miR-223 Regulates mDC-Driven T_H17 Responses in Experimental Autoimmune Encephalomyelitis

Abstract

Myeloid cells play a crucial role in the induction and sustained inflammation in neuroinflammatory disorders such as MS. miR-223, a myeloid cell-specific miRNA, is one of the most upregulated miRNAs in MS patients. We demonstrate that miR-223 KO mice display significantly reduced active and adoptive transfer EAE characterized by reduced numbers of myeloid DCs (mDCs) and T_H17 cells in the CNS. KO mDCs have increased PD-L1 and decreased IL-1β, IL-6 and IL-23 expression and a reduced capacity to drive T_H17, but not T_H1, cell differentiation. Thus miR-223 controls mDC-induced activation of pathologic T_H17 responses during autoimmune inflammation.

Introduction

Blood-derived myeloid cells comprise a significant proportion of the infiltrating perivascular leukocytes in CNS lesions in MS (1) and its animal model, EAE (2, 3). These APCs have the ability to phagocytize myelin debris and to secrete cytokines which promote the expansion and polarization of CD4⁺ T_H1 and T_H17 T cells critical in the initiation and progression of EAE (2).

Ly6c^hiCD11b⁺CD11c⁺ monocyte-derived dendritic cells (Mo-DCs) are a prominent component of CNS-infiltrates cells in EAE (2, 3). Mo-DCs arise from monocytes following their migration into inflamed tissues (4). mDCs are responsible for initiating epitope spreading during EAE within the CNS, inducing the differentiation of T_H17 cells (2, 5, 6). In addition, Ly6C^hi DCs are critical for T_H17 polarization (7).

MicroRNAs (miRNAs) are small non-coding RNAs of 17 to 25 nucleotides that regulate gene expression by inducing mRNA degradation or interfering with mRNA translation (8). More than 60% of protein-coding genes are regulated by miRNAs (9). miRNAs regulate various biological processes including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis (10) and are involved in the pathogenesis of cancer, inflammatory and autoimmune diseases (11).

Expression studies on tissues from MS patients identified miR-223 as one of the top upregulated miRNAs (12, 13). miR-223 expression is mainly confined to myeloid cells (14) has been shown to regulate the proliferation and activation of neutrophils (15) and M1 macrophages (16). Here we demonstrate that miR-223 regulates EAE pathogenesis by controlling mDC-induced activation of pathogenic T_H17 cells.

Materials and Methods

Mice

Female WT C57BL/6 and miR-223^−/− mice were purchased from The Jackson Laboratory and housed in the Northwestern Center for Comparative Medicine SPF facility. Identical results were obtained using either littermate or wildtype C57BL/6 mice as controls.

Active and Transfer EAE

8-10 week-old female mice were injected s.c. with 100 μl of an emulsion containing 200 μg of M. tuberculosis H37Ra (Difco, Detroit, MI) and 200 μg of MOG_35–55 (Genemed, San Francisco, CA) distributed over three sites on the flank. For transfer EAE, spleen and inguinal lymph nodes collected on day 8 post-MOG_35-55 were reactivated with 20 μg/ml of MOG_35–55 and 10 ng/ml recombinant murine IL-12 (R&D Systems) for 72 h. 20×10⁶ cells were transferred to recipient mice. In both models, 200 ng pertussis toxin (List, Campbell, CA) were administered i.p on days 0 and 2. 2-3 independent experiments were performed, with ≥5 mice per group. Clinical signs of EAE were assessed daily as previously described (2).

Isolation of CNS leukocytes

CNS-immune cells were isolated by Percoll gradient centrifugation from homogenized combined brain and spinal cords and enumerated as previously described (2).

Flow Cytometry Analysis

Fc receptors were blocked using anti-mouse CD16/32 (0.25 μg; eBioscience) and cells were then stained for 30 min at 4°C using the specified antibodies (BD Biosciences or eBioscience). Cytokine expression was determined in activated T cells (18 h with 1 μg/ml ionomycin and 20 ng/ml PMA) and APCs (18 h with 10 ng/ml E. coli 0111:B4 LPS (Sigma) in the presence of 2 μg/ml brefeldin A (Sigma). Cells were acquired on a BD Canto II and analyzed using BD FACSDiva version 6.1.

Th1/Th17 cell differentiation

Purified CD4⁺ T cells (≥90%) were cultured with CD4⁻ WT or KO splenocytes (as APCs) plus α-CD3 (1 μg/ml) in Th1- (200 U/ml IL-2, 10 ng/ml IL-12, 1 μg/ml anti-IL-4) or Th17- (10 ng/ml TGF-β, 50 ng/ml IL-6, 10 ng/ml IL-23, 1 μg/ml anti-IFN-γ, 1 μg/ml anti-IL-4, 1 μg/ml anti-IL-2) promoting conditions and analyzed for cytokine expression by flow cytometry.

Bone marrow monocyte isolation

CD11b⁺ Ly6C^hi monocytes were purified (>95%) from BM via magnetic cell sorting (Miltenyi).

Quantitative real-time PCR analysis

CD11b⁺ myeloid cells, B220⁺ B cells and CD90⁺ T lymphocytes were purified using magnetic cell sorting (Miltenyi) and gene expression quantification performed as previously described (15).

Statistical analysis

Statistical analyses were performed using GraphPad PRISM 5.0. Single comparisons of two means were analyzed by Student's t-test. For multiparametric data, two-way ANOVA with a Bonferroni post-test was used. p values < 0.05 were considered significant.

Results and Discussion

miR-223 regulates EAE pathology

We observed a significant delay of the onset and severity of MOG_35-55-induced EAE in miR-223 KO vs. WT mice (Fig. 1A) concomitant with a reduction of CNS infiltration of CD45^hiCD11b⁺Ly6G⁻CD11c⁺ mDCs and CD45^hiCD11b⁻CD3⁺ CD4⁺ T cells (Fig. 1B). The numbers of macrophages/monocytes, neutrophils, and B cells and pDCs did not differ (Supplemental Fig. S1A). We observed a significant reduction of mo-DCs (Ly6^hi) in the CNS of miR-223 KO mice (Fig. 1C) and reduced percentages and numbers of CD4⁺ IL-17⁺, CD4⁺ GM-CSF⁺, and IL-17⁺/IFN-γ⁺ and IL-17⁺/GM-CSF⁺ cells (Fig. 1D, Supplemental Fig. S1B) confirming the importance of Ly6C^hi mDCs in CNS inflammation (2, 6, 17).

FIGURE 1. Deletion of miR-223 reduces EAE clinical scores and CNS infiltration of mDCs and CD4⁺ T cells.

EAE was induced in C57BL/6 WT mice and miR-223 KO mice by active immunization with MOG_35-55. (A) miR-223 KO mice (○) show a significant reduction in clinical scores as compared to WT animals (●). Mean cumulative scores were calculated as the mean of the sum of daily clinical scorers observed for the duration of the experiment (n = 6 mice per group; *, p < 0.05; ***, p < 0.001). (B) Number of CD45^hi CD11b⁺ Ly6G⁻ CD11c⁺ mDCs, CD45^hi CD11b⁺ Ly6G⁻ CD11c⁻ macrophages/monocytes, CD45^hi CD11b⁺ Ly6G⁺ Ly6C⁺ neutrophils, and CD45^hi CD11b⁻ CD3⁺ CD4⁺ T cells in the CNS of EAE miR-223 KO mice (black bars), or WT mice (white bars) at day 18 post-immunization (n = 3 mice per group; *, p < 0.05, **, p < 0.01). (C) Proportion of Ly6C^hi mo-DCs is reduced in the CNS (brain and spinal cord homogenates) of MOG_35-55-immunized miR-223 KO mice (black bar), as compared to WT animals (white bar) at day 18 post-immunization (n = 3 mice per group; *, p < 0.05). (D) Percentages of IL-17, IFN-γ, GM-CSF and TGF-β-expressing CD4⁺ T cells in the CNS of EAE animals revealed a significant reduction of T cells producing IL-17-related cytokines in miR-223 KO animals (black bars), as compared to WT animals (white bar) at day 18 post-immunization (n = 3 mice per group; *p < 0.05). Data shown are representative of three independent experiments performed on ≥5 mice per group.

miR-223 is expressed in myeloid cells

CD11b⁺ myeloid cells had the highest basal level of miR-223 expression (Fig. 2A) and expression is upregulated during EAE with comparable levels at disease onset and peak. miR-223 was not expressed in B and T cells. In transfer EAE, disease onset is delayed and severity significantly reduced in miR-223 recipients of WT MOG_35-55-specific CD4⁺ T cells (Fig. 2B). Collectively, these results indicate a critical role for miR-223 in peripheral and CNS myeloid APCs required for EAE initiation. Interestingly, the fact that miR-223 controls astrocyte expression of CXCL12, a chemokine which attracts CD4+ T cells and myeloid cells (18), may provide a potential mechanistic basis for the reduced disease severity and immune cell infiltration observed in KO mice.

FIGURE 2. miR-223 is expressed in myeloid cells and is upregulated during EAE.

(A) CD11b⁺ myeloid cells, B220⁺ B cells and CD90⁺ T cells were isolated from the spleen of naïve WT mice and EAE mice at onset (day 8 post-immunization) and peak of the disease (day 15 post-immunization) and miR-223 gene expression was quantified by real-time PCR. A significant increase of miR-223 gene expression was observed at EAE onset and peak in the myeloid cell compartment (white bars) (n = 3 to 5 mice per time-point; ***, p < 0.001). (B) 20 × 10⁶ MOG_35-55-reactivated spleen and lymph nodes collected at day 8 post-immunization of WT C57BL/6 animals with MOG_35-55/CFA were injected i.p. into miR-223 KO or WT recipient mice. miR-223 KO mice (○) display a significant reduction in clinical scores as compared to WT animals (●) (***p < 0.001). Data shown are representative of 2 independent experiments performed on ≥5 mice per group.

Reduced T_H17 polarizing cytokines in miR-223 KO mDCs

PD-L1, but not PD-L2, expression was significantly higher on mDCs and BM-monocytes (Fig. 3A, Supplemental Fig. S2A) and CD40 and CD80 expression was enhanced on splenic macrophage/monocytes, but not pDCs, of MOG_35-55-primed KO mice (Supplemental Fig. S2B). These results are in direct accordance with the fact that miR-223 regulates STAT1 expression (19), which is a crucial regulator of PD-L1 expression on mDCs (20). Spleen mDCs from the KO mice expressed significantly lower levels IL-1β, IL-23p19 and IL-6, but control levels of IL-12p40 and TNF (Fig. 3B and Supplemental Fig. S2C). Splenic macrophages/monocytes from miR-223 KO mice had increased expression of TNF, but there were no differences in cytokine expression in splenic pDCs. These data suggest that mDCs from miR-223 KO have a lower capacity to activate T cells due to the PD-L1 increase and, in particular, T_H17 cells due to the decreased expression of T_H17-promoting cytokines (IL-1β, IL-6 and IL-23). These findings are particularly interesting in that miR-223 regulates Roquin-1 expression by binding to the 3’ UTR (21). Roquin-1 is an important regulator of expression of T_H17-related cytokines and T_H17 differentiation (22) and its loss leads to overproduction of IL-6 and increases T_H17 cell differentiation. Mice expressing a M199R mutation in the Roquin protein (Roquin^san/san mice) develop autoimmune pathologies (23) and Roquin-1 was shown to regulate innate and inflammatory responses in autoimmunity (24). In addition, miR-223 regulates genes involved in the inflammasome pathway, cytokine-cytokine receptor interactions, and signaling via the TLR, JAK-Stat, FcεRI, NOD-like receptor, and NF-κb pathway, pathways (25). We are currently analyzing these possibilities with an emphasis on Roquin.

FIGURE 3. Splenic mDCs from miR-223 KO mice express reduced levels of T_H17 polarizing cytokines during EAE.

At day 16 post MOG_35-55 immunization, spleens from miR-223 KO mice and WT mice were homogenized and immune cells were isolated. Cells were gated on CD11b⁺ B220⁻ CD3⁻ CD11c⁺ to analyze the profile of mDCs by flow cytometry. (A) Expression levels of PD-L1 and PD-L2 on splenic mDCs of WT mice (left panel) and miR-223 KO mice (right panel) was examined. (B) IL-23p19, IL-1β and IL-6 expression on splenic mDCs are decreased in miR-223 KO mice (right panels) compared to WT mice (left panels). No difference in TNF and TGF-β were observed. Data shown are representative of 6 mice in 2 independent experiments.

miR-223 KO APCs fail to drive differentiation of T_H17 cells

Naïve CD4⁺ T cells from WT mice were cultured with anti-CD3 and CD4⁻ splenic APCs from naïve WT or miR-223 KO mice in both T_H1 or T_H17 driving conditions and T cell differentiation was assessed at days 1, 3 and 5. In T_H1 skewing conditions, although the percentage of IFN-γ⁺ cells was reduced at day 3 with KO APCs, WT and KO APCs induced equivalent numbers of T_H1 cells at day 5 (Fig. 4A). In contrast, KO APCs were significantly less capable of inducing differentiation of both IL-17- and GM-CSF-producing T_H17 cells with virtually no GM-CSF, a cytokine reported to be essential in the pathogenic process of EAE (26), produced at day 5. The low levels of IFN-γ observed in T_H17 conditions confirm the specific skewing of the T_H17 differentiation.

FIGURE 4. APCs from miR-223 KO mice are deficient in driving differentiation of T_H17 cells.

CD4⁺ T cells were purified from spleens of naïve miR-223 KO mice. The cells were co-cultured with CD4⁻ splenic APCs from naïve WT mice or miR-223 KO mice in (A) T_H1-promoting [IL-2 (200 U/ml), IL-12 (10 ng/ml), and anti-IL-4 (1 μg/ml)] or (B) T_H17-promoting [TGF-β (10 ng/ml), IL-6 (50 ng/ml), IL-23 (10 ng/ml), anti-IL-2, anti-IFN-γ, and anti-IL-4 (1 μg/ml)] conditions. Flow cytometry analyses for IFN-γ, IL-17 and GM-CSF expression by live CD3⁺ CD4⁺ T cells was performed after 1, 3 and 5 days of culture. The origin of the APCs had no effect on differentiation of IFN-γ producing T cells in T_H1-driving conditions. However, in T_H17-driving conditions, CD4⁺ T cells showed a significant decrease in IL-17 and GM-CSF expression when co-cultured with miR-223 KO APCs, as compared to co-culture with WT APCs. Data shown are representative of 3 independent experiments (*p < 0.05; ***p < 0.001).

Collectively, our results uncover a new role for miR-223 in mDCs and emphasize the important role it plays in the development of CNS inflammation by its ability to regulate induction of T_H17 responses by controlling levels of Th17 polarizing cytokines, including IL-1β, IL-6 and IL-23. Additionally, miR-223 may participate in regulation of monocyte differentiation into mo-DCs in inflamed environments. In the absence of miR-223, C57BL/6 mice exhibit significantly lower clinical signs of EAE and associated peripherally-derived CNS-infiltrating inflammatory cells, confirming the importance of this miRNA in the disease pathology and identifying a new potential autoimmune regulatory target.

Abbreviations

mDC

myeloid dendritic cell

miRNAs

micro-RNAs

moDC

monocyte-derived dendritic cell

PMA

phorbol myristate acid