The Role Of Regulatory T Cells In CNS Injury

BackgroundNeuroinflammation is one of the most important mechanisms of the secondary injury after traumatic CNS injury. With the development of neuroimmunology in the past twenty years,the immune response after CNS injury has been extensively studied, but no consistent conclusion has been made. The dogma that CNS is an immune privileged organ and that the infiltration of immune cells into CNS was a detrimental response has been challenged by the finding that neuronal survival could be improved by boosting T cell activity rather than by its suppression. However, the phenotype of protective T cells after CNS injury, and particularly the role of regulatory T(Treg) cells in this process, is still a matter of debate. Naturally occurring Treg cells, which express the transcription factor Foxp3, have been intensively studied for their ability to suppress adaptive immune responses. This subset of T cells, which develops with high avidity to self-antigens, is especially important in controlling autoimmunity. Therefore, it has been proposed that Treg cells mediate their actions by attenuating both protective and detrimental post injury immune responses, and thus either exacerbating or ameliorating neuronal degeneration. Despite these studies, the exact mechanism of their action in the injured CNS remains unclear.Immune response can be divided into two parts, innate immune response and adaptive immune response. Macrophages are a group of important innate immune cells, play an important role in the wound healing process. Recently, the heterogeneity of macrophages has come to light, with two general classes being described as classically or alternatively activated. Although classically activated macrophages express high levels of proinflammatory cytokines such as TNFα and IL-1β, and exhibit a robust respiratory burst, alternatively activated(tissue-building) macrophages express high levels of arginase1 and several factors that play a role in promoting tissue homeostasis and recovery from insults. Several studies have shown the neuroprotective ability of alternatively macrophages in CNS injury, but what leads to, and sustains this phenotype is unclear in the context of CNS trauma.Objective 1. To study the immune response after CNS injury. 2. To investigate the role of regulatory T cells in CNS injury and its underlying mechanism, in order to find a proper way to boost the protective immune response while suppress the detrimental immune response after CNS injury by manipulating regulatory T cells. 3. To observe the difference between microglia and monocyte derived macrophage. Methods 1. The injury models we used are optic nerve crush model and spinal cord contusion model. We assessed neuronal survival after optic nerve injury by counting the Fluro gold labelled retinal ganglion cells, and used Basso Mouse Scale to rate mouse hind limb motor function recovery after spinal cord injury;2. Two different methods were used to establish Treg depletion model. One is i.p. injection of anti-CD25 antibody(clone: PC61). The other is DEREG(Diphtheria toxin receptor is expressed under the foxp3 gene promoter) transgenic mice,with 40μg/kg or 4μg/kg Diphtheria toxin i.p. injected to completely or partially deplete Foxp3-expressing Treg cells; 3. In vitro ATRA induced Treg cells were injected via tail vein to increase the number of circulating Treg cells and boost their activity; 4. Flow cytometry was used to assess the T cell immune response in lymph nodes after CNS injury; 5. Immunohistochemistry and q PCR were used to assess the arginase 1, i NOS and other cytokines expression after CNS injury; 6. Chemeric mice(Bone marrow from UBC-GFP mice was transplanted into radiated WT mice), and CCR2 KO mice were used to study the difference between peripheral monocyte derived macrophage and microglia derived macrophage in CNS injury site, and the role of monocyte derived macrophage in CNS injury. Results 1. Compared to deep cervical lymph nodes(d CLN) of uninjured mice or skin-draining lymph nodes of optic nerve injured mice, CD4+T cells increased in d CLNs, while CD8+T cells decreased after injury. Both Treg cells and Teff cells got increased in d CLNs after injury. The CD4+T cells in d CLNs had increased IL-4 expression and could increase arginase 1 expression in cultured BMDMs; 2. Resection of d CLN 3 weeks before optic nerve injury was detrimental to RGC survival; Glucocorticoids Dexamethasone exacerbated RGC survival after optic nerve injury and the functional recovery after spinal cord injury; 3. Depletion of Treg with 250μg PC61 antibody or with 40μg/kg Diphtheria toxin resulted in decreased neuronal survival and worse functional recovery. Supplement with induced Treg also resulted in decreased neuronal survival after CNS injury. Both depletion and supplement of Treg caused decreased arginase 1 expression and decreased expression of anti-inflammatory cytokines in the injury site; 4. Partially deplete Treg with 25μg PC61 or 4μg/kg Diphtheria toxin significantly increased RGC survival after optic nerve injury, and also had increased arginase-1 expression in the injury site. 5. In the chimeric mice arginase-1 expressed mainly in GFP+ macrophages in the injury site. And arginase-1 hugely decreased in CCR2 KO mice after CNS injury. In CCR2-RFP mice,arginase-1 mainly expressed in RFP+ cells, which were monocytes from the peripheral. 6. Comparing to WT mice, CCR2 KO mice had decreased RGC survival after optic nerve injury and worse functional recovery after spinal cord injury;Conclusion 1. We found that the peripheral immune response after CNS injury happens in d CLNs, which is leading by CD4+T cells in which both Treg cells and Teff cells increased. The immune response after CNS injury is neuroprotective; 2. Arginase-1 mainly expresses in peripheral monocyte-derived macrophages rather than in microglia-derived macrophages in the CNS injury site. And the monocyte-derived macrophages are neuroprotective; 3. Complete depletion of Treg cells may over-unleash the suppression of Treg on Teff response, and lead to decreased neuronal survival and worse functional recovery. While supplement of extra Treg cells may lead to the suppression of neuroprotective immune response and also lead to decreased neuronal survival. And partially depleting Treg cells has an opposite effect, which may because it release the the suppression of Treg on protective T cells response. All of the results remind people to take caution when manipulating Treg cells to treat CNS injury. 4. Treg may exert its effect via affecting the polarization state of macrophages in the injury site.