Headache disorders, including chronic migraine, post-traumatic headache (PTH), and medication overuse headache (MOH), are highly prevalent and debilitating.20,40,57 A substantial proportion of patients remains either unresponsive to current treatment options or intolerant to their side effects. There is an urgent need to understand the disease mechanisms and to develop safer and more effective therapies with mechanisms of action distinct from the existing approaches.
Activation of many proinflammatory immune cells, including meningeal mast cells, macrophages, dendritic cells, and T cells, has been implicated in the pathophysiology of migraine and PTH.9,37,41,48,51 On the contrary, little is known about the involvement of immunosuppressive regulatory T (Treg) cells in headache disorders, let alone their potential as a therapeutic target. Tregs are a specialized subpopulation of CD4+ T cells that express high-level of transcription factor Foxp3 and the high-affinity interleukin-2 (IL2) receptor CD25.52 They possess far-ranging suppressive activity affecting the function of all types of immune cells with multiple mechanisms, therefore are widely implicated in the maintenance of immune homeostasis.52 Many clinical trials use repeated low-dose interleukin-2 (ld-IL2) treatment to selectively expand and activate Treg cells in vivo without activating effector T cells regardless of the disease background.30,49 Ld-IL2 is well tolerated in patients and shows indications of clinical efficacy.49,54
Chronic pain-associated Treg changes have been studied in mouse models of nerve injury and experimental autoimmune encephalomyelitis. Nerve injury reduces Treg cells in mouse spleen25 but increases Treg cell number in the injured nerve as well as the ipsilateral draining lymph node (LN), dorsal root ganglia (DRG), and dorsal horn of the spinal cord.36 Experimental autoimmune encephalomyelitis also results in Treg increases in the brain, spinal cord, LN, and spleen.21 In both disease models, cutaneous mechanical allodynia is exacerbated by the depletion of Treg cells and is attenuated by the increase in Treg cell number.4,21,25,36,38
In this study, we conducted the first preclinical study to test the hypothesis that ld-IL2 treatment may prevent and/or reverse the chronification of migraine and other headache disorders. Repeated injections of nitroglycerin (NTG, a nitric oxide [NO] donor and a reliable trigger of migraine in patients) resulted in a 50% reduction of the ratio of Treg cells among CD3+ T cells in mouse trigeminal ganglia (TG), suggesting that chronic migraine is associated with a deficiency in Treg-mediated immune homeostasis. Ld-IL2 treatment not only completely reversed NTG-induced facial skin hypersensitivity but also blocked the effects of subsequent NTG administrations through endogenous Treg cells. Importantly, ld-IL2 did not alter basal nociceptive responses or induce the development of tolerance. Ld-IL2 also effectively reversed the behavioral sensitization related to MOH and prevented the development of both acute and persistent PTH-related behaviors in a mouse model of mild traumatic brain injury (mTBI). Collectively, this study identifies Treg cell as a promising target for treating chronic migraine. Our results support the potential of ld-IL2 as a safe and effective treatment for multiple headache disorders with a mechanism of action distinct from the existing treatment approaches.
2. Materials and methods
All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis. To avoid social isolation stress, all mice were group housed (2-5 per cage, same sex) in the animal facility of Washington University in St. Louis on a 12-hour light–dark cycle with constant temperature (23-24°C), humidity (45%-50%), and food and water ad libitum. All experiments were performed during the light phase (9 am-4 pm). Adult mice (8-14 weeks old, both male and female) were used in the experiments. Adult Swiss Webster mice were purchased from Charles River. Adult C57BL/6J mice and the breeders of DEREG (depletion of regulatory T cell, 32050-JAX) mice were purchased from the Jackson Laboratory. The DEREG mice contain a transgenic allele that expresses the diphtheria toxin receptor–enhanced green fluorescent protein (DTR-EGFP) fusion protein under the control of the genomic sequences that regulate the expression of endogenous foxp3.35 Heterozygous DEREG mice were generated by crossing the DEREG breeder with C57BL/6J mice. The genotype was determined by polymerase chain reaction of tail DNA. The DTR-EGFP transgenic allele was amplified with the forward primer (5′-CCTACGGCGTGCAGTGCTTCAGCCGC-3′) and the reverse primer (5′-CGGCGAGCTGCACGCTGCCGTCCTC-3′), producing a 300-bp fragment. The polymerase chain reaction conditions were 96°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 33 cycles. Of note, DEREG mice were used as a reporter line for Foxp3+ Treg cells, not for depletion of Treg cells in this study.
2.2. Mouse models of headache disorders
2.2.1. Mouse model of chronic migraine and drug treatments
After measuring baseline nociceptive responses, mice received repetitive intraperitoneal (i.p.) injections of NTG (10 mg/kg in saline with 1% propylene glycol) or vehicle (saline with 1% propylene glycol in saline, 10 mL/kg) every 2 days for 4 or more times as described previously.47 Nocifensive behaviors were measured 2 days after each injection (before the next treatment). NTG (SDM27; Copperhead Chemical, Tamaqua, PA) was freshly diluted from the stock (10% in propylene glycol, aliquoted in airtight glass vials and stored at 4°C) with saline for every injection.
Recombinant mouse IL2 (carrier-free; Biolegend, San Diego, CA) was freshly diluted from the stock (1 mg/mL aliquots at −80°C) every day. Each mouse received daily i.p. injection of 1-µg IL2 in 100-µL saline. The control mice received daily i.p. injections of 100-µL saline. In Treg depletion experiment, mice received 1 i.p. injection of antibodies against CD25 (500 µg/mouse, BP0012; BioXcell, West Lebanon, NH11) or control IgG (BP0088; BioXcell). The antibody binds to CD25 on Treg cell membrane and depletes Treg cells through Fcγ receptor III–mediated antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis.53 Note that on the days that the mouse behaviors were tested, NTG, IL2 and/or antibodies were always injected after completing the behavioral tests.
2.2.2. Mouse model of mild traumatic brain injury–induced post-traumatic headache
Adult Swiss Webster mice (30-35 g) were subjected to mTBI using a modified closed head weight-drop method.43,58 Briefly, mice were anesthetized with 3% isoflurane for 90 seconds and were placed chest down on a foam sponge (4-cm thickness and 0.062-g/cm3 density) directly underneath a hollow cylindrical tube (1.5-cm inner diameter) placed approximately 1 cm vertically over the mouse's head. To induce mTBI, a 30-g weight (1.3-cm diameter and 3.4-cm height) was dropped through the tube from a height of 80 cm, striking the center point between the ears once. All mice regained righting reflex within 2 minutes. No mortality, skull fracture, or motor deficit was observed in any mice. Sham mice were anesthetized with 3% isoflurane for 90 seconds but not subjected to the weight drop. Facial mechanical thresholds were measured before and at various time points after mTBI. Daily ld-IL2 or saline were administered 1 or 7 days after the sham or mTBI procedure. To reveal mTBI-induced hyperalgesia priming, mice received daily i.p. injections of NTG (0.1 mg/kg, i.p.) for 4 to 6 days, starting at 35 days after mTBI. Facial mechanical thresholds were measured between day 35 and 43 days after mTBI. Note that on the days that the mouse behaviors were tested, NTG and/or IL2 were always injected after completing the behavioral tests.
2.2.3. Mouse model of medication overuse headache
After measuring baseline nociceptive responses, mice received daily i.p. injections of sumatriptan succinate (0.6 mg/kg; Fresenius Kabi, Lake Zurich, IL44). Daily ld-IL2 or saline were administered after mice received 2 sumatriptan injections. Facial and hindpaw mechanical thresholds were measured every 2 days. Note that on the days that the mouse behaviors were tested, sumatriptan and/or IL2 were always injected after completing the behavioral tests.
2.3. Behavioral tests
Mice were extensively handled by the experimenters for 2 weeks and were well-habituated to the test room and the test apparatus before each experiment. The experimenters were blinded to the treatments mice received during data collection and analysis.
2.3.1. Open-field test
Mice were habituated in the testing room for 1 hour in their home cages and then tested one at a time. Each mouse was placed in the center of the dimly illuminated, sound-attenuated VersaMax Open Field box (42 × 42 cm; AccuScan Instruments, Columbus, OH) for 1 hour while the experimenter left the room. Horizontal movement and center entries (14 × 14 cm) were recorded and analyzed by the VersaMax software.
2.3.2. Rotarod test
Motor coordination was assessed with the rotarod test 6 days after mTBI. Male mice were habituated in the testing room for 1 hour in their home cages and then underwent 5 training sessions on the Rotarod (Model 7650; Ugo Basile, Trappe, PA) at 4 revolutions/min (rpm) for 5 minutes. Mice that stayed on the rod for at least 2 minutes/session were tested on the accelerating rod (4-40 rpm over 5.5 minutes). The latency to fall from the rod was averaged over 3 trials in individual mice.
2.3.3. Adhesive removal test
We used the adhesive removal test to assess mTBI-induced sensory and motor deficits related to the paw and the mouth.7 The mouse was habituated in a testing box (28.5 × 17.5 × 12 cm) for 60 seconds. Two pieces of adhesive tape strips (0.3 × 0.4 cm; Topcare, Elk Grove Village, IL) were applied with equal pressure on each forepaw to cover the glabrous skin. Tape removal time was defined as the time between the mouse's first reaction to the presence of the tape, either by shaking its paw or bringing its paw to its mouth, and the complete removal of the adhesive strip from the forepaw. Male mice underwent 3 training sessions on post-mTBI day 11 and 2 test sessions the next day. The tape removal times of the left and right forepaws from the 2 test sessions were averaged in individual mice.
2.3.4. Responses to mechanical stimuli on the hindpaw
Mice were habituated in individual clear plexiglass boxes (11 × 11 × 15 cm) for 1 to 2 hours. A series of calibrated von Frey filaments was used to apply mechanical stimuli to the plantar surface of the hindpaws. We used the up–down paradigm to determine the 50% withdrawal threshold.12 The thresholds for both hindpaws were averaged to yield a single value for each mouse.
2.3.5. Cheek acetone test
The acute nocifensive response to acetone-evoked evaporative cooling on mouse cheek was measured as previously described.14 The day before testing, both cheeks were shaved (6.5 × 12 mm) under brief anesthesia (2% isoflurane in 100% oxygen). On the test day, mice were habituated in individual plexiglass cylinders (11-cm diameter, 15-cm height) situated in front of 3-way mirrors for 1 to 2 hours. We applied acetone (12 µL) to the shaved cheeks and immediately returned mice to the cylinders. Time spent wiping the treated area was recorded by a video camera and quantified offline. For individual mice, acetone was applied alternatingly to both cheeks at >10-minute intervals, and the duration of behavior was averaged from 4 applications.
2.3.6. Withdrawal responses to facial mechanical stimuli
The hair on mouse forehead (above and between 2 eyes) was shaved the day before testing. On the test day, the experimenter gently held the mouse on the palm with minimal restraint and applied the calibrated von Frey filament perpendicularly to the shaved skin, causing the filament to bend for 5 seconds. A positive response was determined by the following criteria as previously described: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking.22 The up–down paradigm was used to determine the 50% withdrawal threshold.12
2.4. Blood, spleen, and cervical lymph node cell preparation, antibody staining, flow cytometry, and enzyme-linked immunosorbent assay (ELISA)
Adult male DEREG and C57BL/6J mice received 15 daily injections of saline or IL2. About 100-µL blood was collected from each mouse by submandibular bleeding. Mice used for blood collection were not used in the behavioral tests. Spleen and cervical LN tissues were ground and filtered through a sterile 70-µm cell strainer. After lysis of red blood cells (420301; Biolegend), cells were pelleted and resuspended for antibody staining, ELISA, and ELISPOT assay. Live cells were counted by the Vi-CELL Automated Cell Viability Analyzer (Beckman, Chaska, MN).
About 2 × 106 suspension cells were stained with the following antibodies from Biolegend that recognizes mouse CD3ε (clone 145-2C11), CD4 (clone GK1.5 and RM4-5), CD8 (clone 53-6.7), and CD25 (clone PC61). The percentages of individual cell phenotypes were determined using flow cytometric analysis. Data were collected with FACScan (Becton Dickinson, Franklin Lakes, NJ) and analyzed with CellQuest Pro (Becton Dickinson) and Rainbow X Alias (Cytek) softwares. Table 1 summarizes the markers and assays used to define T-cell subsets in this study.
To quantify interferon γ (IFNγ) secretion, splenocytes were plated in 24-well plates (5 × 106 per well) and stimulated with anti-CD3 and anti-CD28 antibodies (1 and 5 µg/mL) in 1 mL complete RPMI1640 media overnight. IFNγ in the supernatants were quantified by ELISA (430901; Biolegend).
ELISPOT assay was performed to determine the number of INFγ-secreting cells. Splenocytes were plated at 5 × 104 cells per well and incubated overnight with RPMI1640 media containing anti-CD3 and anti-CD28 antibodies (500 and 5 µg/mL) for stimulation. IFNγ was detected using a colorimetric reagent kit (R&D SEL485 and SEL002). Following development, images were captured and analyzed on the ImmunoSpot7.0 plate reader (Cellular Technologies, Kennesaw, GA).
2.5. Isolation and adoptive transfer of Treg cells and CD25−CD4+ T cells
Adult C57BL/6J mice (8 to 12 weeks old) received daily injections of IL2 for 12 days. CD25+CD4+ Treg cells and CD25−CD4+ cells were isolated from splenocytes through CD4+ T-cell–negative selection followed by a CD25+ T-cell–positive selection using EasySep mouse CD4+ T-cell pre-enrichment and CD25-positive selection kits (18783; Stem Cell Technologies, Cambridge, MA).
Adult male C57BL/6J mice (8-14 weeks old) received i.p. injections of 10-mg/kg NTG every 2 days. One day after the second NTG injection, mice were injected with 1 × 106 Treg or CD25−CD4+ cells through the tail vein. The 50% withdrawal threshold to facial mechanical stimuli was measured before the first NTG injection and 2 days after each NTG injections.
2.6. Tissue preparation, immunohistochemistry, and image analysis
Mice were euthanized with i.p. injection of barbiturate (200 mg/kg) and were transcardially perfused with warm 0.1 M phosphate-buffered saline (pH 7.2) followed by cold 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2) for fixation. Trigeminal ganglia, lumbar L4 DRG, and the tissues containing the cervical/medullary dorsal horn (from obex to C3 cervical spinal cord) were collected and sectioned at 15 µm in the transverse plane, collected on Superfrost Plus glass slides in sequence and stored at −20°C.
One in every 4 TG, DRG or cervical/medullary dorsal horn sections were processed for each immunohistochemistry experiment as described previously.27 The dura was carefully dissected from the skull using forceps and stained as whole mount. T cells were identified by the rat anti-CD3 antibody (clone 17A2, 1:200; eBioscience, San Diego, CA), and Treg cells were identified with the chicken anti-EGFP (1:1000; AVES Lab, Tigard, OR) antibody in tissues from DEREG mice. AlexaFluor 568- or 488-conjugated secondary antibodies (Invitrogen) were used at 1:1000 dilution. Immunofluorescence was observed through a 40× objective on a Nikon TE2000S-inverted epifluorescence microscope, and images were captured with a CoolSnapHQ2 camera (Photometrics, Tucson, AZ). To quantify CD3+ T cells and Treg cells on the dura, we took random, nonoverlapping images (10 per mouse) in areas adjacent to the middle meningeal artery (MMA). To quantify T cells and Treg cells in TG, DRG, and cervical/medullary dorsal horn, all cells on individual sections were counted, and the number was multiplied by 4 to obtain the total number of cells per ganglion in each mouse. Images of individual sections were captured by an Olympus NanoZoomer Whole-Slide Imaging System and measured with the SimplePCI software (Hamamatsu) to verify that the total areas of the sections quantified were comparable between individual mice. Representative images were adjusted for contrast and brightness using the same parameter within individual experiments. No other manipulations were made to the images. Image analysis was performed with experimenters blinded to the experimental groups.
2.7. Statistical analysis
For behavioral experiments, power analysis was conducted to estimate sample size with >80% power to reach a significance level of 0.05. The experimenters were blinded to the treatments mice received. For flow cytometry, ELISA and immunohistochemistry experiments, sample sizes were estimated based on our previous experience.
All data are reported as mean ± SE of the mean. The Shapiro–Wilk test was used to check data normality. Statistical significance between experimental groups with normally distributed data was assessed by two-tailed t test, analysis of variance (1-way or 2-way, with or without repeated measures) with the post hoc Bonferroni test where appropriate, using Origin and Statistica softwares (from OriginLab and StatSoft, respectively). The nonparametric Mann–Whitney U test, Friedman test, or Kruskal–Wallis analysis of variance on ranks with multiple comparisons (Student–Newman–Keuls method) was used to analyze the differences in the withdrawal threshold to mechanical stimuli. Differences with P < 0.05 were considered statistically significant. The statistical analysis for individual experiments was described in figure legends.
3.1. Repeated NTG treatment reduces the ratio of Treg cells to total T cells in mouse trigeminal ganglia
To mimic the high-frequency recurring headache in chronic migraine patients, we treated male and female C57BL/6J inbred mice with NTG (10 mg/kg, i.p.) or vehicle every 2 days for 5 times and measured the 50% withdrawal thresholds to von Frey filaments on the hindpaws 2 days after each injection (Fig. 1A). Compared with the baseline thresholds, repetitive NTG injections induced a progressive and sustained mechanical hypersensitivity on the hindpaw of both male and female mice (Fig. 1B), consistent with the previous study.24,47 Two days after the last NTG or vehicle injection, we stained the whole-mount dura as well as the TG and L4 DRG sections with the CD3 antibody to label all T cells. The density of CD3+ T cells in the dura surrounding the MMA or in L4 DRG was not altered by the repeated NTG treatment (Figs. 1C and D). By contrast, the abundance of CD3+ cells was nearly doubled in the TG of NTG-treated mice (Figs. 1D and E). There were on average 2176 ± 288 and 4135 ± 562 CD3+ cells per TG from vehicle- and NTG-treated mice (n = 6/group, same mice as in Fig. 1D), indicating that repeated NTG administration preferentially increases the density of total T cells in mouse TG.
Next, we asked whether repeated NTG treatment alters the number of Treg cells in mouse TG, using the DEREG transgenic mice, which specifically express the DTR-EGFP fusion protein in Treg cells.35 Flow cytometric analysis showed that EGFP signal was present in 85 ± 2% CD4+CD25+ splenocytes from adult DEREG mice (n = 4). We verified that NTG-induced hindpaw mechanical hypersensitivity was not affected by the expression of DTR-EGFP in DEREG mice (Fig. 1F inset). The total number of EGFP+ Treg cells was very low in TG and DRG from vehicle-treated mice (∼30 and 6 per ganglion, respectively), and neither was significantly altered by the repetitive NTG injections (Fig. 1F). Because TG from NTG-treated mice contained twice as many CD3+ cells than those from vehicle-treated mice, the proportion of Treg cells among total T cells in TG was significantly reduced by the repeated NTG administration. We speculate that chronic migraine is associated with a deficiency in Treg-mediated immune homeostasis.
3.2. Daily ld-IL2 prevents the development of NTG-induced skin hypersensitivity
It is well documented that ld-IL2 treatment selectively expands and activates Treg cells in both mice and humans.30,32,50 We treated male DEREG mice with daily ld-IL2 (1 µg/mouse, i.p.56) or saline for 15 days. The frequency of EGFP+ Treg cells in the peripheral blood increased by more than 100% after 5 ld-IL2 injections (Fig. 2A), similar to the magnitude of ld-IL2-induced Treg expansion reported in human clinical trials.26,49 The abundance of Treg cells in the blood was maintained by the subsequent IL2 injections (Fig. 2A). The percentage of EGFP+ Treg cells also increased significantly in the cervical LNs that receive all the lymph from the head and neck (Fig. 2B). We also used CD25 as the marker for Treg cells and saw similar ld-IL2-induced increase in the blood and LN (Figs. 2C and D). Conversely, the frequencies of CD4+ and CD3+ T cells in the blood or LNs were not altered by the ld-IL2 treatment (Figs. 2E and F and Table 1). In wild-type C57BL/6J males, 15 days of ld-IL2 treatment doubled the number and the frequency of CD25+CD4+ Treg cells in the spleen (Figs. 3A–C), whereas the number of CD8+ cells increased only slightly (Figs. 3D and E). The number of splenocytes secreting IFNγ was not altered but the amount of CD3/CD28 stimulation-induced IFNγ secretion was significantly reduced in cells from IL-2-treated mice (Figs. 3F and G, Table 1). These data confirmed that daily ld-IL2 treatment preferentially increases Treg cells in the blood, LNs, and spleen, enhancing immunosuppression in mice.
We proceeded to treat male C57BL/6J mice with daily saline or ld-IL2, starting 5 days before the first NTG injection and continuing throughout the experiment (Fig. 4A). Basal hindpaw mechanical sensitivity was not altered by either 5 or 15 days of ld-IL2 injections (Fig. 4B, vehicle + saline vs vehicle + IL2). Conversely, NTG-induced hindpaw mechanical hypersensitivity was completely blocked by the ld-IL2 pretreatment (Fig. 4B, NTG + saline vs NTG + IL2).
In addition to increasing hindpaw mechanical sensitivity, repeated NTG administration also enhances the behavioral responses to acetone-induced cooling of facial skin in mice,29 and this can be blocked by the daily treatment with topiramate, a migraine preventive drug in humans (Cloud and Cao, manuscript in preparation). After measuring baseline responses to acetone-induced cooling, we repeatedly injected female C57BL/6J mice with NTG and measured the duration of acetone-induced face wiping 2 days after each injection (Fig. 4A). Compared with the vehicle group, there was a persistent increase in the duration of acetone-induced cheek wiping in NTG-treated mice (Fig. 4C, vehicle + saline vs NTG + saline). Pretreatment with ld-IL2 did not change the basal responses to acetone-induced cooling (Fig. 4C, vehicle + saline vs vehicle + IL2) but prevented the development of NTG-induced facial cold hypersensitivity (Fig. 4C, NTG + saline vs NTG + IL2). We conclude that ld-IL2 pretreatment can effectively prevent the development of repetitive NTG-induced skin hypersensitivity without compromising the baseline responses to mechanical or cold stimuli.
As in C57BL/6J females, repetitive NTG induced a gradual increase in the duration of acetone-induced cheek wiping in the outbred female Swiss Webster mice (Fig. 4D). Here, we started daily ld-IL2 treatment after mice received 2 NTG injections, but before the facial cold hypersensitivity was fully established (Fig. 4D). The delayed ld-IL2 treatment also blocked the development of facial cold hypersensitivity (Fig. 4D).
Does ld-IL2 increase the number of Treg cells in dura and TG? After 15 days of ld-IL2 treatment, there was a 4-fold increase in the density of EGFP+ Treg cells in the dura surrounding the MMA in male DEREG mice (Figs. 5A and B), whereas the density of CD3+ T cells was not altered (∼80 cells/mm2, similar to those of Fig. 1C). Consequently, ld-IL2 increased the frequency of Treg cells among CD3+ cells from 5% to 20% (Fig. 5C). The number of EGFP+ Treg cells increased 9-fold in the TG after ld-IL2 (Figs. 5D and E), much higher than NTG-induced 2-fold increase in total CD3+ cells (Fig. 1D). Ld-IL2 treatment also resulted in a 4-fold increase in the number of EGFP+ Treg cells in DRG (Fig. 5F). These data suggest that ld-IL2 pretreatment inhibits chronic migraine-related behaviors through increasing the proportion of Treg cells among total T cells in dura, TG, and DRG.
3.3. NTG-induced persistent skin hypersensitivity is completely reversed by ld-IL2 treatment
We went on to test whether ld-IL2 can reverse the established facial skin hypersensitivity resulting from repeated exposure to NTG in mice. Previous studies indicate that repeated enhancing of NO signaling results in a persistent facial mechanical hypersensitivity that can be blocked by the migraine preventive drug propranolol.6,15 In female Swiss Webster mice, the mechanical threshold at the periorbital region was already substantially reduced 2 days after the first NTG injection (Fig. 6A, NTG + saline group, day 1 vs day 3). The facial mechanical hypersensitivity was sustained during the subsequent NTG injections (Fig. 6A, NTG + saline group, day 3-13). We started ld-IL2 treatment after mice received 2 NTG injections (Fig. 6A). Baseline facial mechanical threshold was not altered by daily ld-IL2 (Fig. 6A, vehicle + saline vs vehicle + IL2). Conversely, NTG-induced mechanical hypersensitivity started to reverse after 5 days of ld-IL2 treatment (Fig. 6A, day 9, NTG + saline vs NTG + IL2). After 7 to 9 days of IL2 injections, the facial mechanical threshold of mice in the NTG + IL2 group was not different from those in the vehicle + saline or vehicle + IL2 groups (Fig. 6A, day 11-13), indicating that ld-IL2 can completely reverse the established persistent facial mechanical hypersensitivity resulting from repeated NTG injections.
We repeated the experiment in female C57BL/6J mice. The development and maintenance of NTG-induced sensitization was similar to that seen in Swiss Webster females (Fig. 6B, NTG + saline group), and 3 injections of ld-IL2 was sufficient to completely reverse the effect of repeated NTG (Fig. 6B, NTG + IL2 group). Moreover, continuous ld-IL2 treatment prevented subsequent NTG administrations from inducing behavioral sensitization (Fig. 6B, day 7-11). We also tested whether ld-IL2 blocks NTG-induced acute reduction of facial mechanical threshold, which models the sensory hypersensitivity during a migraine episode in humans.19,23,31,39 The facial mechanical threshold of naïve C57BL/6J mice was significantly reduced 3 hours after systemic injection of NTG (10 mg/kg, i.p., Fig. 6C day 1). After NTG-induced persistent facial skin hypersensitivity was completely reversed by ld-IL2 (day 11 in Fig. 6B), a subsequent NTG injection did not cause an acute reduction of facial mechanical threshold 3 hours later (Fig. 6C, day 11), suggesting that ld-IL2 prevents the onset of migraine episodes triggered by NO signaling.
Next, we tested how long the effect of ld-IL2 lasts after the cessation of treatment and how NTG-induced facial hypersensitivity responds to the second round of ld-IL2 treatment. In male C57BL/6J mice, facial mechanical hypersensitivity was sustained by repeated NTG injections for at least 4 weeks (Fig. 6D, NTG + saline) and was completely reversed by 7 daily ld-IL2 treatments (Fig. 6D, day 4-10). After cessation of IL2, it took 2 NTG injections (day 11-13) to lower the mechanical threshold to the level comparable to that of the NTG + saline group (Fig. 6D, day 15). We then started the second round of the daily ld-IL2 treatment (Fig. 6D, day 19-22). Notably, fewer ld-IL2 injections (4 vs 7 in the first session) were required to completely reverse NTG-induced mechanical hypersensitivity. We also compared the slope of reversal (change of threshold per day) between day 5 to 11 and day 19 to 23. The slope of ld-IL2-induced reversal was significantly steeper in the second session (10.3 ± 1.3, day 19-23) than the first (5.3 ± 0.9, day 5-11, P < 0.01, two-tailed t test). Upon cessation of the second ld-IL2 treatment, it took 3 NTG injections (day 23-27) to reduce the withdrawal threshold to the level similar to that of the NTG + saline group (Fig. 6B, day 29). Taken together, we conclude that daily ld-IL2 can completely reverse the established persistent facial skin hypersensitivity resulting from repeated NTG administration, and the effect of IL2 occurs faster and lasts longer in mice with previous ld-IL2 treatment.
3.4. Treg cells mediate the therapeutic effect of ld-IL2
To identify the cellular target of ld-IL2, we treated mice with an antibody against CD25, the IL2 receptor α chain that is highly expressed in Treg cells.11 A single injection was sufficient to deplete 99% of Treg cells for 2 weeks without altering the facial mechanical thresholds at basal level or after NTG administration (Fig. 7A). By contrast, the effect of ld-IL2 was completely abolished in Treg-depleted mice (Fig. 7B). NTG-induced facial skin hypersensitivity persisted in CD25 antibody-treated mice throughout the course of ld-IL2 treatment (Fig. 7B, day 7-13), suggesting that endogenous Treg cells mediate the therapeutic effect of ld-IL2.
In addition to Treg cells, CD25 antibody may affect activated T cells in NTG-treated mice. We therefore tested whether adoptive transfer of Treg cells reverses NTG-induced skin hypersensitivity. We treated naïve C57BL/6J donor mice with ld-IL2 for 12 days, as previous work shows that Treg cells from IL2-treated mice exhibit stronger suppressive activity compared with those from saline-treated mice.56 We then enriched CD25+CD4+ Treg cells from IL2-treated mice and adoptively transferred the cells to male C57BL/6J mice that had received 2 NTG injections and exhibited profound facial skin hypersensitivity to mechanical stimuli (Fig. 7C, day 4). The day before Treg transfer, the withdrawal threshold to von Frey filaments was about 14% of the baseline value (Fig. 7C, day 3). One day after the Treg transfer, the threshold was already increased to 70% of the baseline value (Fig. 7C, day 5). The effect of transferred Treg lasted for more than 15 days (Fig. 7C, day 5-21), so that the subsequent 7 NTG injections (day 5-17) completely failed to induce either acute (Fig. 7D) or persistent (Fig. 7C, day 7-19) mechanical hypersensitivity on facial skin. The frequency of CD25+ Treg in the peripheral blood increased to 173 ± 33% of the baseline level on day 6 (n = 4 mice, P < 0.05, one-sample t test), remained at 146 ± 39% on day 18, and returned to baseline level (109 ± 41%) by day 26. In control mice that received adoptive transfer of CD25−CD4+ T cells from IL2-treated mice, repeated NTG injections induced persistent facial skin mechanical hypersensitivity throughout the experiment (Fig. 7C). Similar to ld-IL2, Treg transfer did not alter baseline nociceptive responses to heat, cold, or mechanical stimuli on facial skin or on hindpaw (Figs. 7E–I). This is consistent with the working model that ld-IL2 reverses the effects of repeated NTG administration through targeting endogenous Treg cells.
To verify the purity of the transferred Treg cells, we conducted flow cytometry analysis on the enriched CD25+CD4+ Treg cells from DEREG mouse splenocytes. The preparations consisted of 92 ± 4% live cells, 97 ± 1% CD4+ cells among live cells, 87 ± 0.4% CD25+ cells among CD4+ cells and 90 ± 1% EGFP+ cells among CD25+CD4+ cells (n = 3 preparations, 2 mice per preparation), validating that the transferred cells were enriched in Treg. We also adoptively transferred Treg cells from IL2-treated DEREG mice to C57BL/6J recipient mice and used the EGFP signal to determine the tissue distribution of transferred Treg cells. EGFP+ cells were present in the dura and TG 1 day after the adoptive transfer, and the level remained stable for at least 5 days (Fig. 8). By contrast, the cervical/medullary dorsal horn TCC of recipient mice contained few, if any, EGFP+ cells (0-2 cells/mouse) during this period, indicating that the transferred Tregs are located outside the brain while exerting their antimigraine effects.
3.5. Ld-IL2 treatment inhibits mild traumatic brain injury–induced facial mechanical hypersensitivity and hyperalgesia priming
Post-traumatic headache is a debilitating secondary headache disorder and is the most common medical consequence of mTBI.57 Acute PTH occurs within 7 days of the injury. Persistent PTH lasts longer than 3 months to years after the trauma and often takes on a pattern of daily occurrence in the most severe cases.57 We asked whether ld-IL2 can prevent and/or reverse PTH-related behaviors in a mouse model (Fig. 9A). In both male and female Swiss Webster mice, mTBI resulted in a significant reduction of the facial mechanical threshold (Fig. 9B, mTBI + saline groups, 3-28 days), which is mechanistically related to acute PTH.8,22,43 There was no motor deficit as measured by rotarod, open-field, and adhesive removal tests between 6 and 12 days after mTBI, when facial mechanical hypersensitivity reached the plateau (Fig. 10). After the mechanical thresholds returned to basal level 35 days after mTBI, we treated mice with low-dose NTG (0.1 mg/kg, i.p.) daily for 4 to 6 days to mimic high-frequency persistent PTH and measured the facial mechanical thresholds 1 day after the last NTG treatment.43 Only mice that experienced mTBI developed facial mechanical hypersensitivity to repeated low-dose NTG (Fig. 9C, mTBI + saline groups), which is mechanistically related to mTBI-induced hyperalgesia priming during persistent PTH.
First, we treated female Swiss Webster mice with daily ld-IL2, starting 1 day after mTBI for 9 days (Fig. 4A). This completely prevented the development of mTBI-induced acute facial mechanical hypersensitivity as well as the hyperalgesia priming revealed by repeated low-dose NTG (Figs. 9B and C, mTBI + IL2 groups). Next, we treated male Swiss Webster mice with daily ld-IL2 for 9 days, starting on post-mTBI day 7, when the facial mechanical threshold decreased to about 26% of the baseline value (Fig. 9D). The threshold returned to 87% of the baseline value after 3 days of ld-IL2 (Fig. 9D, day 10, mTBI + IL2 group), and mTBI-induced facial skin hypersensitivity was fully reversed by the end of ld-IL2 treatment (Fig. 9D, day 15). Daily ld-IL2 did not change the facial mechanical sensitivity of sham mice (Fig. 9D, sham + saline vs sham + IL2 groups). Importantly, 3 weeks after the cessation of ld-IL2, repeated low-dose NTG failed to establish persistent facial mechanical hypersensitivity in mice that experienced mTBI (Fig. 9E, mTBI + saline vs mTBI + IL2 groups). Collectively, these results indicate that ld-IL2 treatment after mTBI not only reverses injury-induced facial skin hypersensitivity but also prevents the development of hyperalgesia priming related to persistent PTH.
3.6. Ld-IL2 treatment reverses cutaneous mechanical hypersensitivity in a mouse model of medication overuse headache
Medication overuse headache is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headache, for example, sumatriptan.20 In both male and female C57BL/6J mice, daily administration of sumatriptan (0.6 mg/kg, i.p.) resulted in persistent facial and hindpaw mechanical hypersensitivity (Figs. 11A–D, SUMA + saline groups), consistent with previous reports.2,17,44 Ld-IL2 treatment was initiated after mice received 2 sumatriptan injections, after the establishment of mechanical hypersensitivity (Figs. 11A–D, day 3). In both male and female mice, sumatriptan-induced cutaneous hypersensitivity was significantly attenuated after 4 to 6 ld-IL2 injections (Fig. 11, day 7-9) and was completely reversed after 8 days of IL2 treatment (Fig. 11, day 11). Moreover, with the continuous ld-IL2 treatment, neither male nor female mice developed cutaneous hypersensitivity in response to subsequent sumatriptan injections (Fig. 11, day 13). We conclude that ld-IL2 treatment not only reverses sumatriptan-induced behavioral sensitization but also prevents the recurrence of sensitization by subsequent sumatriptan administrations.
Recent years have seen significant advances in elucidating the mechanisms and in developing new therapy for chronic migraine and other headache disorders. Despite these exciting progresses, many patients remain either unresponsive to available treatments or intolerant to their side effects. Although numerous studies indicate that the activation of many proinflammatory immune cells contributes to the pathophysiology of migraine and PTH,9,37,41,48,51 the involvement of the immunosuppressive Treg cell in the chronification of headache disorders remains unknown. Many preclinical studies have identified ld-IL2 as a safe and effective treatment for multiple autoimmune and neurodegenerative diseases,16,30 but the potential of ld-IL2 as a therapy for headache disorders has not been explored. In this study, we used multiple preclinical models and behavioral endpoints in male and female, inbred and outbred strains of mice to address these knowledge gaps. We report for the first time that repeated NTG administration significantly reduced the ratio of Treg cells among total T cells in TG, suggesting that chronic migraine is associated with a deficiency in Treg-mediated immune homeostasis in TG. Next, we tested the therapeutic use of ld-IL2 in mouse models of chronic migraine, PTH, and MOH. Remarkably, in all these models, ld-IL2 treatment completely reversed the established facial skin hypersensitivity, which reflects the central sensitization under the chronic disease states. Continuous ld-IL2 treatment prevented the development of NTG- and sumatriptan-induced mechanical hypersensitivity related to chronic migraine and MOH, respectively. For behaviors related to mTBI-induced acute and persistent PTH, the therapeutic effect of ld-IL2 persisted after cessation of the treatment. The potential clinical significance of these findings is that it provides the first proof-of-concept that ld-IL2 may constitute an innovative therapeutic strategy for the prevention and reversal of chronic migraine, PTH, and MOH.
NTG administration is a well-established experimental model of migraine.18 In migraineurs, NTG triggers migraine-like headache as well as cutaneous allodynia, and both are responsive to triptan treatment.1 In rodents, both NTG-induced acute cutaneous allodynia and central sensitization can be blocked by sumatriptan and antagonists to calcitonin gene-related peptide (CGRP) receptor, suggesting that NTG-induced changes in rodents reflect the spontaneous migraine with cutaneous allodynia in some patients (Refs. 1, 5, 18 and references within). Repeated administration of NTG and other NO donors in rodents elicits persistent cutaneous allodynia that can be blocked by migraine prophylactics topiramate and propranolol,6,15,47 validating that these behavioral changes model symptoms of chronic migraine in humans. In this study, we found that repeated NTG administration resulted in facial cold and mechanical hypersensitivity as well as hindpaw mechanical hypersensitivity, all of which were prevented by pretreatment with ld-IL2. Starting ld-IL2 treatment after mice received repeated NTG administration completely reversed the established persistent facial mechanical hypersensitivity, regardless of mouse strain and sex. Continuous ld-IL2 treatment prevented subsequent NTG injections from re-establishing either acute or persistent cutaneous hypersensitivity. Collectively, these data predict that ld-IL2 treatment not only blocks trigger-induced migraine episodes, thereby preventing migraine chronification, but also induces the remission of chronic migraine in both males and females. Notably, a recent study reports that neither sumatriptan nor CGRP receptor antagonist olcegepant prevents the persistent cephalic mechanical hypersensitivity induced by repeated administration of NO donor isosorbide dinitrate,15 raising the possibility that ld-IL2 may be effective in reversing chronic migraine that is not responsive to drugs targeting CGRP signaling.
We did not attempt to determine the therapeutic window of ld-IL2 in mice because the dose of IL2 required to activate/expand mouse and human Treg cells is very different, and numerous clinical trials have already established the appropriate dose of ld-IL2 in humans.30,49 The dose of IL2 used in this study has been shown to preferentially expand and activate Treg cells in mice.30,56 Indeed, we observed that daily ld-IL2 preferentially increased the frequency of Treg cells in the blood, LNs, and spleen without altering the frequencies of CD4+ and CD3+ T cells. Moreover, ld-IL2 treatment increased the number of Treg cells in dura and TG by 4 to 9 folds, much higher than that seen in the blood, LNs, or spleen. Results from Treg depletion and adoptive transfer experiments further validated Treg cell as the cellular target of ld-IL2. Depletion of endogenous Treg cells with anti-CD25 antibody did not alter the magnitude of NTG-induced mechanical hypersensitivity, likely due to the high dose of NTG used in our study. Whether Treg depletion alters the time course of NTG-induced behaviors and/or enhances the effects of lower dose of NTG merits further investigation.
In this study, we adoptively transferred Treg cells from IL2-treated mice. Further work will be needed to compare the efficacy of Treg cells from naïve mice and from IL2-treated mice. Notably, many exogenous Treg cells were present in the dura and TG 1 day after the adoptive transfer, whereas few Treg cells could be detected in the cervical/medullary dorsal horn even 5 days after the transfer. Together, these results implicate dura and/or TG as sites of action for the therapeutic effect of ld-IL2 and Treg transfer, despite the fact that systemic NTG administration activates multiple pathways at both central and peripheral levels.18,24 This is consistent with the recent studies indicating that both anti-CGRP antibodies and botulinum toxins act peripherally to reduce the frequency of migraine attacks.28,42
In rodent models of nerve injury and experimental autoimmune encephalomyelitis, mechanical allodynia was exacerbated by the depletion of Treg cells and was attenuated by the increase in Treg cell number.4,21,25,36,38 This study expanded the therapeutic value of ld-IL2 and Treg cells to chronic migraine and other headache disorders. Importantly, neither daily ld-IL2 treatment nor Treg transfer alters basal nociceptive responses, and the second round of ld-IL2 treatment reversed NTG-induced hypersensitivity faster and the effect lasted longer than the first round treatment. These data suggest that there is minimum risk of developing drug tolerance and/or MOH associated with ld-IL2 treatment. In fact, ld-IL2 completely reversed the facial as well as hindpaw hypersensitivity induced by daily injections of sumatriptan in both male and female mice. It is possible that ld-IL2 can reverse MOH resulting from repeated use of sumatriptan as well as other antimigraine drugs, as a recent study shows that chronic exposure of rats to 2 acute headache medications with completely different mechanisms of action induced highly similar transcriptome changes in TG.10
Treg cells are known to use multiple mechanisms to exert suppressive activity and to maintain immune homeostasis, one of which is through enhancing the secretion of transforming growth factor β, interleukin-10, and/or interleukin-35.52 All these cytokines have shown antinociceptive effects in chronic pain models.13,21,33,34 Future study is warranted to determine whether ld-IL2 and Treg cells reverse nociceptive behaviors in various models of headache disorders through common or different mechanisms and whether it engages the secretion of individual cytokines and the regulation of the excitation of dural afferent neurons.
In rodent models of mTBI-induced PTH, the development of both acute and persistent PTH-related behaviors can be prevented by the repeated administration of anti-CGRP antibodies, starting immediately or 2 hours after mTBI.8,45 Discontinuing the anti-CGRP treatment after the resolution of mTBI-induced acute allodynia partially inhibits the development of hyperalgesia priming and administration of anti-CGRP antibody after the resolution of acute allodynia fails to block the development of cutaneous allodynia in response to bright light stress.45 In our model of mTBI-induced PTH, starting ld-IL2 treatment 1 day after mTBI completely prevented the development of mTBI-induced acute facial mechanical hypersensitivity as well as the hyperalgesia priming revealed by repeated low-dose NTG. Notably, starting the 9-day ld-IL2 treatment after the acute hypersensitivity was fully established (7 days after mTBI) could still completely prevent the development of hyperalgesia priming in addition to facilitating the resolution of acute mechanical hypersensitivity. In both cases, 9 days of ld-IL2 treatment was sufficient to protect both male and female mice from developing mTBI-induced hyperalgesia priming for at least 30 days. These results illustrate the potential of ld-IL2 as a novel therapy for mTBI-induced acute and persistent PTH, with a wide therapeutic time window and long-lasting beneficial effect after the cessation of the treatment. In addition to peripheral CGRP signaling, meningeal mast cell is also involved in the development of mTBI-induced hyperalgesia priming.9 Future in-depth studies are needed to elucidate whether the mechanisms of action of ld-IL2 involve interfering with the functions of CGRP and/or mast cells and whether it involves peripheral and/or central sites. In addition, the effectiveness of ld-IL2 in other paradigms of mTBI-induced PTH merits further investigation.
Both reduction of Treg cell number and/or function in peripheral blood and decrease of serum IL2 level have been reported in migraine patients,3,46,55 supporting the clinical relevance of our study. There is abundant evidence from many clinical trials that both ld-IL2 treatment and Treg cell transfer are well tolerated in patients and show indications of efficacy against multiple autoimmune diseases.30,49,54 The promising results for this preclinical study strongly support further clinical assessment of ld-IL2 treatment for multiple headache disorders including chronic migraine, PTH, and MOH and underline the high potential for rapid translation to clinical trials.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Appendix A. Supplemental video content
A video abstract associated with this article can be found at http://links.lww.com/PAIN/A953.
The authors thank members of Cao lab for valuable comments on the manuscript and Ms Zhiyu Zhang for the technical help.
Supported by the National Institute of Neurological Disorders and Stroke grant NS103350-02S1 (to Y.-Q.C.).
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