-104-453; Miltenyi Biotec) according to the manufacturer’s instructions. Naive CD4+ T cells (8 × 105 per well) were activated with plate-coated anti-CD3ε mAb (catalog no. 16-0038-85, 5 μg/ml; Thermo Fisher Scientific) and anti-CD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral conditions (catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher Scientific] and catalog no. 16-7311-38, 10 μg/ml anti–IFN-γ mAb [Thermo Fisher Scientific]). The following conditions were used: Th17 (β) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-β1 [R&D Systems]; catalog no. 406-ML-025, 20 ng/ml IL-6 [R&D Systems]; catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher]; and catalog no. 16-7311-38, 10 μg/ml anti–IFN-γ mAb [Thermo Fisher Scientific]), Th17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6 [R&D Systems]; catalog no. 401-ML-025, 20 ng/ml IL-1β [R&D Systems]; catalog no. 1887-ML-010, 20 ng/ml IL-23 [R&D Systems]; catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher Scientific]; and catalog no. 16-7311-38, 10 μg/ml anti–IFN-γ mAb [Thermo Fisher Scientific]), Th1 condition (catalog no. 419-ML-010, 10 ng/ml IL-12 [R&D Systems] and catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher Scientific]), Treg condition (catalog no. 402-ML-020, 5 ng/ml IL-2 [R&D Systems]; catalog no. 7666-MB-005, 2.5 ng/ml TGF-β1 [R&D Systems]; catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher Scientific]; and catalog no. 16-7311-38, 10 μg/ml anti–IFN-γ mAb [Thermo Fisher Scientific]), and Th0 condition (catalog no. 16-7041-95, 10 μg/ml anti–IL-4 mAb [Thermo Fisher Scientific] and catalog no. 16-7311-38, 10 μg/ml anti–IFN-γ mAb [Thermo Fisher Scientific]). Cells were cultured in complete IMDM medium (Life Technology) or RPMI 1640 medium (Life Technology) supplemented with 10% FBS and 2-ME (1:1000).

Intracellular staining and flow cytometry

Cultured cells from the spleen were washed and stimulated for 4–6 h at 37°C with PMA (50 ng/ml), ionomycin (750 ng/ml), plus GolgiPlug Protein Transport Inhibitor (1:1000). Then cells were stained with Live/Dead BV510 (catalog no. L34955, 1:1000; Thermo Fisher Scientific), and for intracellular staining, cells were fixed and permeabilized (BD Biosciences) or with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and next stained with allophycocyanin-conjugated rat anti-mouse IFN-γ (XMG1.2) (BD Biosciences), allophycocyanin–anti-mouse/rat Foxp3 (BD Biosciences), and PerCP-Cy5.5–conjugated rat anti-mouse IL-17A (TC11-18H10) (BD Biosciences) Abs. Stained cells were harvested and then analyzed using an FACSCalibur Flow Cytometer (BD Biosciences), and data were analyzed with FlowJo software.

Quantitative real-time PCR

Total RNA was extracted from the mouse tissues and cells with TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions, and the cDNA was synthesized and stored at −20°C. The expression of the genes encoding mouse Il7r, Tbet, Ccl3, Icos, Il3, Ccl4, Cxcl3, Lag3, Ccl5, Lgals3, Lrmp, Casp1, IL-22, Stat4, Csf2, Gzmb, and so on were quantified by real-time PCR using the PrimeScript RT Reagent Kit (Takara Bio). All gene expression results were normalized to the basal expression of the housekeeping gene Actb. For details of genes primers, please refer to Supplemental Table III. Amplification of cDNA was done using an ABIprism 7900 HT Cycler (Applied Biosystems). Quantitative real-time PCR (qRT-PCR) was employed with the SYBR green methodology according to the following situations: 95°C for 1 min, then performed with 40 cycles at 95°C for 15 s, and 60°C for 30 s with 40 cycles. Mature miR-22, miR-21, miR-155, and miR-146a were examined with TaqMan Kit from Applied Biosystems, and their expression was normalized to the expression of RNU6.

Induction of EAE by myelin oligodendrocyte glycoprotein immunization or 2D2 transfer

At day 0, 6- to 8-wk-old female or male C57BL/6 mice were immunized s.c. in the back region with 200 μg of the myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (catalog no. 163913-87-9; ChinaPeptides) emulsified with CFA (catalog no. 263910; BD Difco). Pertussis toxin (catalog no. P7208; Sigma-Aldrich) at a dose of 300 ng per mouse in PBS was governed i.v. on the day of induction and once more 48 h later. Mice were monitored daily for clinical signs of disease as previously demonstrated: 0, no clinical signs; 0.5, Weakness of half the tail; 1, limp tail; 2, weakness of hind legs but no paraparesis; 2.5, paraparesis in one hind limb; 3, paraplegia (complete paralysis of both two hind limbs); 4, paraplegia with fore limb and moribund state or death. For histological analysis, the CNS were isolated from MOG-immunized mice and were shortly fixed in 4% paraformaldehyde. Paraffin-embedded projects of CNS were stained using H&E or Luxol Fast Blue and detected using light microscopy. For adoptive transfer EAE, naive 2D2 TCR–transgenic CD4+ T cells were differentiated into pathogenic Th17 cells as described in Th cells differentiation in vitro. The cells are counted and reset to 0.5 × 106 cells per milliliter on days 3 and 5 after initial stimulation. Next, the cells were harvested and washed twice with PBS and resuspended at 10 × 106 cells per milliliter. Two hundred microliters of the cell suspension (2 × 106) cells were injected i.p. into each recipient C57BL/6 wild-type mice with a sublethal dose of radiation. EAE is scored as same as shown above.

RNA sequencing

Naive CD4+ T cells from control and miR-22−/− were differentiated under pathogenic Th17 (23) condition. Total RNA was prepared from these cells using TRIzol reagent (Invitrogen). RNA sequencing (RNA-seq) libraries were prepared using a TruSeq Stranded Total RNA Library Prep Kit (Illumina). Sequencing was performed on an Illumina HiSeq X 10 System in a 150/150-bp paired end mode.

RNA-seq data processing

RNA-seq reads were mapped to mm10 and refGene annotation transcriptome downloaded from UCSC Genome Browser using TopHat2 (25), and low quality mapping reads (MAPQ < 10) were filtered by Samtools, and then gene level expression was counted by htseq-count using all refGenes (26). Differentially expressed genes were called by DESeq2 using false discovery rate 0.05 and fold change >1.3 (27). Gene Ontology analysis showed the following: upregulated genes in miR-22−/− Th17 cells were uploaded onto ebioservice bioinformatics platform (Shanghai, China) (http://enrich.shbio.com/index/ga.asp), which is a similar online Gene Ontology enrichment tool to DAVID.

Immunoblot

A total of 2 × 106 pathogenic Th17 (23) cells were lysed in RIPA buffer (catalog no. 89900; Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor mixture (catalog no. 78441; Thermo Fisher Scientific). Individual cell lysates (10 μg per lane) were separated by 12% SDS-PAGE, and then electrically transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA). After being blocked with SuperBlock T20 PBS blocking buffer (catalog no. 37516; Thermo Fisher Scientific), the membranes were incubated with Abs as follows: β-tubulin rabbit mAb (catalog no. 2128S, diluted 1:1000; Cell Signaling Technology), PTEN rabbit mAb (catalog no. 9559S, diluted 1:1000; Cell Signaling Technology), anti-rabbit IgG, and HRP-linked Ab (catalog no. 7074, diluted 1:3000; Cell Signaling Technology).

Statistical analysis

Unless otherwise specified, all statistical analyses were employed by using an unpaired two-tailed Student t test with GraphPad Prism software. A p value <0.05 is considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001).

Results

miR-22 expression is upregulated under autoimmune condition

Previous reports have shown that miR-22 was involved in the pathogenesis of inflammatory disease, including smoking-related emphysema and inflammatory bowel disease (21, 28). In this study, to examine the role of miR-22 in a pathogenic Th17 cell–mediated autoimmune disease, the EAE model was used. This model is a Th17 cell–mediated autoimmune disease typically derived from either active immunization by myelin-derived proteins or from passively transferred activation of myelin-specific CD4+ T lymphocytes (29). RT-PCR analysis revealed that expression of miR-22 was highly increased in inflamed tissues, which include spleen and spinal cords from mice developed autoimmune EAE compared with control mice (Fig. 1). Moreover, we detected a certain number of classical immune-related miRNAs which have been reported broadly in the field of inflammatory diseases, like miR-155, miR-21, and miR-146a. As shown in (Fig. 1), expression of miR-155 and miR-21 were higher in autoimmune conditions than in naive mice. miR-146a expression was with modest upregulation in the spleen, whereas its expression was significantly upregulated in inflamed CNS. These results suggest that expression of miR-22 along with other immune-related miRNAs were upregulated under autoimmune condition.

FIGURE 1.
FIGURE 1.

miR-22 expression is upregulated under autoimmune condition.

(A) TaqMan quantitative RT-PCR analysis of miR-22, miR-155, miR-21, and miR-146a expression in the spleens of control and EAE model mice, respectively. Each dot represents a mouse. (B) TaqMan RT-PCR analysis of miR-22, miR-155, miR-21, and miR-146a in the CNS of control and EAE model mice. The CNS samples are from the spine cord. The relative miRNA expression was normalized to the expression of U6 (A and B). All data are representative of three independent experiments and shown as mean ± SD and by an unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

miR-22 is highly expressed in in vitro–derived Th17 cells

Because the previous study has demonstrated that miR-22 expression was upregulated in Th17 cells derived in vitro compared with naive CD4+ T cells in an miRNA profiling assay (17), we further examined the expression of miR-22 in Th cells via in vitro differentiation naive CD4+ T cells under pathogenic Th17 [Th17 (23)], nonpathogenic Th17 (Th17[β]), Th1, and induced Treg–polarizing conditions. As expected, we consistently observed that induced pathogenic Th17 cells and nonpathogenic Th17 cells have the highest level of miR-22 expression (Fig. 2A). miR-22 expression in Th1 cells were also increased compared with Th0 cells, as demonstrated in previous studies Th17 and Th1 cells work as important pathogenic T cell subsets contributing to multiple autoimmune diseases (5, 30). The expression of miR-22 in naive CD4+ T, Th0, and Treg subset were much lower. Taken together, we showed that miR-22 was upregulated in polarized Th17 cells in vitro.

FIGURE 2.
FIGURE 2.

miR-22 is highly expressed in in vitro–derived Th17 cells.

(A) TaqMan RT-PCR analysis of miR-22, miR-21, and miR-155 expression on different CD4+ T cell subsets differentiated in vitro for 72 h under different skewing conditions. (B) Validation of miR-22, miR-21, and miR-155 expression in vitro. Naive CD4+ T cells were isolated, differentiated as indicated, and analyzed by TaqMan RT-PCR at 72 h. The bar graphs show the mean ± SEM. The relative miRNA expression was normalized to the expression of U6 (A and B). Figures are summary of three independent experiments. The p values were determined by two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Considering that Th17 cells differentiation in vitro required a combination of cytokines TGF-β1, IL-6 (31) or IL-23, IL-6, and IL-1β (13, 15). Subsequently, we tested which of these cytokines were key to miR-22 induction in pathogenic Th17 cells. To this end, we isolated naive CD4+ T cells from healthy mice and stimulated with anti-CD3e plus anti-CD28 mAbs in the presence of IL-6, IL-1b, IL-23, and TGF-β1, respectively. We found that IL-6 alone could upregulate miR-22 expression in activated CD4+ T cells (Fig. 2B). Consistent to previous reports, IL-6 could upregulate miR-21 and miR-155 expression in activated CD4+ T cells (Fig. 2B). Taken together, these data show that miR-22 was highly expressed in Th17 cells and IL-6 was enough to induce the expression of miR-22.

miR-22 is dispensable for Th17 cell differentiation in vitro

A previous study has shown that miR-22 was upregulated in T follicular helper cells, whereas analysis of miR-22–deficient (miR-22–/–) mice demonstrated that miR-22 was dispensable for T follicular helper cell generation and function (32). To evaluate the role of miR-22 for Th17 cells, we generated germline miR-22−/− mice and examined the effect of miR-22 loss in Th17 cell differentiation in vitro. For this purpose, we differentiated naive CD4+ T cells from miR-22−/− and littermate control mice in vitro under pathogenic Th17 and nonpathogenic Th17-skewing condition. A similar frequency of IL-17A–producing Th17 cells was generated from miR-22−/− and control naive CD4+ T cells (Fig. 3A, 3B). To investigate whether miR-22 deficiency affects other CD4+ T cell subsets differentiation, we next stimulated naive CD4+ T cell from control and miR-22−/− mice under Th1 and Treg-polarizing condition. Similarly, the percentage of Th1 and Treg cells remained unchanged after miR-22 deletion (Fig. 3C, 3D). Conclusively, these data suggest that miR-22 was not required for Th17 cell differentiation nor Th1 or Treg cell differentiation in vitro.

FIGURE 3.
FIGURE 3.

miR-22 is dispensable for Th17 cell differentiation in vitro.

(A) Left, Naive splenic CD4+ T cells from control or miR-22−/− mice were differentiated with TGF-β1 + IL-6 for 72 h in IMDM or RPMI-1640 medium, followed by intracellular staining of IL-17A and IFN-γ. Right, Frequency statistics. (B) Left, Representative flow cytometric analysis of the percentages of pathogenic Th17 (23) cells were differentiated with IL-6 + IL-1β + IL-23 from the naive splenic CD4+ T cells of the control and miR-22−/− mice. Right, Frequency statistics. (C) Left, Naive splenic CD4+ T cells from control or miR-22−/− differentiated with IL-2 + TGF-β1. Cells were analyzed by flow cytometry at 72 h. Right, Frequency statistics. (D) Left, Flow cytometric analysis of the percentage of Foxp3+ Treg cells in the naive splenic CD4+ T cells cultured with IL-12 for 72 h from control and miR-22−/−. Right, Frequency statistics. Data are representative of five (A–C) or three (D) independent experiments mean ± SEM (two-tailed Student t test).

Th17 cell–intrinsic miR-22 deletion exacerbates autoimmune EAE

Given the observation that miR-22 is not required for Th17 cells differentiation in vitro, compelled us to ask whether miR-22 deficiency has an impact on Th17 cell–induced autoimmune disease in vivo. For this purpose, initially we immunized miR-22−/− mice and littermate control with MOG35–55 peptide emulsified in CFA to induce EAE. Clinical scores (based on observed symptoms; shown in Materials and Methods) were measured daily. Previously, we revealed that miR-22 expression was upregulated in the autoimmune EAE model, implying that miR-22 was likely to play a crucial role in the induction, progression, or restoration stage. We compared clinical and histological disease progression in the control and miR-22−/− mice. As expected, miR-22−/− mice exhibited milder symptoms of EAE disease (Fig. 4A, 4B). Altogether, our results indicate that global miR-22 deletion reduce autoimmune disease. Previous work by another group has demonstrated that dendritic cell miR-22 expression through targeting HDAC4 promoted Th17 cell–dependent emphysema (21); however, Th17 cell–intrinsic role of miR-22 in autoimmune disease pathogenesis remains unknown.

FIGURE 4.
FIGURE 4.

Th17 cell–intrinsic miR-22 deletion exacerbates autoimmune EAE.

(A) Mean clinical score (± SEM) of EAE induced in 6- to 8-wk-old control and miR-22−/− mice. (B) Representative H&E staining or Luxol Fast staining of spinal cord sections on day 21 after immunization of the mice in (A). Original magnification ×5. (C and D) Passive EAE is induced as previous described (29). Naive 2D2 or 2D2 miR-22−/− splenic CD4+ T cells were differentiated with IL-6 + IL-1β + IL-23 and transferred into syngeneic wild-type (WT) hosts. (C) Left, Cytokine profile of 2D2 T cells after differentiation at day 5. Right, Frequency statistics. (D) Clinical score of recipient mice undergoing EAE (n = 6). (E) H&E staining or Luxol Fast staining of spinal cord lesions of representative mice in (D) on day 21. Images are original magnification ×5. All data are representative of three independent experiments and shown as mean ± SEM. In unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

To examine Th17 cell–intrinsic role of miR-22 in a Th17-dependent autoimmune model, 2D2 Th17 cell–adoptive transfer model was used (33, 34). For this reason, we generated Th17-specific miR-22–/– mice by crossing 2D2 mice with miR-22–/– mice to generate 2D2 miR-22−/− mice, and we differentiated Th17 (23) in vitro for 5 d from naive MOG peptide–specific 2D2 TCR–transgenic CD4+ T cells and 2D2 miR-22−/− CD4+ T cells. These pathogenic Th17 cells readily induced EAE in recipient mice. Following adoptive transfer of 2D2 Th17 (23) or 2D2 miR-22−/− Th17 (23) cells into sublethally irradiated recipients. A similar percentage of IL-17+ T cells was generated from 2D2 and 2D2 miR-22−/− naive cells (Fig. 4C, Supplemental Fig. 1A), consistent with the observation that miR-22 does not affect Th17 differentiation. We then evaluated a set of parameters in adoptive transfer EAE pathology and observed that the clinical score and amounts of inflammatory infiltration and demyelination were significantly increased in the recipients transferred of 2D2 miR-22−/− Th17 cells (mean maximum score: 2D2 miR-22−/− = 4; 2D2 = 2.5) (Fig. 4D, 4E). Thus, our results suggest that Th17 cell–intrinsic expression of miR-22 plays a pivotal role in restraining Th17 cell pathogenicity and autoimmune disease.

miR-22 represses Th17 cell pathogenicity by targeting PTEN-regulated pathways

We next sought to probe the molecular mechanism underlying Th17 cell–intrinsic role miR-22 in autoimmune diseases. To date, the intracellular mechanisms of the key differences between pathogenic Th17 cells and nonpathogenic Th17 cells are not well understood, and genomic approaches afford a compelling unbiased approach to find such complicated mechanisms (14), as a series of previous studies defined a number of signature genes expression that distinguish pathogenic Th17 cells from nonpathogenic Th17 cells (35, 36). We reasoned that sustained miR-22 expression should antagonize IL-6–, IL-1β–, and IL-23–driven pathogenicity of Th17 cells. To test this hypothesis, we studied the impact of miR-22 on pathogenic Th17 cells differentiated with IL-6, IL-1β, and IL-23 from naive control and miR-22−/− CD4+ T cells by analyzing pathogenic Th17 cell signature genes expression. Interestingly, compared with Th17 (23) cells differentiated from control mice, several effector molecules of pathogenic signature genes were with increased expression in miR-22−/− Th17 (23) cells (including Cxcl3, Il7r, Gzmb, Ccl5, Icos, Stat4, Lag3, and Lgals3) (Fig. 5A) and with decreased nonpathogenic signature genes (like Il10 and Irf4) (Supplemental Fig. 1B). Some other signature genes, including Ccl4, Casp1, Tbx21, Il3, Ccl3, Il22, Csf2, Gpr83, Il24, Ccr4, Il9, Il4ra, and Lrmp, were barely affected by the loss of miR-22 (Fig. 5A, Supplemental Fig. 1B).

FIGURE 5.
FIGURE 5.

miR-22 represses Th17 cell pathogenicity by targeting PTEN-regulated pathway.

(A) Expression profile of pathogenicity signature genes of the pathogenic Th17 cells from control and miR-22−/− are measured by qRT-PCR. Data are representative of three independent experiments. (B) Volcano plot of differentially expressed genes in control (blue points) and miR-22−/− (red points) pathogenic Th17 cells (fold change > 1.3; false discovery rate < 0.05, (Supplemental Table I). (C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes in control and miR-22−/− pathogenic Th17 (23) cells in accordance with biological process. The horizontal axis shows −log10 of the p value. (D) Venn diagram displaying miR-22 computationally predicted to target Pten by three different prediction computational models: Target Scan, HITS-CLIP, and RNA-seq (Supplemental Table II). A schematic representation of the conserved sequences of the miR-22 binding sites in the 3′-untranslated region of Pten (red) is also shown. Underlined sequences are conserved in the human, rat, and mouse genomes of mmu-miR-22-3p (blue). (E) The mRNA expression of predicted target genes in pathogenic Th17 (23) from control and miR-22−/− mice is measured using qRT-PCR. The expression of genes were normalized to β-actin (Actb) mRNA (A, E, and G). Data are representative of three independent experiments. (F) The protein levels of PTEN in pathogenic Th17 (23) as determined by immunoblot. Tubulin was used as an internal loading control. Data are representative of three biological replicates. (G) Normalized PTEN protein level, with tubulin as an internal control (n = 3). (H) qRT-PCR analysis of IL-2 expression in pathogenic Th17 (23) cells. Data are representative of three independent experiments and shown as mean ± SEM. In unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

To further interrogate the general functions of miR-22 in Th17 (23) cells, we performed RNA-seq analysis for Th17 (23) cells, which differentiated in vitro from miR-22−/− mice and control mice. There were 1177 differentially expressed genes between miR-22−/− and control Th17 (23) cells. Seven hundred and thirty-four genes (blue) were highly expressed in control Th17 (23) cells, whereas 443 genes (red) were upregulated in miR-22−/− Th17 cells (Fig. 5B). Moreover, gene oncology and pathway analysis indicated that the top several gene categories altered in miR-22–/– Th17 cells were mammalian target of rapamycin (mTOR) signaling pathway, sphingolipid metabolism, inositol phosphate metabolism (Fig. 5C), which are PTEN-regulated signaling pathways (37, 38).

Given the TargetScan Web site (www.targrtscan.org/vert_71/) was served to predict the potential target genes of miR-22. In addition, high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a genome-wide means of viewing the RNA world from an RNA-protein interactome perspective. We then overlapped the RNA-seq data with predicted targets from TargetScan and Ago2 HITS-CLIP data in CD4+ T cells (39) (Fig. 5D). Four potential target genes including Pten, Ddit4, Ugcg, and Trp53inp1 were identified (Fig. 5E). We focused our attention on PTEN, as it plays an important role in CD4+ T cells subsets (40). To test whether Pten is a functional target of miR-22 in pathogenic Th17 cells. TargetScan was served to predetermine the potential binding sites of miR-22 in the 3′-untranslated region of Pten (Fig. 5D). We further found the increased expression of Pten in miR-22−/− Th17 (23) cells (Fig. 5F, 5G). In a previous work, PTEN was demonstrated to promote Th17 cell differentiation and pathogenicity by controlling IL-2 production, which was consistent with our findings that the expression of IL-2 was decreased in Th17 cells from miR-22−/− (Fig. 5H) (24). Taken together, our results identify an miR-22–Pten pathway that controls Th17 cell pathogenicity intrinsically and represses autoimmune diseases.

Discussion

Th17 cells are an important helper cell population playing critical roles in barrier tissue integrity, host defense, and autoinflammatory diseases (41, 42). Despite the tremendous efforts made, mechanisms of Th17 cells plasticity in vivo are still not well understood. Our study demonstrates that miR-22 is with increased expression in the inflammatory tissues of EAE mice. Correspondingly, germline miR-22−/− mice exhibit milder symptoms of EAE compared with control mice. Interestingly, loss of miR-22 expression affects neither Th17 cells differentiation nor other CD4+ T cells subsets differentiation in vitro, although the expression of miR-22 is elevated in Th17 differentiation in vitro. One of the fascinating findings is the crucial inflammatory cytokine IL-6 acted as a key regulator for activation of Th17 cells and could sufficiently induce miR-22 expression (43). To further explore the Th17-intrinsic role of miR-22, we generate 2D2 miR-22−/− mice, and the adoptive transfer EAE experiment demonstrates that Th17-intrinsic miR-22 deletion aggravates EAE development and increases the fatality rate. Consistently, pathogenic Th17 cell signature genes are with markedly increased expression in miR-22−/− Th17 cells.

A recent report of miR-22 has shown that dendritic cell miR-22 was essential for promoting Th17-dependent emphysema, and anti–miR-22 treatment reversed mouse emphysema (21). However, Th17-instrinsic role of miR-22 is unclear, although a recent study reported miR-22 may play a role in inflammatory bowel disease progression (28). In our study, we clearly demonstrate that Th17-instrinsic miR-22 is a critical regulator of its pathogenicity via targeting PTEN-mediated pathways. Pten is functioned as a driver of Th17 cells differentiation and aggravated symptoms of EAE model by suppressing IL-2 production (24). Together, our findings so far dissected Th17-intrinsic miR-22 in regulating autoimmune EAE disease and expand our understanding of mechanisms of Th17 pathogenicity.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank all of the laboratory staff for providing technical assistance and useful suggestions in this study.

Footnotes

  • This work was supported and funded by grants from the National Nature Science Foundation of China (31630021), a Shanghai Municipal Key Medical Center Construction Project (2017ZZ01024-002), and the Shanghai Sailing Program (17YF1417900). This work is done by an innovative research team of high-level local universities in Shanghai led by N.S.

  • The RNA sequencing datasets presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE134895.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    EAE
    experimental autoimmune encephalomyelitis
    HITS-CLIP
    high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation
    miR-22−/−
    miR-22–deficient
    MOG
    myelin oligodendrocyte glycoprotein
    MS
    multiple sclerosis
    Pten
    phosphatase and tensin homologue
    qRT-PCR
    quantitative real-time PCR
    RNA-seq
    RNA sequencing
    Treg
    T regulatory cell.

  • Received January 29, 2020.
  • Accepted May 20, 2020.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

References

  1. ↵
    1. Dendrou C. A.,
    2. L. Fugger,
    3. M. A. Friese
    . 2015. Immunopathology of multiple sclerosis. Nat. Rev. Immunol. 15: 545–558.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Thompson A. J.
    2008. Multiple sclerosis--a global disorder and still poorly managed. Lancet Neurol. 7: 1078–1079.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Hellings N.,
    2. J. Raus,
    3. P. Stinissen
    . 2002. Insights into the immunopathogenesis of multiple sclerosis. Immunol. Res. 25: 27–51.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Kleinewietfeld M.,
    2. D. A. Hafler
    . 2014. Regulatory T cells in autoimmune neuroinflammation. Immunol. Rev. 259: 231–244.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Fletcher J. M.,
    2. S. J. Lalor,
    3. C. M. Sweeney,
    4. N. Tubridy,
    5. K. H. G. Mills
    . 2010. T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clin. Exp. Immunol. 162: 1–11.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Galli E.,
    2. F. J. Hartmann,
    3. B. Schreiner,
    4. F. Ingelfinger,
    5. E. Arvaniti,
    6. M. Diebold,
    7. D. Mrdjen,
    8. F. van der Meer,
    9. C. Krieg,
    10. F. A. Nimer, et al
    . 2019. GM-CSF and CXCR4 define a T helper cell signature in multiple sclerosis. Nat. Med. 25: 1290–1300.
    OpenUrlCrossRef
  7. ↵
    1. Kebir H.,
    2. K. Kreymborg,
    3. I. Ifergan,
    4. A. Dodelet-Devillers,
    5. R. Cayrol,
    6. M. Bernard,
    7. F. Giuliani,
    8. N. Arbour,
    9. B. Becher,
    10. A. Prat
    . 2007. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat. Med. 13: 1173–1175.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Robinson A. P.,
    2. C. T. Harp,
    3. A. Noronha,
    4. S. D. Miller
    . 2014. The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment. Handb. Clin. Neurol. 122: 173–189.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Harrington L. E.,
    2. R. D. Hatton,
    3. P. R. Mangan,
    4. H. Turner,
    5. T. L. Murphy,
    6. K. M. Murphy,
    7. C. T. Weaver
    . 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6: 1123–1132.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Stockinger B.,
    2. S. Omenetti
    . 2017. The dichotomous nature of T helper 17 cells. Nat. Rev. Immunol. 17: 535–544.
    OpenUrlCrossRef
  11. ↵
    1. Ivanov I. I.,
    2. K. Atarashi,
    3. N. Manel,
    4. E. L. Brodie,
    5. T. Shima,
    6. U. Karaoz,
    7. D. Wei,
    8. K. C. Goldfarb,
    9. C. A. Santee,
    10. S. V. Lynch, et al
    . 2009. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139: 485–498.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Yang Y.,
    2. M. B. Torchinsky,
    3. M. Gobert,
    4. H. Xiong,
    5. M. Xu,
    6. J. L. Linehan,
    7. F. Alonzo,
    8. C. Ng,
    9. A. Chen,
    10. X. Lin, et al
    . 2014. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens. Nature 510: 152–156.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Langrish C. L.,
    2. Y. Chen,
    3. W. M. Blumenschein,
    4. J. Mattson,
    5. B. Basham,
    6. J. D. Sedgwick,
    7. T. McClanahan,
    8. R. A. Kastelein,
    9. D. J. Cua
    . 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201: 233–240.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Gaublomme J. T.,
    2. N. Yosef,
    3. Y. Lee,
    4. R. S. Gertner,
    5. L. V. Yang,
    6. C. Wu,
    7. P. P. Pandolfi,
    8. T. Mak,
    9. R. Satija,
    10. A. K. Shalek, et al
    . 2015. Single-cell genomics unveils critical regulators of Th17 cell pathogenicity. Cell 163: 1400–1412.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Ghoreschi K.,
    2. A. Laurence,
    3. X. P. Yang,
    4. C. M. Tato,
    5. M. J. McGeachy,
    6. J. E. Konkel,
    7. H. L. Ramos,
    8. L. Wei,
    9. T. S. Davidson,
    10. N. Bouladoux, et al
    . 2010. Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling. Nature 467: 967–971.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Bartel D. P.
    2009. MicroRNAs: target recognition and regulatory functions. Cell 136: 215–233.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Ichiyama K.,
    2. A. Gonzalez-Martin,
    3. B. S. Kim,
    4. H. Y. Jin,
    5. W. Jin,
    6. W. Xu,
    7. M. Sabouri-Ghomi,
    8. S. Xu,
    9. P. Zheng,
    10. C. Xiao,
    11. C. Dong
    . 2016. The microRNA-183-96-182 cluster promotes T helper 17 cell pathogenicity by negatively regulating transcription factor Foxo1 expression. Immunity 44: 1284–1298.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Pan W.,
    2. S. Zhu,
    3. D. Dai,
    4. Z. Liu,
    5. D. Li,
    6. B. Li,
    7. N. Gagliani,
    8. Y. Zheng,
    9. Y. Tang,
    10. M. T. Weirauch, et al
    . 2015. MiR-125a targets effector programs to stabilize Treg-mediated immune homeostasis. Nat. Commun. 6: 7096.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Zhou S.,
    2. Y. Wang,
    3. Y. Meng,
    4. C. Xiao,
    5. Z. Liu,
    6. P. Brohawn,
    7. B. W. Higgs,
    8. B. Jallal,
    9. Q. Jia,
    10. B. Qu, et al
    . 2016. In vivo therapeutic success of microRNA-155 antagomir in a mouse model of lupus alveolar hemorrhage. Arthritis Rheumatol. 68: 953–964.
    OpenUrl
  20. ↵
    1. Escobar T. M.,
    2. C. Kanellopoulou,
    3. D. G. Kugler,
    4. G. Kilaru,
    5. C. K. Nguyen,
    6. V. Nagarajan,
    7. R. K. Bhairavabhotla,
    8. D. Northrup,
    9. R. Zahr,
    10. P. Burr, et al
    . 2014. miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression. Immunity 40: 865–879.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Lu W.,
    2. R. You,
    3. X. Yuan,
    4. T. Yang,
    5. E. L. Samuel,
    6. D. C. Marcano,
    7. W. K. Sikkema,
    8. J. M. Tour,
    9. A. Rodriguez,
    10. F. Kheradmand,
    11. D. B. Corry
    . 2015. The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T(H)17 cell-dependent emphysema. Nat. Immunol. 16: 1185–1194.
    OpenUrl
  22. ↵
    1. Wu X. N.,
    2. Y. X. Ye,
    3. J. W. Niu,
    4. Y. Li,
    5. X. Li,
    6. X. You,
    7. H. Chen,
    8. L. D. Zhao,
    9. X. F. Zeng,
    10. F. C. Zhang, et al
    . 2014. Defective PTEN regulation contributes to B cell hyperresponsiveness in systemic lupus erythematosus. Sci. Transl. Med. 6: 246ra99.
  23. ↵
    1. Junker A.,
    2. M. Krumbholz,
    3. S. Eisele,
    4. H. Mohan,
    5. F. Augstein,
    6. R. Bittner,
    7. H. Lassmann,
    8. H. Wekerle,
    9. R. Hohlfeld,
    10. E. Meinl
    . 2009. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain 132: 3342–3352.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kim H. S.,
    2. S. W. Jang,
    3. W. Lee,
    4. K. Kim,
    5. H. Sohn,
    6. S. S. Hwang,
    7. G. R. Lee
    . 2017. PTEN drives Th17 cell differentiation by preventing IL-2 production. J. Exp. Med. 214: 3381–3398.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Kim D.,
    2. G. Pertea,
    3. C. Trapnell,
    4. H. Pimentel,
    5. R. Kelley,
    6. S. L. Salzberg
    . 2013. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14: R36.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Anders S.,
    2. P. T. Pyl,
    3. W. Huber
    . 2015. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31: 166–169.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Love M. I.,
    2. W. Huber,
    3. S. Anders
    . 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15: 550.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Pei X. F.,
    2. L. L. Cao,
    3. F. Huang,
    4. X. Qiao,
    5. J. Yu,
    6. H. Ye,
    7. C. L. Xi,
    8. Q. C. Zhou,
    9. G. F. Zhang,
    10. Z. L. Gong
    . 2018. Role of miR-22 in intestinal mucosa tissues and peripheral blood CD4+ T cells of inflammatory bowel disease. Pathol. Res. Pract. 214: 1095–1104.
    OpenUrl
  29. ↵
    1. Jäger A.,
    2. V. Dardalhon,
    3. R. A. Sobel,
    4. E. Bettelli,
    5. V. K. Kuchroo
    . 2009. Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes. J. Immunol. 183: 7169–7177.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Garber K.
    2012. Anti-IL-17 mAbs herald new options in psoriasis. Nat. Biotechnol. 30: 475–477.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Mangan P. R.,
    2. L. E. Harrington,
    3. D. B. O’Quinn,
    4. W. S. Helms,
    5. D. C. Bullard,
    6. C. O. Elson,
    7. R. D. Hatton,
    8. S. M. Wahl,
    9. T. R. Schoeb,
    10. C. T. Weaver
    . 2006. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 441: 231–234.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Liu W. H.,
    2. S. G. Kang,
    3. Z. Huang,
    4. C. J. Wu,
    5. H. Y. Jin,
    6. C. J. Maine,
    7. Y. Liu,
    8. J. Shepherd,
    9. M. Sabouri-Ghomi,
    10. A. Gonzalez-Martin, et al
    . 2016. A miR-155-Peli1-c-Rel pathway controls the generation and function of T follicular helper cells. J. Exp. Med. 213: 1901–1919.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Lafaille J. J.,
    2. K. Nagashima,
    3. M. Katsuki,
    4. S. Tonegawa
    . 1994. High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice. Cell 78: 399–408.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Bettelli E.,
    2. M. Pagany,
    3. H. L. Weiner,
    4. C. Linington,
    5. R. A. Sobel,
    6. V. K. Kuchroo
    . 2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. J. Exp. Med. 197: 1073–1081.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Lee Y.,
    2. A. Awasthi,
    3. N. Yosef,
    4. F. J. Quintana,
    5. S. Xiao,
    6. A. Peters,
    7. C. Wu,
    8. M. Kleinewietfeld,
    9. S. Kunder,
    10. D. A. Hafler, et al
    . 2012. Induction and molecular signature of pathogenic TH17 cells. Nat. Immunol. 13: 991–999.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Wang C.,
    2. N. Yosef,
    3. J. Gaublomme,
    4. C. Wu,
    5. Y. Lee,
    6. C. B. Clish,
    7. J. Kaminski,
    8. S. Xiao,
    9. G. Meyer Zu Horste,
    10. M. Pawlak, et al
    . 2015. CD5L/AIM regulates lipid biosynthesis and restrains Th17 cell pathogenicity. Cell 163: 1413–1427.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Song M. S.,
    2. L. Salmena,
    3. P. P. Pandolfi
    . 2012. The functions and regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol. 13: 283–296.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Bu S.,
    2. B. Kapanadze,
    3. T. Hsu,
    4. M. Trojanowska
    . 2008. Opposite effects of dihydrosphingosine 1-phosphate and sphingosine 1-phosphate on transforming growth factor-beta/Smad signaling are mediated through the PTEN/PPM1A-dependent pathway. J. Biol. Chem. 283: 19593–19602.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Loeb G. B.,
    2. A. A. Khan,
    3. D. Canner,
    4. J. B. Hiatt,
    5. J. Shendure,
    6. R. B. Darnell,
    7. C. S. Leslie,
    8. A. Y. Rudensky
    . 2012. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol. Cell 48: 760–770.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Kälin S.,
    2. M. Becker,
    3. V. B. Ott,
    4. I. Serr,
    5. F. Hosp,
    6. M. M. H. Mollah,
    7. S. Keipert,
    8. D. Lamp,
    9. F. Rohner-Jeanrenaud,
    10. V. K. Flynn, et al
    . 2017. A Stat6/Pten axis links regulatory T cells with adipose tissue function. Cell Metab. 26: 475–492.e7.
    OpenUrl
  41. ↵
    1. Lee Y. K.,
    2. J. S. Menezes,
    3. Y. Umesaki,
    4. S. K. Mazmanian
    . 2011. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. USA 108(Suppl. 1): 4615–4622.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Hernández-Santos N.,
    2. A. R. Huppler,
    3. A. C. Peterson,
    4. S. A. Khader,
    5. K. C. McKenna,
    6. S. L. Gaffen
    . 2013. Th17 cells confer long-term adaptive immunity to oral mucosal Candida albicans infections. Mucosal Immunol. 6: 900–910.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Samoilova E. B.,
    2. J. L. Horton,
    3. B. Hilliard,
    4. T. S. Liu,
    5. Y. Chen
    . 1998. IL-6-deficient mice are resistant to experimental autoimmune encephalomyelitis: roles of IL-6 in the activation and differentiation of autoreactive T cells. J. Immunol. 161: 6480–6486.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section