Active immunization with myelin-derived altered peptide ligand reduces mechanical pain hypersensitivity following peripheral nerve injury

Abstract

Background: T cells have been implicated in neuropathic pain that is caused by peripheral nerve injury. Immunogenic myelin basic protein (MBP) peptides have been shown to initiate mechanical allodynia in a T cell-dependent manner. Antagonistic altered peptide ligands (APLs) are peptides with substitutions in amino acid residues at T cell receptor contact sites and can inhibit T cell function and modulate inflammatory responses. In the present study, we studied the effects of immunization with MBP-derived APL on pain behavior and neuroinflammation in an animal model of peripheral nerve injury.

Methods: Lewis rats were immunized subcutaneously at the base of the tail with either a weakly encephalitogenic peptide of MBP (cyclo-MBP87-99) or APL (cyclo-(87-99)[A(91),A(96)]MBP87-99) in complete Freund's adjuvant (CFA) or CFA only (control), following chronic constriction injury (CCI) of the left sciatic nerve. Pain hypersensitivity was tested by measurements of paw withdrawal threshold to mechanical stimuli, regulatory T cells in spleen and lymph nodes were analyzed by flow cytometry, and immune cell infiltration into the nervous system was assessed by immunohistochemistry (days 10 and 30 post-CCI). Cytokines were measured in serum and nervous tissue of nerve-injured rats (day 10 post-CCI).

Results: Rats immunized with the APL cyclo-(87-99)[A(91),A(96)]MBP87-99 had significantly reduced mechanical pain hypersensitivity in the ipsilateral hindpaw compared to cyclo-MBP87-99-treated and control rats. This was associated with significantly decreased infiltration of T cells and ED1+ macrophages in the injured nerve of APL-treated animals. The percentage of anti-inflammatory (M2) macrophages was significantly upregulated in the APL-treated rats on day 30 post-CCI. Compared to the control rats, microglial activation in the ipsilateral lumbar spinal cord was significantly increased in the MBP-treated rats, but was not altered in the rats immunized with the MBP-derived APL. In addition, immunization with the APL significantly increased splenic regulatory T cells. Several cytokines were significantly altered after CCI, but no significant difference was observed between the APL-treated and control rats.

Conclusions: These results suggest that immune deviation by active immunization with a non-encephalitogenic MBP-derived APL mediates an analgesic effect in animals with peripheral nerve injury. Thus, T cell immunomodulation warrants further investigation as a possible therapeutic strategy for the treatment of peripheral neuropathic pain.

Figure 1

A scheme of the experimental paradigm. Male Lewis rats underwent CCI of the left sciatic nerve or sham operation on day 0 followed by subcutaneous immunization at the base of the tail. They were tested for pain hypersensitivity for 4 weeks (n = 6/group). On 10 and 30 days post-CCI, sciatic nerves (SNs), DRGs, and spinal cords (SCs) were collected for immunohistochemistry (n = 4/group) and spleens and lymph nodes for flow cytometry. For cytokine assay, SNs, DRGs, SCs, and serum were isolated from nerve-injured rats at 10 days post-CCI and from naïve rats (n = 6 to 7/group).

Figure 2

Immunization with cyclo-(87-99)[A ⁹¹ ,A ⁹⁶ ]MBP _87-99 significantly decreases mechanical pain hypersensitivity following CCI. (A-D) Time course of mechanical withdrawal threshold of the hindpaws (in grams) in nerve-injured and sham-operated rats treated with MBP_87-99 (native peptide), APL cyclo-(87-99)[A⁹¹ _,A⁹⁶]MBP_87-99 and CFA only (vehicle control) in (A) the ipsilateral hindpaw and (B) the contralateral hindpaw of the rats with CCI and (C) ipsilateral hindpaw and (D) contralateral hindpaw of the sham-operated rats. +(P < 0.05), ++(P < 0.01), and ⁺⁺⁺(P < 0.001) represent significant differences between the MBP- and APL-treated rats, *(P < 0.05), **(P < 0.01), ***(P < 0.001), and ****(P < 0.0001) indicate significant differences between the APL-treated and control rats. ^#(P < 0.05) and ^##(P < 0.01) represent significant differences between MBP-treated and control rats (n = 6 per group; mean ± SEM; two-way RM-ANOVA with Tukey’s multiple comparison test). Arrows indicate the time point of CCI/sham and immunization.

Figure 3

Immunohistochemistry of T cells in the sciatic nerve, DRGs, and spinal cord. The number of TCRαβ immunoreactive cells at the site of injury, proximal to the injury and distal to the injury in the left sciatic nerve (LSN) and uninjured right sciatic nerve (RSN) at 10 days (A) and 30 days (B) post-CCI. The number of TCRαβ immunoreactive cells in the L4 and L5 ipsilateral DRG (L-DRG) and contralateral DRG (R-DRG) at 10 days (C) and 30 days (D) post-CCI. The number of TCRαβ immunoreactive cells in the ipsilateral (left) and contralateral (right) lumbar spinal cord at 10 days (E) and 30 days (F) post-CCI. ^## P < 0.01 represents a significant difference between the APL-treated and control rats at the site of injury at 10 days post-CCI in the LSN. ****P < 0.0001 denotes significant differences between MBP-treated rats as compared to the APL-treated and control rats at 30 days post-CCI at the site of injury (n = 4 per group; mean ± SEM; two-way ANOVA with Tukey’s multiple comparison test). Representative images of (G) T cells in the uninjured nerve, (H) T cells in control, (I) T cells in MBP-treated and (J) T cells in APL-treated LSN at the site of injury. Scale bar = 50 μm.

Figure 4

Immunohistochemistry of ED1+ macrophages in the sciatic nerve. The average of ED1+ area density at the site of injury, proximal to the injury and distal to the injury in the left sciatic nerve (LSN) and right uninjured nerve (RSN) at 10 days (A) and 30 days (B) post-CCI. *P < 0.05 represents significant difference between APL-treated and control rats at the site of injury and ⁺ P < 0.05 between MBP- and APL-treated rats distal to the injury at 10 days post-CCI. At 30 days post-CCI, *P < 0.05 represents significant difference between the APL and control group and ⁺ P < 0.05 between MBP and APL group proximal to the injury. ****P < 0.0001 represents significant differences between the APL and control group, ⁺⁺⁺⁺ P < 0.0001 between MBP and APL group at the site of injury and distal to the injury; (n = 4 per group; mean ± SEM; two-way ANOVA with Tukey’s multiple comparison test). Representative images of (C) ED1+ cells in uninjured RSN, (D) ED1+ cells in CFA-treated control, (E) ED1+ cells in MBP-treated, and (F) ED1+ cells in the APL-treated LSN at the site of injury. Scale bar = 50 μm.

Figure 5

Immunohistochemistry of M1- and M2-like macrophages in the injured nerve. Representative immunofluoroscent images of triple labeled cells for (A) ED1+ (green) iNOS+ (red) DAPI (blue) and (B) Iba-1+ (red) Arginase-1+ (green) DAPI (blue) cells at the site of injury in the LSN. Scale bar = 100 μm.

Figure 6

Analysis of M1 and M2-like macrophages in the injured nerve. The percentage of (A) ED1+ and iNOS+ cells (M1 macrophages) and (B) Iba-1+ and Arginase + cells (M2 macrophages) at 10 and 30 days post-CCI in the LSN. *P < 0.05 represents a significant difference between APL-treated and control rats (n = 3 per group; mean ± SEM; two-way ANOVA with Tukey’s multiple comparison test).

Figure 7

Immunohistochemistry of microglia and astrocytes in the lumbar spinal cord following CCI and immunization. The percentage of Iba-1+ area density in the dorsal and ventral lumbar spinal cord at 10 days (A) and at 30 days (B) post-CCI and the percentage of GFAP+ area density in the dorsal and ventral lumbar spinal cord at 10 days (C) and at 30 days (D) post-CCI. Representative images of Iba-1+ cells in the spinal cord of the control (E), MBP-treated (F), APL-treated (G), and GFAP+ cells in the spinal cord of the control (H), MBP-treated (I), and APL-treated (J) rats. ⁺ P < 0.05 and ⁺⁺ P < 0.01 represent significant differences between the MBP-treated and control rats, and **P < 0.01 and ****P < 0.0001 indicate significant differences between MBP- and APL-treated rats at 30 days post-CCI (n = 4 per group; mean ± SEM; two-way ANOVA with Tukey’s multiple comparison test).

Figure 8

Flow cytometry analysis of CD4+ CD25+ FoxP3+ expressing Treg cells in the spleen and lymph nodes following CCI and immunization. (A) Representative forward scatter (FSC) vs. side scatter (SSC) plot showing the acquired events and lymphocyte gate from rat spleen. (B) Representative histogram of counts vs. CD4+ cell population selected for further gating. (C) Representative dot plots of Treg cells in the spleen of the control (left), MBP-treated (middle), and APL-treated (right) rats at 10 days post-CCI. (D) The percentage of CD4+ CD25+ FoxP3+ cells in the spleen and lymph nodes at 10 and 30 days post-CCI. **P < 0.01 indicates significant differences between APL-treated and MBP-treated and control rats at 10 days post-CCI (n = 4 per group; mean ± SEM; two-way ANOVA with Tukey’s multiple comparison test).

Figure 9

Bio-plex analysis of cytokine expression profile in the nervous tissue and serum at 10 days post-CCI. Heat maps illustrating the summary of fold change expression of cytokines in the (A) LSN, (B) left DRG, (C) spinal cord, and (D) serum of immunized nerve-injured rats compared to control uninjured naïve rats at 10 days post-CCI. Yellow asterisks indicate significant differences in cytokine concentrations in treatment groups as compared to the uninjured control group (n ≥ 4 per group; one-way ANOVA with Dunnet’s multiple comparison test compared to the uninjured group).

Figure 10

IL-1α and IL-β profile in the injured sciatic nerve at 10 days post-CCI. The concentrations of IL-1α (A) and IL-1β (B) in the LSN of immunized nerve-injured rats at 10 days post-CCI. *P ≤ 0.05 indicates a significant difference between MBP- and APL-treated rats (n = 6 per group; mean ± SEM; one-way ANOVA with Dunnet’s multiple comparison test compared to the APL-treated group).