Characterization of immune response to neurofilament light in experimental autoimmune encephalomyelitis
© Puentes et al.; licensee BioMed Central Ltd. 2013
Received: 8 July 2013
Accepted: 8 September 2013
Published: 22 September 2013
Autoimmunity to neuronal proteins occurs in several neurological syndromes, where cellular and humoral responses are directed to surface as well as intracellular antigens. Similar to myelin autoimmunity, pathogenic immune response to neuroaxonal components such as neurofilaments may contribute to neurodegeneration in multiple sclerosis.
We studied the immune response to the axonal protein neurofilament light (NF-L) in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. To examine the association between T cells and axonal damage, pathology studies were performed on NF-L immunized mice. The interaction of T cells and axons was analyzed by confocal microscopy of central nervous system tissues and T-cell and antibody responses to immunodominant epitopes identified in ABH (H2-Ag7) and SJL/J (H2-As) mice. These epitopes, algorithm-predicted peptides and encephalitogenic motifs within NF-L were screened for encephalitogenicity.
Confocal microscopy revealed both CD4+ and CD8+ T cells alongside damaged axons in the lesions of NF-L immunized mice. CD4+ T cells dominated the areas of axonal injury in the dorsal column of spastic mice in which the expression of granzyme B and perforin was detected. Identified NF-L epitopes induced mild neurological signs similar to the observed with the NF-L protein, yet distinct from those characteristic of neurological disease induced with myelin oligodendrocyte glycoprotein.
Our data suggest that CD4+ T cells are associated with spasticity, axonal damage and neurodegeneration in NF-L immunized mice. In addition, defined T-cell epitopes in the NF-L protein might be involved in the pathogenesis of the disease.
KeywordsNeurofilament light Axonal damage Neurodegeneration Experimental autoimmune encephalomyelitis Multiple sclerosis
Multiple sclerosis (MS) is a chronic demyelinating and neurodegenerative disease of the central nervous system (CNS) widely considered due to aggressive, autoreactive T cells and antibodies to myelin [1–3]. However, accumulating evidence shows that immune responses to neuronal and axonal proteins are also present in a wide range of neurodegenerative disorders including MS [4–6]. That these responses may contribute to axonal and neuronal damage, pathological hallmarks of MS, is supported by observations that immunization with neuronal antigens and transfer of antibodies directed to neuronal and axonal proteins induce neuronal damage in animals [7–10].
The lack of expression of molecules of the major histocompatibility complex (MHC) class II on neurons indicates that neurons cannot activate CD4+ cells in an antigen-specific manner. However, neurons constitutively express or readily upregulate expression of MHC class I during inflammation, indicating that neurons may become targets for CD8+ T cells . Conceptually, both CD4+ and CD8+ T cells could mediate attack on axons and neurons, either by direct contact via antigen-independent interactions or as a result of collateral damage . Activated T cells in the CNS are reported to produce cytotoxic molecules as well as glutamate, nitric oxide and reactive oxygen species that could contribute to the damage and progressive neurodegeneration observed in MS and other neurodegenerative diseases in which inflammation has been described [13–15]. In addition, activation of B cells might lead to the production of specific antibodies to neuronal antigens that could also contribute to the damage and progressive neurodegeneration .
To examine the mechanisms of autoimmunity to neurons we have developed a model of autoimmune induced axonal and neuronal damage following immunization of mice with the neuronal cytoskeletal protein neurofilament light (NF-L) . Whether T-cell responses to neuroaxonal components are pathogenic in MS is as yet unknown; although we have recently shown that NF-L is phagocytosed by MHC class II+ microglia/macrophages in MS brain lesions , indicating a potential source by which autoreactive T cells could become reactivated in MS. In mice, we have shown that autoimmunity to NF-L causes spasticity and neurodegeneration and that axonal damage is a direct consequence of such responses [7, 8]. Infiltration of CD3+ T cells and B220+ cells mainly localized in the dorsal column of the spinal cord of NF-L immunized mice was also observed . Likewise, immunoglobulin deposits were observed into the axons in mice immunized with NF-L protein .
In the present study, we characterized the T-cell infiltrates in the CNS of spastic mice. Both CD4+ and CD8+ T cells were found in close association to axons, although CD4+ T cells dominated the infiltrates in lesions. In addition, increased perforin expression and cells positive for granzyme B could be observed in the spinal cord of mice immunized with NF-L. Furthermore, NF-L peptides were screened for T-cell and B-cell responses in ABH (H2-Ag7) and SJL/J (H2-As) mice, and the pathogenic potential of these peptides and predicted binding motifs to H2-Ag7 present in the NF-L sequence were investigated.
In summary, our study reveals that T cells associated with the expression of cytotoxic molecules are present in lesions in the dorsal columns of spastic mice immunized with NF-L and we show, for the first time, that active immunization with defined NF-L peptides induced neurological disease in ABH mice.
Materials and methods
Male and female 10-week-old Biozzi ABH (H-2dq1) and SJL/J (H-2s) mice were obtained from Harlan (Bicester, UK) and Charles River laboratories (Kent, UK) or were bred at QMUL (London, UK). All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act (1986) and approved by the local ethics committee. All procedures were performed following Institutional ethical review in accordance to the United Kingdom Animals (Scientific Procedures) Act (1986) and European Union Directive 2010/63/EU. Animals were housed and monitored consistent with the principles of the ARRIVE guidelines as described previously .
Sequences of mouse neurofilament-light peptides a
1 to 16
273 to 288
9 to 24
281 to 296
17 to 32
289 to 304
25 to 40
297 to 312
33 to 48
305 to 320
41 to 56
313 to 328
49 to 64
321 to 336
57 to 72
329 to 344
65 to 80
337 to 352
73 to 88
345 to 360
81 to 96
353 to 368
89 to 104
361 to 376
97 to 112
369 to 384
105 to 120
377 to 392
113 to 128
385 to 400
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393 to 408
129 to 144
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441 to 456
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449 to 464
185 to 200
457 to 472
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473 to 488
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489 to 504
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497 to 512
233 to 248
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241 to 256
513 to 528
249 to 264
521 to 536
257 to 272
529 to 544
265 to 280
Induction of experimental autoimmune encephalomyelitis
Mice were injected subcutaneously with 1 mg SCH, 200 μg rmNF-L, 200 μg MOG35–55 or rmNF-L and MOG35–55 (1:1), or pools of 30 μg each peptide emulsified with incomplete Freund’s adjuvant (Difco Laboratories, (Detroit, Michigan, USA) UK) supplemented with 48 μg Mycobacterium tuberculosis and 6 μg Mycobacterium butyricum (Difco Laboratories) on day 0 and day 7 as described previously . Control mice were immunized with complete Freund’s adjuvant (CFA) only. All mice were injected with 200 ng Bordetella pertussis toxin (Sigma St. Louis, Missouri, USA) intraperitoneally, immediately after immunization and 24 hours later.
To identify encephalitogenic epitopes, four to six mice were immunized with rmNF-L, individual or pooled peptides. To optimize identification, sequences containing motifs that bind to or interact with H2-Ag7 were selected as described previously . The Rankpep server was additionally used to predict binding to H2-Ag7.
Mice were monitored daily and scored according to a neurological scale: 0, normal; 0.5, partial loss of tail tone; 1, paralysis or spasticity of the tail; 2, impaired righting reflex; 3, paralysis or spastic paresis of one limb; 4, paralysis or spastic paresis of two limbs; and 5, moribund [7, 18]. Mice were sacrificed by carbon dioxide inhalation and brains and spinal cords snap-frozen in liquid nitrogen or processed for pathology .
Sections (3 μm) from snap-frozen spinal cord tissues were fixed with acetone and incubated overnight at 4°C with mAb directed to CD4 (YTS 191.1.2), CD8 (YTS 169AG; ImmunoTools, Friesoythe, Germany), MHC-I antigens (HM1091; Hycult Biotech, Plymouth Meeting, PA, USA) or biotinylated MHC-II (OX 6, a kind gift of Jack van Horssen, VU University Medical Center) diluted in antibody diluent (Immunologic; Duiven, The Netherlands). After washing, endogenous peroxidase was blocked with 0.3% H2O2 in PBS. Sections stained for CD4, CD8 and MHC-I were incubated with biotinylated rabbit anti-rat Ig (Dako, Glostrup, Denmark) for 1 hour, followed by peroxidase-coupled avidin–biotin complex (ABC kit; Vector Laboratories, Burlingame, CA, USA). Sections stained with biotinylated MHC-II were incubated with streptavidin–horseradish peroxidase complex (Dako) for 1 hour. All secondary antibodies were visualized with 3,3′-diaminobenzidine. Antibodies were prescreened on brain, liver, lung, spleen and tonsil tissues and isotype control mAb served as negative control. The percentage of CD4+ and CD8+ T cells were counted at 25× objective at three levels of the spinal cord.
For immunofluorescence, sections were incubated with blocking solution (CleanVision IHC/ICC; Immunologic) containing 10% normal goat serum for 2 hours, washed in PBS and incubated with mAb to NF-L (10H9), SMI-32 (Covance, Princeton, NJ, USA) or NeuN (Merck Millipore; Darmstadt, Germany) and CD3 (CD3-12; Serotec, Oxford, UK), CD4 (YTS 191.1.2) or CD8 (YTS 169AG; ImmunoTools) overnight at 4°C. After washing in PBS, sections were incubated with goat anti-mouse IgG1 Alexa 594 or goat anti-rat IgG Alexa 488 (Invitrogen; Paisley, UK) for 60 minutes at room temperature. Sections were viewed using confocal laser scanning microscopy (Leica DMI6000; Rijswijk, The Netherlands). Image processing was performed using NIH Image J software .
Granzyme B staining was performed on paraffin-embedded sections (4 μm). In brief, sections were deparaffinized and rinsed in H2O. Subsequently, endogenous peroxidase was blocked as described above. After rinsing in PBS, antigen retrieval in Tris–ethylenediamine tetraacetic acid buffer (pH 9.0) was performed in a microwave followed by incubation with 10% normal goat serum. Sections were incubated overnight at 4°C with polyclonal rabbit anti-granzyme B (ab4059; Abcam; Cambridge, UK) in 1% BSA. Subsequently, sections were washed in PBS and incubated with secondary antibody Envision anti Rabbit labeled with horseradish peroxidase (K4002; Dako) for 30 minutes and visualized with 3,3′-diaminobenzidine.
Reverse transcriptase polymerase chain reaction
Spinal cords from control and NF-L immunized ABH mice were dissolved in lysis buffer (NucleoSpin RNA/Protein kit; Machery-Nagel GmbH, Düren, Germany) and homogenized with 1.4 mm ceramic beads (Precellys 24; Peqlab Biotechnologie GmbH, Erlangen, Germany) at 5000 rpm for 15 seconds. Subsequently, RNA was isolated using NuceloSpin (Macherey-Nagel) according to the manufacturer’s recommendations. Purity was confirmed using 260:280 OD ratios (Nano-Drop 1000; Peqlab Biotechnologie GmbH). RT reactions were performed with the MMLV RT-kit and random hexanucleotide primers (Invitrogen) and gene expression was measured using Taq-Polymerase (Biomol GmbH, Hamburg, Germany).
Primers for perforin amplification (sense, 5′-CTGCCACTCGGTCAGAATG-3′; antisense, 5′-CGGAGGGTAGTCACATCCAT-3′) were used at annealing temperature of 59°C, amplifying an 88-base-pair fragment. Expression levels of the reference gene hypoxanthine guanine phosphoribosyl transferase were used as control. Primers for hypoxanthine guanine phosphoribosyl transferase amplification (sense, 5′-GCTGGTGAAAAGGACCTCT-3′; antisense: 5′-CACAGGACTAGAACACCTGC-3′) were used at an annealing temperature of 60°C, amplifying a 248-base-pair fragment, and primers for 18sRNA amplification (sense, 5′-CGGCTACCACATCCAAGGAA-3′; antisense, 5′-GCTGGAATTACCGCGGCT-3′) were used at an annealing temperature of 60°C, amplifying a 187-base-pair fragment. Gene expression was performed using RT-PCR technology (Bio-Rad; Munich, Germany) with SYBR green (SensiMix™; Bioline; Luckenwalde, Germany), as published previously .
T-cell proliferation assays
ABH and SJL/J mice were immunized with rmNF-L in CFA or CFA only (n = 4 per group). Spleen cells were collected 10 days after priming and single-cell suspensions (3 × 105/ml) cultured in RPMI medium (Gibco, Invitrogen; Paisley, UK) with 5% FCS (Gibco, Invitrogen), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 mM Hepes, and 5 × 10–5 M 2-mercaptoethanol (Gibco, Invitrogen). Cells were incubated with NF-L peptides or rmNF-L for 72 hours. Proliferation was determined by incorporation of [3H]-thymidine (GE Healthcare, Uppsala, Sweden). Stimulation indices (SIs) were calculated as the proliferative response in the presence of antigen divided by the response in the absence of antigen. SI in the CFA control group ranged from 0.6 to 1.1. Positive stimulation was defined as SI >1.5.
Enzyme-linked immunosorbent assay
To identify B-cell epitopes in NF-L, Biozzi ABH and SJL/J mice (n = 4 per group) were immunized with rmNF-L protein in complete adjuvant and serum was collected on day 15 post immunization. The mouse immune sera were tested for their reactivity to NF-L overlapping peptides. Nunclon plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 10 μg/ml mouse NF-L peptides or rmNF-L protein, in carbonate buffer. Plates were washed twice in PBS and blocked for 1 hour at 37°C with 2% BSA/PBS. After blocking, ABH or SJL/J immune sera, diluted 1:100 in 1% (BSA/PBS), were added and incubated for 1 hour at room temperature. After washing in PBS–Tween 0.1%, the plates were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated rabbit anti-mouse Ig (Dako). The reaction product was developed with TMB substrate (Thermo Fisher Scientific; Loughborough, UK) and stopped by the addition of 2 M hydrochloric acid. The absorbance was measured at 450 nm, using a Synergy HT microplate reader (Bio-Tek instruments; Winooski, Vermont, USA). Background values were obtained by the reactivity of immune serum on peptide uncoated wells. An absorbance above the mean plus three standard deviations of the background reactivity was taken as positive.
For comparison of clinical experimental autoimmune encephalomyelitis (EAE) scores, significance between the groups was determined by nonparametric Mann–Whitney rank-sum test (SigmaStat; Systat Software Inc., San Jose, CA, USA). Data represent the mean ± standard error of the mean (**P <0.01, *P <0.05).
CD4+ and CD8+ T cells are associated with neuronal/axonal damage in spastic mice
Cytotoxic molecules are present in the spinal cord of NF-L immunized mice
Autoimmunity to NF-L exacerbates MOG35–55 experimental autoimmune encephalomyelitis in ABH mice
Immunodominant B-cell and T-cell epitopes of neurofilament light in mice
Pathogenicity of immunodominant epitopes
Pathogenic neurofilament-light peptides in ABH mice a
Number with EAE
Mean group scoreb
Mean EAE scorec
Mean day of onset
4.0 ± 0.0*
4.0 ± 0.0
15.3 ± 0.3
0.7 ± 0.3*
0.9 ± 0.2
31.0 ± 1.4
241 to 256
0.1 ± 0.1
0.5 ± N/A
21.0 ± N/A
249 to 264
0.9 ± 0.3*
0.9 ± 0.3
25.6 ± 6.4
257 to 272
0.4 ± 0.2
0.7 ± 0.2
29.3 ± 0.6
265 to 280
273 to 288
345 to 360
0.7 ± 0.3
1.2 ± 0.3
29.3 ± 0.3
353 to 368
0.5 ± 0.2
0.8 ± 0.2
25.7 ± 4.4
129 to 144
0.2 ± 0.1
0.5 ± 0.0
18.5 ± 6.5
297 to 312
0.2 ± 0.1
0.5 ± 0.0
30.5 ± 0.5
313 to 328
113 to 128, 281 to 296, 289 to 304, 321 to 336 (4)
0.9 ± 0.4
1.2 ± 0.3
28.7 ± 0.7
201 to 216, 377 to 392, 457 to 472 (3)
Additionally, the capacity of NF-L-specific T cells to transfer spasticity was tested in ABH mice. Lymph node and spleen cells from rmNF-L-immunized ABH mice were collected 12 days after immunization and stimulated for 10 days in vitro with rmNF-L. Activated cells were adoptively transferred into irradiated naïve recipients and followed until day 25. Mild signs of disease (score 0.5) were observed in two of seven mice revealing the pathogenic potential of T cells to NF-L (data not shown).
Accumulating evidence indicates that as well as myelin-specific T cells, neuronal-specific T cells may gain access to the CNS and contribute to neurodegeneration. Immune responses to neurons are reported in Rasmussen’s encephalitis , Alzheimer’s disease , Parkinson’s disease  and paraneoplastic disorders , underscoring the potential role of pathogenic neuronal-reactive T cells in MS in which axonal damage correlates with disability. To examine the role of T cells in neurodegeneration, we have studied autoimmune-meditated neurodegeneration in mice immunized with NF-L in which spasticity and paralysis, clinical features characteristic of MS, are observed [7, 8]. In previous studies, infiltration of CD3+ and B220+ cells and immunoglobulin deposits were observed in spinal cord lesions of NF-L immunized mice .
In this study, we aimed to further investigate the cellular and humoral immune response in the mouse model of NF-L-induced neurodegeneration. Immunohistochemistry revealed the association of both CD4+ and CD8+ T cells with neuronal damage and the expression of cytotoxic molecules in the CNS. We also identify immunodominant regions and encephalitogenic epitopes on the NF-L protein. Together, our data support the accumulating evidence that T cells play a role in neuronal and axonal damage in neurodegenerative disorders. Adoptive transfer experiments to test the ability of NF-L peptide-specific T cells to induce disease would be of interest in future studies.
A pre-requisite for antigen-specific CD8+ cytotoxic damage of neurons is the expression of MHC class I molecules. Strong evidence exists of MHC class I on neurons, neurites and axons, revealing differential expression depending on neuronal subtype, development stage and the inflammatory stimulus. MHC class I expression is highly upregulated on neurons following IFNγ treatment but not TNFα . Intriguingly, CD8+ T cells directed to NF-L generated from spastic mice produce high levels of IFNγ , indicating that T cells to NF-L could trigger progressive neurodegeneration. Further support for T-cell-mediated neuronal damage comes from the finding that, in EAE, myelin-reactive T cells are activated by neurofilament peptides . Such interaction between T cells and neurons may induce antigen-specific lysis of neurons  or dysfunction by impairing electrical signaling  and induction of rapid microtubule axonal destabilization . In the spastic model it is unlikely that antigen-specific CD8+ T-cell activation leads to neuronal damage since MHC class I was not present on neurons, either in the lesions or in normal appearing tissue. While it is unlikely that CD4+ T cells induce neuronal death in an antigen-specific fashion, as neurons do not express MHC class II, pathogenic CD4+ T cells expressing NKG2C could injury neurons and axons expressing HLA-E, as reported for oligodendrocytes in MS .
T-cell-mediated neuronal damage may alternatively occur via antigen-independent interactions involving Fas–FasL, TRAIL, CD11a and CD40. In culture, polyclonally activated CD4+ and CD8+ T cells are cytotoxic to human neurons [30, 32], underscoring the potential pathogenic role of both CD4+ and CD8+ T cells. In line with these studies, our results show that granzyme B and perforin are present in the lesions of spastic mice, indicating that neuronal damage is more likely due to antigen-independent mechanisms. The predominance of CD4+ T cells in the lesions of NF-L immunized mice may be due to selective recruitment or selective depletion of CD8+ T cells . Moreover, the ratio between the T-cell subsets in the lesions in our model is similar to myelin-induced EAE . Further studies on the expression of cytotoxic molecules in T-cell subpopulations in the CNS of NF-L immunized mice will be necessary.
Our observations that co-immunization with MOG and NF-L leads to exacerbated disease is pertinent to what might occur in MS during myelin damage, in which neurons may be more vulnerable to immune responses to neuronal antigens. In this way, the course of neuronal degeneration might be accelerated in the presence of NF-L reactive T cells  and antibodies to NF in MS .
EAE studies have been instrumental in identifying the role of T cells and antibodies to myelin in disease. These have revealed important information about peptide:MHC:T-cell receptor interactions crucial for development of tolerance regimes using altered peptide ligands or tolerogenic routes for delivery of pathogenic peptides to modulate myelin-specific T cells [37, 38]. These approaches have proved effective in chronic EAE models, preventing the clinical relapses, but do not control progressive disease , indicating that mechanisms other than myelin autoimmunity, such as autoimmunity to neuronal proteins, are involved in neurodegeneration in MS.
Pathogenic peptides associated with neurological disease in ABH mice
In conclusion, we show that peptide epitopes of NF-L induce neurological disease and that potentially pathogenic CD4+ T cells dominate the lesions of NF-L immunized mice, a model for immune-mediated neurodegeneration. Our data suggest that, similar to peptide therapies targeting myelin responses , immune therapies targeting neuronal-specific T cells could thus be beneficial in reducing neurodegeneration in inflammatory disorders such as MS.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images.
Prof. David Baker and Prof. Sandra Amor share the senior authorship.
Bovine serum albumin
Central nervous system
Complete Freund’s adjuvant
Experimental autoimmune encephalomyelitis
Major histocompatibility complex
Myelin oligodendrocyte glycoprotein
Polymerase chain reaction
Spinal cord homogenate
The authors thank Bert van het Hof for technical assistance. This work was supported by Stichting MS Research, The Netherlands, grant number 07–627, the DANA foundation and by the MS Society of Great Britain and Northern Ireland, grant number NSCG-1F7R.
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