PROJECT SUMMARY/ABSTRACT Chronic migraine is highly debilitating, poorly understood, and with limited treatment options. Although the activation of many pro-inflammatory immune cells has been shown to contribute to migraine pathophysiology, the involvement of CD3+ T lymphocytes, especially the immunosuppressive regulatory T (Treg) cells, in migraine chronification and reversal remains unknown. We will address the knowledge gap in this application. Some migraine patients exhibit numerical or functional impairment of Treg cell in the peripheral blood. In a mouse model of chronic migraine, repeated administration of nitroglycerin (NTG, a reliable trigger of migraine in patients) not only induced persistent behavioral sensitization, but also doubled the number of CD3+ T cells in the trigeminal ganglia (TG) without altering the number of Treg cells, again suggesting a loss of balance between immune activation and suppression. Repeated NTG also increased the number of TG neurons that can be activated by neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase- activating polypeptide (PACAP), indicating the sensitization of TG neurons. Low-dose interleukin-2 (ld-IL2) treatment, which preferentially expands and activates endogenous Treg cells, completely reversed chronic migraine-related behavioral sensitizations without altering basal nociceptive responses. Ld-IL2 also effectively reduced the number of CGRP- and PACAP-responsive TG neurons in NTG-treated mice to the control level. Mechanistically, we found that both peripheral transforming growth factor beta (TGF) and interleukin-10 (IL10) signaling are required for ld-IL2 to reverse chronic migraine-related behavioral and cellular sensitizations. In this application, we propose to test the hypothesis that neuro-immune interactions contribute to the development and resolution of chronic migraine, likely through regulating the sensitivity of TG neurons in the trigeminovascular pathway. First, we will selectively deplete CD4+ or CD8+ T cells to determine which T cell subset(s) contribute to the development and resolution of chronic headache-related sensitizations. We will then use flow cytometry to further investigate which T cell subtype(s) within CD4+ and CD8+ cells are altered by repeated NTG. Secondly, to further elucidate how Tregs and ld-IL2 reverses migraine chronification, we will examine whether ld-IL2 increases TGFβ1 and IL10 in Treg cells in TG and dura. Treg-selective gene knockout strategy and adoptive transfer of Treg cells will be employed to determine whether Treg cells are the main source of TGF1 and IL10 that mediate the effects of ld-IL2. Lastly, we will test whether repeated NTG increases CGRP and/or PACAP peptide expression in TG neurons. Conditional KO mice will be used to selectively eliminate CGRP or PACAP signaling in primary afferent neurons. We will ask whether headache chronification is entirely or partially mediated through CGRP and PACAP signaling in TG neurons. Collectively, results from this study will not only shed light on how neuro-immune interactions regulate migraine chronification and reversal, but also facilitate mechanism-based drug discovery.

PROJECT NARRATIVE Our previous work shows that restoring the balance between regulatory T cells and other immune cells can effectively reverse chronic migraine-related behavioral and cellular sensitizations in mice. In this application, we propose to test the hypothesis that neuro-immune interactions contribute to the chronification and resolution of migraine headache through regulating the activity of primary afferent neurons in the trigeminovascular pathway. This will elucidate the mechanisms underlying migraine chronification and help developing new therapeutic strategies.