The mechanism of action of Granulocyte Macrophage-Colony Stimulating Factor in an animal model of Multiple Sclerosis

摘要 多发性硬化症(MS)是一种中枢神经系统(CNS)炎症性脱髓鞘疾病，是 西半球年轻人中非创伤性神经功能障碍的最常见原因。 耗尽淋巴细胞或阻止其迁移到中枢神经系统的疾病修饰剂(DMA)已经被 在3期临床试验中显示可减少多发性硬化症复发的频率。然而，这些药物都不是 治愈的和无效的对所有患者都有效。粒细胞巨噬细胞集落刺激因子 髓系细胞生长因子已成为MS产生GM-CSF T细胞的有希望的治疗靶点 在未经治疗的复发缓解期MS患者中发生的频率相对较高，并已在 多发性硬化症脑组织。GM-CSF缺乏或中和可抵抗实验性自身免疫 多发性硬化的动物模型--脑脊髓炎--GM-CSF在自身免疫中的作用机制 脱髓鞘还没有得到明确的证明。在初步研究中，我们发现GM-CSF信号 是导致EAE小鼠慢性神经功能障碍的必需因素 髓鞘特异性Th17细胞的转移。GM-CSF的阻断或缺乏导致数量的减少 CD88(C5a受体)表达 髓系细胞明显增多。我们发现CNS CD88+单核细胞和髓系树突状细胞(MDC)很少 髓鞘反应性T细胞的抗原提呈细胞。在癌症和哮喘的动物模型中，CD88+髓系 细胞具有免疫调节特性。基于这些观察，我们建议研究两个潜在的 GM-CSF在EAE效应期的作用机制。在目标1中，我们将测试我们的假设 GM-CSF缺乏加速EAE浸润物中促炎症iNOS+髓系细胞转化为 CD88+精氨酸酶-1+免疫抑制表型。我们将比较细胞表面标志物的表达， GM-CSF受体缺陷的抗原提呈能力、细胞因子谱和免疫调节特性 (GM-CSFR-/-)与从GM-CSFR-/-/WT混合骨髓分离的WT髓系细胞的比较 嵌合小鼠处于EAE高峰期。我们还将确定GM-CSFR-/-领养转移的治疗 使用CD88拮抗剂的受者会加剧EAE，并促进慢性残疾。在《目标2》中我们将 研究GM-CSF在中性粒细胞募集中的作用，中性粒细胞介导血脑屏障的破坏， 送到中枢神经系统。我们将确定CXCL1(一种吸引中性粒细胞趋化因子)的中枢神经系统强制表达，或者 重组粒细胞集落刺激因子(G-CSF)治疗预防GM-EAE缓解 CSFR-/-主机。这项拟议的研究可以增加我们对EAE免疫发病机制的理解 和多发性硬化症，并最终导致新的髓系细胞调节药物的开发，用于治疗 对淋巴细胞靶向DMA无反应的多发性硬化患者。

Abstract Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), is the most common cause of non-traumatic neurological disability in young adults in the Western Hemisphere. Disease modifying agents (DMA) that deplete lymphocytes, or block their migration to the CNS, have been shown to reduce the frequency of MS relapses in Phase 3 clinical trials. However, none of those drugs are curative and none are effective in all patients. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a myeloid cell growth factor, has emerged as a promising therapeutic target in MS. GM-CSF producing T cells occur at a relatively high frequency in untreated relapsing remitting MS patients, and have been identified in MS brain tissue. Deficiency or neutralization of GM-CSF confers resistance against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The mechanism of action of GM-CSF in autoimmune demyelination has yet to be definitively demonstrated. In preliminary studies we found that GM-CSF signaling is necessary for the development of chronic neurological deficits in mice with EAE induced by the adoptive transfer of myelin-specific Th17 cells. GM-CSF blockade or deficiency resulted in a decrease in the numbers and percentages of CNS-infiltrating donor T cells and granulocytes, while CD88 (C5a receptor) expressing myeloid cells were enriched. We found that CNS CD88+ monocytes and myeloid dendritic cells (mDC) are poor antigen presenting cells for myelin-reactive T cells. In animal models of cancer and asthma, CD88+ myeloid cells have immunoregulatory properties. Based on these observations, we propose to investigate two potential mechanisms of action of GM-CSF during the effector phase of EAE. In Aim 1, we will test our hypothesis that GM-CSF deficiency accelerates the conversion of pro-inflammatory iNOS+ myeloid cells in EAE infiltrates to an immunosuppressive CD88+ arginase-1+ phenotype. We will compare cell surface marker expression, antigen presenting capacity, cytokine profiles and immunoregulatory properties of GM-CSF receptor deficient (GM-CSFR-/-) versus WT myeloid cells isolated from the CNS of GM-CSFR-/-/ WT  WT mixed bone marrow chimeric mice at peak EAE. We will also determine whether treatment of GM-CSFR-/- adoptive transfer recipients with CD88 antagonists exacerbates EAE and promotes chronic disability. In Aim 2 we will investigate the role of GM-CSF in the recruitment of neutrophils, that mediate blood-brain-barrier breakdown, to the CNS. We will determine if forced CNS expression of CXCL1 (a neutrophil attracting chemokine), or treatment with recombinant granulocyte-colony stimulating factor (G-CSF), prevents EAE remission in GM- CSFR-/- hosts. The proposed research could increase our understanding of the immunopathogenesis of EAE and MS, and eventually lead to the development of novel myeloid cell modulating drugs for the treatment of MS patients who do not respond to lymphocyte-targeting DMA.