You are here
Effects of active immunisation with myelin basic protein and myelin-derived altered peptide ligand on pain hypersensitivity and neuroinflammation
Journal of Neuroimmunology, September 2015, Pages 59 - 70
Neuropathic pain is a debilitating condition in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Specific myelin basic protein (MBP) peptides are encephalitogenic, and myelin-derived altered peptide ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active immunisation with a weakly encephalitogenic epitope of MBP (MBP87–99) and its mutant APL (Cyclo-87–99[A91,A96]MBP87–99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II expression and microglia activation in the spinal cord, without developing clinical signs of paralysis. Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising the important role of neuroimmune crosstalk in neuropathic pain.
- MBP87–99 immunisation initiates pain sensitivity without causing paralytic disease.
- MBP87–99 induces T cell, MHC II and microglia activation in the spinal cord.
- Co-immunisation with APL decreases mechanical and thermal pain hypersensitivity.
- Co-immunisation with APL reduces T cells and MHC II expression in the spinal cord.
Keywords: Neuropathic pain, Experimental autoimmune encephalomyelitis, Pain hypersensitivity, Myelin basic protein, Altered peptide ligand, Neuroinflammation.
Neuropathic pain is a common consequence of injury or autoimmune diseases in the central nervous system (CNS), such as multiple sclerosis (MS). It is a highly debilitating condition characterised by an array of symptoms including spontaneous pain, abnormal sensations (e.g., burning, tingling), allodynia (increased sensitivity to non-painful stimuli) and hyperalgesia (increased sensitivity to noxious stimuli) ( Baron et al., 2010 ). Recently, neuropathic pain has been considered as a neuroimmune disorder, where immune and inflammatory responses play a crucial role in the underlying pathogenesis (Austin and Moalem-Taylor, 2010 and Moalem and Tracey, 2006). In particular, activation of T lymphocytes and pro-inflammatory responses has been shown to contribute significantly to the development and maintenance of neuropathic pain ( Moalem et al., 2004 ).
Accumulating evidence suggests myelin basic protein (MBP) as a novel mediator of pain ( Liu et al., 2012 ). In an animal model of peripheral nerve injury, the level of Schwann cell-mediated MBP degradation and associated neuroinflammation were correlated to the development of mechanical nociception after nerve damage, and inhibition of the MBP-degrading matrix metalloproteinases (MMPs) resulted in sustained MBP preservation and attenuation of mechanical allodynia ( Kobayashi et al., 2008 ). MMP-mediated fragmentation of MBP as a consequence of Wallerian degeneration has been shown to expose immunodominant MBP peptide epitopes inducing mechanical allodynia in both a T cell-dependent and independent manner ( Liu et al., 2012 ).
MBP is one of the major auto-antigens involved in inducing T cell activation in MS patients and is capable of inducing experimental autoimmune encephalomyelitis (EAE), the animal model of MS, in rodents ( Matsoukas et al., 2005 ). EAE is characterised by various degrees of ascending paralysis (tail to hind limb to fore limb paralysis) and continuing loss of body weight of the animal ( Encinas et al., 2001 ). Activated T cells specific against MBP have been identified in the blood and cerebrospinal fluid of MS patients and are thought to be associated with initiation of the disease (Martin et al, 1991 and Steinman, 1996). Animals with EAE that were subjected to immunisation with myelin-derived peptides, such as immunodominant MBP, develop symptoms of neuropathic pain, including tactile and cold allodynia, and mechanical and thermal hyperalgesia (Aicher et al, 2004, Olechowski et al, 2009, and Thibault et al, 2011). While several fragments of myelin antigens have been used to induce EAE in susceptible rodents, the disease severity and the degree of motor and sensory disturbances greatly depend on the species, strain and the epitope of the myelin antigen used. For example, MBP87–99 is known to be a weak EAE agonist whereas myelin oligodendrocyte glycoprotein (MOG)35–55 and MBP72–85 are strong EAE agonists in Lewis rats (Olechowski et al, 2009, Tselios et al, 2001, and Tian et al, 2013a).
Induction of EAE in susceptible animals with myelin-derived antigens results in proliferation of peripheral T cells expressing antigen-specific T cell receptor (TCR). These autoreactive T cells are activated in response to the presentation of antigen bound to major histocompatibility complex (MHC) molecules, and subsequently migrate to the CNS. In the CNS, these cells synthesise and secrete inflammatory mediators including cytokines and chemokines that initiate an inflammatory cascade involving other immune cells and the activation of resident microglia. This leads to myelin damage, demyelination and subsequent neurodegeneration, which mimics the clinical and histopathological features of MS in humans (Schreiner et al, 2009 and Constantinescu et al, 2011).
Despite the selective specificity of the TCR, T cells can respond to a range of related peptides with varying potency. Variant peptides can be generated by altering the amino acid sequence to induce altered T cell responses, and those analogues are defined as altered peptide ligands (APLs). APLs are structurally different from the native peptide by one or more amino acid substitutions to the essential TCR contact residues (Kersh and Allen, 1996 and Sloan-Lancaster and Allen, 1996). APLs are capable of modulating the T cell immune response by acting as antagonists, partial, or weak agonists, thus resulting in inhibition of T cell activation by the native immunogenic peptide ( Katsara et al., 2008b ) or T cell anergy (functional inactivation following an antigen encounter) (Sloan-Lancaster et al, 1994 and Tseveleki et al, 2015). Furthermore, APLs may induce functional changes to T cells by activating different signalling pathways resulting in deviation of a T helper (TH)1 mediated pro-inflammatory milieu towards a TH2 mediated anti-inflammatory milieu ( Nicholson et al., 1995 ). Therefore, APLs have been exploited to manipulate autoreactive T cell responses in various animal models of autoimmune disease including EAE ( Katsara et al., 2008a ). They have been shown to ameliorate clinical EAE (Katsara et al, 2009, Tselios et al, 2001, and Nicholson et al, 1995), reduce depressive behaviour ( Lewitus et al., 2009 ), and promote recovery following incomplete spinal cord injury ( Hauben et al., 2001 ) in experimental rodents.
We have previously demonstrated that active immunisation with MBP-derived APL (cyclo-[A91,A96]MBP87-99) suppresses the severity of guinea pig-MBP-induced clinical EAE along with significantly diminished mechanical pain hypersensitivity in rats ( Tian et al., 2013a ). A subsequent study revealed that active immunisation with the same MBP-derived APL significantly reduced mechanical pain hypersensitivity and neuroinflammation in nerve-injured rats ( Perera et al., 2015 ). In the present study, we investigated the effects of active immunisation with weakly encephalitogenic MBP87–99 and MBP-derived APL on pain hypersensitivity, locomotor function, neuroinflammation and prevalence of systemic regulatory T (Treg) cells in uninjured Lewis rats.
2. Materials and methods
Adult male Lewis rats 8–10 weeks old (Animal Resource Centre, Perth, WA, Australia) were used for immunisation. Rats were group-housed with water and food ad-libitum and maintained on a 12:12 hour light/dark cycle. A constant room temperature and humidity level was maintained in the animal facility, and the animals were monitored daily throughout the experiments. All animal experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales, Sydney, Australia and performed in compliance with guidelines issued by the International Association for the Study of Pain.
Linear human MBP87–99, which is homologues to rat MBP87–99, was used as the native peptide for immunisation. It has been reported that this region of MBP is highly conserved in mammalian species including humans and rodents ( Jones et al., 1992 ). Cyclo-(87–99)[A91,A96]MBP87–99 was used as the APL for co-immunisation with MBP87–99. The amino acid sequence of linear MBP (VHFFKNIVTPRTP) was replaced by Alanine (A) at positions 91 and 96 in the APL (VHFFANIVTARTP). Previous research demonstrated that amino acids K91, T95 and P96 interacted with the TCR and substitution with alanine at these positions was able to inhibit the T-cell response in vitro (Karin et al, 1994 and Katsara et al, 2008c). Cyclic peptides have been shown to be more stable in vivo with increased metabolic stability, potency, receptor selectivity and bioavailability relative to their linear counterparts (Matsoukas et al, 2005, Tselios et al, 2001, and Katsara et al, 2006). Therefore, the APL [A91,A96]MBP87–99 was cyclised using a head-to-tail method. Peptides were custom synthesised by Mimotopes (Pty) Ltd, Clayton, VIC, Australia.
2.3. Active immunisation and assessment of clinical symptoms
Rats were anesthetised with 5% isofluorane (Delvet Pty Ltd., Seven Hills, NSW, Australia) and were maintained with 2–3% of isoflurane in oxygen. They were immunised subcutaneously at the base of the tail with either 200 μl of Complete Freund's Adjuvant (CFA; emulsion containing 1 mg/mL Mycobacterium tuberculosis as control; n = 6) or 200 μl of MBP (200 μg MBP87–99 in CFA; n = 6) or 200 μl of emulsion containing MBP and APL (200 μg MBP87–99+ 250 μg cyclo-(87–99)[A91,A96]MBP87–99 in CFA; n = 6). Immunised rats were assessed daily for clinical signs of EAE for 4 weeks using the following scale: 0—normal rats; 1—flaccid tail; 2—weak hind limbs with ataxia; 3—hind limb paralysis; 4—paraplegia with forelimb paralysis. In addition, body weight was also recorded during the experimental period.
2.4. Pain hypersensitivity testing
Animals were habituated to the pain behavioural testing apparatus for 1 h prior to the initial testing in a quiet and well controlled environment. Thereafter, tests were carried out 3 times a week, after habituating the animals in the apparatus for at least 30 min. Baseline data prior to immunisation and up to 4 weeks post-induction were collected for mechanical withdrawal threshold and thermal withdrawal latency in hindpaws.
For mechanical pain hypersensitivity testing, rats were housed in a persplex test cage on an elevated mesh and the mid-plantar surface of the hindpaw was stimulated using a dynamic von Frey anesthesiometer (Ugo Basil, Comero, Italy). Thermal hyperalgesia was assessed by exposing the plantar surface of the hindpaw to a radiant heat using a plantar analgesia metre (Ugo Basile, Varese, Italy). To prevent tissue damage a cut-off point was set at 20 s.
Paw withdrawal thresholds and latencies were measured automatically from the initiation of heat or mechanical stimulus to withdrawal of the paw, defined as a sudden withdrawal of paw away from the stimulus. The left and right hindpaws were tested 3 times in each testing session with a 3–5 min interval and then the average latency and threshold from the two hindpaws was calculated.
2.4.1. Open field test
Locomotion and rearing were assessed on day 17 post-induction by placing the rats into a photo beam activity system — open field chamber (16″ × 16″) (SD Instruments, San Diego, USA), enclosed within a customised white Perspex box to block visual distractions, with no auditory distractions and stable lighting for 5 min. The total distance travelled (locomotion) and number of rearings (exploratory behaviour) were interpreted from laser beam breaks using the manufacturer's supplied software.
2.5. Tissue harvesting
Rats were sacrificed and tissues were removed for immunohistochemistry and flow cytometry at the peak of disease i.e. 17 days post induction (dpi). Rats were deeply anaesthetised with isoflurane, and popliteal and inguinal lymph nodes and spleens were collected in phosphate-buffered saline (PBS). Rats were then injected with sodium pentobarbital and perfused with heparinised saline (0.9% NaCl) followed by 4% paraformaldehyde (PFA). Lumbar spinal cords (segment L4–L6) were dissected and post-fixed in 4% PFA overnight at 4 °C, and then transferred to 30% sucrose + 0.1% sodium azide solution and stored at 4 °C until sectioning.
Spinal cords were embedded in Tissue Tek® O.C.T. compound (Sakura, AJ Alphen aan den Rijn, The Netherlands), frozen at − 20 °C and sectioned using a cryostat. Spinal cords were sectioned coronally (20 μm thick) from rostral to caudal and collected directly onto superfrost plus slides, so that each slide contained six sections distributed evenly in L4–L6 segment. Slides were air dried and stored at − 20 °C until used. For staining, slides were first incubated with 100% ethanol for 10 min at room temperature followed by 2 washes with distilled (d)H2O for 5 min each. For T cells only, slides were incubated in acetone for 5 min. After washing with PBS for 3 min, sections were blocked for 30 min at room temperature with PBS containing 0.05% Tween 20 and 5% normal donkey serum (Jackson Immune Research, Westgrove, PA). Sections were then incubated with primary antibodies diluted in PBS containing 5% bovine serum albumin (Sigma-Aldrich, Sydney, Australia) + 0.05% Tween-20 ± 0.05% Triton-X for 1 h at room temperature. Primary antibodies used; mouse anti-rat TCRαβ (T cells; 1:250, clone R73, BD Bioscience), mouse anti-rat RT1B (MHC class II; 1:100, clone OX6; Serotec, Abacus, QLD, Australia), rabbit anti-rat ionized calcium binding adaptor molecule 1 (Iba-1) (microglia; 1:2000; Wako Chemicals USA, Richmond, VA, USA), mouse anti-rat glial fibrillary acidic protein (GFAP) (astrocytes; 1:2000, Chemicon, Temecula, CA, USA), and rabbit anti-mouse/rat aspartoacylase (ASPA) (oligodendrocytes; 1:500, kindly gifted by Dr Georg Von Jonquieres and A/Prof Matthias Klugmann, Translational Neuroscience Facility, UNSW, Sydney, Australia). Sections were rinsed 4 times in PBS and incubated for 1 h with the secondary antibodies; Alexa Fluor 488 conjugated donkey anti-mouse (1:250, Jackson Immuno Research Laboratories) or Cy3 conjugated donkey anti-rabbit (1:400, Jackson Immuno Research Laboratories) in the same buffer as the primary antibody. For Isolectin B4 (IB4) staining, sections were first washed with 50% ethanol for 10 min followed by twice wash in water for 5 min each. Sections were then washed in PBS for 5 min, blocked with 5% goat serum in PBS for 30 min, and incubated with IB4 conjugated to FITC (1:100, Sigma Aldrich, Sydney, Australia) for 2 h at room temperature prior to being washed 4 times in PBS. Prolong gold anti-fade reagent with 4′, 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Mulgrave, VIC, Australia) was applied before slides were cover slipped.
2.5.2. Immunofluorescent image analysis and quantification
Slides were visualised with an Olympus fluorescence microscope and images captured using an Olympus DP73 camera and Cellstandard software (Olympus, Tokyo, Japan). For each animal, slides containing each of six spinal cord sections were stained for a given antibody. Images of the dorsal and ventral regions were taken from both left and right sides of 6 different L4–L6 coronal sections per slide. All images were captured at x10 magnification. Images of the T cells were captured from the dorsal and ventral white matter only, since no T cells were observed in the dorsal and ventral horns. Images for MHC class II were taken from both the dorsal and ventral white matter and horns. Images for Iba-1, GFAP and ASPA were taken from the dorsal and ventral horns of the spinal cord. The spinal cord areas for analysis were determined according to the presence of cells, and/or the sciatic territories in the dorsal and ventral horns since pain behaviour was tested in the mid-plantar surface of the hindpaw, which falls within the sciatic nerve distribution. Cells were then either counted manually (for TCR αβ) using the cell counter plug-in, or where their numbers were too numerous, were analysed by densitometry (GFAP, MHC class II and Iba-1) using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Densitometry measurements were made by adjusting the threshold of each image and measuring the percentage of immunopositive areas. Cell counts or densitometry measurements from dorsal and ventral regions were quantified and were averaged for each animal. The data were plotted as the mean of 3–4 rats, and expressed according to cell count or percentage area of immunoreactivity. For IB4 + immunoreactivity, mean intensity was measured using ImageJ. Additional lower magnification images were captured from scanned slides using Aperio ScanScope XT slide scanner and Aperio eslide manager software for representative purpose (Leica Biosystems, Buffalo grove, IL, USA).
2.6. Flow cytometry
Flow cytometry was performed on lymphocytes collected from the spleen and lymph nodes on 17 dpi. Each tissue was processed separately to achieve single cell suspensions by pressing the spleen and lymph nodes through a 40 μm cell strainer (BD Bioscience, Franklins Lakes, NJ, USA) in PBS. Cell suspensions were centrifuged for 4–5 min at 800 ×g at 4 °C, before discarding the supernatant. Red blood cell (RBC) lysis was performed for spleen samples by resuspending the cells in RBC Lysis Buffer (eBioscience, San Diego, CA, USA) for 4–5 min with occasional shaking. Cell suspensions were first washed in PBS, with a second wash in RPMI (Roswell Park Memorial Institute) media (Invitrogen, Mulgrave, VIC, Australia). Cells were incubated at 37 °C in RPMI/10% foetal bovine serum (Invitrogen) for 1 h, to allow adherence and removal of monocytes. Following incubation, cells were counted and re-suspended in flow cytometry staining buffer (eBioscience). Cell surface markers were stained for 30 min at 4 °C, with the following combination of antibodies: mouse anti-rat CD4-FITC (eBioscience) and mouse anti-rat CD25-APC (eBioscience) or suitable isotype controls. Cells were then washed 3 times with MACS buffer (MiltenylBiotec,San Diego, CA) before being fixed overnight at 4 °C with fixation/permeabilisation solution (eBioscience). The following day, samples were washed twice with permeabilisation buffer (eBioscience), and stained with rat anti-mouse/rat Foxp3-PE or isotype control antibodies in permeabilisation buffer for 30 min at 4 °C. Finally, samples were washed 3 times with permeabilisation buffer and re-suspended in flow cytometry staining buffer (eBioscience). Cells were then acquired and analysed on a BDFACS CantoII flow cytometer using BDFACS DIVA software (Becton Dickinson (BD) Biosciences, Franklins Lakes, NJ, USA). A minimum of 100,000 events were acquired for each sample.
2.7. Data analysis
The number of animals used was n = 6 per group for pain behavioural testing and n = 3–4 per group for immunohistochemistry and flow cytometry based on previous studies from our group and others (Moalem et al, 2004, Moalem-Taylor et al, 2007, Austin et al, 2012, Kim and Moalem-Taylor, 2011, Liu et al, 2012, and Perera et al, 2015). The experimenter was aware of the treatments during testing. Statistical analysis was performed using Graphpad prism 6.0 software. For EAE clinical scores, data were analysed with a non-parametric Friedman test with Dunn's post-test. Behavioural results were analysed with two-way repeated measure analysis of variance (RM-ANOVA) followed by Tukey's multiple comparisons post-test. Immunohistochemistry and flow cytometry results were analysed with ordinary one way-ANOVA with Tukey's post-test. P < 0.05 was considered as statistically significant.
3.1. Immunisation with MBP87–99 induced very mild clinical EAE that was significantly reduced by co-immunisation with APL
We determined the effects of immunisation with MBP87–99 or co-immunisation of MBP87–99 together with APL cyclo-(87–99)[A91,A96]MBP87–99 (MBP + APL) on the development of EAE in Lewis rats. CFA was used as a control and animals were monitored for EAE symptoms for 4 weeks post-immunisation ( Fig. 1 ). First clinical signs emerged with the development of tail flaccidity (score of 1) in the MBP-treated group on 7 dpi, followed by very mild EAE until day 22. Only 4/6 rats developed a flaccid tail, and only 2 developed signs of hindlimb weakness (clinical score of 2) in the MBP-treated group. Rats developed a maximum mean clinical score of 0.6 at 17 dpi and completely recovered by day 24 ( Fig. 1 A). In contrast, in the APL + MBP-treated group, 2 animals developed signs of tail flaccidity emerging from 12 dpi, and showed a short duration of mild clinical disease until 22 dpi. As expected, rats treated with CFA alone did not exhibit any signs of clinical/neurological deficits. Clinical scores of MBP-treated rats were significantly greater than those of control (P < 0.001) and APL + MBP-treated (P < 0.05) rats.
Furthermore, body weight changes of the immunised rats were recorded ( Fig. 1 B). MBP-treated rats demonstrated significantly reduced weight gain compared to APL + MBP-treated (P < 0.01) and control (P < 0.01) rats. Body weight gain of MBP-immunised rats started to decline at 10 dpi while the APL + MBP-treated and control rats showed steady weight gain throughout the experimental period.
As an additional measure of behavioural function, we assessed locomotor behaviour using an open field test at 17 dpi and data were analysed between the treatment groups ( Fig. 1 C, D). Overall, there were no significant differences between the treatment groups in total distance travelled ( Fig. 1 C) or rearing count ( Fig. 1 D).
3.2. Immunisation with MBP87–99 induced mechanical and thermal pain hypersensitivity that was significantly reduced by co-immunisation with APL
Symptoms of neuropathic pain have been reported in both MS patients and animal models of EAE ( Tian et al., 2013b ). Therefore, the changes in mechanical and thermal pain hypersensitivity in the hindpaws of rats were assessed following immunisation with (i) MBP87–99; (ii) MBP87–99 together with APL (cyclo-(87–99)[A91,A96]MBP87–99); and (iii) CFA control for 4 weeks post-immunisation. Interestingly, despite developing an extremely mild form of EAE ( Fig. 1 ), MBP-immunised rats developed a robust mechanical allodynia and thermal hyperalgesia in the hindpaws ( Fig. 2 ). More specifically, mechanical allodynia ( Fig. 2 A) was observed at the same time of onset of clinical deficits, whereas thermal hyperalgesia ( Fig. 2 B) became evident as early as 3 days following immunisation, which was prior to the appearance of any clinical symptoms. Co-immunisation with APL significantly increased the mechanical withdrawal threshold and thermal withdrawal latency in the hindpaws when compared to the MBP-treated rats.
MBP-treated rats demonstrated significantly reduced withdrawal threshold (P < 0.001–0.0001) to mechanical stimuli compared to MBP + APL-treated and control rats at 11, 14, 17 and 22 dpi. No significant differences were seen in mechanical withdrawal threshold between the MBP + APL-treated and control rats. The mechanical paw withdrawal thresholds of MBP + APL-treated rats and CFA-treated control rats remained within a range of 16–20 g post immunisation, whereas MBP-treated rats demonstrated the lowest mechanical withdrawal threshold of 8 g at disease peak (17 dpi). Although the mechanical withdrawal threshold in the hindpaws of MBP-treated rats started to increase after 17 dpi, it was still significantly reduced compared to the other 2 groups during the monitoring period ( Fig. 2 A).
In MBP-treated rats, paw withdrawal latency to the thermal stimuli began to drop as early as 3 dpi and continued until 14 dpi. The lowest withdrawal latency of 6.9 s was exhibited at 11 dpi in the MBP-treated group, before normalising thereafter. However, the paw withdrawal latency to thermal stimuli in MBP-treated rats was significantly decreased compared to the MBP + APL-treated (P < 0.0001) and control (P < 0.0001) rats on days 4, 7, 11, 14 and 17 ( Fig. 2 B).
3.3. Immunisation with MBP87–99 induced T cell infiltration and MHC class II + cell expression in the spinal cord that were significantly reduced by co-immunisation with APL
T cells have been implicated in animal models of neuropathic pain due to nerve injury and EAE (Moalem et al, 2004, Olechowski et al, 2009, and Jones et al, 2008). To investigate the effects of immunisation with MBP87–99 and MBP-derived APL on T cell infiltration, spinal cord sections were immunostained with anti-rat TCRαβ at 17 dpi and the captured images were analysed ( Fig. 3 ). We found a significantly greater number of T cells in the dorsal column (P < 0.05) and ventral (P < 0.05) white matter of the spinal cord in MBP-treated rats ( Fig. 3 A, D) compared to the MBP + APL-treated ( Fig. 3 A, E) and control ( Fig. 3 A, F) rats. In the dorsal column at 17 dpi, the mean T cell number in the MBP-treated group was 147.9 ± 60.1, which was significantly different from that of MBP + APL-treated (25.1 ± 13.1) and control (1.6 ± 0.8) rats. T cell infiltration into the ventral white matter was considerably lower in all treatment groups compared to the dorsal white matter; however, the mean T cell number was still significantly higher (P < 0.05) in the MBP-treated group compared to the other two groups ( Fig. 3 A right). T cells were almost absent in the control CFA-treated rats ( Fig. 3 A, F). The pattern of T cell immunoreactivity revealed a distinct pathway of T cells migrating into the spinal cord ( Fig. 3 C). It was noticeable that most of the T cells appeared to enter the spinal cord through the central canal and dorsal marginal zone, with a significantly greater number of T cells seen at the central canal. However, T cells were absent in the dorsal and ventral horns of the spinal cord.
In addition, we analysed MHC class II + immunoreactivity in the dorsal and ventral regions of the spinal cord of immunised rats at 17 dpi ( Fig. 3 B, G–I). MBP-treated rats ( Fig. 3 B, G) demonstrated significantly increased MHC class II + cell presence (P < 0.001) in both the dorsal and ventral white matter and horns as compared to the MBP + APL co-immunised rats ( Fig. 3 B, H) and CFA-treated control rats ( Fig. 3 B, I). The percentages of MHC II + cell density in MBP-treated rats were 1.3 ± 0.3 (dorsal horn + column) and 1.2 ± 0.09 (ventral horn + white matter), whereas in MBP + APL-treated rats they were 0.4 ± 0.1 (dorsal column) and 0.5 ± 0.04 (ventral white matter) and in control rats 0.4 ± 0.06 (dorsal column) and 0.4 ± 0.04 (ventral white matter) ( Fig. 3 B). There was no MHC II + cell reactivity in the dorsal and ventral horns of the spinal cord in MBP + APL-treated and control rats.
3.4. Immunisation with MBP87–99 increased spinal microglial activation that was significantly reduced by co-immunisation with APL
Reactivity of glial cells in the spinal cord has been documented in previous studies of neuropathic pain and EAE in rodent models (Olechowski et al, 2009 and Austin et al, 2012). To observe changes in activation of microglia and astrocytes in the spinal cord of immunised rats, we performed immunohistochemistry using antibodies against Iba-1 (microglia) and GFAP (astrocytes) at 17 dpi ( Fig. 4 ). We found significantly greater Iba-1 + cell reactivity in the spinal cord in MBP-treated rats ( Fig. 4 A, D) as compared to the MBP + APL-treated ( Fig. 4 A, E) and control ( Fig. 4 A, F) rats. The higher density of Iba-1 + staining was more pronounced in the dorsal horn of the spinal cord relative to the ventral horn in MBP-treated rats ( Fig. 4 C). The percentage of Iba-1 + cell density was significantly increased in MBP-treated rats as compared to the MBP + APL-treated (P < 0.001) and control (P < 0.0001) rats in the dorsal horn of the spinal cord ( Fig. 4 A left). MBP + APL-treated rats also demonstrated significantly enhanced (P < 0.05) Iba-1 + cell density relative to the control rats in both dorsal and ventral horns ( Fig. 4 A). In the ventral horn, immunisation with MBP led to an increased microglial reactivity as compared to control rats (P < 0.01). However, there was no significant difference between the MBP-treated and MBP + APL-treated rats in the ventral horn ( Fig. 4 A right).
Moreover, there was no significant difference in astrocyte activation between the treatment groups ( Fig. 4 B), although a decreasing trend of GFAP + immunoreactivity was noted, albeit not statistically significant, in both dorsal and ventral horns in the MBP + APL-treated rats ( Fig. 4 B, H) compared to the MBP-treated ( Fig. 4 B, G) and control ( Fig. 4 B, I) rats.
3.5. Immunisation with MBP87–99 and co-immunisation with APL had no effects on spinal IB4 + immunoreactivity, myelin expression and oligodendrocyte numbers
The subpopulation of non-peptidergic primary afferents responsible for pain transmission that express binding sites for IB4 has been shown to play a role in the development of neuropathic pain. Nerve injury results in a decrease in IB4 binding in the superficial dorsal horn of the spinal cord, suggesting either loss of IB4-binding nociceptors or decreased binding due to altered expression of the IB4 binding site (Bailey and Ribeiro-Da-Silva, 2006 and Munglani et al, 1995). Thus, we performed immunostaining for IB4 to identify non-peptidergic nociceptive terminals in the dorsal horn of the spinal cord of immunised rats at 17 dpi ( Fig. 5 C–E). We observed IB4 staining in both left and right dorsal horns of the spinal cord of immunised rats that was limited to laminae I–II of the superficial dorsal horn ( Fig. 5 C–E). However, there was no significant difference between the treatment groups in the mean intensity of the IB4 labelling ( Fig. 5 A). Further, we labelled spinal cord sections with anti-MBP antibody to assess whether there were changes in myelin expression between the treatment groups at the peak of disease. No obvious visible loss of myelin was evident in any of the treatment groups (data not shown). Oligodendrocytes in the CNS synthesise myelin and have recently been implicated in neuropathic pain ( Gritsch et al., 2014 ). Therefore, we analysed oligodendrocyte staining with anti-ASPA antibody in spinal cord sections of immunised rats. No significant differences were apparent between the treatment groups in the number of immunoreactive ASPA + cells ( Fig. 5 B, F–H).
3.6. Immunisation with MBP87–99 and co-immunisation with APL had no effects on the prevalence of regulatory T cells in the spleen and lymph nodes
APLs are known to induce immunosuppressive Treg cells (CD4+CD25+FoxP3+ phenotype) in experimental animal models ( Nicholson et al., 1997 ). Thus, the effect of immunisation with APL (cyclo-(87–99)[A91,A96]MBP87–99) on the prevalence of Treg cells in the spleen and lymph nodes at 17 dpi in immunised rats were analysed using flow cytometric analysis. There were no statistical significant differences in the percentages of CD4+CD25+FoxP3+ Treg cells between the treatment groups in any of the tissues analysed ( Fig. 6 A, B).
Myelin-derived APLs have been shown to confer beneficial effects in various animal models of autoimmune diseases including EAE (Karin et al, 1994 and Tian et al, 2013a), experimental models of spinal cord injury (Rodriguez-Barrera et al, 2013 and Ibarra et al, 2013) and peripheral nerve injury ( Perera et al., 2015 ). Our previous study demonstrated that co-immunisation of guinea pig MBP with the APL cyclo-(87–99)[A91,A96]MBP87–99 not only ameliorated the clinical disease course, but also normalised mechanical pain sensitivity in Lewis rats with EAE ( Tian et al., 2013a ). While this effect of the APL was observed in rats with a severe paralytic disease, the present study investigated the effects of APL treatment on pain hypersensitivity in MBP87–99-immunised rats with extremely mild clinical EAE and no confounding effects of hind limb paralysis, and further explored changes in neuroinflammatory responses. Here, we show that immunisation with the native peptide MBP87–99 induced mechanical and thermal pain hypersensitivity in the hindpaws, spinal cord T cell infiltration, MHC class II + cell expression and microglial activation, without causing severe neurological and locomotor impairment in Lewis rats. In agreement with our findings, a recent study using a newly developed mouse model of EAE demonstrated that EAE-affected mice exhibited a mild clinical disease course with temporal development of mechanical allodynia in the hindpaws and a significantly increased spinal glial activation and T cell infiltration, without developing severe motor deficits ( Khan et al., 2014 ). Further, we demonstrate that co-immunisation with the APL, cyclo-(87–99)[A91,A96]MBP87–99 significantly reduced the already mild clinical symptoms of EAE, decreased mechanical and thermal pain hypersensitivity, and lessened neuroinflammation in the spinal cord. These findings are in line with previous studies showing protective effects of myelin-derived APLs on disease severity and duration in EAE (Tian et al, 2013a, Gaur et al, 1997, Nicholson et al, 1995, Fischer et al, 2000, Santambrogio et al, 1998, Ruiz et al, 1999, Karin et al, 1994, and Brocke et al, 1996).
The native peptide, MBP87–99 is a weak encephalitogenic epitope in Lewis rats; it has been shown that administration of several doses of MBP87–99 is required to induce EAE, and animals present with mild clinical signs of disease ( Tselios et al., 2001 ). In agreement with this property of the native peptide, we found that most immunised rats developed tail weakness/paralysis and only two out of six rats developed mild signs of hindlimb weakness. In addition, none of the animals developed substantial locomotion deficits or alterations in exploratory behaviour ( Fig. 1 C, D). However, the MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity in the hindpaws following immunisation, suggesting a possible nociceptive effect of MBP87–99 without causing severe paralytic disease. MBP is now considered a novel conciliator of pain, since intraneural administration of the immunodominant peptide MBP84–104 into naïve sciatic nerve produced robust mechanical allodynia with increased gene expression of the antigen presentation pathway and T cell activation in the injected nerve and the corresponding levels of the spinal cord. The ability of MBP84–104 to initiate allodynia and activation of inflammatory pathways was diminished in athymic nude rats, suggesting a T cell-dependent mechanism in MBP-induced pain facilitation ( Liu et al., 2012 ). It is thus logical to assume that MBP-specific T cells may have contributed to the increased pain hypersensitivity in MBP-treated rats in the present study.
T cells have been implicated in several studies of neuropathic pain (Austin and Moalem-Taylor, 2010 and Moalem et al, 2004), and EAE is thought to be associated with a TH1 and TH17 cell phenotype ( Duffy et al., 2014 ). Since chronic neuropathic pain is common in MS patients and in animals with EAE, we investigated T cell infiltration into the spinal cord of immunised rats. In the present study, we noted a significant T cell infiltration in the dorsal column of the spinal cord in MBP-treated rats, in agreement with a previous study using mice with MOG-induced EAE ( Jones et al., 2008 ). The dorsal column of the spinal cord is known to be involved in neuropathic pain, particularly in mediating mechanical allodynia through large diameter low threshold Aβ fibres, which transmit afferent input to supraspinal sites through the dorsal column pathways. Indeed, tactile allodynia was completely blocked in rats with spinal nerve ligation by lesions of the dorsal column at spinal cord T8 level, or by applying lidocaine into the ipsilateral gracile nucleus, which receives projections from the dorsal column ( Ossipov et al., 2000 ). We found a significant decrease in T cell numbers in the spinal cord in rats co-immunised with MBP + APL, which also showed diminished pain hypersensitivity. This suggests an inhibitory effect of myelin-derived APL on T cell activation. It has been reported that APLs can affect TCR signal transduction through alteration of protein tyrosine phosphorylation, kinase activity, and transcriptional activation of key cytokine promoters thereby inhibiting T cell activation or modifying T cell function ( Nel and Slaughter, 2002 ). APLs can also exert an antagonistic effect on T cell activation by loss of H-bond contacts between the peptide and TCR, thereby causing partial or complete inactivation of T cells (Tselios et al, 2001 and Degano et al, 2000).
A previous study has demonstrated that T cell infiltration and interferon (IFN)-γ signalling in the adult dorsal spinal cord is a major contributor to neuropathic pain hypersensitivity ( Costigan et al., 2009 ). Following partial peripheral nerve injury in adult rats, significant infiltration of T cells and up-regulation of IFN-γ were observed in the spinal cord, and IFN-γ receptor-deficient animals displayed significantly less mechanical allodynia compared to controls ( Costigan et al., 2009 ). In addition, infiltrating CD4 + T cells in the lumbar spinal cord following peripheral nerve injury were shown to express cytokines of the proinflammatory TH1 phenotypes, such as IFN-γ, TNF and GM-CSF, that may contribute to the pathogenesis of neuropathic pain ( Draleau et al., 2014 ). Thus, peripheral suppression of proinflammatory T cell activation and reduced spinal T cell infiltration could be one of the possible mechanisms by which APL improved neuropathic pain behaviour in the immunised rats in our study.
Involvement of MHC class II expressing cells in neuropathic pain has been studied in several animal models of neuropathic pain ( Sweitzer et al., 2002 ). MHC Class II complexes on antigen presenting cells interacting with infiltrating lymphocytes following nerve injury are responsible for introducing antigenic peptides to T cells. In addition, increased glial expression of MHC II has been described in several peripheral nerve injury models including facial nerve transection, sciatic nerve crush injury, thoracic dorsal column contusion, spinal nerve transection and injury to the nerve root (Kreutzberg, 1996, Hashizume et al, 2000, and Popovich et al, 1993). Interestingly, inhibition of glial MHC II expression was effective in improving pain behaviours following nerve injury (Sweitzer and Deleo, 2002 and Hashizume et al, 2000). Further, MHC class II knockout mice exhibit diminished mechanical allodynia following peripheral nerve transection ( Sweitzer et al., 2002 ). In the present study, we demonstrate a significantly greater expression of MHC II immunoreactivity in the lumbar spinal cord of MBP-treated rats as compared to those of MBP + APL-treated and control rats. APLs can block the formation of MHC-Peptide-TCR complexes and thereby inhibit the development of autoimmunity ( Mantzourani et al., 2006 ). Thus, APL-induced blocking of both T cell and MHC II + antigen presenting cell activation may have contributed to the analgesic effect in the co-immunised animals.
Microglial activation in the spinal cord is a key player in the pathogenesis of neuropathic pain ( Austin and Moalem-Taylor, 2010 ). Accumulating evidence suggests inflammation and reactive gliosis are key features of EAE (Olechowski et al, 2009 and Lu et al, 2012). Herein, we observed a significantly enhanced Iba-1 + immunoreactivity in the dorsal and ventral horns of the lumbar spinal cord of MBP-treated rats compared to the co-immunised and control rats. It has been shown that intraspinal administration of activated microglia into naïve rats is sufficient for the induction of neuropathic pain ( Tsuda et al., 2003 ). Indeed, activated glial cells can modulate spinal excitability and facilitate pain hypersensitivity via synthesis and secretion of cytokines and growth factors, such as brain-derived neurotrophic factor (Coull et al, 2005 and Deleo and Yezierski, 2001). In addition, increased expression of P2X4 ATP receptors and Toll-like receptors (TLRs) in microglia has been reported in neuropathic pain. TLR4 knockout mice exhibited significantly diminished pain hypersensitivity with downregulation of microglial markers following L5 nerve transection ( Tanga et al., 2005 ), and blocking of ATP signalling through P2X4 receptors ameliorated tactile allodynia (Tsuda et al, 2003 and Tsuda et al, 2009). Moreover, the chemokine CCL2 has been implicated as a key mediator in microglial activation in neuropathic pain conditions, and is also upregulated in conditions like MS and spinal cord injury ( Thacker et al., 2009 ). Thus, down regulation of one or more of the above mentioned signalling pathways by co-immunisation with APL might have contributed to the analgesic mechanism of APL. However, it is noteworthy that there are conflicting reports regarding the role of microglia in neuropathic pain in both humans and animal models. Using novel imaging techniques, Loggia et al. provided an evidence for brain glial activation in chronic pain patients. The thalamic levels of translocator protein, presumably exerting pain-protective effects, were negatively correlated with clinical pain and levels of circulating proinflammatory cytokines ( Loggia et al., 2015 ). Recent clinical trials assessing the efficacy of glial modulators, such as low-dose naltrexone ( Younger and Mackey, 2009 ), propentophylline ( Landry et al., 2012 ) and minocycline ( Martinez et al., 2013 ) in neuropathic pain patients showed contrasting results. In animals, a dissociation between microglia activation and pain hypersensitivity has been demonstrated in several models including peripheral nerve injury ( Colburn et al., 1997 ), chemotherapy-induced peripheral neuropathy ( Zheng et al., 2011 ) and central neuropathic pain due to oligodendrocyte ablation ( Gritsch et al., 2014 ). In addition, previous studies reported significantly enhanced GFAP + astrocyte activation in MOG-induced EAE ( Olechowski et al., 2009 ), and astrocytes have been shown to play a role in neuropathic pain pathogenesis ( Austin and Moalem-Taylor, 2010 ). In the MBP-treated rats, we did not observe enhanced spinal astrocyte activation, which may be explained by the extremely mild clinical EAE ( Fig. 1 ). Our study suggests that astrocytes do not play a role in the pain hypersensitivity of the MBP-immunised rats. Indeed, several studies have reported findings of neuropathic pain behaviours without profound astrocyte reactivity in animal models including chemotherapy-induced peripheral neuropathy ( Ledeboer et al., 2007 ), genetic ablation of oligodendrocytes ( Gritsch et al., 2014 ) and peripheral nerve injury (Moalem-Taylor et al, 2011 and Mika et al, 2009).
The involvement of oligodendrocytes in autoimmune-mediated neuropathic pain remains unknown. Gritsch et al. have recently demonstrated that perturbation of oligodendrocyte functions that maintain axonal integrity can lead to central neuropathic pain ( Gritsch et al., 2014 ). In our study, however, no change in oligodendrocyte numbers in the spinal cord was observed in the MBP-immunised rats. In addition, demyelination has been associated with neuropathic pain ( Wallace et al., 2003 ), and several animal studies have demonstrated demyelination in the CNS following immunisation with encephalitogenic epitopes (Liu et al, 2013 and Gupta et al, 2013). Here, in accordance with the extremely mild EAE, we did not observe any clear signs of myelin loss (determined by MBP staining) in the white matter of the spinal cord. These results suggest that oligodendrocytes and myelin integrity are not likely to play a role in this animal model. While non-peptidergic IB4-binding C fibre staining has been shown to decrease in neuropathic pain (Bailey and Ribeiro-Da-Silva, 2006 and Munglani et al, 1995), an increased IB4 + immmunoreactivity in the spinal cord of proteolipid protein (PLP)139–151-immunised SJL/J mice exhibiting neuropathic pain behaviours has been reported ( Lu et al., 2012 ), and our results show no change in IB4 immunoreactivity in the spinal cord. Thus, the role of non-peptidergic IB4-binding C fibres in neuropathic pain associated with EAE appears unclear.
APLs are known to facilitate bystander suppression by inducing protective Treg cells in animal models of EAE. Nicholson et al. demonstrated that APL derived from myelin PLP induced Treg cells in SJL mice with EAE ( Nicholson et al., 1997 ). Contrary to that study, we did not find a significant effect of co-immunisation with MBP87–99 and APL (cyclo-(87–99)[A91,A96]MBP87–99) on the prevalence of Treg cells in lymphoid organs when compared to MBP-treated and control rats. Differences in the native peptide and APL, animal species, time point and tissue used may be possible reasons for the differential effects. Treg cells have been shown to play a role in inhibiting neuropathic pain behaviours in animal models of peripheral nerve injury and experimental autoimmune neuritis ( Austin et al., 2012 ). In the present study, APL immunisation had no effect on Treg cells suggesting alternative mechanisms by which co-immunisation produced the observed reduction in pain hypersensitivity.
In summary, we demonstrate that active immunisation with MBP87–99 induces mechanical and thermal pain hypersensitivity without causing severe motor deficit in Lewis rats, suggesting that MBP-associated autoimmunity can result in neuropathic pain. This finding highlights the emerging concept of an autoimmune basis for chronic pain. For example, there are many autoimmune conditions that are associated with neuropathic pain such as chronic inflammatory demyelinating polyradiculoneuropathy ( Boukhris et al., 2007 ) and multiple sclerosis ( Moulin et al., 1988 ). Additionally, autoantibodies targeting voltage-gated potassium channel complexes, which cause neuronal hyperexcitability, have recently been detected in chronic idiopathic pain patients ( Klein et al., 2012 ). Our results also show that co-immunisation with the non-encephalitogenic APL (cyclo-(87–99)[A91,A96]MBP87–99) ameliorates the clinical disease with significantly improved mechanical and thermal pain hypersensitivity. This was associated with reduced spinal cord neuroinflammation, in particular downregulation of T cell infiltration and decreased activation of antigen presenting cells and microglia in APL-treated rats. However, further studies are required to elucidate the mechanisms underlying MBP-induced pain hypersensitivity and APL (cyclo-(87–99)[A91,A96]MBP87–99)-induced analgesia. The translation of APL-mediated immunotherapy from animal models of EAE to human trials has been challenging. Clinical trials in MS patients using [A91]MBP83–99 were terminated due to the occurrence of adverse reactions in 9% of patients (Kappos et al, 2000 and Bielekova et al, 2000). Nevertheless, future studies on APL treatment using careful titration of the dosage, a suitable safe adjuvant, and the correct route of administration may lead to a promising immunotherapy.
Myelin basic protein
experimental autoimmune encephalomyelitis
central nervous system
myelin oligodendrocyte glycoprotein
repeated measure of analysis of variance
Glial fibrillary acidic protein
ionized calcium-binding adapter molecule 1
major histocompatibility complex
T cell receptor
altered peptide ligand
standard error of mean
Complete Freund's adjuvant
fluorescent associated cell analysis
Phosphate buffered saline
Roswell Park Memorial Institute
photo beam activity system
days post induction/immunisation
Conflict of interest statement
The authors of this publication declare that they have no potential conflicts of interest.
The studies reported in this publication were funded by a grant from the National Health and Medical Research Council of Australia (ID # APP1045343) to Gila Moalem-Taylor. The authors would like to thank the BRIL Flow Cytometry Facility and the Histology and Microscopy Unit at the University of New South Wales, Australia for assisting with flow cytometry experiments and slide scanning, respectively.
- Aicher et al., 2004 S.A. Aicher, M.B. Silverman, C.W. Winkler, B.F. Bebo Jr. Hyperalgesia in an animal model of multiple sclerosis. Pain. 2004;110:560-570 Crossref
- Austin and Moalem-Taylor, 2010 P.J. Austin, G. Moalem-Taylor. The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines. J. Neuroimmunol.. 2010;229:26-50 Crossref
- Austin et al., 2012 P.J. Austin, C.F. Kim, C.J. Perera, G. Moalem-Taylor. Regulatory T cells attenuate neuropathic pain following peripheral nerve injury and experimental autoimmune neuritis. Pain. 2012;153:1916-1931 Crossref
- Bailey and Ribeiro-Da-Silva, 2006 A.L. Bailey, A. Ribeiro-Da-Silva. Transient loss of terminals from non-peptidergic nociceptive fibers in the substantia gelatinosa of spinal cord following chronic constriction injury of the sciatic nerve. Neuroscience. 2006;138:675-690 Crossref
- Baron et al., 2010 R. Baron, A. Binder, G. Wasner. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol.. 2010;9:807-819 Crossref
- Bielekova et al., 2000 B. Bielekova, B. Goodwin, N. Richert, I. Cortese, T. Kondo, G. Afshar, B. Gran, J. Eaton, J. Antel, J.A. Frank, H.F. Mcfarland, R. Martin. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83–99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat. Med.. 2000;6:1167-1175
- Boukhris et al., 2007 S. Boukhris, L. Magy, M. Khalil, P. Sindou, J.M. Vallat. Pain as the presenting symptom of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). J. Neurol. Sci.. 2007;254:33-38 Crossref
- Brocke et al., 1996 S. Brocke, K. Gijbels, M. Allegretta, I. Ferber, C. Piercy, T. Blankensteini, R. Martin, U. Utz, N. Karin, D. Mitchell, T. Veromaa, A. Waisman, A. Gaur, P. Conlon, N. Ling, P.J. Fairchild, D.C. Wraith, A. O'Garra, C.G. Fathman, L. Steinman. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature. 1996;379:343-346 Crossref
- Colburn et al., 1997 R.W. Colburn, J.A. Deleo, A.J. Rickman, M.P. Yeager, P. Kwon, W.F. Hickey. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J. Neuroimmunol.. 1997;79:163-175 Crossref
- Constantinescu et al., 2011 C.S. Constantinescu, N. Farooqi, K. O'Brien, B. Gran. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br. J. Pharmacol.. 2011;164:1079-1106 Crossref
- Costigan et al., 2009 M. Costigan, A. Moss, A. Latremoliere, C. Johnston, M. Verma-Gandhu, T.A. Herbert, L. Barrett, G.J. Brenner, D. Vardeh, C.J. Woolf, M. Fitzgerald. T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity. J. Neurosci.. 2009;29:14415-14422 Crossref
- Coull et al., 2005 J.A. Coull, S. Beggs, D. Boudreau, D. Boivin, M. Tsuda, K. Inoue, C. Gravel, M.W. Salter, Y. De Koninck. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature. 2005;438:1017-1021 Crossref
- Degano et al., 2000 M. Degano, K.C. Garcia, V. Apostolopoulos, M.G. Rudolph, L. Teyton, I.A. Wilson. A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. Immunity. 2000;12:251-261 Crossref
- Deleo and Yezierski, 2001 J.A. Deleo, R.P. Yezierski. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain. 2001;90:1-6 Crossref
- Draleau et al., 2014 K. Draleau, S. Maddula, A. Slaiby, N. Nutile-Mcmenemy, J. De Leo, L. Cao. Phenotypic identification of spinal cord-infiltrating CD4 T lymphocytes in a murine model of neuropathic pain. J. Pain Relief. 2014;(Suppl. 3):003
- Duffy et al., 2014 S.S. Duffy, J.G. Lees, G. Moalem-Taylor. The contribution of immune and glial cell types in experimental autoimmune encephalomyelitis and multiple sclerosis. Mult. Scler. Int.. 2014;2014:285245
- Encinas et al., 2001 J.A. Encinas, M.B. Lees, R.A. Sobel, C. Symonowicz, H.L. Weiner, C.E. Seidman, J.G. Seidman, V.K. Kuchroo. Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis. Int. Immunol.. 2001;13:257-264 Crossref
- Fischer et al., 2000 F.R. Fischer, L. Santambrogio, Y. Luo, M.A. Berman, W.W. Hancock, M.E. Dorf. Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression. J. Neuroimmunol.. 2000;110:195-208 Crossref
- Gaur et al., 1997 A. Gaur, S.A. Boehme, D. Chalmers, P.D. Crowe, A. Pahuja, N. Ling, S. Brocke, L. Steinman, P.J. Conlon. Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms. J. Neuroimmunol.. 1997;74:149-158 Crossref
- Gritsch et al., 2014 S. Gritsch, J. Lu, S. Thilemann, S. Wörtge, W. Möbius, J. Bruttger, K. Karram, T. Ruhwedel, M. Blanfeld, D. Vardeh, A. Waisman, K.-A. Nave, R. Kuner. Oligodendrocyte ablation triggers central pain independently of innate or adaptive immune responses in mice. Nat. Commun.. 2014;5
- Gupta et al., 2013 S. Gupta, R. Utoft, H. Hasseldam, A. Schmidt-Christensen, T.D. Hannibal, L. Hansen, N. Fransen-Pettersson, N. Agarwal-Gupta, B. Rozell, A. Andersson, D. Holmberg. Global and 3D spatial assessment of neuroinflammation in rodent models of multiple sclerosis. PLoS One. 2013;8:e76330 Crossref
- Hashizume et al., 2000 H. Hashizume, M.D. Rutkowski, J.N. Weinstein, J.A. Deleo. Central administration of methotrexate reduces mechanical allodynia in an animal model of radiculopathy/sciatica. Pain. 2000;87:159-169 Crossref
- Hauben et al., 2001 E. Hauben, E. Agranov, A. Gothilf, U. Nevo, A. Cohen, I. Smirnov, L. Steinman, M. Schwartz. Posttraumatic therapeutic vaccination with modified myelin self-antigen prevents complete paralysis while avoiding autoimmune disease. J. Clin. Invest.. 2001;108:591-599
- Ibarra et al., 2013 A. Ibarra, M. Sosa, E. Garcia, A. Flores, Y. Cruz, H. Mestre, S. Martinon, B.A. Pineda-Rodriguez, G. Gutierrez-Ospina. Prophylactic neuroprotection with A91 improves the outcome of spinal cord injured rats. Neurosci. Lett.. 2013;554:59-63 Crossref
- Jones et al., 1992 R.E. Jones, D. Bourdette, H. Offner, A.A. Vandenbark. The synthetic 87–99 peptide of myelin basic protein is encephalitogenic in Buffalo rats. J. Neuroimmunol.. 1992;37:203-212 Crossref
- Jones et al., 2008 M.V. Jones, T.T. Nguyen, C.A. Deboy, J.W. Griffin, K.A. Whartenby, D.A. Kerr, P.A. Calabresi. Behavioral and pathological outcomes in MOG 35–55 experimental autoimmune encephalomyelitis. J. Neuroimmunol.. 2008;199:83-93 Crossref
- Kappos et al., 2000 L. Kappos, G. Comi, H. Panitch, J. Oger, J. Antel, P. Conlon, L. Steinman. Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial. The altered peptide ligand in relapsing MS study group. Nat. Med.. 2000;6:1176-1182
- Karin et al., 1994 N. Karin, D.J. Mitchell, S. Brocke, N. Ling, L. Steinman. Reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: T cell receptor antagonism and reduction of interferon gamma and tumor necrosis factor alpha production. J. Exp. Med.. 1994;180:2227-2237 Crossref
- Katsara et al., 2006 M. Katsara, T. Tselios, S. Deraos, G. Deraos, M.T. Matsoukas, E. Lazoura, J. Matsoukas, V. Apostolopoulos. Round and round we go: cyclic peptides in disease. Curr. Med. Chem.. 2006;13:2221-2232
- Katsara et al., 2008a M. Katsara, G. Deraos, T. Tselios, M.-T. Matsoukas, I. Friligou, J. Matsoukas, V. Apostolopoulos. Design and synthesis of a cyclic double mutant peptide (cyclo(87–99)[A91, A96]MBP87–99) induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of multiple sclerosis. J. Med. Chem.. 2008;52:214-218
- Katsara et al., 2008b M. Katsara, G. Minigo, M. Plebanski, V. Apostolopoulos. The good, the bad and the ugly: how altered peptide ligands modulate immunity. Expert. Opin. Biol. Ther.. 2008;8:1873-1884 Crossref
- Katsara et al., 2008c M. Katsara, E. Yuriev, P.A. Ramsland, G. Deraos, T. Tselios, J. Matsoukas, V. Apostolopoulos. A double mutation of MBP(83–99) peptide induces IL-4 responses and antagonizes IFN-gamma responses. J. Neuroimmunol.. 2008;200:77-89 Crossref
- Katsara et al., 2009 M. Katsara, E. Yuriev, P.A. Ramsland, T. Tselios, G. Deraos, A. Lourbopoulos, N. Grigoriadis, J. Matsoukas, V. Apostolopoulos. Altered peptide ligands of myelin basic protein (MBP87–99) conjugated to reduced mannan modulate immune responses in mice. Immunology. 2009;128:521-533 Crossref
- Kersh and Allen, 1996 G.J. Kersh, P.M. Allen. Structural basis for T cell recognition of altered peptide ligands: a single T cell receptor can productively recognize a large continuum of related ligands. J. Exp. Med.. 1996;184:1259-1268 Crossref
- Khan et al., 2014 N. Khan, T.M. Woodruff, M.T. Smith. Establishment and characterization of an optimized mouse model of multiple sclerosis-induced neuropathic pain using behavioral, pharmacologic, histologic and immunohistochemical methods. Pharmacol. Biochem. Behav.. 2014;126:13-27
- Kim and Moalem-Taylor, 2011 C.F. Kim, G. Moalem-Taylor. Detailed characterization of neuro-immune responses following neuropathic injury in mice. Brain Res.. 2011;1405:95-108 Crossref
- Klein et al., 2012 C.J. Klein, V.A. Lennon, P.A. Aston, A. Mckeon, S.J. Pittock. Chronic pain as a manifestation of potassium channel-complex autoimmunity. Neurology. 2012;79:1136-1144 Crossref
- Kobayashi et al., 2008 H. Kobayashi, S. Chattopadhyay, K. Kato, J. Dolkas, S. Kikuchi, R.R. Myers, V.I. Shubayev. MMPs initiate Schwann cell-mediated MBP degradation and mechanical nociception after nerve damage. Mol. Cell. Neurosci.. 2008;39:619-627 Crossref
- Kreutzberg, 1996 G.W. Kreutzberg. Microglia: a sensor for pathological events in the CNS. Trends Neurosci.. 1996;19:312-318 Crossref
- Landry et al., 2012 R.P. Landry, V.L. Jacobs, E.A. Romero-Sandoval, J.A. Deleo. Propentofylline, a CNS glial modulator does not decrease pain in post-herpetic neuralgia patients: in vitro evidence for differential responses in human and rodent microglia and macrophages. Exp. Neurol.. 2012;234:340-350 Crossref
- Ledeboer et al., 2007 A. Ledeboer, B.M. Jekich, E.M. Sloane, J.H. Mahoney, S.J. Langer, E.D. Milligan, D. Martin, S.F. Maier, K.W. Johnson, L.A. Leinwand, R.A. Chavez, L.R. Watkins. Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats. Brain Behav. Immun.. 2007;21:686-698 Crossref
- Lewitus et al., 2009 G.M. Lewitus, A. Wilf-Yarkoni, Y. Ziv, M. Shabat-Simon, R. Gersner, A. Zangen, M. Schwartz. Vaccination as a novel approach for treating depressive behavior. Biol. Psychiatry. 2009;65:283-288 Crossref
- Liu et al., 2012 H. Liu, S.A. Shiryaev, A.V. Chernov, Y. Kim, I. Shubayev, A.G. Remacle, S. Baranovskaya, V.S. Golubkov, A.Y. Strongin, V.I. Shubayev. Immunodominant fragments of myelin basic protein initiate T cell-dependent pain. J. Neuroinflammation. 2012;9:119
- Liu et al., 2013 C. Liu, Y. Li, J. Yu, L. Feng, S. Hou, Y. Liu, M. Guo, Y. Xie, J. Meng, H. Zhang, B. Xiao, C. Ma. Targeting the shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil. PLoS One. 2013;8:e54841 Crossref
- Loggia et al., 2015 M.L. Loggia, D.B. Chonde, O. Akeju, G. Arabasz, C. Catana, R.R. Edwards, E. Hill, S. Hsu, D. Izquierdo-Garcia, R.R. Ji, M. Riley, A.D. Wasan, N.R. Zurcher, D.S. Albrecht, M.G. Vangel, B.R. Rosen, V. Napadow, J.M. Hooker. Evidence for brain glial activation in chronic pain patients. Brain. 2015;138:604-615
- Lu et al., 2012 J. Lu, M. Kurejova, L.N. Wirotanseng, R.A. Linker, R. Kuner, A. Tappe-Theodor. Pain in experimental autoimmune encephalitis: a comparative study between different mouse models. J. Neuroinflammation. 2012;9:233 Crossref
- Mantzourani et al., 2006 E.D. Mantzourani, T.V. Tselios, S.G. Grdadolnik, J.A. Platts, A. Brancale, G.N. Deraos, J.M. Matsoukas, T.M. Mavromoustakos. Comparison of proposed putative active conformations of myelin basic protein epitope 87–99 linear altered peptide ligands by spectroscopic and modelling studies: the role of positions 91 and 96 in T-cell receptor activation. J. Med. Chem.. 2006;49:6683-6691 Crossref
- Martin et al., 1991 R. Martin, M.D. Howell, D. Jaraquemada, M. Flerlage, J. Richert, S. Brostoff, E.O. Long, D.E. Mcfarlin, H.F. Mcfarland. A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J. Exp. Med.. 1991;173:19-24 Crossref
- Martinez et al., 2013 V. Martinez, B. Szekely, J. Lemarié, F. Martin, M. Gentili, S. Ben Ammar, J.F. Lepeintre, C. Garreau De Loubresse, M. Chauvin, D. Bouhassira, D. Fletcher. The efficacy of a glial inhibitor, minocycline, for preventing persistent pain after lumbar discectomy: a randomized, double-blind, controlled study. Pain. 2013;154:1197-1203 Crossref
- Matsoukas et al., 2005 J. Matsoukas, V. Apostolopoulos, H. Kalbacher, A.-M. Papini, T. Tselios, K. Chatzantoni, T. Biagioli, F. Lolli, S. Deraos, P. Papathanassopoulos, A. Troganis, E. Mantzourani, T. Mavromoustakos, A. Mouzaki. Design and synthesis of a novel potent myelin basic protein epitope 87–99 cyclic analogue: enhanced stability and biological properties of mimics render them a potentially new class of immunomodulators †. J. Med. Chem.. 2005;48:1470-1480 Crossref
- Mika et al., 2009 J. Mika, M. Osikowicz, E. Rojewska, M. Korostynski, A. Wawrzczak-Bargiela, R. Przewlocki, B. Przewlocka. Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain. Eur. J. Pharmacol.. 2009;623:65-72 Crossref
- Moalem and Tracey, 2006 G. Moalem, D.J. Tracey. Immune and inflammatory mechanisms in neuropathic pain. Brain Res. Rev.. 2006;51:240-264 Crossref
- Moalem et al., 2004 G. Moalem, K. Xu, L. Yu. T lymphocytes play a role in neuropathic pain following peripheral nerve injury in rats. Neuroscience. 2004;129:767-777 Crossref
- Moalem-Taylor et al., 2007 G. Moalem-Taylor, H.N. Allbutt, M.D. Iordanova, D.J. Tracey. Pain hypersensitivity in rats with experimental autoimmune neuritis, an animal model of human inflammatory demyelinating neuropathy. Brain Behav. Immun.. 2007;21:699-710 Crossref
- Moalem-Taylor et al., 2011 G. Moalem-Taylor, M. Li, H. Allbutt, A. Wu, D. Tracey. A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent neuropathic injury. Mol. Pain. 2011;7:1 Crossref
- Moulin et al., 1988 D.E. Moulin, K.M. Foley, G.C. Ebers. Pain syndromes in multiple sclerosis. Neurology. 1988;38:1830-1834
- Munglani et al., 1995 R. Munglani, A. Bond, G.D. Smith, S.M. Harrison, P.J. Elliot, P.J. Birch, S.P. Hunt. Changes in neuronal markers in a mononeuropathic rat model relationship between neuropeptide Y, pre-emptive drug treatment and long-term mechanical hyperalgesia. Pain. 1995;63:21-31 Crossref
- Nel and Slaughter, 2002 A.E. Nel, N. Slaughter. T-cell activation through the antigen receptor. Part 2: role of signaling cascades in T-cell differentiation, anergy, immune senescence, and development of immunotherapy. J. Allergy Clin. Immunol.. 2002;109:901-915 Crossref
- Nicholson et al., 1995 L.B. Nicholson, J.M. Greer, R.A. Sobel, M.B. Lees, V.K. Kuchroo. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity. 1995;3:397-405 Crossref
- Nicholson et al., 1997 L.B. Nicholson, A. Murtaza, B.P. Hafler, A. Sette, V.K. Kuchroo. A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens. Proc. Natl. Acad. Sci. U. S. A.. 1997;94:9279-9284 Crossref
- Olechowski et al., 2009 C.J. Olechowski, J.J. Truong, B.J. Kerr. Neuropathic pain behaviours in a chronic-relapsing model of experimental autoimmune encephalomyelitis (EAE). Pain. 2009;141:156-164 Crossref
- Ossipov et al., 2000 M.H. Ossipov, J. Lai, T.P. Malan, F. Porreca. Spinal and supraspinal mechanisms of neuropathic pain. Ann. N. Y. Acad. Sci.. 2000;909:12-24
- Perera et al., 2015 C.J. Perera, S.S. Duffy, J.G. Lees, C.F. Kim, B. Cameron, V. Apostolopoulos, G. Moalem-Taylor. Active immunization with myelin-derived altered peptide ligand reduces mechanical pain hypersensitivity following peripheral nerve injury. J. Neuroinflammation. 2015;12:28
- Popovich et al., 1993 P.G. Popovich, W.J. Streit, B.T. Stokes. Differential expression of MHC class II antigen in the contused rat spinal cord. J. Neurotrauma. 1993;10:37-46 Crossref
- Rodriguez-Barrera et al., 2013 R. Rodriguez-Barrera, A.M. Fernandez-Presas, E. Garcia, A. Flores-Romero, S. Martinon, V.Y. Gonzalez-Puertos, H. Mestre, C. Flores-Dominguez, V. Rodriguez-Mata, M. Konigsberg, S. Solano, A. Ibarra. Immunization with a neural-derived peptide protects the spinal cord from apoptosis after traumatic injury. Biomed. Res. Int.. 2013;2013:827517
- Ruiz et al., 1999 P.J. Ruiz, H. Garren, D.L. Hirschberg, A.M. Langer-Gould, M. Levite, M.V. Karpuj, S. Southwood, A. Sette, P. Conlon, L. Steinman. Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis. J. Exp. Med.. 1999;189:1275-1284 Crossref
- Santambrogio et al., 1998 L. Santambrogio, M.B. Lees, R.A. Sobel. Altered peptide ligand modulation of experimental allergic encephalomyelitis: immune responses within the CNS. J. Neuroimmunol.. 1998;81:1-13 Crossref
- Schreiner et al., 2009 B. Schreiner, F.L. Heppner, B. Becher. Modeling multiple sclerosis in laboratory animals. Semin. Immunopathol.. 2009;31:479-495 Crossref
- Sloan-Lancaster and Allen, 1996 J. Sloan-Lancaster, P.M. Allen. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol.. 1996;14:1-27 Crossref
- Sloan-Lancaster et al., 1994 J. Sloan-Lancaster, B.D. Evavold, P.M. Allen. Th2 cell clonal anergy as a consequence of partial activation. J. Exp. Med.. 1994;180:1195-1205 Crossref
- Steinman, 1996 L. Steinman. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell. 1996;85:299-302
- Sweitzer and Deleo, 2002 S.M. Sweitzer, J.A. Deleo. The active metabolite of leflunomide, an immunosuppressive agent, reduces mechanical sensitivity in a rat mononeuropathy model. J. Pain. 2002;3:360-368 Crossref
- Sweitzer et al., 2002 S.M. Sweitzer, K.A. White, C. Dutta, J.A. Deleo. The differential role of spinal MHC class II and cellular adhesion molecules in peripheral inflammatory versus neuropathic pain in rodents. J. Neuroimmunol.. 2002;125:82-93 Crossref
- Tanga et al., 2005 F.Y. Tanga, N. Nutile-Mcmenemy, J.A. Deleo. The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy. Proc. Natl. Acad. Sci. U. S. A.. 2005;102:5856-5861 Crossref
- Thacker et al., 2009 M.A. Thacker, A.K. Clark, T. Bishop, J. Grist, P.K. Yip, L.D. Moon, S.W. Thompson, F. Marchand, S.B. Mcmahon. CCL2 is a key mediator of microglia activation in neuropathic pain states. Eur. J. Pain. 2009;13:263-272 Crossref
- Thibault et al., 2011 K. Thibault, B. Calvino, S. Pezet. Characterisation of sensory abnormalities observed in an animal model of multiple sclerosis: a behavioural and pharmacological study. Eur. J. Pain. 2011;15:231.e1-231.e16 Crossref
- Tian et al., 2013a D.H. Tian, C.J. Perera, V. Apostolopoulos, G. Moalem-Taylor. Effects of vaccination with altered peptide ligand on chronic pain in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Front. Neurol.. 2013;4:168
- Tian et al., 2013b D.H. Tian, C.J. Perera, G. Moalem-Taylor. Neuropathic pain in animal models of nervous system autoimmune diseases. Mediat. Inflamm.. 2013;2013:298326
- Tselios et al., 2001 T. Tselios, V. Apostolopoulos, I. Daliani, S. Deraos, S. Grdadolnik, T. Mavromoustakos, M. Melachrinou, S. Thymianou, L. Probert, A. Mouzaki, J. Matsoukas. Antagonistic effects of human cyclic MBP87-99 altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation. J. Med. Chem.. 2001;45:275-283
- Tseveleki et al., 2015 V. Tseveleki, T. Tselios, I. Kanistras, O. Koutsoni, M. Karamita, S.S. Vamvakas, V. Apostolopoulos, E. Dotsika, J. Matsoukas, H. Lassmann, L. Probert. Mannan-conjugated myelin peptides prime non-pathogenic Th1 and Th17 cells and ameliorate experimental autoimmune encephalomyelitis. Exp. Neurol.. 2015;267:254-267
- Tsuda et al., 2003 M. Tsuda, Y. Shigemoto-Mogami, S. Koizumi, A. Mizokoshi, S. Kohsaka, M.W. Salter, K. Inoue. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature. 2003;424:778-783 Crossref
- Tsuda et al., 2009 M. Tsuda, K. Kuboyama, T. Inoue, K. Nagata, H. Tozaki-Saitoh, K. Inoue. Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays. Mol. Pain. 2009;5:28 Crossref
- Wallace et al., 2003 V.C. Wallace, D.F. Cottrell, P.J. Brophy, S.M. Fleetwood-Walker. Focal lysolecithin-induced demyelination of peripheral afferents results in neuropathic pain behavior that is attenuated by cannabinoids. J. Neurosci.. 2003;23:3221-3233
- Younger and Mackey, 2009 J. Younger, S. Mackey. Fibromyalgia symptoms are reduced by low-dose naltrexone: a pilot study. Pain Med.. 2009;10:663-672 Crossref
- Zheng et al., 2011 F.Y. Zheng, W.H. Xiao, G.J. Bennett. The response of spinal microglia to chemotherapy-evoked painful peripheral neuropathies is distinct from that evoked by traumatic nerve injuries. Neuroscience. 2011;176:447-454 Crossref
a School of Medical Sciences, UNSW Medicine, UNSW Australia, Sydney, NSW 2052, Australia
b Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, Melbourne, VIC Australia
⁎ Corresponding author at: Neuropathic Pain Research Group, Translational Neuroscience Facility, School of Medical Sciences, Wallace Wurth Building, Level 3, Room 355B, The University of New South Wales, UNSW Sydney, NSW 2052, Australia.
© 2015 Elsevier B.V., All rights reserved.