Low-dose Interleukin-2 Reverse Behavior Sensitization In Mouse Of Pain Through Regulating Treg Cell In Peripheral Nervous System

BackgroundHeadache and neuropathic pain(NP)are highly prevalent neurological disorder with complex pathophysiology and limited treatment options,which are belong to the most debilitating diseases.A substantial proportion of patients remains either unresponsive to current treatment options or intolerant to their side effect.Previous studies indicate that many proinflammatory immune cells contribute to headache and neuropathic pain pathophysiology,but little is known about the involvement of immunosuppressive regulatory T(Treg)cells in them.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.Repeated low-dose interleukin-2(Ld-IL2)treatment selectively expands and activates Treg cells in vivo without activating effector T cells.In a mouse model of chronic migraine,we observed that repeated nitroglycerin(NTG,a reliable trigger of migraine in patients)administration doubled the number of CD3+T cells in the trigeminal ganglia(TG)without altering the number of Treg cells,suggesting a deficiency in Treg-mediated immune homeostasis.Thus,we hypothesized that Treg cells play a key role in the development of headache as well as NP and enhancing Treg function may be effective to treat multiple headache disorders and neuropathic pain.Objectives1.To explore the role of Treg cells in the pathophysiology of headache and NP.2.To investigate the therapeutic effect of Ld-IL2 on headache and NP.Methods1.Animal models and behavior testsWe use adult male and female mice from the strain of C57BL/6J?Swiss Webster and DEREG(depletion of regulatory T cell)to build Chronic migraine(CM)model,medication over use headache(MOH)model,posttraumatic headache(PTH)model and neuropathic pain(NP)model.Through various behavior experiments to test the mice's response to punctate or brush stimuli,heat or cold stimuli.Motor coordination is assessed with the rotarod test.The adhesive removal test is used to assess mTBI-induced sensory and motor deficits related to the paw and the mouth.Open field test is used to determine general activity levels,gross locomotor activity,and exploration habits of mice.2.Immune cells and cytokine detectionTrigeminal ganglia,dura,sciatic nerve and lumbar L4 DRG were collected and sectioned at 15mm in the transverse plane,collected on Super frost Plus glass slides in sequence and stored at-20?.Using immunohistochemistry experiment to stain the CD3+T cell and Treg cell.Use flow cytometry to detect the number of immune cells in blood,spleen,and cervical lymph node.Use enzyme-linked immunosorbent assay(ELISA)to detect the cytokine secretion from the spleen cells.Results1.Repeated NTG treatment reduces the ratio of Treg cells to total T cells in mouse TG.2.Daily Ld-IL2 prevents the development of NTG-induced skin hypersensitivity,and completely reversed the established allodynia through Treg cells.3.Ld-IL2 treatment inhibits mTBI-induced facial mechanical hyper-sensitivity and hyperalgesia priming.4.Ld-IL2 treatment reverses cutaneous mechanical hyper-sensitivity in a mouse model of MOH.5.Ld-IL2 treatment increases the ratio of Treg/CD3+T cells in the injured sciatic nerve,relieve the skin hypersensitivity and dynamic mechanical allodynia on hind paw in the mouse model of NP.Conclusions1.The occurrence and development of nitroglycerin-induced chronic migraine in mice is associated with a decrease in the ratio of Treg/CD3+T cells in the trigeminal ganglion.2.Using Ld-IL2 or Treg adoptive transfer expands the cell number of Treg in TG,and attenuates the headache induced skin hypersensitivity.3.Ld-IL2 shows similar therapeutic effect in model of MOH and PTH.4.Ld-IL2 treatment preferentially enhances Treg-mediated suppression in the injured nerves by increasing the ratio of Treg cells to total CD3+T cells,and then,prevents and reverses the skin hypersensitivity and dynamic mechanical allodynia in the mouse model of NP.