CD4+CD25+ Treg cells regulate microglia and provide neuroprotection in ALS

描述（由申请人提供）：CD4+CD25+ Treg细胞调节小胶质细胞并在肌萎缩侧索硬化症（ALS）中提供神经保护，肌萎缩侧索硬化症（ALS）是最常见的运动神经元疾病，其特征是大脑和脊髓中运动神经元的广泛丧失。ALS的病因尚不清楚，也没有已知的治疗方法。神经炎症，以激活的小胶质细胞和浸润的免疫细胞为特征，是ALS患者和ALS小鼠的病理标志。目前的证据表明，小胶质细胞/巨噬细胞的激活可能是一把双刃剑。大量研究表明，选择性活化的巨噬细胞（M2）具有保护作用，而经典活化的巨噬细胞（M1）具有毒性。CD4+ T细胞，特别是CD4+CD25+ T调节性（Treg）细胞可调节M1/M2巨噬细胞表型。然而，关于小胶质细胞/单核细胞激活状态与ALS中CD4+CD25+ Treg细胞之间的相互作用知之甚少。我们对mSOD1G93A小鼠的初步研究表明，小胶质细胞在疾病缓慢进展的早期可能表现为M2表型，在疾病迅速进展的终末期可能表现为M1表型。我们的体内数据显示，CD4+ t细胞的缺失加速了mSOD1小鼠的疾病进展。mSOD1G93A/CD4-/-小鼠脊髓中M2标记物Ym1及神经营养因子表达降低，炎症因子明显升高。与mSOD1G93A CD4+CD25- T-细胞相比，我们确定mSOD1G93A CD4+CD25+ Treg细胞通过NOX2表达来抑制成年mSOD1G93A小胶质细胞的活化。此外，CD4+CD25+Foxp3+ Treg细胞在疾病进展较慢的阶段扩增。因此，我们的假设是CD4+CD25+ Treg细胞发挥调节作用，随着疾病的进展，延迟小胶质细胞/单核细胞从保护性（M2）状态向毒性（M1）状态的转变，CD4+CD25+ Treg细胞的过继转移将延长保护性M2状态，延缓mSOD1转基因小鼠的疾病进展。因此，Specific Aim 1将通过定量RT-PCR、流式细胞术和使用M1和M2标记物的免疫组织化学，鉴定mSOD1G93A小鼠不同疾病阶段小胶质细胞/单核细胞的不同激活状态。特异性目的2将在体外研究不同小胶质细胞/单核细胞激活状态与CD4+CD25+ Treg细胞之间的相互作用。此外，将在体内treg缺失的mSOD1G93A小鼠的脊髓和血液中检测到M1和M2表型。特异性Aim 3将确定CD4+CD25+ Treg细胞的过继转移是否延长mSOD1G93A小鼠M2小胶质细胞表型并具有有益作用。将在移植小鼠中检查疾病进展、运动神经元丧失、小胶质细胞/单核细胞的激活状态。本项目将提供CD4+CD25+ Treg细胞调节M2小胶质细胞/单核细胞并在ALS小鼠中提供神经保护的体外和体内证据。由于ALS患者只有在发病后才会就医，因此迫切需要针对减缓疾病进展的治疗，例如利用CD4+ CD25+ Treg细胞。

DESCRIPTION (provided by applicant): CD4+CD25+ Treg cells regulate microglia and provide neuroprotection in ALS Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is characterized by the extensive loss of motoneurons in the brain and spinal cord. The cause of ALS is unknown, and there is no known cure. Neuroinflammation, characterized by activated microglia and infiltrating immune cells, is a pathological hallmark in both ALS patients and ALS mice. Current evidence suggests that microglial/macrophage activation may be a double-edged sword. Numerous studies have concluded that alternatively activated macrophages (M2) are protective, while classically activated macrophages (M1) are toxic. The M1/M2 macrophages phenotypes have been shown to be modulated by CD4+ T-cells, especially CD4+CD25+ T regulatory (Treg) cells. However, little is known about the interaction between microglia/monocyte activation states and CD4+CD25+ Treg cells in ALS. Our preliminary studies with mSOD1G93A mice demonstrated that microglia may exhibit an M2 phenotype at early phase when disease was slowly progressing and an M1 phenotype at end stage when disease was rapidly progressing. Our in vivo data showed that the absence of CD4+ T-cells accelerates disease progression of mSOD1 mice. In the spinal cords of mSOD1G93A/CD4-/- mice, expression of an M2 marker, Ym1, as well as neurotrophic factors were decreased, while inflammatory factors were significantly increased. We determined that mSOD1G93A CD4+CD25+ Treg cells inhibited activation of adult mSOD1G93A microglia, as measured by NOX2 expression, when compared with mSOD1G93A CD4+CD25- T- cells. Moreover, CD4+CD25+Foxp3+ Treg cells expanded during slower disease progression phase of disease. Therefore, our hypothesis is that CD4+CD25+ Treg cells play a regulatory role delaying the microglia/monocyte shift from a protective (M2) to a toxic (M1) state as disease progresses and that the adoptive transfer of CD4+CD25+ Treg cells will prolong the protective M2 state and delay disease progression of mSOD1 transgenic mice. Thus, Specific Aim 1 will identify distinct activation states of microglia/monocytes at different disease phases in mSOD1G93A mice by quantitative RT-PCR, flow cytometry and immunohistochemistry using M1 and M2 markers. Specific Aim 2 will examine the interaction between different microglia/monocyte activation states and CD4+CD25+ Treg cells in vitro. Additionally, M1 and M2 phenotypes will be detected in the spinal cords and blood of Treg-depleted mSOD1G93A mice in vivo. Specific Aim 3 will determine if the adoptive transfer of CD4+CD25+ Treg cells prolongs the M2 microglial phenotype and has beneficial effects in mSOD1G93A mice. Disease progression, motoneuron loss, activation states of microglia/monocytes will be examined in the transferred mice. This project will provide in vitro and in vivo evidence that CD4+CD25+ Treg cells regulate M2 microglia/monocytes and provide neuroprotection in ALS mice. Since ALS patients seek medical attention only after disease onset, therapies directed at slowing disease progression, such as utilizing CD4+ CD25+ Treg cells, are critically needed. PUBLIC HEALTH RELEVANCE: ALS is a horrific, devastating neurodegenerative disease in which patients watch themselves deteriorate over a very short period of time, and despite extensive basic investigations, there is minimal effective therapy. Our own efforts to develop meaningful therapies have focused upon the roles of the innate and adaptive immune systems. Recently, T cells have been shown to have the ability to improve neurological function and life expectancy in ALS models - since T cells are readily accessible in ALS patients, defining the specific populations mediating neuroprotection in the ALS models is translatable into our ultimate goal of using T cell therapies in ALS patients to slow disease progression and improve their quality of life.