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. 2010 May;24(5):1583–1592. doi: 10.1096/fj.09-137323

Genetic deficiency of Irgm1 (LRG-47) suppresses induction of experimental autoimmune encephalomyelitis by promoting apoptosis of activated CD4+ T cells

Hongwei Xu *,†,§,1, Zhi-Ying Wu *,†,††,1, Fang Fang *,†, Lan Guo *,†, Doris Chen *,†, John Xi Chen ‖, David Stern , Gregory A Taylor #,**, Hong Jiang ‡, Shirley ShiDu Yan *,†,2
PMCID: PMC2879948  PMID: 20056715

Abstract

The immunity-related GTPase Irgm1, also called LRG-47, is known to regulate host resistance to intracellular pathogens through multiple mechanisms that include controlling the survival of T lymphocytes. Here, we address whether Irgm1 also plays a role in the pathogenesis of experimental autoimmune encephalitis (EAE). We find that Irgm1/LRG-47 is a significant factor in the progression of EAE and multiple sclerosis (MS). Expression of Irgm1 was robustly elevated in MS-affected lesions and in the central nervous system (CNS) of myelin basic protein (MBP)-induced EAE mice, especially in cells of lymphoid and mononuclear phagocyte origin. Homozygous Irgm1 null mice were resistant to MBP-induced EAE, and CD4+ T cells in spleen and CNS of these mice displayed decreased proliferative capacity, increased apoptosis, and up-regulated interferon (IFN)-γ induction. Therefore, Irgm1-induced survival of autoreactive CD4+ T cells contributes significantly to the pathogenesis of EAE. Blockade of Irgm1 may be a potential therapeutic strategy for halting multiple sclerosis.—Xu, H., Wu, Z.-Y., Fang, F., Guo, L., Chen, D., Chen, J. X., Stern, D., Taylor, G. A., Jiang, H., Yan, S. S. Genetic deficiency of Irgm1 (LRG-47) suppresses induction of experimental autoimmune encephalomyelitis by promoting apoptosis of activated CD4+ T cells.

Keywords: IFN-γ-mediated apoptosis, EAE/MS, animal model

Multiple sclerosis (MS) is the most common autoimmune disorder of the central nervous system. Its pathogenesis involves the production of T lymphocytes that are reactive against components of myelin sheaths, which is thought to play a key pathological role in the disease process (1,2,3). Experimental autoimmune encephalomyelitis (EAE), a widely used rodent model of human MS, is induced by immunization of mice with encephalitogenic myelin antigens in the presence of specific adjuvants (4). EAE is characterized by central nervous system (CNS) inflammation and is associated with demyelination and infiltration of inflammatory mononuclear cells, including a subset of encephalitogenic myelin antigen-specific CD4+ T cells. Interaction of activated CD4+ T cells and myelin antigens seemingly provokes a massive inflammatory response and promotes continued proliferation of T cells and activation of macrophages that subsequently lead to demyelination and CNS destruction. The mechanisms underlying these cycles of disease reactivation that result in the exacerbation and progression of autoimmunity remain elusive. Extinction of the immune response either by limiting expansion and persistence of autoreactive T cells or by inhibiting inflammation by blocking cytokine induction is an important and promising therapeutic approach to EAE/MS.

Interferon (IFN)-γ has a central role in host resistance to infection and immune system regulation. The multiple functions of IFN-γ during the immune response include modulating CD4 T-cell homeostasis, particularly activation-induced apoptosis and autophagic cell death (5,6,7,8). In the setting of EAE, IFN-γ-deficient mice display a progressive and fatal clinical course associated with increased proliferation and decreased apoptosis of activated CD4 T cells in response to antigen (7). These observations favor the likelihood that downstream effectors of IFN-γ may have important roles in immunobiology. These effectors could include a family of IFN-γ-inducible intracellular 47-kDa GTPases [immunity-related GTPases (IRG)] that has been shown to contribute to resistance to intracellular pathogens (9,10,11,12,13,14). In the mouse, one member of this family, the immunity-related GTPase Irgm1 (also known as LRG-47), is essential for resistance to multiple pathogens that include Mycobacterial tuberculosis and Toxoplasma gondii, whereas other members of this protein family are involved in the response to distinct spectra of pathogens (4,5,6,7,8,9). The human ortholog IRGM may serve a similar role in intracellular microbial killing, phagosome maturation, and autophagy (15). However, the roles of Irgm1 and IRGM in MS/EAE are unknown.

We recently demonstrated that receptor for advanced glycation endproducts (RAGE), a multiligand member of the immunoglobulin superfamily, mediates induction of EAE under certain conditions. In the myelin basic protein (MBP) model, blockade of RAGE prevents symptomatic EAE induction at least in part by inhibiting immunocyte migration into the CNS (16). To probe underlying mechanisms, we performed microarray analysis to elucidate differences in gene expression between EAE-induced mice and their wild-type (WT) counterparts. Prominent up-regulation of Irgm1 was observed in EAE-induced animals treated with MBP compared with naive mice. In the present studies, we analyze the role of Irgm1 in the pathogenesis of EAE relevant to MS. Our results reveal a role for Irgm1 in the survival of autoreactive CD4 T cells in EAE; we provide in vivo and in vitro evidence that Irgm1 deficiency suppresses EAE in Irgm1-deficient mice by preventing expansion of the activated CD4 T-cell population and promoting apoptosis in these cells. The absence of Irgm1-induced negative regulation of IFN-γ, after initiating the autoimmune response, is responsible for autoreactive CD4+ T-cell death leading to attenuation of EAE development and progression.

MATERIALS AND METHODS

Mice

Irgm1/LRG-47-knockout mice (Irgm1−/−) were generated as described previously (14). Mice were backcrossed into the B10PL background (n>10 generations). WT littermates (in B10PL) served as controls.

Human MS tissue

Frozen spinal cord specimens from MS patients were obtained from the Human Brain and Spinal Fluid Resource Center, VA West Los Angeles Healthcare Center (Los Angeles, CA, USA), which is sponsored by the National Institute of Neurological Disorders and Stroke, the National Institute of Mental Health, the National Multiple Sclerosis Society, and the Department of Veterans Affairs (n=5 for MS and n=4 for age-matched controls). Fixed and embedded MS tissue specimens were obtained from the Pathology Department of Columbia University (n=5 for MS and n=5 for age-matched controls).

Induction of EAE

EAE was induced as described previously (16) using a protocol approved by the Columbia University Institutional Animal Care and Use Committee. Induction of EAE using the MBP model was performed by injecting B10PL mice (subcutaneously) with 1–9NAc MBP in complete Freund’s adjuvant (CFA) on d 0. Clinical symptoms were scored from 1 to 5 as described previously (10).

Microarray analysis

The Clontech Atlas mouse 1.2 array was utilized according to manufacturer’s directions (PT3140–1; Clontech, Palo Alto, CA, USA). Briefly, gene expression profiles were determined for 1176 independent genes gridded on the mouse Atlas 1.2 nylon array. Total RNA was isolated from both EAE and WT mouse spinal cord, respectively, using Atlas Pure Total RNA labeling system (cat. no. 634562; Clontech); 2 μg RNA was then radiolabeled with α-32P-dATP (Amersham Biosciences, Piscataway, NJ, USA). cDNA probes were synthesized and hybridized to separate Atlas Mouse cDNA Expression Array membranes. Hybridized membranes were analyzed by autoradiography followed by image analysis using Atlas Image 1.5 software (Clontech). The strength of the signal for each gene was calculated by subtraction of the background signal for each segment and expressed in intensity units.

Isolation of mononuclear cells from the CNS

To analyze cellular infiltrates in the CNS, mice were deeply anesthetized, and intracardiac perfusion with PBS (30 ml) was performed. The brain and spinal cord were minced and digested for 45 min at 37°C in HBSS containing 0.2 U of liberase R1 (Roche, Mannheim, Germany) and DNase I (50 μg/ml). Softened tissue fragments were forced through a 40-μm cell strainer, and homogenates were resuspended in 70% Percoll (Sigma, St. Louis, MO, USA) and overlaid with 30% Percoll. Gradients were centrifuged at 2000 g for 30 min. Mononuclear cells in the CNS were collected from the Percoll interface.

Flow cytometry

Cells were washed with FACS buffer [PBS containing 0.1% (w/v) sodium azide and 2% (v/v) fetal calf serum] and preincubated with CD16/CD32 monoclonal antibody (clone 2.4G2; Pharmingen, San Jose, CA, USA) for 15 min at 4°C to block nonspecific binding to Fc receptors. Fluorochrome-conjugated monoclonal antibodies (rat anti-mouse CD4-PerCP, rat anti-mouse CD44-PE, and appropriate isotype controls) were purchased from Pharmingen. After incubation, cells were washed twice with FACS buffer and data were acquired on a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and then analyzed using FLOWJO software (Treestar, San Carlos, CA, USA).

Proliferation assays

For T-cell proliferation, cells isolated from spleens and lymph nodes were cultured in serum-free medium with 1–9NAc MBP (5 μg/ml) for 72 h. 3H-thymindine (1 μCi/well) was added 16 h before harvesting. For the in vivo 5-bromo-2′ deoxyuridine (BrdU; Sigma) uptake assay, mice were injected intraperitoneally with 1 mg of BrdU twice (once each on d 13 and 14) during the course of EAE. CNS mononuclear cells were isolated and stained with anti-CD4-PerCP (Pharmingen). BrdU staining was performed according to the manufacturer’s instruction (Pharmingen). BrdU incorporation was analyzed on gated CD4+ T cells.

Assays for apoptosis

Apoptosis was measured in vitro using 2 different methods. The annexin V assay was used to detect cells in the early stages of apoptosis. Splenic and CNS cells were stained with anti-CD4-allophycocyanin IgG and resuspended in annexin binding buffer. Cells were stained for 15 min with 5 μl of FITC-labeled annexin V (Pharmingen) at room temperature in the dark. Analysis was performed by flow cytometry within 1 h. In situ detection of apoptotic cells was performed on MBP-activated CD4 T cells cultured on chamber slides using the in situ Cell Death Detection Kit (Roche). Briefly, air-dried cell samples were fixed with a freshly prepared fixation solution for 1 h at 25°C and then incubated in permeabilization solution for 2 min on ice. Terminal deoxinucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were detected according to the manufacturer’s instructions (Roche). The percentage of TUNEL-positive cells is defined as the percentage of the number of TUNEL-positive cells divided by the total number of cells per field.

Quantitative real-time PCR

Total RNA was extracted from lymph nodes and spinal cord using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA synthesis was performed using random hexamer primers and the TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Samples were subjected to real-time PCR analysis on an ABI Prism 7700 Sequence Detection System under standard conditions. Relative mRNA abundance was normalized against 18S RNA (the endogenous control). The primers and probe for human IRGM and mouse Irgm1 were designed using Primer Express software (Applied Biosystems) and purchased from Applied Biosystems: human IRGM based on GeneBank accession no. XM_293893, BC038539, or BC128168 (5′-GGAACTTGCCAGAGGTGATCTC-3′, reverse primer 5′-GCCTTACCCTCATGTCCTGTGT-3′, and probe TCATCAGTGCCCTTCGA) and mouse Irgm1 based on GeneBank accession no. NM_008326 or U19119 (5′-TGGCAATGGCATGTCATCTT-3′, reverse primer 5′-AGTACTCAGTCCGCGTCTTCGT-3′, and probe ACTTCGAGTCATCGGC).

Northern blotting

Samples for Northern blotting were collected from MBP-immunized WT mice. Total RNA was isolated using TRIzol (Invitrogen) as described previously (17). For each sample, 10 μg of total RNA was electrophoresed through 1% agarose-6% formaldehyde gels. RNAs were transferred to Hybond N+ membranes and then hybridized with 32P-labeled mouse Irgm1 cDNA or β-actin cDNA (the latter used as a control probe) by QuikHyb solution (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions at 68°C for 1 h. The cDNA probe was labeled with 32P using a random primer labeling kit (Stratagene).

Generation of antibody to human IRGM

We made an affinity purified antibody specific to human IRGM (GeneBank accession no. XM_293893 or BC038539) corresponding to aa 57–74 (GHEGKASPPTELVKATQR) and 135–152 (EDMGKKFYIVWTKLDMDL) in rabbit. The affinity-purified antibody was generated over resin with both human IRGM peptides (aa 57–74 and 135–152).

ELISA for cytokine detection

CD4 T cells were isolated from MBP-immunized mouse spleen/lymph nodes and purified using magnetic beads conjugated with anti-CD4 mAb and a magnetic column (Miltenyi Biotec, Inc., Auburn, CA, USA). CD4 T cells were cultured in T-cell medium (Click’s medium with 10% FCS, 0.01 M HEPES, 5×10−5 M 2-ME, 2 mM l-glutamine, and antibiotics) with MBP (5 μg/ml) and irradiated splenic cells as antigen-presenting cells. Supernatants were collected at the time points indicated. Analyses of mouse IFN-γ were performed on both supernatants and serum samples, collected at d 14 post-EAE induction using commercially available ELISA kits (R&D, Minneapolis, MN, USA).

Histology and immunostaining

Paraffin-embedded sections were stained with hematoxylin and eosin (H&E). Inflammatory infiltrates were assessed semiquantitatively by evaluating the percentage area occupied by nuclei in H&E-stained sections per high-power field (5 fields/slide) using the morphometric option of the Universal Imaging System (16, 18). Histological analysis was performed by a researcher blinded to experimental group and mouse ID number. To localize Irgm1 antigen in spinal cord, double immunostaining with anti-Irgm1 and anti-CD4 or CD11b IgG was performed. Cryopreserved spinal cord sections were fixed with 4% paraformadehyde in PBS for 30 min. Slides were permeabilized and blocked in PBS containing 5% horse serum and 0.2% saponin for 60 min. They were then incubated with goat anti-Irgm1 IgG (A-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-CD4, or rat anti-CD11b mAbs (BD Pharmingen) followed by secondary antibodies (donkey anti-goat IgG conjugated-FITC and rabbit anti-rat IgG conjugated-TRITC; Sigma). Sections were then examined by confocal microscopy.

Statistical analysis

Results are expressed as means ± se. When 2 groups were compared, a 2-tailed Student’s t test was used. When multiple groups were compared, ANOVA was used. Fisher’s t test was used for post hoc comparisons. Significance of the EAE score was analyzed utilizing nonparametric methods (Mann-Whitney and Wilcoxon test) in addition to the ANOVA and was followed by Dunnett’s post hoc tests using StatView 5.0 software (http://www.citewise.com). A value of P < 0.05 was considered statistically significant.

RESULTS

Increased expression of Irgm1 in mouse EAE

Based on microarray comparison of spinal cords from MBP-induced EAE to those from WT mice, we identified a significant increase in Irgm1 mRNA in EAE mice vs. naive controls (Fig. 1) as well as other gene products (Table 1). To verify the microarray results, we performed quantitative PCR analysis and Northern blotting to evaluate Irgm1 expression levels in EAE mice. Utilizing quantitative PCR, it was found that Irgm1 mRNA increased ∼30-fold (normalized to total 18S RNA) in spinal cord tissue (a time when symptomatology was approaching a peak (14–18 d; P<0.001) after treatment with MBP as compared with controls (untreated animals or those receiving only CFA; Fig. 2A). Furthermore, levels of Irgm1 mRNA increased by ∼9-fold and ∼2.5-fold when normalized to CD4 and CD11b mRNA, respectively (Fig. 2B, C). Northern blot analysis on spinal cord tissue from animals subjected to MBP-induced EAE was performed at several time points; a trend toward higher levels of Irgm1 transcripts was first noted by d 10 post-MBP treatment, and a statistically significant increase was seen by d 14, clinical score 2–3 (Fig. 2D, E). These data indicate a close correlation between Irgm1 expression and symptomatic EAE. Irgm1 antigen was detected in CD4 T cells (Fig. 2F2) and mononuclear phagocytes (Fig. 2G2) in spinal cords from EAE-induced mice on d 16 by confocal microscopy with double immunostaining of Irgm1 and CD4 or CD11b; Irgm1 antigen was colocalized in a subpopulation of cells also staining for CD4 (Fig. 2F3) and CD11b (Fig. 2G3).

Figure 1.

Figure 1.

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Microarray results show an increase in Irgm1/LRG-47 mRNA (arrow) in spinal cord tissue of MBP-induced EAE mice (B) compared with WT naive mice (A).

TABLE 1.

Gene expression in EAE mice

Gene name Expression pattern (EAE vs. naïve)
Interferon inducible protein 1 (LRG-47) ↑
Interferon regulatory factor 1 (IRF1) ↑
Thymus cell antigen 1 ↓
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Figure 2.

Figure 2.

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Expression of Irgm1 in EAE mice. A–C) Total RNA was isolated from spinal cords of WT, CFA-treated, or MBP-induced EAE mice. Quantitative real-time PCR shows levels of mouse Irgm1 mRNA normalized to 18S RNA (A), CD4 RNA (B), or CD11b RNA (C), respectively, in spinal cord tissue from each group on d 14–18 after EAE induction (this time point corresponded to peak symptoms in the MBP-treated group). n = 3–5 mice/group. Data present fold-increased mRNA expression in EAE- and CFA-treated groups vs. naive mice. D) Northern blotting analysis of spinal cord tissue for Irgm1 transcripts. Time points indicate number of days after immunization with MBP-derived peptide. Bottom panel: Northern blotting for β-actin probe as a control for the equal amount of RNA used in each lane. E) Quantification of Northern blotting images. n = 3/group. F, G) Localization of Irgm1 antigen in spinal cord tissue of EAE mice. Spinal cord sections were costained with goat anti-Irgm1 and rat anti-CD4 or rat anti-CD11b followed by rabbit anti-goat IgG conjugated with FITC and anti-rat conjugated with TRITC. Irgm1 antigen (green, F2, G2) was detected in CD4+ T cells (red, F1) and CD11b-positive mononuclear cells (red, G1). View: ×600 (F1–3); ×400 (G1–3). *P < 0.001.

Increased expression of IRGM in MS-affected spinal cords

We also examined whether IRGM expression is modulated in MS patients’ brain and spinal cord lesions. As predicted from the murine EAE model, expression of IRGM was significantly increased in the MS-affected spinal cord tissues from MS patients. Quantitative real-time PCR demonstrated increased levels of human IRGM transcripts in MS-affected spinal cord tissue, as compared with age-matched normal controls (Fig. 3A). Immunostaining with specific anti-IRGM IgG demonstrated the presence of Irgm1 especially in mononuclear phagocytes and CD4+ T cells as shown by costaining for CD68 and CD4+ antigens, respectively (Fig. 3B, C). Negative controls were used to determine the specificity of the immune reaction. The specific staining patterns were not present when the IRGM antibody was omitted, replaced by nonimmune IgG or preabsorbed by its antigen (IRGM peptides; Fig. 3D, E).

Figure 3.

Figure 3.

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Expression of IRGM in human MS tissues. A–C) Quantitative real-time PCR (A) and immunoblotting of human spinal cord tissue from MS patients and age-matched normal controls for IRGM (B, C). Spinal cord tissue from MS patients was subjected to sequential immunostaining with anti-IRGM IgG (B1, C1) followed by anti-CD68 (B2) or anti-CD4 (C2). D–F) Specific staining pattern was lost when the IRGM antibody was replaced by nonimmune IgG (NI IgG; D), preadsorbed with IRGM peptides (E), or omitted (F). Scale bars = 5 μm. *P < 0.01.

Deficiency of Irgm1 protects animal from EAE

The close association of EAE with Irgm1 mRNA/protein expression led us to explore whether this mediator plays a pathogenic role in EAE [using homozygous mutant mice devoid of Irgm1 (Irgm1−/−) in the B10PL strain]. For these studies, we used the MBP model in which Irgm1−/− or age/strain-matched WT B10PL littermate mice were immunized with an acylated N-terminal peptide comprised of nine residues from MBP (1–9NAc MBP; ref. 10). Compared with WT controls, Irgm1−/− mice exhibited strikingly reduced incidence and severity of disease (mean clinical score: 2.85±0.4 se on d 16–21 and 3.2±0.5 se on d 29–32 in WT EAE mice vs. 0.1–0.2±0.2 se in Irgm1−/− EAE mice, respectively, P<0.01; Fig. 4A, C). Heterozygous Irgm1+/− mice showed a slight reduction in the severity of symptomatic disease compared with strain-matched WT controls but not at a statistically significant level (Fig. 4A). Histological analysis of spinal cord tissue harvested at the time of peak symptoms (d 18) post-MBP treatment demonstrated a striking reduction in inflammatory cell infiltrates in Irgm1−/− mice, as compared with other genotypes (Fig. 4B). Inflammatory infiltrates were assessed semiquantitatively by evaluating the percent area occupied by nuclei in H&E-stained sections per high power field (5 fields/slide), using the morphometric option of the Universal Imaging System (16, 18).

Figure 4.

Figure 4.

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Effects of gene deletion of Irgm1 on the induction of EAE. A) Irgm1 heterozygous mutant (Irgm1+/−), homozygous mutant (Irgm1−/−) mice, and WT littermates (Irgm1+/+) were immunized with 1–9NAc MBP-derived peptide. Symptoms were assessed according to a clinical scoring system (*P<0.01 vs. Irgm+/+ mice). B) Bottom: representative H&E-stained spinal cord sections from EAE-induced mice of the indicated genotype (Irgm1+/+, Irgm1–47+/−, and Irgm1−/−) on d 21. Top: analysis of area occupied by nuclei from samples derived from the above groups of mice. *P < 0.01, #P < 0.05; n = 5–7/group. C) Percentage of each group of mice developing disease.

Irgm1 deficiency enhances apoptosis of CD4+ T cells during EAE

To analyze the mechanisms through which Irgm1 affected on the pathogenesis of EAE, we examined the CD4 T-cell population in Irgm1−/− and control mice. We found that the percentage of CD4 T cells in CNS (brain and spinal cord) eluates from Irgm1−/− mice 18–20 d after MBP treatment was reduced ∼50%, compared with WT mice (22.7 and 46.6% for Irgm1−/− and controls, respectively; Fig. 5A). This reduction was especially pronounced in the activated CD4 T-cell population that expresses high levels of CD44 (CD44hi); activated CD4 T cells comprised only 8–9% of cells in spinal cord eluates from EAE-induced Irgm1−/− mice vs. 32% in EAE-induced WT animals (Fig. 5A). Most striking was the ∼3-fold increase in apoptotic cells, based on annexin V staining, in the CD4 T-cell population harvested on d 20 from the CNS of Irgm1−/− mice as compared with controls (30 vs. 9%, for Irgm1−/− and controls, respectively; Fig. 5A). Similarly, the percentage of CD44hi was significantly reduced in the lymphocyte population from lymph nodes of Irgm1−/− mice compared with WT controls (Fig. 5B). Apoptosis, as shown by TUNEL-positive cells, was increased in lymph nodes from Irgm1−/− mice, compared with WT littermates (Fig. 5C). To more fully understand the relationship between Irgm1 expression and cell proliferation in CD4 cells in vivo, mice were treated with BrdU intraperitoneally, and FACS analysis was performed on cells isolated from the CNS using anti-CD4 and anti-BrdU IgG. Compared with WT mice, cellular eluates from the CNS of Irgm1−/− mice displayed a strong reduction in the percentage of CD4 T cells incorporating BrdU (4.69% in LRG-47−/− vs. 33% in WT mice; Fig. 5D).

Figure 5.

Figure 5.

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Irgm1-knockout mice (Irgm1−/−) display reduced accumulation and enhanced apoptosis of activated CD4 T cells on EAE induction. A) Infiltrating cells were isolated from the CNS of Irgm1−/− mice and WT littermates on d 18–20 (clinical score 2.5–3 in WT animals), and were immunostained with antibodies to CD4 conjugated with PerCP, CD44 conjugated with PE, or annexin V conjugated with FITC. FACS analyses were used to determine the percentage of cells staining positively for each of the markers, CD4, CD44hi, and annexin V. CD44hi and annexin V were gated for CD4. *P < 0.05; n=3–5/group. B) FACS analysis of activated CD4 T cells isolated from lymph nodes of EAE mice. Lymphocytes isolated from lymph nodes of LRG−/− mice and WT littermates were stained with anti-CD4 conjugated with PerCP and CD44 conjugated with PE. Percentage of CD44hi-positive cells was determined by FACS gated for CD4 cells. *P < 0.01; n=3–5/group. C) Lymphocytes from lymph nodes of EAE mice were subjected to TUNEL staining. TUNEL-positive cells were quantified using Universal Imaging software. *P < 0.05. D) In vivo proliferation of CD4+ T cells isolated from EAE mice. Mononuclear cells isolated from the CNS were stained with anti-CD4 PerCP and anti-BrdU followed by FACS analysis. Cells were pooled from 5 mice/group. BrdU incorporation was expressed as the percentage of BrdU-positive CD4+ T cells gated in CD4+ T cells.

These in vivo observations concerning a possible relationship between Irgm1 expression and the proliferative potential of CD4 T cells led us to perform the following in vitro experiments. T cells were purified from spleens and lymph nodes of mice immunized 7 d previously with MBP and cultured in the presence of MBP. We found that T cells from Irgm1−/− mice showed diminished proliferation in response to MBP, as compared with T cells from WT controls (Fig. 6A). Correspondingly, the percentage of annexin V-positive cells was increased in Irgm1−/− vs. WT mice (Fig. 6B). Further analysis of CD4 T cells isolated from lymph nodes of EAE mice after MBP treatment revealed an increase in the percentage of TUNEL-positive CD4 T cells from Irgm1−/− mice as compared with WT mice (Fig. 7C).

Figure 6.

Figure 6.

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Effects of Irgm1 deficiency on T-cell proliferation and apoptosis of CD4 T cells in response to MBP: in vitro studies. A) 3H-thymidine incorporation was performed on lymphocytes and splenocytes isolated from the indicated mice on d 7 after immunization with MBP (5 μg/ml). *P<0.01 vs. other groups; n=3–5/group. B) Splenocytes from Irgm1−/− and WT littermates were stained with anti-CD4 conjugated to PE and annexin V conjugated to FITC after stimulation with MBP (5 μg/ml) for 48 and 72 h, respectively. FACS analyses show percentage of annexin-positive cells gated in the CD4+ T-cell population.

Figure 7.

Figure 7.

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Irgm1 deficiency increases IFN-γ production and enhances apoptosis. A) IFN-γ production in CD4 T cells from WT and Irgm1−/− mice. CD4 T cells were isolated from spleens of WT mice and Irgm1−/− mice, and cultured with MBP (5 μg/ml) for 24, 48, and 72 h. B) ELISA for measurement of serum levels of IFN-γ in WT and Irgm1−/− EAE mice (d 14–18); n = 3–5 per group. C) Percentage of TUNEL-positive cells in MBP-induced Irgm1-deficient CD4+ cells, WT CD4+ T cells, WT CD4+ T cell plus IFN-γ (100 ng/ml), or Irgm1-deficient CD4+ T cells plus anti-IFN-γ (10 μg/ml) for 48 h.

Effect of Irgm1 on IFN-γ induction

To further understand the apparently central role of Irgm1 in regulation of CD4 T-cell function and survival in the setting of pathogenic autoimmunity, we examined how Irgm1 might impact expression of IFN-γ in active CD4+ T cells. CD4+ T cells isolated from lymph nodes/spleens of Irgm1−/− mice 7 d after immunization with MBP-derived peptide were restimulated with MBP. Levels of IFN-γ were increased in the cultured supernatants from Irgm1-deficient CD4+ T cells compared with WT CD4+ T cells (Fig. 7A). In addition, levels of IFN-γ were elevated in sera of Irgm1−/− mice compared with WT mice that were MBP-induced to develop EAE (Fig. 7B).

Since IFN-γ has been shown to function as a negative regulator of lymphocyte proliferation, eliminating activated CD4+ T cells by apoptosis during the immune response (5,6,7, 19), we next examined whether Irgm1-deficiency-mediated induction of IFN-γ contributes to the apoptosis of CD4+ T cells in response to MBP. Irgm1-deficient CD4+ T cells and WT CD4 T cells were stimulated with MBP alone or MBP plus anti-IFN-γ IgG (5–10 μg/ml) for 48 h. In the presence of MBP, Irgm1-deficient CD4+ T cells displayed a significant higher level of TUNEL-positive cells than WT CD4+ T cells. Neutralization of IFN-γ by the addition of anti-IFN-γ IgG to the cultured medium completely blocked the effect of increased IFN-γ (due to the lack of Irgm1) on MBP-induced apoptosis as shown by a reduction in TUNEL-positive cells (Fig. 7C). To determine the effect of IFN-γ on apoptosis, MBP-induced CD4+ T cells were incubated with IFN-γ (100 ng/ml) for 2 d. IFN-γ-treated cells demonstrated a significant increase in numbers of TUNEL-positive apoptotic cells as compared with vehicle-treated cells. Of note, in the absence of MBP stimulation there was no significant difference in the levels of TUNEL-positive cells between Irgm1-deficient CD4+ T cells and WT CD4+ T cells, suggesting that the lack of Irgm1 does not affect physiological cellular function in the absence of stimulators/stressors.

DISCUSSION

The present studies offer new insights into the mechanisms underlying Irgm1-mediated activation and/or expansion of autoreactive CD4+ T-cell populations in the EAE animal model. The substantial expression of Irgm1 in autoreactive CD4+ T cells and macrophages of both MS- and EAE-affected spinal cord tissue indicates a role for this mediator in the pathogenesis of EAE and a potentially significant role in MS. Indeed, as it is shown that Irgm1-knockout mice exhibit milder EAE symptomology than WT mice, the current studies provide substantial evidence of a protective effect of Irgm1 blockade in the development and progression of EAE. It is of note that strong up-regulation of Irgm1 transcripts did not occur until 14 d after the initial induction of EAE. This observation suggests that Irgm1 expression probably occurs as a distal consequence of activation of cytokine/chemokine signal transduction mechanisms. The nature of the signals leading to Irgm1 expression in this setting remains to be elucidated by future studies.

Apoptosis is an important mechanism for maintaining homeostasis of lymphocytes in the immune system. During the immune response, larger numbers of activated T-cell effectors are generated. Most of these cells are then eliminated by apoptosis to restore homeostasis to the T-cell compartment. Thus, apoptosis is an important physiological mechanism for immune system regulation and is responsible for elimination of autoreactive T lymphocytes (T cells), B lymphocytes (B cells), and monocytes from the circulation and prevention of their transport into the CNS (20, 21). Apoptosis of infiltrating T cells in lesions has been noted during clinical recovery in autoimmune diseases of the nervous system, including animal models of MS (22).

T-cell death occurs in the CNS of mice with EAE at the time of spontaneous remission from paralytic disease (22,23,24). Failure to suppress expansion of activated CD4+ T-cell populations will result in accumulation of large numbers of activated CD4 T cells in the spleen and cerebrospinal fluid during EAE and leads to disease progression. For example, IFN-γ-knockout mice and normal mice treated with anti-IFN-γ antibody paradoxically show more severe encephalitis after immunization with myelin proteins (compared with normal mice) due to diminished IFN-γ-mediated apoptosis of T cells (7, 25, 26). In addition, recent studies (27, 28) showed that osteopontin induces relapse and progression of autoimmune brain disease through enhanced survival of activated T cells. The present results demonstrate that after initiating autoimmune response during EAE, blockade of Irgm1 enhances apoptosis of active CD4+ T cells in EAE-affected tissues (lymph node and CNS), as well decreasing the levels of infiltrating T cells in EAE-induced Irgm1-knockout mice. We found more TUNEL- and annexin-V-positive CD4+ T-cell infiltration in the CNS and lymph nodes of Irgm1-knockout mice with EAE than in those of WT mice with EAE. Thus, the absence of Irgm1 resulted in enhanced death of autoreactive T cells. It is likely that death of encephalitogenic CD4+ T cells, as well as diminished proliferation capacity, accounts for the relatively mild course of EAE in Irgm1-knockout mice. Thus, the lack of Irgm1-induced apoptotic elimination of autoreactive T cells may be one of mechanisms underlying alleviation of clinical progression of EAE.

It is possible that genetic deletion of Irgm1 may affect normal physiological function as well as cause neuropathological changes. For example, a recent report (8) indicates that impaired progenitor cell proliferation was observed in Irgm1−/− mice. Based on our past studies, homozygous Irgm1−/− mice in B10.PL background do not display an obvious phenotype in the absence of stressors; they grow and develop normally and display normal fertility in both genders. Furthermore, cultured CD4+ T cells isolated from Irgm1-knockout mice grow well and show no evidence of toxicity as shown by thymidine incorporation in the absence of MBP stimulation (Fig. 6A), as compared with lack of same in CD4+ T cells isolated from WT littermate controls. Taken together, these data suggest that the protective effects of Irgm1 deficiency on induction of EAE are unlikely to be due to impaired progenitor cell proliferation in Irgm1−/− mice as recently reported (8).

IFN-γ has been shown to function as a negative regulator of lymphocyte expansion, eliminating activated CD4 T cells by promoting apoptosis during the immune response (5,6,7, 19). This may be one of the mechanisms underlying the exacerbation of EAE observed in IFN-γ-knockout mice. Although Irgm1 is an inducible IFN-γ protein, surprisingly the present results demonstrate that IFN-γ levels were significantly elevated in CD4+ T cells lacking Irgm1, as well as in sera from Irgm1-knockout mice exhibiting EAE. These observations suggest that IFN-γ directly triggers the death of Irgm1-deficient CD4+ T cells during pathogen-driven T-helper 1 (Th1) responses.

Based on these findings, we propose that blockade of Irgm1 interrupts a negative feedback loop, the latter triggered after the immune response is initiated, thereby limiting subsequent expression of IFN-γ. As a result, reexpression of IFN-γ leads to levels that attenuate the ongoing immune response. While Irgm1 has been shown to be a contributor to the host response to intracellular bacterial infection (14, 29, 30) and has been suggested to function as a survival factor in the context of mycobacterial infection (31, 32), our data indicate that it is also present in CD4+ T cells, where it may promote survival of pathogenic self-reactive T cells by preventing T-cell apoptosis. We propose that induction of EAE by activation of autoreactive MBP-specific T cells leads to early (d 7) secretion of IFN-γ, thereby initiating a delayed type hypersensitivity response in the CNS. Induction of Irgm1 by IFN-γ facilitates the survival of the expanded autoreactive T-cell population and may play an important role in the pathogenesis of autoimmune diseases, such as EAE.

Our data indicate the potential significance of Irgm1 in modulating the properties of CD4 T cells. As recently reported, Irgm1/LRG-47 promotes the expansion of activated CD4+ T-cell populations via prevention of IFN-γ-induced cell death (33). Given that Irgm1 is involved in the cellular autophagy machinery for IFN-γ-activated macrophages controlling the fate of pathogens (9, 15) and for CD4+ T cells during pathogen-driven Th1 response (8, 33), it will be of interest (as part of future investigations) to determine whether autophagy-related mechanisms underlying the effects of Irgm1 contribute to survival of autoreactive CD4+ T cells during EAE. The protective role of Irgm1 deficiency for encephalitogenic CD4 T cells in EAE (thereby enhancing pathogenicity) contrasts with its role in buttressing a protective host response to certain pathogens (9, 12, 30, 31). Understanding how Irgm1 fits into cytokine networks in lymphocytes and mononuclear phagocytes (i.e., through the regulation of IFN-γ expression) will likely provide insight into this apparent dichotomy.

In conclusion, the current findings clearly demonstrated the protective effect of Irgm1 deletion on the induction of EAE through promoting autoreactive CD4 T-cell apoptosis. Irgm1 blockade-mediated induction of IFN-γ contributes to autoreactive CD4+ T-cell apoptosis during EAE. These mechanisms eliminate autoreactive T cells, directly linking Irgm1 deletion to the alleviation of EAE and implicating this GTPase in the pathogenesis of autoimmune disorders. Thus, Irgm1 homologues may be a potential target for therapy for MS, the human counterpart of EAE.

Acknowledgments

These studies were supported by National Institute of Neurological Disorders and Stroke grant NINDS 042855. The authors thank A. P. Hays (Department of Pathology, Columbia University) for providing human MS samples and suggestions. G.T. is supported by U.S. National Institutes of Health grant AI-57831 and a Veterans Affairs Merit Review grant.

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