Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Feb 1.
Published in final edited form as: J Immunol. 2019 Dec 18;204(3):531–539. doi: 10.4049/jimmunol.1801113

IL-9 controls CNS autoimmunity by suppressing GM-CSF production

Satoshi Yoshimura *,†,1, Rodolfo Thome *,1, Shingo Konno *,‡, Elisabeth R Mari *, Javad Rasouli *, Daniel Hwang *, Alexandra Boehm *, Yanhua Li *, Guang-Xian Zhang *, Bogoljub Ciric *, Abdolmohamad Rostami *
PMCID: PMC7197375  NIHMSID: NIHMS1544283  PMID: 31852750

Abstract

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are inflammatory diseases of the central nervous system (CNS) in which T helper (Th) 17 cells play a major role in the disease pathogenesis. Th17 cells that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) are pathogenic and drive inflammation of the CNS. IL-9 is a cytokine with pleiotropic functions, and it has been suggested that it controls the pathogenic inflammation mediated by Th17 cells, and IL-9R−/− mice develop more severe EAE compared to wild-type (WT) counterparts. However, the underlying mechanism by which IL-9 suppresses EAE has not been clearly defined. Here we investigated how IL-9 modulates EAE development. By using mice knockout for IL-9 receptor (IL-9R), we show that more severe EAE in IL-9R−/− mice correlates with increased numbers of GM-CSF+ CD4+ T cells and inflammatory dendritic cells (DCs) in the CNS. Furthermore, DCs from IL-9R−/− mice induced more GM-CSF production by T cells and exacerbated EAE upon adoptive transfer than did WT DCs. Our results suggest that IL-9 reduces autoimmune neuroinflammation by suppressing GM-CSF production by CD4+ T cells through the modulation of DCs.

Introduction

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are neuro-inflammatory diseases mediated by leukocytes that infiltrate the central nervous system (CNS), activate microglia and astrocytes leading to axonal loss (1-3). T helper (Th) 17 cells that produce granulocyte-macrophage colony-stimulating factor (GM-CSF) are primary drivers of CNS inflammation by activating CNS-infiltrating myeloid cells, such as monocytes and dendritic cells (DCs), and perpetuating inflammation (4, 5). The observation that CSF2−/− mice are resistant to EAE confirms the central role of GM-CSF in EAE (6, 7). Thus, suppression of GM-CSF-producing cells and activated myeloid cells is needed to reduce inflammation and disease progression in EAE.

IL-9 is a cytokine initially associated with Th2 responses in helminth infection, asthma and chronic obstructive pulmonary disease (8-11). In addition to Th2 cells, Th17, Treg, and mast cells secrete IL-9 as well (12). Although it was initially reported that IL-9 has a deleterious role in multiple inflammatory diseases, more recent evidence shows that IL-9 limits pathogenic autoimmune responses (13-16). For example, adoptive EAE experiments with polarized MOG35-55-specific Th9 and Th17 cells showed that transfer of Th9 cells induced a milder EAE compared to the transfer of Th17 cells (17). Likewise, IL-9R−/− mice develop a much more severe EAE compared to WT controls (13). These observations show that IL-9 has an immunoregulatory role in the context of EAE; however, the mechanisms by which IL-9 suppresses disease are not well understood.

Here we confirm that absence of IL-9 or its receptor worsens EAE symptoms compared to WT mice, likely due to increased frequency of GM-CSF+ Th cells and activation of DCs in the CNS. We further show that administration of IL-9 delays disease development by suppressing GM-CSF production by Th cells and by modulating DCs. Lastly, adoptive transfer of IL-9R−/− DCs into WT mice led to an increase in disease severity by increasing numbers of GM-CSF+ Th cells while adoptive transfer of WT DCs to IL-9R−/− mice did not affect EAE development. In summary, these results show that IL-9 controls EAE by modulating GM-CSF production by T cells through DCs.

Materials and Methods

Mice

Eight to 12 weeks old C57BL/6 mice and 2D2 mice (C57BL/6 background) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Dr. J.-C. Renauld (Ludwig Institute, Brussels, Belgium) and Dr. A.N.J McKenzie (MRC Laboratory of Molecular Biology, Cambridge, UK) kindly provided homozygous breeding pairs of IL-9R−/− and IL-9−/− mice on the C57BL/6 background. For all experiments, age and sex-matched mice were used. Every effort was made to minimize the suffering of mice. Thomas Jefferson University’s Institutional Animal Care and Use Committee approved all experiments described in this study.

EAE induction and evaluation

Anesthetized mice were subcutaneously injected with 200 μg of MOG35-55 peptide (MEVGWYRSPFSRVVHLYRNGK, Genscript, NJ, USA), and an equal volume of Complete Freund’s adjuvant supplemented with 10 mg/mL of heat-killed Mycobacterium tuberculosis H37Ra (Difco Lab, Detroit, MI, USA) at two sites on the back. Additionally, mice were intraperitoneally injected with 200 ng of pertussis toxin (Sigma-Aldrich, St. Lous, MO, USA) in PBS on days 0 and 2 p.i.. Disease development was analyzed daily and scored according to the following scales: 0, no clinical sign; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and front limb weakness; 5, full paralysis/death. A cumulative score was calculated as the sum of all daily scores of each mouse divided by the number of mice in each group.

IL-9 treatment

Mice were intraperitoneally (i.p.) injected with 1 μg of recombinant murine IL-9 (R&D Systems, Minneapolis, USA) dissolved in 0.2 mL of PBS on days 1, 4 and 7 p.i.. Control mice received 0.2 mL of PBS only.

Isolation of CNS cells and flow cytometry

To analyze cells that infiltrated the CNS of EAE mice, anesthetized mice were perfused with ice-cold PBS, and the brains and spinal cords were collected. CNS tissue was enzymatically digested in Liberase TL (Sigma-Aldrich, USA) at 37°C for 30 minutes and the CNS infiltrating cells were enriched through centrifugation in an isosmotic 70%/30% Percoll gradient. Mononuclear cells were recovered from the Percoll interface, washed and stimulated in complete Iscove’s Modified Dulbecco’s Medium (IMDM) containing PMA (50 ng/mL), ionomycin (500 ng/mL) in the presence of GolgiPlug (1 μg/1×106 cells) for 4 h at 37°C. Cells were then surface stained with fluorochrome-conjugated Abs against CD45 (30-F11), CD3 (17A2), CD4 (RM4-5, BD Biosciences), CD11b (M1/70), CD11c (N418), CD25 (PC61), CD40 (3/23), CD80 (16-10A1), CD86 (GL1, BD- Biosciences), and MHC-II (M5/114.15.2) for 20 minutes at 4°C. For intracellular staining, cells were fixed and permeabilized using commercial reagents (ThermoFischer) and incubated with Abs against IFN-γ (XMG 1.2, BD Biosciences), IL-6 (MP5-20F3), IL-9 (RM9A4), IL-10 (JES5-16E3), IL-12/23p40 (C15.6), IL-17 (TC11-18H10.1), GM-CSF (MP1-22E9) and Foxp3 (FJK-16s, eBioscience) for 30 minutes at 4°C. Unless otherwise stated, all antibodies were from Biolegend. Samples were acquired on a FACSAria Fusion (BD Bioscience) and analyzed using FlowJo Software (Tristar Inc.).

Antigen-presentation assays

Spleens were collected from EAE mice and dissociated through 70 μm cell strainer to prepare single cell suspensions. After treatment with RBC lysis buffer (Biolegend, CA, USA) cells were extensively washed with complete IMDM by centrifugation at 1300 rpm for 5 minutes at 4°C. Splenic CD11c+ DCs were isolated by positive selection with magnetic beads (CD11c+ cell isolation kit, Miltenyi Biotec, CA, USA) and seeded in a 96-well U-bottom plate at a concentration of 20,000 DCs/well. Bead isolated MOG35-55-reactive naïve CD4+ T cells were seeded over DCs at 200,000 T cells/well. Additionally, MOG35-55 peptide was added to cultures at a 25 μg/mL concentration. Cultures were incubated for 72 h at 37°C, and dye decay was analyzed by flow cytometry in CD4+ T cells.

Gene expression analysis by real-time PCR

RNA extraction and cDNA synthesis were conducted using commercially available kits (RNeasy Mini kit, Qiagen; and High Capacity cDNA Reverse Transcriptional Kit, Thermo). Gene expression of IL1B (Mm00434228_m1), IL6 (Mm00446190_m1), IL10 (Mm01288386_m1), IL12A (Mm00434165_m1), IL17A (Mm00439618_m1), IL23A (Mm00518984_m1), IL27 (Mm00461162_m1), IFNG (Mm00801778_m1), IDO1 (Mm00492590_m1), FOXP3 (Mm01268569_m1) and IL-9R (Mm0119343_g1) were analyzed in comparison to GAPDH (Mm99999915_g1, used as an endogenous control) by TaqMan real-time PCR (Applied Biosystems, Foster City, CA). Gene expression levels are shown as a relative copy number by using the method of delta threshold (2-ΔΔCt).

Adoptive transfer of WT and IL-9R−/− DCs

For adoptive transfer experiments, we anesthetized WT and IL-9R−/− mice and injected through the retro-orbital route 1.5 million CD11c+ DCs per mouse. The mouse was allowed to recover for three days and was subsequently subjected to EAE induction. CD11c+ DCs were isolated from spleens of WT and IL-9R−/− mice as described above. Disease development was evaluated daily.

Statistical analysis

Comparisons of daily clinical scores among experimental groups with EAE were carried out by Two Way ANOVA and Bonferroni post-test. Analyses between three or more groups were performed with One-Way ANOVA and post-tested with Bonferroni. Comparisons between two groups were carried out with Studentś t test. Values of p < 0,05 were considered significant.

Results

IL-9R deficiency aggravates EAE by increasing the frequencies of pathogenic GM-CSF-producing CD4+ T cells and inflammatory DCs in the CNS

To study the immunomodulatory role of IL-9 in CNS autoimmunity, we immunized WT and IL-9R−/− mice to induce EAE and checked them daily for signs of disease. IL-9R−/− mice developed earlier and more sever disease compared to WT mice (Fig. 1A and B). Interestingly, disease in IL-9R−/− mice was more severe until the beginning of chronic phase (around day 22 p.i.) when it became similar to disease in WT mice (Fig. 1A). Our results slightly contrast those from Elyaman et al. wherein EAE in IL-9R−/− mice was more aggressive than in WT mice throughout the course of the disease (13). Although the finding that IL-9R−/− mice develop more severe EAE in both studies is similar, we credit this minor discrepancy to the amount of the immunizing agent used in both studies. Given that myelin degeneration plays a major role in the chronic phase of MOG35-55-induced EAE whereas inflammation is more prevalent in the acute phase, this result suggests that the effect of IL-9 in CNS autoimmunity is limited to leukocytes and inflammation rather than tissue degeneration. Accordingly, at day 15 p.i. the CNS of IL-9R−/− mice had significantly more CD45+ leukocytes than WT mice (Fig. 1C). Numbers of CD4+ T cells and CD11c+ DCs obtained from the CNS of IL-9R−/− mice were greater than from the CNS of WT mice, but this trend did not reach statistical significance (Fig. 1C). Histological analysis confirmed that IL-9R−/− mice had more infiltrating cells in the spinal cords than WT mice (Supplementary Figure 1). An analogous experiment conducted with IL-9−/− mice gave us similar results (data not shown).

Figure 1. IL-9R−/− mice develop earlier and more severe EAE with increased frequency of GM-CSF+CD4+ T cells in the CNS.

Figure 1.

Open in a new tab

WT and IL-9R−/− mice (n = 5 each group) were immunized with MOG35-55 to develop EAE. (A) Daily clinical scores of disease severity, (B) Cumulative scores of disease severity. (C) The numbers of CD45+ CNS-infiltrating cells, CD4+ T cells and DCs, and frequency of DCs were determined by flow cytometry at day 15 post-immunization (p.i.). (D) Flow cytometry analysis of IFN-γ, IL-9, IL-10, IL-17, GM-CSF and Foxp3+ CD4+ T cells from the CNS of EAE mice. (E) RNA was isolated from the spinal cords at day 15 p.i. and analyzed for the expression of IL1B, IL6, IL10, IL12A, IL17A, IL23A, IL27, IFNG, FOXP3 and IDO1. Bar graphs depict mean ± standard error mean (SEM). Values of P < 0.05 (*), P < 0.01 (**), P < 0.001 (***),were considered statistically significant. NS, not significant.

Given that EAE is mainly a Th cell-driven disease, we analyzed the phenotype of CD4+ T cells in the CNS of EAE mice. The frequencies of GM-CSF+ CD4+ T cells were significantly increased in IL-9R−/− mice compared to WT mice, while no difference was observed in IL-17+, IFN-γ+, IL-10+ and Foxp3+ CD4+ T cells (Fig. 1D). Study of the gene expression of inflammatory mediators in the CNS tissue of EAE mice at day 15 p.i. revealed that IL-9R−/− mice had greater expression of IL1B, IL6, IL12A, IL17 and IFNG in IL-9R−/− mice than WT mice (Fig. 1E). Given that Indoleamine 2,3 Di-oxygenase (IDO) plays an important role in suppression of the T cell responses , we also analyzed IDO (encoded by IDO1 gene) and we found that IL-9R−/− mice had a reduced expression of IDO1 compared to WT mice (Fig. 1E). GM-CSF is a hallmark cytokine of pathogenic Th17 cells in EAE (4, 5). Thus an increased frequency of GM-CSF+ CD4+ T cells in IL-9R−/− mice suggests that IL-9 negatively regulates GM-CSF production by T cells. Inflammatory cytokines produced primarily by APCs (IL1B, IL6, IL12A) were among the most upregulated genes in the CNS tissue of IL-9R−/− mice, suggesting that IL-9 regulates the activation of APCs.

To test this hypothesis, we analyzed the phenotype of CNS-infiltrating DCs of IL-9R−/− and WT EAE mice (Fig. 2A). Although levels of CD40, CD80 and CD86 were similar between IL-9R−/− and WT mice, CD11c+ cells of IL-9R−/− mice expressed significantly more of MHC class II (Fig. 2B). Further analysis revealed that DCs from IL-9R−/− EAE mice produced significantly more IL-6 than DCs from WT EAE mice (Fig. 2C). No significant difference in the production of IL-10 and IL12/23p40 was observed (Fig. 2C).

Figure 2. Lack of IL-9R signaling increases inflammatory DCs in the CNS.

Figure 2.

Open in a new tab

WT and IL-9R−/− mice (n = 5 each group) were immunized with MOG35-55 to induce EAE. (A) Mice were dissected at day 15 p.i. DCs were identified by a surface marker combination as CD11c+ MHCII+. (B) Flow cytometry analysis of the expression of MHC II, CD40, CD80 and CD86 on DCs. (C) Flow cytometry analysis of IL-6, IL-10 and IL-12/23+ DCs in the CNS of EAE mice. Bar graphs depict mean ± standard error mean (SEM). Values of P < 0.05 (*) were considered statistically significant. NS, not significant.

To investigate the inflammatory profile of IL-9R−/− DCs, we immunized IL-9R−/− and WT mice with MOG35-55 and analyzed the gene expression of cytokines involved in antigen presentation by splenic DCs at day 10 p.i. IL-9R−/− DCs expressed significantly more IL1B, IL6 and IL23A compared to WT DCs (Fig. 3A). Conversely, IL27 expression was significantly decreased in IL-9R−/− DCs compared to WT DCs (Fig. 3A). When cultivated with naïve MOG35-55-reactive CD4+ T cells from 2D2 mice, IL-9R−/− DCs induced IFN-γ production by T cells more efficiently than WT DCs (Fig. 3B).

Figure 3. IL-9R−/− DCs from EAE mice induce IFN-γ production by CD4+ T cells.

Figure 3.

Open in a new tab

IL-9R−/− and WT mice (n = 3 per group) were immunized with MOG35-55 to induce EAE. At day 10 p.i., CD11c+ DCs in the spleen were isolated using magnetic beads. (A) Gene expression of IL1B, IL6, IL10, IL12A, IL23A and IL27. (B) Isolated DCs were co-cultured with 2D2 naïve CD4+ T cells in the presence of MOG at 37°C. After 72 h, cells were analyzed for production of IFN-γ, IL-10, IL-17 and GM-CSF by CD4+ T cells by flow cytometry. Bar graphs depict mean ± standard error mean (SEM). Values of P < 0.05 (*), P < 0.01 (**) were considered statistically significant. NS, not significant.

Exogenous IL-9 reduces EAE by suppressing pathogenic GM-CSF-producing CD4+ T cells and inflammatory DCs in the CNS

Our data showed that absence of IL-9R or IL-9 promotes EAE development by increasing the influx of pathogenic leukocytes in the CNS. To test our hypothesis that IL-9 negatively modulates the activity of DCs and T cells, we immunized WT mice and administered recombinant (r) IL-9 in the priming phase of the disease at days 1, 4 and 7 p.i. rIL-9 delayed disease onset and significantly suppressed disease severity in the acute phase compared to PBS-treated EAE mice (Fig. 4A and B). Ultimately, at the beginning of chronic phase, disease in mice treated with rIL-9 and PBS became similar (not shown); rIL-9 was unable to suppress EAE when administered at the chronic phase of disease (Supplementary Figure 2) indicating that the effect of IL-9 is limited to the inflammatory phase of EAE. Given that absence of IL-9R led to an increase in the influx of cells in the CNS, we investigated whether administration of rIL-9 to WT mice would have the opposite effect. We found that EAE mice receiving rIL-9 had similar numbers of CD45+ cells in the CNS compared to PBS-treated EAE mice (Fig. 4C). Although numbers of CD4+ T cells and CD11c+ DCs were not significantly different among groups, rIL-9-treated EAE mice had reduced total numbers and frequency of CD11c+ DCs compared to PBS-treated controls (Fig. 4C). Histological analysis confirmed that rIL-9 treatment significantly reduced the infiltration of leukocytes in the spinal cords (Supplementary Figure 3).

Figure 4. IL-9 treatment suppress EAE.

Figure 4.

Open in a new tab

WT mice (n = 5 per group) were immunized to develop EAE. At days 1, 4, and 7 p.i., mice were i.p. injected with 1 μg of recombinant IL-9 (rIL-9) or PBS for control. (A) Daily clinical scores of disease severity, (B) Cumulative scores of disease severity, (C) Mice were sacrificed at day 21 p.i. and the numbers of CD45+ cells, CD4+ T cells and DCs in the CNS were determined by flow cytometry. (D) Flow cytometry analysis of IFN-γ, IL-9, IL-10, IL-17, GM-CSF and Foxp3+ CD4+ T cells in the CNS of EAE mice. (E) Flow cytometry analysis of expression of MHC class II, CD40, CD80 and CD86 in CNS DCs. (F) Flow cytometry analysis of IL-6, IL-10 and IL-12/23+ DCs in the CNS of EAE mice. Bar graphs depict mean ± standard error mean (SEM). Values of p < 0.05 (*), p < 0.01 (**) were considered statistically significant. NS, not significant.

Analysis of the phenotype of infiltrating CD4+ T cells in the CNS revealed that rIL-9 significantly reduced the frequencies of GM-CSF+ and IFN-γ+ CD4+ T cells compared to PBS-treated mice (Fig. 4D). Consistent with a previous report (13), the rIL-9 treatment increased the frequencies of IL-9+ CD4+ T cells (Fig. 4D). Given that GM-CSF is a hallmark cytokine of pathogenic Th17 cells in the CNS of EAE mice, we hypothesize that the role of IL-9 in GM-CSF suppression is indirect, most likely through modulation of APCs. In line with our hypothesis, analysis of DC maturation markers showed that rIL-9 significantly reduced the expression of MHC class II but had no significant effect on CD40, CD80 and CD86 expression (Fig. 4E). No significant differences were observed in IL-6-, IL-10- and IL-12/23p40-producing DCs among groups (Fig. 4F).

To investigate the effect of IL-9 on DC function, we treated MOG35-55-immunized WT mice with rIL-9 at days 1, 4 and 7 p.i. and analyzed the expression of cytokines involved in antigen-presentation by DCs isolated from spleens. rIL-9 suppressed the expression of IL1B, IL6 and IL12A compared to DCs from the PBS-treated groups (Fig. 5A). Interestingly, rIL-9 did not increase the expression of IL10 and IL27 in DCs, which suggests that IL-9 has a suppressive rather than immunomodulatory effect on DCs. When co-cultured with naïve T cells from 2D2 mice, DCs from rIL-9-treated mice stimulated significantly lower production of IFN-γ and GM-CSF by T cells, and greater IL-10 production compared to DCs from PBS-treated mice (Fig. 5B). Of note, rIL-9 augmented the expression of its receptor in DCs in a positive feedback loop (Fig. 5C). Our data suggest that rIL-9 impacted disease via T cell/DC modulation.

Figure 5. IL-9 suppresses inflammatory DCs.

Figure 5.

Open in a new tab

(A) WT mice (n = 3 per group) were immunized to induce EAE and treated with rIL-9 or PBS as described above. At day 7 p.i., CD11c+ DCs in the spleen were isolated and the gene expression levels of IL1B, IL6, IL10, IL12A, IL23A and IL27 were analyzed. (B) Isolated DCs were co-cultured with naïve CD4+ T cells from 2D2 mice in the presence of MOG35-55 at 37°C. After 72 h, cells were analyzed by flow cytometry for production of IFN-γ, IL-10, IL-17, and GM-CSF in CD4+ T cells. (C) Relative expression of IL-9R in isolated DCs. Bar graphs depict mean ± standard error mean (SEM). Values of P < 0.05 (*), P < 0.01 (**) were considered statistically significant. NS, not significant.

Absence of IL-9 signaling in DCs is sufficient to increase inflammation and aggravate EAE

Our data suggest that IL-9 has a protective effect in EAE by modulating the phenotype of T cells and DCs. The fact that IL-9 modulates DCs in the context of CNS inflammation and its underlying mechanism has not been described before. Therefore, we tested if the adoptive transfer of WT DCs will suppress EAE in IL-9R−/− mice and vice versa, if IL-9R−/− DCs would enhance disease in WT mice. For that purpose, we transferred WT or IL-9R−/− DCs to IL-9R−/− mice and immunized them to induce EAE. In parallel, WT mice received WT or IL-9R−/− DCs. WT and IL-9R−/− mice that did not receive DC served as controls. IL-9R−/− DCs transferred into WT mice significantly increased EAE severity, whereas transfer of IL-9R−/− DCs to IL-9R−/− mice did not change disease course (Fig. 6A and B).

Figure 6. IL-9R−/− DCs aggravate EAE by stimulating GM-CSF+ CD4+ T cells.

Figure 6.

Open in a new tab

WT and IL-9R−/− mice (n = 5 per group) were adoptively transferred with CD11c+ DCs from WT or IL-9R−/− mice. 72 h later, the mice were immunized to induce EAE. Simultaneously, naïve WT (n = 5) and IL-9R−/− mice (n = 4) were immunized and served as controls. (A) Daily clinical scores of disease severity, (B) Cumulative scores of disease severity, (C) Mice were dissected on day 21 p.i. and the numbers of CD45+ cells and DCs in the CNS were determined by flow cytometry. (D) Flow cytometry analysis of IFN-γ, IL-9, IL-10, IL-17, GM-CSF and Foxp3+ CD4+ T cells in the CNS of EAE mice. (E) Expression of MHC class II, CD40, CD80 and CD86 in DCs in the CNS were determined by flow cytometry. (F) Flow cytometry analysis of IL-6, IL-10 and IL-12/23+ DCs in the CNS of EAE mice. Bar graphs depict mean ± standard error mean (SEM). Values of P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****) were considered statistically significant. NS, not significant.

Analysis of CNS infiltrating cells revealed that there was no difference in total numbers of CD45+ cells among different experimental groups; however, mice that received IL-9R−/− DCs had a significantly greater proportions of CNS-infiltrating DCs compared to controls (Fig. 6C). Analysis of the phenotype of CD4+ T cells showed that transfer of IL-9R−/− DCs to WT mice increased the frequency of pathogenic GM-CSF+ CD4+ T cells, whereas IL-9+ CD4+ T cells were decreased in the CNS compared to the other groups (Fig. 6D). Notably, adoptive transfer of WT DCs to IL-9R−/− mice did not influence the numbers of GM-CSF+CD4+ T cells in the CNS compared to other groups of IL-9R−/− mice (Fig. 6D). These results further support our hypothesis that IL-9 suppresses inflammatory DCs instead of stimulating modulatory DCs.

Analysis of CNS infiltrating DCs revealed that recipients of IL-9R−/− DCs had a significant increase in the expression of MHC class II, CD40, CD80, and CD86, whereas transfer of WT DCs to IL-9R−/− mice reduced the expression of these molecules to levels found in WT mice (Fig. 6E). Finally, DCs from mice that received IL-9R−/− DCs had a significant increase in IL-6 production compared to mice that received WT DCs (Fig. 6F). Collectively, our results show that IL-9/IL-9R signaling has a protective role in EAE by suppressing GM-CSF-production by T cells through the modulation of DCs.

Discussion

Pathogenic Th17 cells play a significant role in MS and EAE pathogenesis and GM-CSF is a hallmark cytokine of these cells (4, 5, 18). Although IL-9 was initially associated with Th2 responses, its role is broader reaching from tumor immunity to Th17 cell-mediated inflammation (12, 19, 20). IL-9 increases the suppressive function of Treg cells and lack of IL-9R worsens EAE (13). Here we show that the lack of IL-9R aggravates EAE by increasing the frequencies of pathogenic GM-CSF-producing T cells and inflammatory DCs in the CNS. Likewise, IL-9 treatment ameliorated EAE by suppressing GM-CSF production by T cells and DC activation. Furthermore, IL-9-treated DCs repressed GM-CSF production while increasing IL-10-production by MOG35-55-reactive T cells. Ultimately, adoptive transfer of IL-9R−/− DCs to WT mice recapitulated the phenotype of EAE in IL-9R−/− mice and increased GM-CSF-producing T cells in the CNS.

IL-17, IL-1β, IL-6, IL-23, and IL-22 are Th17-related cytokines frequently increased in serum of patients with MS and other autoimmune diseases. More recently, we showed that GM-CSF is a signature cytokine of pathogenic Th17 cells in CNS inflammation in EAE and that GM-CSF+ T cells are present in MS lesions of untreated patients and their numbers decrease in IFN-β-treated patients (5, 18). Notably, CSF2R, the receptor for GM-CSF, is increased in MS lesions (21). CSF2−/− mice are resistant to EAE and antibody depletion of GM-CSF results in amelioration of disease while its overexpression by T cells causes spontaneous neuroinflammation (7, 22). These observations show that modulation of GM-CSF may be an efficient venue to reduce EAE/MS severity. Here we confirmed a previous finding that IL-9R−/− mice develop a more severe EAE compared to WT mice (13) and furthered our understanding of the underlying mechanism by showing that disease worsening correlated with an increase in the frequencies of GM-CSF+ T cells and inflammatory DCs.

DCs that infiltrate the CNS of EAE mice are inflammatory and reactivate invading lymphocytes; also the adoptive transfer of myelin-pulsed DCs induces EAE in naïve mice (23-25). Conversely IFN-β, first-line drug therapy for MS, suppress disease by modulating DCs (26-28). The maturation phenotype and different subsets of DCs may explain their multifaceted role in CNS autoimmunity. Dermal CD103+ DCs prime T cells after MOG35-55 immunization and their ablation confers EAE resistance due to insufficient Th17 cell activation (29). Meanwhile, spleen resident CD11b+CD103+ DCs are tolerogenic and induce Treg cells while suppressing Th17 cells in EAE via a mechanism that is partially dependent on IL-27 (30-32). Intravenous delivery of the MOG35-55 antigen stimulates tolerogenic splenic CD103+ DCs while inducible expression of MOG driven by the CD11c marker also confers tolerance to EAE in mice (32, 33). These observations place DCs as essential cell types in CNS autoimmunity; thus their characterization and modulation are much needed to develop new therapeutic approaches in MS/EAE. Here we show that, while IL-9R−/− DCs had an increase in IL-6 production, administration of rIL-9 suppressed DC maturation and reduced the expression of IL-6 at the protein and gene levels. IL-6 signals through STAT3 and is a necessary factor for Th17 cell development (34-36); also, IL-6 induces GM-CSF and, in a positive feedback loop, GM-CSF induces IL-6 (37, 38). We hypothesized that reduced IL-6 in IL-9-treated DCs could negatively influence the production of GM-CSF by T cells and we found that DCs from rIL-9-treated mice suppressed GM-CSF production by CD4+ T cells.

Treg cells are necessary to control undesired immune responses by acting through cell contact and cytokine secretion mechanisms. In EAE and MS, Treg cells are reduced or inefficient (39, 40). It has been proposed that IL-9 improves the fitness of Treg cells and that exacerbation of EAE in IL-9R−/− mice is a result of a defective Treg cell pool (13). In this paper, we did not evaluate the suppressive function of Treg cells. However, we observed that IL-9R−/− mice had no significant difference in Treg cell numbers compared to WT mice. Moreover, rIL-9 treatment did not influence the numbers of Treg cells. DCs from rIL-9-treated mice failed to induce the differentiation of Treg cells in vitro. These observations led us to believe that IL-9 controls EAE by suppressing inflammatory T cells rather than stimulating regulatory T cells. To further investigate if IL-9 signaling in DCs suppresses EAE, we adoptively transferred WT DCs to IL-9R−/− mice and analyzed disease development following immunization. The adoptive transfer of WT DCs had no significant effect on EAE severity and did not influence the frequencies of Treg cells and GM-CSF+ T cells in the CNS of IL-9R−/− mice. Conversely, we found that transfer of IL-9R−/− DCs to WT mice led to an exacerbated disease development that correlated with increased GM-CSF+ T cells in the CNS.

Given that DCs fine-tune the immune responses by stimulating Treg cells and suppressing inflammatory cells, the use of tolerogenic DCs to treat EAE has yielded promising results (41, 42). Understanding the mechanisms by which DCs control inflammation could be leveraged for the development of new therapeutic approaches to treat EAE/MS and other autoimmune diseases. Collectively, our results show that IL-9 controls neuroinflammation by indirectly modulating GM-CSF production through DCs. IL-9 is a cytokine with pleiotropic functions and further investigations to ascertain its role in the control of autoimmune inflammation are warranted.

Supplementary Material

1

Key points:

IL-9R−/− mice develop severe EAE with increased frequency of GM-CSF+ CD4+ T cells.

IL-9R−/− DCs induce GM-CSF in CD4+ T cells and contribute to disease worsening.

Acknowledgments

This work was supported by the National Multiple Sclerosis Society (RG-4745A2/3 to Dr. Rostami and FG-1608-25579 to Dr. Thome) and the National Institutes of Health (NIH, 5R01NS088729 to AMR).

References

  • 1.Kroenke MA, Carlson TJ, Andjelkovic AV, and Segal BM. 2008. IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 205: 1535–1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, and Cua DJ. 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201: 233–240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Korn T, Anderson AC, Bettelli E, and Oukka M. 2007. The dynamics of effector T cells and Foxp3+ regulatory T cells in the promotion and regulation of autoimmune encephalomyelitis. J Neuroimmunol 191: 51–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Codarri L, Gyulveszi G, Tosevski V, Hesske L, Fontana A, Magnenat L, Suter T, and Becher B. 2011. RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation. Nat Immunol 12: 560–567. [DOI] [PubMed] [Google Scholar]
  • 5.El-Behi M, Ciric B, Dai H, Yan Y, Cullimore M, Safavi F, Zhang GX, Dittel BN, and Rostami A. 2011. The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF. Nat Immunol 12: 568–575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.McQualter JL, Darwiche R, Ewing C, Onuki M, Kay TW, Hamilton JA, Reid HH, and Bernard CC. 2001. Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med 194: 873–882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ponomarev ED, Shriver LP, Maresz K, Pedras-Vasconcelos J, Verthelyi D, and Dittel BN. 2007. GM-CSF production by autoreactive T cells is required for the activation of microglial cells and the onset of experimental autoimmune encephalomyelitis. J Immunol 178: 39–48. [DOI] [PubMed] [Google Scholar]
  • 8.Shimbara A, Christodoulopoulos P, Soussi-Gounni A, Olivenstein R, Nakamura Y, Levitt RC, Nicolaides NC, Holroyd KJ, Tsicopoulos A, Lafitte JJ, Wallaert B, and Hamid QA. 2000. IL-9 and its receptor in allergic and nonallergic lung disease: increased expression in asthma. J Allergy Clin Immunol 105: 108–115. [DOI] [PubMed] [Google Scholar]
  • 9.Sismanopoulos N, Delivanis DA, Alysandratos KD, Angelidou A, Vasiadi M, Therianou A, and Theoharides TC. 2012. IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis. PLoS One 7: e33271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.McNamara PS, and Smyth RL. 2005. Interleukin-9 as a possible therapeutic target in both asthma and chronic obstructive airways disease. Drug News Perspect 18: 615–621. [DOI] [PubMed] [Google Scholar]
  • 11.Turner JE, Morrison PJ, Wilhelm C, Wilson M, Ahlfors H, Renauld JC, Panzer U, Helmby H, and Stockinger B. 2013. IL-9-mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation. J Exp Med 210: 2951–2965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Noelle RJ, and Nowak EC. 2010. Cellular sources and immune functions of interleukin-9. Nat Rev Immunol 10: 683–687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Elyaman W, Bradshaw EM, Uyttenhove C, Dardalhon V, Awasthi A, Imitola J, Bettelli E, Oukka M, van Snick J, Renauld JC, Kuchroo VK, and Khoury SJ. 2009. IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells. Proc Natl Acad Sci U S A 106: 12885–12890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Stephens GL, Swerdlow B, Benjamin E, Coyle AJ, Humbles A, Kolbeck R, and Fung M. 2011. IL-9 is a Th17-derived cytokine that limits pathogenic activity in organ-specific autoimmune disease. Eur J Immunol 41: 952–962. [DOI] [PubMed] [Google Scholar]
  • 15.Ding X, Cao F, Cui L, Ciric B, Zhang GX, and Rostami A. 2015. IL-9 signaling affects central nervous system resident cells during inflammatory stimuli. Exp Mol Pathol 99: 570–574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Li H, Nourbakhsh B, Cullimore M, Zhang GX, and Rostami A. 2011. IL-9 is important for T-cell activation and differentiation in autoimmune inflammation of the central nervous system. Eur J Immunol 41: 2197–2206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Jager A, Dardalhon V, Sobel RA, Bettelli E, and Kuchroo VK. 2009. Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes. J Immunol 183: 7169–7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rasouli J, Ciric B, Imitola J, Gonnella P, Hwang D, Mahajan K, Mari ER, Safavi F, Leist TP, Zhang GX, and Rostami A. 2015. Expression of GM-CSF in T Cells Is Increased in Multiple Sclerosis and Suppressed by IFN-beta Therapy. J Immunol 194: 5085–5093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Purwar R, Schlapbach C, Xiao S, Kang HS, Elyaman W, Jiang X, Jetten AM, Khoury SJ, Fuhlbrigge RC, Kuchroo VK, Clark RA, and Kupper TS. 2012. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat Med 18: 1248–1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lu Y, Hong S, Li H, Park J, Hong B, Wang L, Zheng Y, Liu Z, Xu J, He J, Yang J, Qian J, and Yi Q. 2012. Th9 cells promote antitumor immune responses in vivo. J Clin Invest 122: 4160–4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Imitola J, Rasouli J, Watanabe F, Mahajan K, Sharan AD, Ciric B, Zhang GX, and Rostami A. 2018. Elevated expression of granulocyte-macrophage colony-stimulating factor receptor in multiple sclerosis lesions. J Neuroimmunol 317: 45–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Spath S, Komuczki J, Hermann M, Pelczar P, Mair F, Schreiner B, and Becher B. 2017. Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System. Immunity 46: 245–260. [DOI] [PubMed] [Google Scholar]
  • 23.Weir CR, Nicolson K, and Backstrom BT. 2002. Experimental autoimmune encephalomyelitis induction in naive mice by dendritic cells presenting a self-peptide. Immunol Cell Biol 80: 14–20. [DOI] [PubMed] [Google Scholar]
  • 24.Sagar D, Lamontagne A, Foss CA, Khan ZK, Pomper MG, and Jain P. 2012. Dendritic cell CNS recruitment correlates with disease severity in EAE via CCL2 chemotaxis at the blood-brain barrier through paracellular transmigration and ERK activation. J Neuroinflammation 9: 245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Paterka M, Siffrin V, Voss JO, Werr J, Hoppmann N, Gollan R, Belikan P, Bruttger J, Birkenstock J, Jung S, Esplugues E, Yogev N, Flavell RA, Bopp T, and Zipp F. 2016. Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation. EMBO J 35: 89–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Chen M, Chen G, Deng S, Liu X, Hutton GJ, and Hong J. 2012. IFN-beta induces the proliferation of CD4+CD25+Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells in the treatment of multiple sclerosis. J Neuroimmunol 242: 39–46. [DOI] [PubMed] [Google Scholar]
  • 27.Pennell LM, and Fish EN. 2017. Interferon-beta regulates dendritic cell activation and migration in experimental autoimmune encephalomyelitis. Immunology 152: 439–450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Huang YM, Adikari S, Bave U, Sanna A, and Alm G. 2005. Multiple sclerosis: interferon-beta induces CD123(+)BDCA2- dendritic cells that produce IL-6 and IL-10 and have no enhanced type I interferon production. J Neuroimmunol 158: 204–212. [DOI] [PubMed] [Google Scholar]
  • 29.King IL, Kroenke MA, and Segal BM. 2010. GM-CSF-dependent, CD103+ dermal dendritic cells play a critical role in Th effector cell differentiation after subcutaneous immunization. J Exp Med 207: 953–961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Thome R, Moore JN, Mari ER, Rasouli J, Hwang D, Yoshimura S, Ciric B, Zhang GX, and Rostami AM. 2017. Induction of Peripheral Tolerance in Ongoing Autoimmune Inflammation Requires Interleukin 27 Signaling in Dendritic Cells. Front Immunol 8: 1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Mari ER, Rasouli J, Ciric B, Moore JN, Conejo-Garcia JR, Rajasagi N, Zhang GX, Rabinovich GA, and Rostami A. 2016. Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis. Eur J Immunol 46: 1783–1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Li H, Zhang GX, Chen Y, Xu H, Fitzgerald DC, Zhao Z, and Rostami A. 2008. CD11c+CD11b+ dendritic cells play an important role in intravenous tolerance and the suppression of experimental autoimmune encephalomyelitis. J Immunol 181: 2483–2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yogev N, Frommer F, Lukas D, Kautz-Neu K, Karram K, Ielo D, von Stebut E, Probst HC, van den Broek M, Riethmacher D, Birnberg T, Blank T, Reizis B, Korn T, Wiendl H, Jung S, Prinz M, Kurschus FC, and Waisman A. 2012. Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity 37: 264–275. [DOI] [PubMed] [Google Scholar]
  • 34.Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, and Kuchroo VK. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238. [DOI] [PubMed] [Google Scholar]
  • 35.McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, and Cua DJ. 2007. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol 8: 1390–1397. [DOI] [PubMed] [Google Scholar]
  • 36.Yang XO, Panopoulos AD, Nurieva R, Chang SH, Wang D, Watowich SS, and Dong C. 2007. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 282: 9358–9363. [DOI] [PubMed] [Google Scholar]
  • 37.Evans R, Shultz LD, Dranoff G, Fuller JA, and Kamdar SJ. 1998. CSF-1 regulation of Il6 gene expression by murine macrophages: a pivotal role for GM-CSF. J Leukoc Biol 64: 810–816. [DOI] [PubMed] [Google Scholar]
  • 38.Rollwagen FM, Davis TA, Li YY, Pacheco ND, and Zhu XL. 2004. Orally administered IL-6 induces elevated intestinal GM-CSF gene expression and splenic CFU-GM. Cytokine 27: 107–112. [DOI] [PubMed] [Google Scholar]
  • 39.Viglietta V, Baecher-Allan C, Weiner HL, and Hafler DA. 2004. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 199: 971–979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen TR, Backstrom BT, Sobel RA, Wucherpfennig KW, Strom TB, Oukka M, and Kuchroo VK. 2007. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 13: 423–431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Thome R, Bonfanti AP, Rasouli J, Mari ER, Zhang GX, Rostami A, and Verinaud L. 2018. Chloroquine-treated dendritic cells require STAT1 signaling for their tolerogenic activity. Eur J Immunol. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Takenaka MC, and Quintana FJ. 2017. Tolerogenic dendritic cells. Semin Immunopathol 39: 113–120. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1544283-supplement-1.pdf (191.3KB, pdf)

RESOURCES