Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization

Abstract

Background: Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment.

Methods: Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca²⁺-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking.

Results: CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls.

Conclusions: These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.

Keywords: C-C motif chemokine receptor 2 (CCR2); C-C motif ligand 2 (CCL2); Calcitonin gene-related peptide (CGRP); Facial mechanical hypersensitivity; Low-dose interleukin-2 (LD-IL-2); Medication overuse headache; Regulatory T (Treg) cell.

Fig. 1

CCL2 and CCR2 mRNA expression in dura or TG after repeated sumatriptan administration. A Timeline of experiments. B-C Relative CCL2 mRNA expression levels in dura (B) and TG (C) of female CD-1 mice after 4 or 12 daily i.p. injections of saline or sumatriptan (suma, n = 5–11/group). The abundance of CCL2 mRNA was normalized to that of GAPDH in individual samples. ^*p < 0.05, two-tailed t-test. D-E Relative CCR2 mRNA expression levels in dura (D) and TG (E) after 4 or 12 daily injections of saline or sumatriptan (same mice as in B-C). The abundance of CCR2 mRNA was normalized to that of GAPDH in individual samples

Fig. 2

CCL2 and CCR2 global KO mice still exhibit repeated sumatriptan-induced behavioral sensitization. A Timeline of experiments. Note that drugs were always injected after the completion of behavioral tests on the same day. B-C Sumatriptan-induced facial skin hypersensitivity was comparable in wild-type, male CCL2 KO, and female CCR2 KO mice (n = 6–9/group). ^###p < 0.001, compared with the day 1 baseline threshold within individual groups. D-E Sumatriptan-induced acute sensitization and hyperalgesic priming were also comparable between wild-type, female CCL2, and male CCR2 global KO mice (n = 6–8/group). ^###p < 0.001, compared with the day 1 baseline threshold within individual groups. ^!!!p < 0.001, compared with the day 20 threshold within individual groups. F Repeated sumatriptan-induced acute sensitization and hyperalgesic priming were not altered by the CCL2 neutralizing antibody (n = 7/group). ^###p < 0.001, compared with the day 1 baseline threshold within individual groups. ^!!!p < 0.001, compared with the day 20 threshold within individual groups

Fig. 3

Repeated sumatriptan induces acute sensitization and hyperalgesic priming in CGRPα global KO mice. A Timeline of experiments. Note that sumatriptan and NTG were always injected after the completion of behavioral tests on the same day. B-C Both female (B) and male (C) CGRPα KO mice exhibited sumatriptan-induced facial skin hypersensitivity and hyperalgesic priming to that of wild-type control mice (n = 6–8/group). Wild-type mice in B and C are the same as mice in 2D and 2E, respectively. ^###p < 0.001, compared with the day 1 baseline threshold within individual groups. ^!!!p < 0.001, compared with the day 20 threshold within individual groups

Fig. 4

Repeated sumatriptan does not increase the number of CGRP-R and/or PACAP-R neurons in TG. A Timeline of experiments. B The percentages of CGRP-R, PACAP-R and CGRP-R&PACAP-R neurons in TG cultures from female CD-1 mice after repeated saline (sal) or sumatriptan (suma) administration (n = 4 mice/group, a total of 128 and 153 neurons were measured in saline and sumatriptan groups, respectively). C Venn diagrams of the percentages of CGRP-R and PACAP-R TG neurons in saline and sumatriptan groups (same neurons as in B), respectively. D The percentages of CGRP-R, PACAP-R and CGRP-R&PACAP-R neurons in TG cultures from female CD-1 mice after repeated saline or sumatriptan administration (n = 5 mice/group, a total of 100 and 195 neurons were measured in sal and suma groups, respectively). E Venn diagrams of the percentages of CGRP-R and PACAP-R TG neurons in saline and sumatriptan groups (same neurons as in D), respectively

Fig. 5

Loss of CCL2 or CCR2 abolishes the effect of LD-IL-2 on sumatriptan-induced behavioral sensitization. A Timeline of experiments. Note that all drugs were always injected after the completion of behavioral tests on the same day. B LD-IL-2 reversed sumatriptan-induced facial skin hypersensitivity in wild-type males but not CCL2 KO mice (n = 8/group). ^*p < 0.05, ^***p < 0.001, wild-type versus CCL2 KO; ^###p < 0.001, compared with the day 1 baseline threshold within the wild-type group; ^††p < 0.01, ^†††p < 0.001, compared with the baseline threshold within the CCL2 KO group. C The effect of LD-IL-2 on sumatriptan-induced behavioral sensitization was absent in female CCR2 KO mice (n = 6–7/group). ^*p < 0.05, ^**p < 0.01, wild-type versus CCR2 KO; ^###p < 0.001, compared with the day 1 baseline threshold within the wild-type group; ^†††p < 0.001, compared with the baseline threshold within the CCR2 KO group. D CCL2 neutralizing antibody blocked the effect of LD-IL-2 on sumatriptan-induced behavioral sensitization in male C57BL/6J mice (n = 7–8/group). ^***p < 0.001, control IgG versus CCL2 ab; ^††p < 0.01, ^†††p < 0.001, compared with the day 1 baseline threshold in control IgG group; ^###p < 0.001, compared with the baseline threshold in CCL2 ab group; ^!!!p < 0.001, compared with the day 20 threshold in the CCL2 ab group. E The effect of LD-IL-2 on sumatriptan-induced behavioral sensitization was abolished by the CCL2 neutralizing antibody in female C57BL/6J mice (n = 7/group). ^***p < 0.001, control IgG versus CCL2 ab; ^†††p < 0.001, ^###p < 0.001, compared with the day 1 baseline threshold in control IgG and CCL2 ab groups, respectively; ^!!!p < 0.001, compared with the day 20 threshold in CCL2 ab group

Fig. 6

LD-IL-2 does not rely on CGRPα to reverse MOH-related behavioral sensitization. A-B LD-IL-2 reversed sumatriptan-induced facial skin hypersensitivity in male (A, n = 6–9/group) and female (B, n = 6–8/group) CGRPα KO mice. ^###p < 0.001, ^†††p < 0.001, compared with the baseline threshold within the wild-type and CGRPα KO groups, respectively

Fig. 7

LD-IL-2 increases the number of CCR2-expressing, CD25⁺T cells in dura and TG. A Repeated diphtheria toxin (DT) administration abolished the effect of LD-IL-2 on sumatriptan-induced facial mechanical hypersensitivity in DEREG mice (saline group: 4 males and 4 females; DT group: 5 males and 3 females). ^***p < 0.001, saline versus DT groups. ^#p < 0.05, ^###p < 0.001, compared with the day 1 threshold in saline group; ^†††p < 0.001, compared with the day 1 threshold in the DT group. Note that all drugs were injected after the completion of behavioral tests on the same day. Both sumatriptan (day 1–2) and LD-IL-2 (day 3–12) were injected daily. B Venn diagrams of the percentages of EGFP⁺ and CD25⁺ cells among CD3⁺ T cells in dura surrounding the MMA and in TG tissues from naïve CCR2 HZ mice (replot of data published in [10]). C Representative images of EGFP⁺ and CD25⁺ cells in dura and TG tissues from CCR2 HZ mice after 12 daily injections of saline or LD-IL-2 (1 µg/mouse/day, i.p.). Arrowheads indicate CD25⁺ cells. D-E The density of EGFP⁺CD25⁺ cells in dura (D) and the number of EGFP⁺CD25⁺ cells in TG (E) of female CCR2 HZ mice after 12 daily injections of saline or LD-IL-2 (n = 3–4/group). ^**p < 0.01, two-tailed t test

Fig. 8

LD-IL-2-induced Treg cell increase in dura and TG is significantly reduced in CCL2 KO mice. A The density of EGFP⁺ Treg cells in dura surrounding the MMA from female DEREG (WT) and DEREG_CCL2KO (KO) mice at baseline and after 12 daily injections of saline or LD-IL-2 (n = 3–8/group). ^*p < 0.05, ^**p < 0.01. B The number of EGFP⁺ Treg cells in TG from DEREG and DEREG_CCL2KO mice at baseline and after 12 daily saline or LD-IL-2 (same mice as in B). ^*p < 0.05, ^**p < 0.01. C Gating strategy for individual T cell subpopulations. D-E The frequencies of EGFP⁺CD25⁺ (D) and EGFP⁺ (E) Treg cells in the peripheral blood from female DEREG and DEREG_CCL2KO mice at baseline and after 5 daily saline or LD-IL-2 injections (n = 6/group), respectively. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001