IL-7Rα confers susceptibility to experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is a neurological disorder that causes paralysis in young adults and affects women more frequently than men. The etiology of MS is not known, but it is generally viewed as an autoimmune disease of the central nervous system (CNS), influenced by genetic and environmental factors. Recent studies have identified interleukin 7 receptor alpha (IL-7Rα) as a risk factor for multiple sclerosis. But the role of IL-7Rα in experimental autoimmune encephalomyelitis (EAE) model of MS is not known. In this study we demonstrate that IL-7Rα deficient (IL-7Rα^−/−) mice remain resistant to MOGp35-55-induced EAE. When compared to C57BL/6 wild type mice, IL-7Rα^−/− mice showed less severe inflammation and demyelination in the CNS. The attenuation of EAE in IL-7Rα^−/− mice was associated with a decrease in T helper (Th) 1 and Th17 responses in the CNS and lymphoid organs. IL-7Rα^−/− mice also showed an increase in Th2 response and CD4⁺Foxp3⁺ regulatory T cells. These findings highlight that IL-7Rα confers susceptibility by influencing autoimmune Th1/Th17 responses in EAE model of MS.

Introduction

Multiple sclerosis (MS) is a neurological disorder that affects more than one million people worldwide. The disease usually begins in young adults and affects women more frequently than men.^1,2 About 30% of MS patients develop clinical paralysis and become wheel chair-bound for the rest of their lives, causing immense health and socioeconomic problems. In most patients MS begins as relapsing-remitting disease and over time it progresses to chronic progressive disease. While relapsing MS is characterized by acute focal sclerotic lesions with myelin loss and infiltration of inflammatory cells in the central nervous system (CNS), axonal degeneration and brain atrophy contribute to irreversible long-term disability in chronic progressive MS.^2,3 Although the etiology of MS is not known, it is generally viewed as a myelin antigen (Ag)-reactive T helper (Th) 1 and Th17 cell-mediated autoimmune disease influenced by genetic and environmental factors.⁴ Thus, many of the current treatments used to inhibit the clinical symptoms of MS target the immune and inflammatory responses of the brain.

While the human leukocyte Ag (HLA) has long been considered a susceptibility gene for autoimmune diseases, recent studies have identified mutations in the interleukin 7 receptor alpha (IL-7Rα) chain as additional risk factors for MS.^5–11 Genomic convergence analyses identified a nonsynonymous coding single nucleotide polymorphism (SNP) in IL-7Rα (rs6897932; T244I) that affects alternative splicing and subsequent skipping of exon 6.^5,6 This may increase the ratio of soluble to membrane bound IL-7R for individuals with this polymorphism.⁶ A similar analysis reported that MS patients have a significant increase of interleukin 7 (IL-7) and IL-7R in the cerebrospinal fluid (CSF) compared to patients with other neurological diseases.^7,8 Interestingly, IL-7Rα is downregulated in human CD4⁺CD25⁺ regulatory T cells (Tregs) that have suppressive activity on effector T cells in MS and other autoimmune diseases.^12,13 IL-7 is a cytokine produced by stromal epithelial cells in the thymus that signals through IL-7R complex to promote B and T cell development and rescues thymocytes from apoptotic cell death.^14,15 The IL-7R is comprised of the common gamma chain (γc; CD132) and the IL-7R alpha chain (IL-7Rα, CD127). The γc is shared by receptors for the cytokines IL-2, IL-4, IL-9, IL-15 and IL-21 and mutation causes severe combined immunodeficiency (XSCID) in humans and mice.^16,17 IL-7Rα deficient (IL-7Rα^−/−) mice have reduced number of thymic precursors with decreased survival and response to TCR-dependent and independent stimuli.^14,18 Thymic stromal lymphopoietin (TSLP) is another cytokine that signals through IL-7Rα/TSLP receptor complex and stimulates the activation and differentiation of B cells, peripheral CD4⁺ T cells and thymic myeloid dendritic cells.^19,20 While IL-7Rα^−/− mice will be defective in both IL-7 and TSLP induced responses,²¹ its significance to pathogenesis of CNS inflammation and demyelination is not known.

In this study we examined the impact of IL-7Rα deficiency on the development of experimental autoimmune encephalomyelitis (EAE) model of MS. We found that the IL-7Rα^−/− male mice remained resistant to EAE in association with reduced neural Ag-induced Th1/Th17 responses, suggesting a role for IL-7Rα in conferring susceptibility to multiple sclerosis.

Materials and Methods

Animals

The C57BL/6 mice and IL-7Rα^−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in the animal care facility at Methodist Research Institute. Four to six week old male and female mice were used in the experiments. Animal protocols were approved by the Methodist Research Institute Animal Care and Use Committee.

Reagents

The 21 amino acid peptide [MEVGWYRSPFSRVVHLYRNGK] corresponding to mouse MOGp35-55 (96.81% pure) was obtained from Genemed Synthesis Inc. (San Francisco, CA). WST-1 was purchased from Roche Diagnostics (Indianapolis, IN). Recombinant mouse IFNγ, IL-17, IL-4 and IL-10 were purchased from R&D Systems (Minneapolis, MN). Anti-IL-17 antibodies were obtained from BD Pharmingen (Franklin Lakes, NJ). The anti-IFNγ antibodies were obtained from Endogen (Rockford, IL). The antibodies specific to CD28, IL-4, IL-10 and the fluorochrome-conjugated antibodies specific to CD4, CD25, Foxp3, IL-17, IFNγ and isotype controls were obtained from e-Biosciences (San Diego, CA). The anti-CD3 mAb was purified from ascitic fluid collected from nude mice following transplantation of 2C11 hybridoma cells. The RNeasy Mini kit was purchased from Qiagen (Valencia, CA). TaqMan reverse transcription kit, fast optical 96-well plates and RT-PCR primers were purchased from Applied Biosystems (Foster City, CA). Concanavalin A (ConA), avidin alkaline-phosphatase, P-nitrophenylphosphate and diethanolamine were obtained from Sigma (St. Louis, MO).

Induction and evaluation of EAE

To induce EAE, four to six week old C57BL/6 (wild-type) and IL-7Rα^−/− male and female mice were immunized with 100 μg MOGp35-55 peptide Ag in 150 μl emulsion of Complete Freund's Adjuvant (CFA) (Sigma Chemicals, St Louis, MO) in the lower dorsum on days 0 and 7. The mice also received (i.p.) 100 ng of pertussis toxin (Sigma Chemicals, St. Louis, MO) on days 0 and 2. The clinical symptoms were scored every day from day 0 to 30 as follows: 0, normal; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5 paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4 moribund; 5, death. Mean clinical score (MCS) was calculated by adding the clinical scores for one group and dividing by the total number of mice in that group.

Histological analysis

The IL-7Rα^−/− and wild-type mice induced to develop EAE were sacrificed on day 30 by CO₂ asphyxiation and perfused by intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Spinal cords were dissected and fixed in 10% formalin in PBS. Decalcified spinal cords were subjected to transverse sectioning of cervical, upper thoracic, lower thoracic, and lumbar regions and 10 μm sections were stained with Luxol Fast Blue and hematoxylin and eosin at the Indiana University Pathology Department. Inflammation and demyelination in the spinal cord sections were assessed under a microscope in a blinded manner. The spinal cord sections were viewed as anterior, posterior and two lateral columns (4 quadrants). Each quadrant displaying the infiltration of mononuclear cells or loss of myelin was assigned a score of one inflammation or one demyelination, respectively. Thus, each animal has a potential maximum score of 16 and this study represents the analyses of spinal cords from at least 5 mice per group. The pathological score from each group is expressed as percent positive over total number of quadrants examined.²² Images were acquired using a fluorescence microscope with a 10× aperture and 10× objective lens (Leitz DMRB, Leica) and a digital camera (SPOT RT, SPOT Advanced software, Diagnostic Instruments, Inc.).

T cell Proliferation assay

Spleens cells were isolated from IL-7Rα^−/− and wild-type mice on day 14 following immunization with MOGp35-55 and cultured in 96 well tissue culture plates in RPMI medium (2×10⁵/200 μl/well) with 0, 2.5, 5 and 10 μg/ml MOGp35-55 peptide for 72 hrs. WST-1 reagent (10 μl/well) was added and OD at 450 nm measured after 4 hrs using a titer-plate reader (Alpha Diagnostics, San Antonio TX). Spleens cells were also isolated from wild-type and IL-7Rα^−/− mice before (naïve), and after (day 14) immunization with CFA alone or in CFA+ MOGp35-55. The cells (5×10⁶/ml) were incubated in phosphate buffered saline (PBS) with 0.2 μM carboxy-fluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) for 5 min at 37 °C. Cells were washed and resuspended in complete RPMI medium (2.5×10⁶/ml) and cultured with 0, 2.5, 5 or 10 μg/ml MOGp35-55 peptide. Alternatively, cells were cultured with 2 μg/ml anti-CD3 plus 1 μg/ml anti-CD28 Abs or 0, 1 or 2.5 μg/ml ConA, for positive controls. After 72 hrs, cells were harvested, stained for CD4-PE and resuspended in PBS/0.1% bovine serum albumin (BSA) containing 10 μg/ml propidium iodide. Live cells were gated on CD4 and the percentage of proliferating CD4⁺ cells were determined based on CFSE dilution by flow cytometry.

Quantitative real-time PCR

The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ABI Prism 7900 fast sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Brain samples were isolated from wild-type and IL-7Rα^−/− mice on day 14 following induction of EAE, flash frozen and stored at −80 °C until used. Five to six brain samples per group from two different experiments were pooled and total RNA was extracted using the RNeasy Mini kit according to the manufacturer's instructions (Qiagen, Valencia, CA). The RNA samples (500 ng/20 μl reaction) were reverse transcribed into cDNA (RT-PCR) using random hexamer primers and TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA (100 ng/sample) was subjected to qPCR analysis in quadruplicate using forward and reverse primers, TaqMan Universal Master Mix, and probe (10 μl/reaction) in fast optical 96-well plates. The data were analyzed using the ABI Prism 7900 relative quantification (ΔΔCt) study software (Applied Biosystems, Foster City, CA). In this study we have used primer sets for 10 selected inflammatory genes with GAPDH as internal control (Applied Biosystems, Foster City, CA). The expression levels of inflammatory genes normalized to GAPDH are presented as arbitrary fold changes compared with control samples.

Cytokine ELISA

Spleen cells were isolated from IL-7Rα^−/− and wild-type mice on day 14 following induction of EAE and cultured in 12 well tissue culture plates in RPMI medium (1×10⁶/ml/well) with 0, 2.5, 5 and 10 μg/ml MOGp35-55 peptide. The culture supernatants were collected after 48 hrs and the presence of secreted IFNγ, IL-17, IL-4 and IL-10 analyzed by Enzyme-Linked Immunosorbant Assay (ELISA). Flat-bottomed 96-well Nunc plastic plates were coated with capture antibody at 4°C overnight. Plates were washed and subsequently blocked with 3% bovine serum albumin (BSA) in PBS for 1 hr at room temperature. Recombinant cytokine standards and the culture supernatant samples were added and incubated at 4°C overnight. After washing in PBS with 1% BSA, the plates were incubated with detection antibody for 1 hr at room temperature and then with Avidin-alkaline phosphatase for 30 min, followed by colorimetric assessment with 1 mg/ml p-nitrophenylphosphate prepared in 1 M diethanolamine. Optical density (OD) was measured at 405 nm after 4 hrs using a titer-plate reader (Alpha Diagnostics, San Antonio TX). Results were interpolated from a standard curve and expressed as ng/ml or pg/ml.

Immunostaining and flow cytometry

For Treg analysis, spleen cells (2×10⁶/sample) from wild-type and IL-7Rα^−/− mice were isolated on day 14 following induction of EAE. The cells were incubated with anti-CD16/CD32 Fc receptor blocker (30 min at 22–25°C) and subjected to surface staining (30 min at 4°C), followed by fixation and permeabilization with BD Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ) or Foxp3 Fixation/Permeabilization kit (eBioscience, San Diego, CA) according to the manufacturer's instructions. Next, cells were washed and stained with anti-Foxp3 antibody at 4°C for 1 hr. For intracellular cytokine analysis, spleen cells (2×10⁶/sample) from wild-type and IL-7Rα^−/− mice were isolated on day 14 following induction of EAE and restimulated with 1 μg/ml ionomycin and 50 nM phorbol 12-myristate 13-acetate (PMA) for 1 h at 37°C followed by BD GolgiStop (containing monensin) for an additional 5 h. The cells were subjected to surface staining (30 min at 4°C) followed by fixation and permeabilization with BD Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. Next, cells were washed and stained (1 hr at 4°C) for the intracellular cytokines IL-17 and IFNγ. T cells were gated based on forward scatter/side scatter properties. Spectral overlap was eliminated by appropriate compensation based on single stained controls, and gating parameters were determined using isotype controls. The cells were analyzed using a BD FACSCalibur™ or a BD LSRII™ flow cytometer (Becton Dickinson, San Jose, CA) and analyzed with FlowJo software (Ashland, OR).

Statistical analyses

To determine statistical significance, unpaired t tests, Wilcoxon matched pairs tests or one-way analysis of variance tests were performed by Bonferronni's multiple comparison test using GraphPad Prism v5.0 (GraphPad Software, La Jolla, CA). Post-hoc analyses were performed using Dunnett's correction. Bar graph values are means ± SEM.

Results

IL-7Rα is essential for the development of EAE model of MS

To elucidate the role of IL-7Rα in the pathogenesis of MS, we first studied the development of EAE in IL-7Rα^−/− mice. Immunization of wild-type female mice with MOGp35-55 induced the onset of EAE with a mean clinical score (MCS) of 0.2 on day 12 that progressed to a MCS of 2.0 by day 20 and remained high with an area under the curve (AUC) of 27.2 and an ending MCS of 1.8 on day 30 (Fig. 1; Table I). Interestingly, IL-7Rα^−/− female mice developed less frequent paralysis with delayed disease onset (day 15) that peaked with a MCS of 0.7 (3 fold decrease compared to wild-type females, p<0.05) on day 20, an AUC of 6.0 and an ending MCS of 0.3 on day 30. The wild-type male mice immunized with MOGp35-55 developed clinical signs on day 11 (MCS = 0.2) that progressed to a more severe disease reaching a peak MCS of 2.8 on day 19 and remained high with an AUC of 34.4, and an ending MCS of 1.9 on day 30. Interestingly, IL-7Rα^−/− male mice immunized with MOGp35-55 developed no clinical signs of EAE (AUC = 0). Of the twelve IL-7Rα^−/− female mice tested, two developed EAE with clinical scores as high as the wild-type female mice, while the remaining ten mice developed no detectable clinical signs of EAE throughout the period of study (Table I). These results demonstrate that IL-7Rα^−/− female mice are less likely to develop EAE when compared to wild-type mice, while the IL-7Rα^−/− male mice remained completely resistant to EAE. However, a one-way ANOWA using the Bonferronni's multiple comparison test revealed no statistically significant gender difference in the clinical outcome of EAE. Thus our findings suggest that IL-7Rα confers susceptibility to EAE model of MS.

FIGURE 1.

Development of EAE in IL-7Rα deficient mice. Four to six week old wild-type female (■) (n=12), IL-7Rα^−/− female (•) (n=12), wild-type male (■) (n=9) and IL-7Rα^−/− male (•) (n=9), mice were induced to develop EAE by immunization with MOGp35-55 Ag. The clinical symptoms were scored every day in a blinded manner and presented as mean ± SEM for each group. The data represent 4 combined experiments.

Table I.

Clinical analyses of EAE in wild-type and IL-7Rα^−/− mice^a

Analysis	Females		Males
	IL-7Rα+/+	IL-7Rα−/−	IL-7Rα+/+	IL-7Rα−/−
MMCS	2.1	0.7*	2.8	0***
AMCS	0.9	0.2***	1.1	0***
MCS, day 30	1.8	0.3*	1.9	0*
MCS ≥ 1, days	16	0	18	0
AUC	27.2	6.0	34.4	0
DI	12/12	2/12	9/9	0/9

^aThe mean clinical score (MCS) of EAE was calculated for each day by adding the clinical score for all mice in a group and then dividing by the total number of mice. The mean maximum clinical score (MMCS) is the MCS at the peak of the disease. Average mean clinical score (AMCS) was calculated by adding the MCS for all days (from day 0 to 30) and then dividing by 30. MCS, day 30 was the MCS on the last day (day 30). The mean clinical score more than one (MCS≥1) was obtained by counting the number of days with MCS greater than or equal to one. Analyses were calculated using GraphPad Prism 5.0 software. (AUC = area under the curve, DI = disease incidence)

^*p<0.05

^***p<0.001 compared to the IL-7Rα^+/+ group of the same gender using a one-way ANOVA with a Bonferroni's Multiple Comparison test. The MMCS, AMCS and MCS Day 30 did not significantly differ between IL-7Rα^+/+ male versus IL- 7Rα^+/+ female animals or IL-7α^−/− male versus IL-7Rα^−/− female animals.

We then examined whether the attenuation of paralytic symptoms in the IL-7Rα^−/− mice was associated with decreased inflammation and demyelination in the CNS. Wild-type female mice immunized with MOGp35-55 showed 84% of the spinal cord quadrants positive for lesions with infiltration of mononuclear cells (MNCs) and extensive myelin loss on day 30 (Fig. 2). Interestingly, IL-7Rα^−/− female mice showed only 9% of spinal cord quadrants positive for lesions with infiltrating cells and myelin loss (9 fold decrease, p<0.001) on day 30. Similarly, the wild-type male mice immunized with MOGp35-55 showed 88% and 89% of spinal cord quadrants positive for lesions with infiltrating MNCs and myelin loss, respectively. In contrast, the spinal cords from IL-7Rα^−/− male mice showed no detectable lesions with infiltrating MNCs (p<0.001) or myelin loss (p<0.001). These results suggest that the IL-7Rα^−/− mice remained resistant to EAE in association with reduced or no detectible pathological signs of inflammation or demyelination in the CNS.

FIGURE 2.

Development of CNS inflammation and demyelination in IL-7Rα deficient mice. The spinal cords isolated from C57BL/6 wild type and IL-7Rα^−/− mice following induction of EAE were stained with Luxol Fast Blue or hematoxylin and eosin to examine demyelination or inflammation, respectively. The spinal cord sections from cervical, upper thoracic, lower thoracic and lumbar regions (four sections per animal) were analyzed. Each quadrant of the spinal cord sections with loss of myelin or infiltration of mononuclear cells received a score of one demyelination or inflammation, respectively. The pathological score for each treatment group was expressed as the percentage positive quadrants over the total number of quadrants examined. The values are mean ± SEM *** p<0.001 compared to wild-type and this study includes the analysis of ~80 spinal cord quadrants per group.

IL-7Rα is not critical for myelin Ag-induced CD4⁺ T cell expansion in EAE model of MS

To understand the role of T cells in the attenuation of EAE in IL-7Rα^−/− mice, we examined T cell proliferation in culture. Whole spleen cells isolated from wild type female mice on day 14 following immunization with MOGp35-55 displayed a dose-dependent increase in proliferation, reaching a maximum of 10.6 fold increase at 10 μg/ml Ag (Fig. 3A). Although the spleen cells from IL-7Rα^−/− female mice immunized with MOGp35-55 also showed a dose-dependent increase in proliferation, this effect was significantly diminished (p<0.001) when compared to wild type mice, reaching a maximum of 2.3 fold increase at 10 μg/ml Ag. Moreover, the MOGp35-55 immune spleen cells from wild-type male mice showed a strong proliferative response reaching a maximum of 32 fold increase at 10 μg/ml Ag (Fig. 3A). In contrast, spleen cells from the IL-7Rα^−/− male mice immunized with MOGp35-55 showed a complete attenuation (p<0.001) of neural Ag-induced proliferation in culture. To determine the difference in CD4⁺ T cell response to neural antigen, spleen cells were isolated from male and female wild type and IL-7Rα^−/− mice on day 0 (naïve) and 14 following immunization with CFA alone or in combination with MOGp35-55 and analyzed by CFSE assay. Antigen-induced proliferation was determined by calculating the percentage of CD4⁺/CFSE^dilute cells. The CD4⁺ T cells from naïve mice (Supplemental Fig. 1) and CFA-immunized control mice (Supplemental Fig. 2) did not proliferate in response to MOGp35-55 in culture. A subtle increase in CD4⁺ T cell proliferation was observed in response to 10 μg/ml MOGp35-55 Ag in both wild-type and IL-7Rα^−/− mice. Specifically, in the wild-type and IL-7Rα^−/− female mice there was a 1.3- and 1.4-fold increase over medium control, respectively (Fig. 3B; Supplemental Fig. 3). Also, in the wild-type and IL-7Rα^−/− male mice there was a 9.4- and 2.9-fold increase over medium control, respectively (Fig. 3B; Supplemental Fig. 3). Since the proliferating CD4⁺ T cell population in both WT and IL-7Rα^−/− mice following culture with MOGp35-55 for 3 days was quite small we then examined the nonspecific CD4⁺ T cell proliferation in the IL-7Rα^−/− mice. Spleen cells from naïve male WT and IL-7Rα^−/− mice were cultured with anti-CD3 plus anti-CD28 Abs or the T cell mitogen concanavalin A (ConA). The CD4⁺ T cells from both WT and IL-7Rα^−/− mice showed proliferation in response to ConA stimulation and TCR activation with a more robust response in the wild-type (Supplemental Fig. 4). Stimulation of spleen cells from WT mice with 1 and 2.5 μg/ml ConA resulted in the proliferation of 58% and 81% CD4⁺ T cells, respectively. Whereas, the spleen cells from IL-7Rα^−/− mice with 1 and 2.5 μg/ml ConA caused the proliferation of only 35% and 45% of CD4⁺ cells, respectively. Similarly, TCR activation using anti-CD3 Ab resulted in the proliferation of 89% of CD4⁺ cells in WT mice compared to only 62% proliferating CD4⁺ T cells in IL-7Rα^−/− mice. These results suggest that the attenuation of EAE in IL-7Rα^−/− mice was not contributed solely through changes in neural antigen-specific CD4⁺ T cell proliferation but by defective cellularity and associated autoimmune responses.

FIGURE 3.

Neural Ag-induced T cell proliferation in wild-type and IL-7Rα^−/− mice. A) The proliferative response of whole spleen cells was measured by WST-1 assay. Whole spleen cells isolated from C57BL/6 wild type and IL-7Rα^−/− female or male mice on day 14 following immunization with MOGp35-55 Ag were stimulated with 0, 2.5, 5 or 10 ng/ml of MOGp35-55 Ag. The values are mean ± SEM of triplicates for each data point and the figures are representative of 4 independent experiments. *** p<0.001 compared to wild-type. B) The proliferative response of CD4⁺ spleen cells was determined by CFSE staining. Spleen cells isolated from wild type and IL-7Rα^−/− male mice on day 14 following immunization with MOGp35-55 Ag were stained with 0.2 μ CFSE and cultured with 0, 2.5, 5 or 10 ng/ml of MOGp35-55 Ag. Harvested cells were stained with anti-CD4-PE antibody, resuspended in 10 μg/ml propidium iodide and analyzed by flow cytometry. The values are percentages of live CD4⁺ cells.

IL-7Rα^−/− mice induced to develop EAE express altered levels of inflammatory cytokines in the CNS

To define the mechanism for attenuated EAE in the IL-7Rα^−/− mice, we examined the mRNA expression of Th1 and Th17 cell-derived inflammatory cytokines in the CNS of wild-type and IL-7Rα^−/− mice on day 14 following induction of EAE (Fig. 4). The levels of IFNγ and IL-17 mRNA were significantly reduced in IL-7Rα^−/− male and female mice when compared to wild-type mice. Interestingly, the level of the Th1 transcription factor T-bet was reduced in male IL-7Rα^−/− mice while T-bet was significantly increased in female IL-7Rα^−/− mice, compared to wild-type. The levels of IFNγ and IL-17 in the brain correlate with disease severity. The anti-inflammatory cytokines IL-4 and IL-10 were undetectable in all groups (data not shown).

FIGURE 4.

Expression of effector T cell-derived inflammatory cytokines in the CNS of wild-type and IL-7Rα deficient mice. Brain tissue from five to six wild type and IL-7Rα^−/− mice from three experiments was isolated on day 14 following induction of EAE by immunization with MOGp35-55 Ag. Total RNA was extracted from brain and the expression of IL-17, IFNγ and T-bet was analyzed by qRT-PCR using GAPDH as internal control. The fold changes in the expression of cytokines in EAE mice were calculated based on wild-type as control and are shown as the mean ± SEM of quadruplicates. ** p<0.01, *** p<0.001 compared to wild-type. ND = not detectable. This analysis has been repeated three times.

To further define the mechanism for reduced EAE in the IL-7Rα^−/− mice we examined the mRNA expression of the IL-12 family cytokines in the CNS of wild-type and IL-7Rα^−/− mice (Fig. 5). Interestingly, several inflammatory cytokines were differentially regulated between the male and female IL-7Rα^−/− mice when compared to their respective wild-type controls. Relative quantities of IL-27p28 and IL-12p35 were decreased in the IL-7Rα^−/− male mice while these cytokines were unchanged in the IL-7Rα^−/− female mice. Additionally, IL-23p19 was increased in the IL-7Rα^−/− male mice but unchanged in the females, and IL-12p40 was undetectable in the IL-7Rα^−/− female mice, but unchanged in the males. These results suggest that the altered expression of IL-12 family cytokine could be another mechanism by which IL-7Rα^−/− mice developed defective Th1/Th17 responses and remained resistant to EAE.

FIGURE 5.

Expression of APC-derived inflammatory cytokines in the CNS from wild-type and IL-7Rα^−/− mice. Brain tissue from five to six wild type and IL-7Rα^−/− mice from three experiments was isolated on day 14 following induction of EAE by immunization with MOGp35-55 Ag. Total RNA was extracted from brain and the expression of IL-12p35, IL-12p40, IL-23p19 and IL-27p28 was analyzed by qRT-PCR using GAPDH as internal control. The fold changes in the expression of cytokines in EAE mice were calculated based on wild-type as control and are shown as the mean ± SEM of quadruplicates. * p<0.05, *** p<0.001 compared to wild-type. ND = not detectable. This analysis has been repeated three times.

IL-7Rα is essential for IFNγ and IL-17 production in EAE model of MS

To further determine the mechanisms in the attenuation of EAE in IL-7Rα^−/− mice, we examined neural Ag-induced Th1 and Th17 responses in IL-7Rα^−/− mice (Fig. 6). Spleen cells from wild-type female mice sensitized with MOGp35-55 displayed a dose-dependent increase in the secretion of IFNγ and IL-17, reaching a maximum of 16 fold and 5 fold increase at 10 μg/ml Ag, respectively. Whereas, spleen cells from IL-7Rα^−/− female mice sensitized with MOGp35-55 showed significant decrease in the secretion of IFNγ (p<0.001) and IL-17 (p<0.001) when compared to wild type mice. Moreover, MOGp35-55 sensitized spleen cells from wild-type male mice also showed significant increase in the secretion of IFNγ and IL-17 reaching 9 and 4.4 fold increase at 10 μg/ml Ag, respectively. MOGp35-55 immune spleen cells from IL-7Rα^−/− male mice also showed significant decrease in the secretion of IFNγ (p<0.001) and IL-17 (p<0.001) in response to Ag. However, both the IL-7Rα^−/− female and male mice showed a decrease in the percentage of CD4⁺ cells when compared to wild-type controls. This is consistent with previously published reports of decreased CD4⁺ splenocyte cellularity in naïve IL-7Rα^−/− mice^{14, 23}. It is possible that the observed decrease in cytokine secretion in the IL-7Rα^−/− mice is due to a smaller proportion of CD4⁺ cells and not directly related to alterations in cytokine secretion. To examine this we performed intracellular cytokine staining on the CD4⁺ T cell population to determine if the decrease in cytokine secretion was a result of decreased Th1 and Th17 differentiation or simply a consequence of reduced CD4⁺ splenocyte cellularity. The percentage of CD4⁺/IFNγ⁺ and CD4⁺/IL-17⁺ T cells was similar in MOG-immunized IL-7Rα^−/− mice compared to wild-type mice (Fig. 7C). However, we observed Th17, not Th1 differentiation in the IL-7Rα^−/− mice as a result of MOGp35-55 immunization and the percentage of CD4⁺/IFNγ⁺ cells did not change as a result of CFA or CFA+MOGp35-55 immunization in the IL-7Rα^−/− mice (Fig. 7A–C). Therefore, the secretion of inflammatory cytokines is reduced in IL-7Rα^−/− mice as a result of fewer CD4⁺/IL-17⁺ and CD4⁺/IFNγ⁺ splenocytes and deficient Th1 cell differentiation.

FIGURE 6.

Neural Ag-induced IFNγ, IL-17, IL-4 and IL-10 secretion in IL-7Rα deficient mice. Whole spleen cells were isolated from three wild-type or IL-7Rα^−/− mice per group on day 14 following immunization with MOGp35-55 Ag. The cells were stimulated with 0, 2.5 or 10 μg/ml of MOGp35-55 in culture. The levels of IFNγ, IL-17, IL-4 and IL-10 in the culture supernatants were determined via ELISA. The values are mean ± SEM of triplicates for each data point and the figures are representative of three to four independent experiments. * p<0.05, ** p<0.01, *** p<0.001 compared to wild-type.

FIGURE 7.

Intracellular IFNγ and IL-17 in immune cells from wild-type and IL-7Rα^−/− male mice. Whole spleen cells were isolated from three mice per group of naive (a) or on day 14 following immunization with CFA (b) or CFA+MOGp35-55 Ag (c). The cells were cultured with PMA and ionomycin for 1 h followed by GolgiStop (containing monensin) for an additional 5 h before staining with CD4, IL-17- and IFNγ-specific antibodies and analyzing by flow cytometry. The values are percentages of CD4⁺ cells that express either IFNγ or IL-17. The figures are representative of four independent experiments

IL-7Rα is a critical regulator of IL-4 and IL-10 production in EAE model of MS

Since splenocytes from IL-7Rα^−/− mice produce less IL-17 and IFNγ we questioned if the anti-inflammatory cytokines were modulated as well. Thus, we examined neural Ag-induced IL-4 and IL-10 secretion in whole spleen cultures (Fig. 6, lower panels). Spleen cells from IL-7Rα^−/− female mice sensitized with 10 μg/ml MOGp35-55 showed a 3.9 and 2.2 fold increase in the secretion of IL-4 (p<0.01) and IL-10 (p<0.01), respectively, compared to wild type mice. Moreover, spleen cells sensitized with 10 μg/ml MOGp35-55 from IL-7Rα^−/− male mice also showed a 2.3 and 3.2 fold increase in the secretion of IL-4 (p>0.05) and IL-10 (p<0.01), respectively, compared to wild type mice (Fig. 6, lower panels). It is possible that the increased secretion of IL-4 in response to MOGp35-55 is not only from activated CD4⁺ Th2 cells, but may also in fact be released from IL-4-secreting mast cells. While the increase in anti-inflammatory IL-4 and IL-10 in IL-7Rα^−/− mice may not be specific to activated CD4⁺ T cells, our findings suggest the involvement of IL-4 and IL-10 in the regulation of Th1/Th17 responses and in conferring resistance to EAE in IL-7Rα^−/− mice.

IL-7Rα modulates the expansion of regulatory T cells in EAE model of MS

We then examined whether the attenuation of Th1/Th17 response and increase in IL-4 and IL-10 secretion in EAE was associated with increased Treg activity in IL-7Rα^−/− mice. Spleen cells from wild-type female mice immunized with MOGp35-55 displayed 3.8% CD4⁺CD25⁺Foxp3⁺ T cells (conventional Tregs, _convTregs) and 5.5% CD4⁺CD25⁻Foxp3⁺ T cells (inducible Tregs, _iTregs) (Fig. 8D). Whereas, spleen cells from MOGp35-55 sensitized IL-7Rα^−/− female mice showed 7.3% _convTregs and 17.7% _iTregs (Fig 8D). Moreover, MOGp35-55 sensitized spleen cells from wild-type male mice showed 3.6% _convTregs and 6.2% _iTregs. Whereas, IL-7Rα^−/− male mice immunized with MOGp35-55 Ag showed 8.3% _convTregs and 13.5% _iTregs. Representative dot plots for naïve, CFA immunized and CFA+MOGp35-55 immunized mice are shown in Figure 8 A–C. Compared to wild type, the IL-7Rα^−/− mice induced to develop EAE showed an increase in _convTregs and _iTregs. These results suggest that the increased CD4⁺/Foxp3⁺ Treg activity could be another mechanism by which IL-7Rα^−/− mice develop resistance to EAE.

FIGURE 8.

Treg cells in IL-7Rα^−/− mice. Whole spleen cells were isolated from three mice per group of naive (a) or on day 14 following immunization with CFA (b) or CFA+MOGp35-55 Ag (c). Cells were stained for surface Ag (CD4 and CD25), fixed and stained for intracellular transcription factor Foxp3. Representative FACS dot plots for Foxp3 and CD25 expression gated on CD4⁺ splenocytes are shown (a–c). The percentage of CD25⁺Foxp3⁺ conventional Treg (_convTreg) or CD25⁻Foxp3⁺ inducible Treg (_iTreg) cells among the gated CD4 splenocytes from MOG-immunized mice (d) is shown as the mean ± SEM for 5 animals in the male groups or 4 animals in the female groups. * p<0.05, ** p<0.01 compared to wild-type. The figure is a representative of three independent experiments.

Discussion

EAE is an autoimmune disease model of MS that has been used to study the mechanism of MS pathogenesis and to test the efficacy of potential therapeutic agents for the treatment of MS.^24–27. The pathogenesis of EAE involves activation of immune cells, differentiation of neural Ag-induced Th1 and Th17 cells and secretion of myelinotoxic inflammatory cytokines in the CNS. In this study we have used the EAE model to determine the physiological role of IL-7R in the regulation of MS. We have demonstrated that the IL-7Rα^−/− female mice were less likely to develop EAE compared to wild-type controls while the IL-7Rα^−/− male mice remained completely resistant to EAE. Of the twelve IL-7Rα^−/− female mice tested, only two developed EAE with clinical scores as high as the wild-type female mice, while the remaining ten mice developed no detectable clinical signs of EAE throughout the period of study (Table I). However, a one-way ANOVA using the Bonferronni's multiple comparison test revealed no statistically significant differences in MMCS, AMCS and MCS day 30 between the male and female IL-7Rα knockout and wild type groups. Thus, our findings suggest the influence of IL-7Rα in mediating susceptibility to EAE. Epidemiological studies have suggested the existence of gender differences in the incidence of MS among patient populations. While women are more likely to develop the disease (2:1), men progress faster from relapsing remitting to secondary progressive MS and develop severe disease.^28,29 Both female and male IL-7Rα^−/− mice showed significantly reduced splenocyte proliferation in response to neural Ag compared to the wild type mice (Fig. 3A). While neural antigen-specific CD4⁺ T cell proliferation was similar in wild-type and IL-7Rα^−/− mice (Fig. 3B, Supplemental Fig. 3), reduced cellularity and other associated autoimmune responses could be the mechanisms by which IL-7Rα regulates gender differences and susceptibility to EAE and MS.

It has been well recognized that differentiation of Th1 and Th17 cells and the secretion of IFNγ and IL-17, respectively, associates with the development of EAE.³⁰ In this study we found that the spleen cells from IL-7Rα^−/− female and male mice secreted lower levels of IFNγ and IL-17 in response to neural Ag than the wild type controls. This attenuation is associated with a decrease in the number of CD4⁺/IL-17⁺ and CD4⁺/IFNγ⁺ cells in the IL-7Rα^−/− mice. Furthermore, the percentage of CD4⁺/IFNγ⁺ cells in IL-7Rα^−/− naïve mice remained unchanged following induction of EAE (Fig. 7), suggesting that deficient Th1 differentiation may also contribute to the decreased clinical and pathological signs of EAE in IL-7Rα^−/− mice. Earlier studies have shown that adoptive transfer of encephalitogenic T cells cultured in the presence of rIL-7 resulted in severe and prolonged EAE compared to the control and in vitro treatment with IL-7 resulted in increased IFNγ secretion and Ag-specific T cell proliferation.³¹ Our findings are consistent with earlier reports showing upregulation of IFNγ and IL-17 production by IL-7 in a model of rheumatoid arthritis.^32,33 Additionally, IFNγ and IL-17 mRNA levels in the brains of IL-7Rα^−/− mice were significantly reduced compared to wild-type controls. This decrease was accompanied by diminished T-bet expression in the IL-7Rα^−/− male mice. The lower levels of secreted IFNγ and IL-17 from Ag stimulated spleen cells and reduced mRNA levels in the brain suggest that the regulation of Th1/Th17 responses could be another mechanism by which IL-7Rα confers susceptibility to EAE. IL-12 and IL-23 are APC-derived proinflammatory cytokines that can induce Th1 and Th17 responses and secretion of IFNγ and IL-17, respectively. We observed significant decrease in the expression of IL-12/IL-23p40 in both male and female IL-7Rα^−/− mice. Targeted disruption of IL-12p40 but not IL-12p35 causes resistance to EAE indicating that the p40 subunit shared by IL-12 and IL-23 is critical for disease pathogenesis.^34,35 Interestingly, male IL-7Rα^−/− mice had decreased levels of IL-12p35 and IL-27p28 brain mRNA whereas female IL-7Rα^−/− mice did not. IL-27 synergistically enhances the IL-12-induced production of IFNγ and has been shown to be upregulated in the CNS and lymph nodes of mice throughout the course of EAE.^36,37 The altered expression of IL-12p35, IL-12p40, IL-23p19 and IL-27p28 mRNA in the brain at the onset of EAE could be a mechanism by which IL-7Rα^−/− mice develop resistance to EAE.

IL-4 and IL-10 are protective anti-inflammatory cytokines that regulate EAE and MS.^38–41 We found that IL-4 and IL-10 secretion from whole spleen culture was increased in IL-7Rα^−/− mice. Despite fewer CD4⁺ T cells, the IL-7Rα^−/− mice secrete elevated levels of IL-4, suggesting an increase in neural antigen-induced Th2 differentiation. However, the contribution of other cell types such as mast cells can not be excluded. The increased secretion of IL-4 occurs concurrently with a decrease in IFNγ secretion (Fig. 6). IFNγ has been shown to induce apoptosis in developing mast cells.⁴² Therefore, the decrease in proinflammatory IFNγ secretion in the IL-7Rα^−/− mice may have promoted the survival of IL-4-secreting mast cells. Moreover, we observed increased IL-10 secretion from whole spleen culture in IL-7Rα^−/− mice and increased CD4⁺CD25⁺ T cells and Forkhead box P3 (Foxp3) expression (Fig. 6, Fig. 8). CD4⁺CD25⁺ T cells may convert CD4⁺ helper T cells into regulatory cells expressing IL-10 and/or TGF-β.^43–45 Additionally, IL-10 has been shown to maintain Foxp3 expression and Treg activity in the presence of colitis-induced inflammation.⁴⁶ Future studies will determine if blocking IL-10 directly alters CD4⁺CD25⁺ T cell production or Foxp3 expression in the IL-7Rα^−/− mice.

Earlier studies have shown that IL-7Rα is down regulated in human Tregs and is a marker for identifying and purifying Treg populations¹². In this study we found that IL-7Rα^−/− mice induced to develop EAE show an increased proportion of Treg cells (Fig. 8; Table 2). Foxp3 is a transcription factor that serves as a marker for Treg cells.^47–50 Defects in the Foxp3 gene expression disrupt the development of naturally occurring Tregs leading to fatal autoimmune and inflammatory diseases.⁵¹ The role played by IL-7Rα in Foxp3 expression is somewhat controversial. In one study the percentage of CD4⁺Foxp3⁺ splenocytes was reduced in the IL-7Rα^−/− naïve mice compared to controls.⁵² In contrast, Bayer and colleagues reported that the percentage of CD4⁺Foxp3⁺ splenocytes was similar between IL-7Rα^−/− naïve and wild-type mice²³. In our study the percentage of CD4⁺ splenocytes expressing Foxp3 were significantly increased in the IL-7Rα^−/− mice compared to the wild-type mice (Fig. 8). Thus despite a reduction in total numbers of T cells in IL-7Rα^−/− mice, the ratio of Teff cells to Treg cells is lower in the IL-7Rα^−/− mice (Table 2). While conventional Tregs are defined as CD4⁺CD25⁺ cells that express Foxp3, recent studies have reported CD4⁺CD25⁻-inducible Tregs (iTregs) and CD4⁺Foxp3⁺ Tregs with dynamic CD25 expression.^53,54 Therefore, we have analyzed both CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁻Foxp3⁺ populations. Although the regulatory properties of these cells remain to be determined, it is possible that one or both populations may contribute to confer resistance to EAE in IL-7Rα^−/− mice.

Table 2.

Regulatory T cell analysis in wild-type and IL-7Rα^−/− mice

Genotype	Gender	Immunity	EAE score	Total cells Mean ± SE	CD4+ cells Mean ± SE	CD4+/Foxp3+ Treg cells Mean ± SE	CD4+/Foxp3- Teffcells	Teff:Tregratio
aIL-7Rα+/+	Female	Naïve	0	248	18.23	2.34	15.88	6.8:1
aIL-7Rα−/−	Female	Naïve	0	31	0.63	0.17	0.46	2.7:1
bIL-7Rα+/+	Female	CFA	0	505	25.60	4.38	21.23	4.8:1
bIL-7Rα−/−	Female	CFA	0	86	1.05	0.39	0.66	1.7:1
cIL-7Rα+/+	Female	MOG	1.3	296 ± 33	38.07 ± 8.47	2.34 ± 0.57	35.73	15.3:1
cIL-7Rα−/−	Female	MOG	0	124 ± 27	4.90 ± 0.06	1.07 ± 0.16	3.83	3.6:1
aIL-7Rα+/+	Male	Naïve	0	255	17.44	2.26	15.18	6.7:1
aIL-7Rα−/−	Male	Naïve	0	41	0.33	0.08	0.25	3.0:1
bIL-7Rα+/+	Male	CFA	0	633	39.88	6.47	33.41	5.2:1
bIL-7Rα−/−	Male	CFA	0	106	2.14	0.83	1.31	1.6:1
cIL-7Rα+/+	Male	MOG	1	425 ± 200	40.80 ± 11.05	3.63 ± 1.06	37.17	10.2:1
cIL-7Rα−/−	Male	MOG	0	164 ± 18	5.90 ± 2.46	1.16 ± 0.08	4.74	4.1:1

Total spleen cells were isolated from female and male

^anaive mice (N=1 per group) or on day 14 following immunization with

^bCFA alone (N=1 per group) or in combination with

^cMOGp35-55 (N=3 per group).

EAE score is the average clinical score of the animals at the time of sacrifice. Total cells are the number of spleen cells obtained from the individual animal. The number of CD4⁺ and CD4⁺Foxp3⁺ Treg cells in the spleen was determined by flow cytometry. The number of CD4⁺/Foxp3-Teff cells was calculated by subtracting the number of CD4⁺Foxp3⁺ Treg cells from the number of CD4⁺ cells. Cell numbers are given in millions (cells × 10⁶) and mean ± standard error (SE) are reported for animals immunized with ^cMOGp35-55. Teff:Treg ratio is the ratio of total Teff cells to total Treg cells in the spleen.

In conclusion, our data present evidence for the first time that IL-7Rα^−/− mice remain resistant to EAE model of MS in association with a decrease in Th1/Th17 and an increase in Th2/Treg responses. The decreased frequency of MOGp35-55-specific CD4⁺ T cells and the defective Th1/Th17 responses might have contributed to confer resistance to EAE in the IL-7Rα^−/− mice. Altered expression of IL-12 family cytokines that influence the differentiation of Th1/Th17 responses could be additional mechanisms by which IL-7Rα^−/− mice remain resistant to EAE. Moreover, elevated levels of IL-4 and IL-10 and increased frequency of CD4⁺Foxp3⁺ regulatory T cells could have also contribute to confer resistance to EAE in IL-7Rα^−/− mice. Thus, IL-7Rα is critical in the pathogenesis of EAE and targeting IL-7Rα-mediated signaling networks may prove to be effective in the treatment of MS and other autoimmune diseases.

Supplementary Material

Supp Figure 1

Neural antigen-induced proliferation of CD4⁺ T cells from naive IL-7Rα^−/− mice. Spleen cells isolated from naive WT and IL-7Rα^−/− mice were stained in 0.2 μM CFSE and cultured with 0, 2.5, 5 or 10 μg/ml of MOGp35-55 for 72 hrs. The cells were then stained with anti-CD4-PE Ab, resuspended in 10 μg/ml propidium iodide and analyzed by flow cytometry. The values are percentages of CD4⁺ T cells and the figure is a representative of three independent experiments.

Supp Figure 2

Neural antigen-induced proliferation of CD4⁺ T cells from CFA-sensitized IL-7Rα^−/− mice. Spleen cells isolated from WT and IL-7Rα^−/− mice on day 14 following immunization with CFA were stained in 0.2 μM CFSE and cultured with 0, 2.5, 5 or 10 μg/ml of MOGp35-55 for 72 hrs. The cells were then stained with anti-CD4-PE Ab, resuspended in 10 μg/ml propidium iodide and analyzed by flow cytometry. The values are percentages of CD4⁺ T cells and the figure is a representative of three independent experiments.

Supp Figure 3

Neural antigen-induced proliferation of CD4⁺ T cells from CFA+MOGp35-55-sensitized IL-7Rα^−/− mice. Spleen cells isolated from WT and IL-7Rα^−/− mice on day 14 following immunization with CFA+MOGp35-55 were stained in 0.2 μM CFSE and cultured with 0, 2.5, 5 or 10 μg/ml of MOGp35-55 for 72 hrs. The cells were then stained with anti-CD4-PE Ab, resuspended in 10 μg/ml propidium iodide and analyzed by flow cytometry. The values are percentages of CD4⁺ T cells and the experiment is a representative of three independent experiments.

Supp Figure 4

Proliferation of CD4⁺ T cells in IL-7Rα^−/− mice. Spleen cells isolated from naive WT and IL-7Rα^−/− mice were stained with 0.2 μM CFSE and cultured in medium, anti-CD3+anti-CD28 Ab, or 1 and 2.5 μg/ml ConA for 72 hrs. The cells were then stained with anti-CD4-PE Ab, resuspended in 10 μg/ml propidium iodide and analyzed by flow cytometry. The values are percentages of CD4⁺ cells and the figure is a representative of two independent experiments.

Abbreviations used in this paper

AMCS

average mean clinical score

Ag

antigen

AUC

area under the curve

BSA

bovine serum albumin

CFSE

carboxy-fluorescein succinimidyl ester

CNS

central nervous system

CSF

cerebrospinal fluid

CFA

Complete Freund's Adjuvent

DI

disease incidence

ELISA

Enzyme-Linked Immunosorbant Assay

EAE

experimental autoimmune encephalomyelitis

Foxp3

Forkhead box P3

HLA

human leukocyte Antigen

IL-7

interleukin 7

IL-7Rα

interleukin 7 receptor alpha

MCS

mean clinical score

MMCS

mean maximum clinical score

MNC

mononuclear cells

MS

multiple sclerosis

MOG

myelin oligodendrocyte glycoprotein

PBS

phosphate buffered saline

qRT-PCR

quantitative real-time polymerase chain reaction

Treg

regulatory T cells

SNP

single nucleotide polymorphism

Th

T helper

TSLP

thymic stromal lymphopoietin