Differentiation of Pathogenic Th17 Cells Is Negatively Regulated by Let-7 MicroRNAs in a Mouse Model of Multiple Sclerosis

Abstract

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Keywords: CCR2; CCR5; CD4; EAE; IL-1R1; IL-23R; miRNA.

Figure 1

Downregulation of let-7 miRNAs upon activation is required for CD4⁺ T cell pathogenicity in EAE. (A) Mean clinical scores in vehicle- (no dox) treated wild-type (WT) (n = 3) and Let-7Tg (n = 4) mice or doxycycline- (+ dox) treated WT (n = 7) and Let-7Tg (n = 7) mice immunized with MOG₃₅₋₅₅ in complete Freund's adjuvant (CFA) and pertussis toxin (60 ng). (B) Number of total mononuclear cells at the peak of the disease (day 9–15 post-immunization) in the CNS of vehicle- (no dox) or doxycycline- (+ dox) treated WT vs. Let-7Tg mice. (C) Number of CNS-infiltrated CD4⁺ T cells at the peak of the disease (day 9–15 post-immunization) in vehicle- (no dox) or doxycycline- (+ dox) treated WT vs. Let-7Tg mice as analyzed by flow cytometry. (D) Intracellular staining of CD4⁺ T cells from the CNS of vehicle- (no dox) or doxycycline- (+ dox) treated WT vs. Let-7Tg mice (left). Numbers indicate the frequencies of cytokine-positive cells within the indicated gates. Quantification of the numbers of cytokine-positive cells as assessed by flow cytometry for each staining strategy (right). *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 (A–D), employing two-way ANOVA (A) or compared with WT using two-tailed Student's t-test (B–D). Data are from two combined independent experiments (A–C; mean ± S.E.M. of each population from all mice), or from one experiment representative of two independent experiments (D; mean ± S.E.M. of each population from all mice).

Figure 2

Let-7 miRNAs negatively regulate CD4⁺ T cell pathogenicity in a cell-intrinsic manner in EAE. (A) Mean clinical scores in Rag2KO recipient mice that received 2D2Rag2KO WT (n = 7), 2D2Rag2KO Let-7Tg (n = 7) or 2D2Rag2KO Lin28Tg (n = 8) naïve CD4⁺ T cells (2–2.5 × 10⁶ cells/recipient) and that were subsequently immunized with MOG₃₅₋₅₅ in complete Freund's adjuvant (CFA) and pertussis toxin (60 ng). (B) Number of total mononuclear cells at the peak of the disease (day 9 post-immunization) in the CNS of Rag2KO recipients that received 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg cells. (C) Number of CNS-infiltrated 2D2Rag2KO CD4+ T cells at the peak of the disease (day 9 post-immunization) in Rag2KO recipients transferred with 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg cells as analyzed by flow cytometry. (D) Intracellular staining of donor CD4⁺ T cells from the CNS of Rag2KO recipients that received 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg cells (left). Numbers indicate the frequencies of cytokine-positive cells within the indicated gates. **p < 0.01, ***p < 0.001, ****p < 0.0001 (A–C), compared with WT employing two-way ANOVA (A) or using two-tailed Student's t-test (B,C). Data are from two combined independent experiments (A–C; mean ± S.E.M. of each population from all mice) or from one experiment representative of two independent experiments (D).

Figure 3

Let-7 miRNAs control the proliferation of CD4⁺ T cells by negatively regulating metabolic reprogramming and cell cycle progression. (A) Survival rate of WT, Let-7Tg and Lin28Tg CD4⁺ T cells activated in vitro for 3 days with α-CD3 and α-CD28 mAbs (5 μg/mL each) as analyzed by trypan blue exclusion. (B) Proliferation of Cell-Trace Violet-labeled WT, Let-7Tg and Lin28Tg CD4⁺ T cells activated in vitro for 3 days α-CD3 and α-CD28 mAbs (5 μg/mL each) as analyzed by flow cytometry. Numbers indicate the cell frequencies within the indicated gates for each genotype. (C) Quantitative RT-PCR analysis of the cell cycle regulators, cyclin D2 (Ccnd2), cyclin-dependent kinase 6 (Cdk6), cell division cycle 25a phosphatase (Cdc25a), and ubiquitin-conjugating enzyme E2 Cdc34 (Cdc34) in naïve CD4⁺ T cells activated with plate-bound α-CD3 and α-CD28 mAbs (5 μg/mL each) for increasing time periods as indicated, presented relative to results obtained for the ribosomal protein Rpl13a (control). (D) Quantitative RT-PCR analysis of the transcription factors Myc (Myc) and AP-4 (Tfap4), as well as Myc direct target genes involved in glycolysis and protein synthesis, glucose transporter 3 (Glut3), hexokinase 2 (Hk2), lactate dehydrogenase A (Ldha), glutamyl-tRNA synthetase (Qars) and tyrosyl-tRNA synthetase (Yars) in naïve CD4⁺ T cells activated with plate-bound α-CD3 mAbs and α-CD28 mAbs (5 μg/mL each) for 48 h, presented relative to results obtained for the ribosomal protein Rpl13a (control). *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 (A,C,D), compared with WT using two-tailed Student's t-test. Data are from one experiment representative of at least two independent experiments (A,C,D; mean ± S.E.M. of technical triplicates of each population from all mice) or from two independent experiments (B).

Figure 4

Let-7 miRNAs inhibit the acquisition of pathogenic Th17 phenotype. (A) Intracellular staining of CD4⁺ T cells from 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg mice polarized in vitro toward the pathogenic Th17 lineage with IL-6, IL-1β, and IL-23. Numbers indicate the frequencies of cytokine-positive cells within the indicated gates. (B) Quantitative RT-PCR analysis of the cytokines IL-17A (Il17a) and GM-CSF (Csf2), the cytokine receptors IL-1R1 (Il1r1) and IL-23R (Il23r), and the transcription factor Bhlhe40 (Bhlhe40) in in vitro-generated pathogenic Th17 cells from 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg mice from (A), presented relative to results obtained for the ribosomal protein Rpl13a (control). (C) Diagram positioning in silico-identified let-7 binding sites (black vertical lines) within the mouse and human mRNA sequences of the cytokine receptors IL1-R1 (Il1r1 and IL1R1, respectively) and IL-23R (Il23r and IL-23R, respectively). *p < 0.05, ****p < 0.0001 (B), compared with WT using two-tailed Student's t-test. Data are from one experiment representative of two independent experiments (A) or from two independent experiments (B; mean ± S.E.M. of technical triplicates of each population from all mice).

Figure 5

Let-7 miRNAs prevent the chemokine-dependent migration of in vitro-generated pathogenic Th17 cells by suppressing the expression of the chemokine receptors CCR2 and CCR5. (A) Quantitative RT-PCR analysis of the chemokine receptors CCR2 (Ccr2) and CCR5 (Ccr5) in in vitro-generated pathogenic Th17 cells from 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg mice, presented relative to results obtained for the ribosomal protein Rpl13a (control). (B) Diagram positioning in silico-identified conserved (red vertical lines) and non-conserved (black vertical lines) let-7 binding sites within the mouse and human mRNA sequences of the chemokine receptors CCR2 (Ccr2 and CCR2, respectively) and CCR5 (Ccr5 and CCR5, respectively). (C) Luciferase reporter assay of let-7 targeting in-silico-identified let-7-binding sites in mouse Ccr2 or Ccr5 mRNA, in NIH/3T3 cells transfected with a luciferase reporter vector containing either the wild-type or mutated variants of these binding sites, or either the wild-type or a mutated variant of the antisense seed sequence of let-7g (controls). Results are presented as relative luminescence units (RLU), calculated by normalization of Firefly luciferase activity to Renilla luciferase activity (control). (D) Transwell migration assay of in vitro-generated pathogenic Th17 cells from 2D2Rag2KO WT, 2D2Rag2KO Let-7Tg, and 2D2Rag2KO Lin28Tg mice from (A) in response to the chemokines CCL2 (50 ng/mL) and CCL4 (50 ng/mL) alone or in combination (50 ng/mL or 10 ng/mL each). Results are presented as percentage of cell migration in media only control, defined as 100%. (E) Transwell migration assay of in vitro-generated pathogenic Th17 cells from 2D2Rag2KO WT and 2D2Rag2KO Let-7Tg mice, transduced with empty vector (solid bars), Ccr2-overexpression vector (horizontally-striped bars), and Ccr5-overexpression vector (diagonally-striped bars) in response to the chemokines CCL2 (50 ng/mL) and CCL4 (50 ng/mL) alone. Results are presented as percentage of cell migration in media only control, defined as 100%. *p < 0.05, **p < 0.01; ***p < 0.001, ****p < 0.0001 (A,C,D,E), compared with WT using two-tailed Student's t-test. Data are from one experiment representative of at least two independent experiments (A,C,D,E; mean ± S.E.M. of technical triplicates).