Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion
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Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelinspecific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca2+ binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG35–55) immunization, Aif-1-deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG35–55-induced EAE and as a potential therapeutic target in MS.
Multiple sclerosis (MS) is a chronic progressive disorder caused by the formation of inflammatory plaques in the brain and spinal cord (1). Experimental autoimmune encephalomyelitis (EAE) shares both neuropathological and clinical features of MS (2). Studies of MS and EAE provide evidence that T lymphocytes specific for myelin antigens contribute to disease pathogenesis (3). Inflammation in EAE is mediated by major histocompatibility complex (MHC) class II-restricted, Thl-type CD4+ myelin reactive, and Th17-type T cells (4, 5, 6). Autoreactive T cells activate in the periphery, cross the blood-brain barrier to enter the central nervous system (CNS) and serve as important disease initiators, affecting both the local cytokine milieu and the recruitment and activation of various effector cells (7, 8, 9). Microglia and macrophages also contribute to EAE; they produce cytokines that promote inflammation during induction, but also phagocytose and clear apoptotic cell bodies, debris and inhibitory substances that limit remyelination and axon regeneration (10,11). The molecular mechanisms that control expansion, activation and CNS trafficking of myelin-specific autoreactive T cells and the complex functions of microglia and macrophages in EAE are incompletely understood.
Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca2+ binding adapter-1 [Iba-1]) is a 17-kDa, interferon (IFN)-γ-inducible, EF hand motif protein encoded within the class III region of the MHC (human chromosome 6p21.3, mouse chromosome 17B1) in an area densely clustered with inflammatory response genes (12,13). Largely similar gene products arising from the same locus have been named Iba1, microglial response factor-1 (MRF1) and daintain; Iba1 in particular is a well-known histologic marker of microglia and of their activation in pathological CNS conditions. Aif-1 is differentially expressed in various mouse and human tissues (14,15) and in multiple leukocyte types including macrophages and T cells at basal levels (16, 17, 18). In inflammatory disease models, upregulated Aif-1 expression has been reported in microglia, macrophages, T cells, synoviocytes, pancreatic β-cells and adipocytes under various pathological conditions representing encephalomyelitis, uveitis, neuritis, arteriopathies, arthritis and diabetes, respectively (19).
The significance of increased Aif-1 expression in neuroinflammatory diseases such as EAE (20,21) has not been characterized. Overexpression of Aif-1 in MOLT-4 T cells increases proliferation, migration and activation (17) and in macrophage cell lines enhances production of interleukin (IL)-6, IL-12 and IL-10 (22). On the other hand, impaired Aif-1 function decreases microglial phagocytosis (23). Extrapolation from these in vitro findings suggests that Aif-1 deficiency might ameliorate EAE by limiting T cell and macrophage inflammatory activity, but could also allow cellular debris to accumulate, secondarily exacerbating inflammation and neurotoxicity and impairing regenerative processes. We recently developed an Aif-1-deficient mouse line (24) that can be used to determine the net effect of loss of Aif-1 in disease models.
Materials and Methods
Aif-1-deficient mice were generated through a homologous recombination gene targeting strategy (24). The targeted aif-1 allele was backcrossed onto the C57BL/6 strain for eight generations, and the corresponding knockout (aif-1−/−) and wild-type (wt) littermates were bred in-house as homozygous or heterozygote lines in the barrier facility at the Albert Einstein College of Medicine. All experiments involving live animals were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine and complied with the Guide for the Care and Use of Laboratory Animals (25).
Induction of EAE and Evaluation of Clinical Disease
EAE was induced in mice as previously described (26). Briefly, 10- to 12-wk-old male mice were immunized subcutaneously in the lower dorsum with 300 µg myelin oligodendrocyte glycoprotein (MOG35–55) peptide (MEVGWYRSPFSRVVHLYRNGK; Celtek Bioscience, Nashville, TN, USA) in a 200 µL emulsion of incomplete Freund adjuvant (IFA) containing 5 mg/mL Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA). Subsequent to immunization, the mice received intraperitoneal injections of pertussis toxin (500 ng; List Biological Laboratories, Campbell, CA, USA) on the first day of sensitization and again after 2 d. We considered the day after MOG immunization as d 1. The EAE disease activity was scored as follows: 0, no symptoms; 1, floppy tail; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death.
Histologic and Immunofluorescence Analysis of Spinal Cords
For pathological analysis, EAE mice were anesthetized at the time points indicated and perfused with phosphate-buffered saline (PBS) via cardiac puncture. The spinal cord was flushed by hydrostatic pressure by using PBS. The lumbar spinal cord was postfixed overnight with 4% paraformaldehyde, and the tissues were paraffin embedded. To assess infiltration, coronal sections (6-µm thickness) were stained with hematoxylin and eosin (H&E) and examined by using an Axioskop II microscope with an MRc camera (Zeiss, Thornwood, NY, USA) in the Albert Einstein College of Medicine Analytical Imaging Facility. The extent of infiltration was represented as an infiltrated area, which was quantified by measuring the individual area covered by positive hematoxylin staining (representing single or clustered nuclei) in the submeningium of each spinal cord section by using Photoshop CS3, version 10 software (Adobe, San Jose, CA, USA); the average was normalized to total white matter area and expressed as a percentage.
To assess demyelination, paraffin-embedded spinal cord sections were deparaffinized and blocked with 10% donkey serum for 1 h at room temperature followed by antigen retrieval. The sections were incubated with anti-mouse myelin basic protein (MBP) (BioLegend, San Diego, CA, USA) overnight at 4°C and incubated with donkey anti-mouse Alexa 548 (Life Technologies [Thermo Fisher Scientific Inc., Waltham, MA, USA]) for 1 h at room temperature. The counterstained slides were mounted in aqueous mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) dihydrochloride hydrate (Electron Microscopy Sciences, Hatfield, PA, USA) and examined by using an Olympus IX 81 microscope (Olympus America, Center Valley, PA, USA) with motorized stage and a Cooke Sensicam QE air-cooled charged-coupled device (CCD) camera in the Albert Einstein College of Medicine Analytical Imaging Facility. The extent of demyelination was quantified (Adobe Photoshop) by measuring the area of non-MBP-stained white matter, normalized to total white matter area, and expressed as a percentage.
Isolation of Mononuclear Cells from the CNS
Spinal cords were perfused and flushed by hydrostatic pressure, and the recovered tissues were homogenized and digested with collagenase A (2 mg/mL; Roche Diagnostics, Indianapolis, IN, USA) in RPMI 1640 at 37°C for 15 min. The digested tissues were filtered through a 100-µm cell strainer to obtain a single cell suspension and centrifuged at 500g for 5 min. Cell pellets from two mice in each group were pooled, resuspended in 70% Percoll (Sigma-Aldrich, St. Louis, MO, USA), overlaid with 30% Percoll and centrifuged at 200g for 15 min. The cell monolayer at the 70–30% interphase was collected and stained with various antibodies for flow cytometry, as described below.
Flow Cytometry Analysis
At d 16 after EAE induction, spleen and peripheral lymph node cells were isolated, depleted of erythrocytes, blocked for Fc receptors RII/III with antibodies specific for CD16/CD32 (BD Biosciences, San Jose, CA, USA) and stained for surface markers with the following antibodies: anti-CD3-APC, anti-CD4-FITC, anti-CD8-PerCP, anti-B220-Pacific blue, anti-CD69-PE, anti-CD25-APC, anti-Foxp3-PE, anti-Gr1-PerCP, F4/80-PE (all from BD Biosciences), anti-CD45-Pacific blue (BioLegend) and anti-CD11b-APC (eBiosciences, San Diego, CA, USA). The stained cells were analyzed by fluorescence-activated cell sorting (FACS) (LSRII, BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Ashland OR, USA).
T-Cell Activation and Proliferation
To evaluate T-cell activation, splenocytes from naive 10-wk-old wt and aif-1−/− mice were isolated and enriched for CD4 T cells by using an EasySep positive selection kit (Stemcell Technologies, Vancouver, BC, Canada). CD4 T cells were seeded in a 12-well plate (3.5 × 106/well) and stimulated with either dimethyl sulfoxide or phorbol myristic acid (10 ng/mL) and ionomycin (500 ng/mL) in the presence of a protein transport inhibitor (GolgiPlug™, BD Biosciences; 1 µg/mL/106 cells) for 5 h. Cells were harvested and subjected to intracellular staining with anti-IL-2-FITC (BD Biosciences) and analyzed by FACS (LSRII). Data were analyzed using FlowJo software. To assess T-cell proliferation, splenocytes were stimulated with either α-CD3 (200 ng/mL) or MOG35–55 (20 µg/mL) for 72 h, and proliferation was measured by adding [3H]thymidine (25 µCi/mL) for the last 24 h of the assay. Incorporated [3H]thymidine was measured by using a β-counter and expressed as counts per minute.
Cytokine Expression Analysis
Mononuclear cells were isolated from spleens of d-16 EAE-induced mice, and single cell suspensions were prepared in RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine and β-mercaptoethanol. Splenocytes (4 × 105 cells/well) were stimulated with MOG35–55 peptide (10 and 20 µg/mL) for 72 h. Cytokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) by using antibodies to IL-6, IL-2, IFN-γ (BD Biosciences) and IL-12p40 (R&D Systems, Minneapolis, MN, USA).
Real-Time Quantitative Polymerase Chain Reaction
Spleen tissues were homogenized with TRIzol (Life Technologies [Thermo Fisher Scientific]), and total RNA was extracted by using chloroform and precipitated with isopropanol. Synthesis of cDNA was performed by using 2 µg RNA using a reverse transcription system (Life Technologies [Thermo Fisher Scientific]). Real-time polymerase chain reaction was performed by using a Roche 480 light cycler using SYBR green quantitative master mix (Roche Applied Sciences, Indianapolis, IN, USA). The relative expression of cytokine and iNOS genes was determined in comparison to that of gapdh. Data were analyzed by using the Pfaffl method (27). The following primers were used: 5′-GCTACCAAACTGGATATAATCAGGA-3′ and 5′-CCAGGTAGCTATGGTACTCCAGAA-3′ (IL-6); 5′-GATTCAGACTCCAGGGGACA-3′ and 5′-TGGTTAGCTTCTGAGGACACATC-3′ (IL-12p40); 5′-CCATCAGCAGATCATTCTAGACAA-3′ and 5′-CGCCATTATGATTCAGAGACTG-3′ (IL-12p35); 5′-GCTGTTGATGGACCTACAGGA-3′ and 5′-TTCAATTCTGTGGCCTGCTT-3′ (IL-2); 5′-CATCGGCATTTTGAACGAG-3′ and 5′-CGAGCTCACTCTCTGTGGTG-3′ (IL-4); 5′-ATCTGGAGGAACTGGCAAAA-3′ and 5′-TTCAAGACTTCAAAGAGTCTGAGGTA-3′ (IFN-γ); 5′-TCTTCTCATTCCTGCTTGTGG-3′ and 5′-GGTCTGGGCCATAGAACTGA-3′ (TNF-α); 5′-CAGGGAGAGCTTCATCTGTGT-3′ and 5′-GCTGAGCTTTGAGGGATGAT-3′ (IL-17); 5′-TCCCTACTAGGACTCAGCCAAC-3′ and 5′-TGGGCATCTGTTGGGTCT-3′ (IL-23p19); 5′-GGGCTGTCACGGAGATCA-3′ and 5′-CCATGATGGTCACATTCTGC-3′ (iNOS); 5′-CAGAGCCACATGCTCCTAGA-3′ and 5′-GTCCAGCTGGTCCTTTGTTT-3′ (IL-10); 5′-CCTCTGACCCTTAAGGAGCTTAT-3′ and 5′-CGTTGCACAGGGGAGTCT-3′ (IL-13).
Data are represented as mean ± standard error of the mean (SEM). Two-tailed Student t test, two-way analysis of variance and Mann-Whitney U test were used to assess statistical significance. P values <0.05 were considered statistically significant. Quantitative analyses were performed with Prism for Mac OSX (GraphPad Software).
All supplementary materials are available online at https://doi.org/www.molmed.org.
Mice Lacking Aif-1 Show Lower Incidence and Reduced Clinical Severity of EAE
Development of EAE in wt and aif-1−/− mice.
Day of onseta
Days to peak clinical diseaseb
Mean clinical score
Cumulative disease index (%)c
8.1 ± 0.61
10.1 ± 0.84
1.46 ± 0.13
28 ± 4.5
2.6 ± 0.26
8.6 ± 1.3
10.6 ± 0.84
0.67 ± 0.05***
13 ± 4.5*
1.5 ± 0.33*
Aif-1 Deficiency in Mice Decreases EAE-Associated CNS Leukocyte Infiltration and Demyelination
Aif-1 Deficiency Reduces CNS Infiltration by CD4 T Cells
Aif-1 Promotes Expansion and Activation of Encephalitogenic CD4 T Cells in Spleen
To test this idea directly, we challenged splenocytes from previously immunized wt and aif-1−/− mice with either anti-CD3 or MOG35–55. With both anti-CD3, as a general T-cell activator, or MOG35–55 as a specific antigen rechallenge, cells from aif-1−/− mice proliferated less than wt control (Figures 4C, D). On the other hand, activation of T cells from naive wt and aif-1−/− mice by phorbol ester and ionomycin was equivalent (Supplementary Figure S3), which shows that the Aif-1 deficiency affects acquired but not basal T-cell responsiveness. Collectively, these data suggest that Aif-1 promotes myelinspecific CD4 T-cell expansion in the spleen, which in turn supports CD4 T-cell infiltration and demyelination of the spinal cord in EAE. Because antigen stimulation also promotes immune cell recruitment to and activation in lymph nodes, we further determined the effect of Aif-1 deficiency on lymph node populations after MOG35–55 immunization; these studies showed no significant differences between wt and aif-1−/− mice in lymph node populations including T-cell subsets, B cells and monocytes (Supplementary Figure S4).
Aif-1 Deficiency Promotes Th1 to Th2 Bias in Spleen
Studies of both MS and EAE implicate myelin-specific T cells, macrophages and microglia as mediators of disease activity (3). Expression of Aif-1 increases above basal levels in these cell types (16, 17, 18) in neuro-inflammatory disease models (19). Conceivably, increased expression of Aif-1 during EAE pathogenesis (20,21) could promote T cell, macrophage and/or microglial proinflammatory functions and overall disease activity. Alternatively, Aif-1-dependent microglial phagocytosis (23) and clearance of cellular debris could be important as means to limit inflammation and enhance regenerative processes (11). We recently developed an Aif-1-deficient mouse model (24) that can be used to gain insight into the significance of Aif-1 expression in disease models. These mice do not show growth or fertility defects, or evidence of immunodeficiency in normal barrier housing conditions. To assess the relative importance of these different Aif-1 functions in the EAE model in vivo, we immunized wt and aif-1−/− littermate mice with MOG35–55 peptide and characterized their clinical and pathological response.
Although baseline peripheral, splenic, thymic lymphocyte and CNS microglial populations were similar in wt and aif-1−/− mice, the Aif-1-deficient mice had lower incidence and severity of induced disease. This result corresponded to reduced CNS leukocyte infiltration and demyelination and was associated with impaired expansion and activation of myelin-specific CD4 T cells and decreased proinflammatory cytokine production in the periphery. These findings suggest that Aif-1-dependent proinflammatory activities are dominant in this setting, whereas its phagocytotic and clearance functions are less critical.
Interestingly, the effect of Aif-1 deficiency on leukocyte populations after immunization was relatively modest, with a decrease of ∼10% in the number of both CD3+CD4+ and CD4+CD69+ T cells; on the other hand, proliferation of splenocytes lacking Aif-1 in response to either general anti-CD3 or specific MOG35–55 antigen challenge was reduced by ∼50%. This markedly impaired proliferative response was accompanied by a substantial reduction in several important Th1 cytokines, including IL-6, IFN-γ, IL-12 and IL-2, plus an increase in the Th2 cytokine IL-4, suggesting that loss of Aif-1 limits Th1-type immune responses while enhancing Th2-type immune responses. We found no differences in the iTreg cell population or in expression of markers of Th17 differentiation, including IL-23 p19 and IL-17. Nevertheless, because IL-12 p40 combines with IL-23 p19 to form functional IL-23 heterodimers, reduction of the former could limit IL-23 activity, which has been shown to be critical for EAE (33), particularly during disease induction (34).
The mechanistic basis for Aif-1-dependent effects on cytokine expression is not yet clear. Aif-1 localizes primarily to the cytoplasm, and molecular functions ascribed to Aif-1 to date include actin bundling activity (35); it has also been linked to signaling via the small GTPase Rac (36,37), which itself is well known to affect actin cytoskeletal dynamics (38). The Aif-1 protein structure is notable for a conserved EF hand motif, although there are conflicting reports as to whether this is capable of binding Ca2+ (39,40). Broad manipulations of the actin cytoskeleton, as with cytochalasin D or latrunculin B treatment, have been shown to affect relevant gene expression, including activity of the IL-2 gene promoter and Ca2+-activated NFAT (nuclear factor of activated T-cells) transcription factors (41,42). It is possible that effects on actin cytoskeletal remodeling, Rac activation and/or Ca2+ handling due to loss of Aif-1 impinge negatively on signaling pathways that support T-cell inflammatory gene expression. Alternatively, a recent study shows proinflammatory activity of recombinant Aif-1 added to monocytes (43). Although Aif-1 lacks a classical secretory motif, this observation suggests that it could have important effects as a released extracellular factor.
These findings identify Aif-1 as a molecule that promotes Th1-helper CD4 T-cell expansion, activation and a proinflammatory cytokine milieu in MOG35–55-induced EAE. Future studies with selective inactivation of Aif-1 will be required to assess the relative importance of Aif-1 in monocyte/macrophage and T lymphocyte lineages and to define the molecular mechanisms by which Aif-1 promotes autoreactive CD4 T-cell expansion, activation and inflammatory cytokine expression. Regardless of the specific mechanism at play, our results to date implicate Aif-1 as a potential target for MS therapy and provide support for the idea that inhibitors of Aif-1 expression or function may have beneficial effects on MS disease activity.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank the staff of the Albert Einstein College of Medicine Analytical Imaging Facility, FACS and Histology core facilities of Albert Einstein College of Medicine for their expert assistance. This work was supported in part by Pilot Project PP1500 from the National Multiple Sclerosis Foundation and by grant HL67944 from the National Institutes of Health.
- 25.Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, Division on Earth and Life Studies, National Research Council of the National Academies. (2011) Guide for the Care and Use of Laboratory Animals. 8th edition. Washington (DC): National Academies Press.Google Scholar
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