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）是中枢神经系统（CNS）的炎性脱髓鞘疾病， 是西半球年轻人非创伤性神经残疾的最常见原因。 消耗淋巴细胞或阻断其向CNS迁移的疾病修饰剂（DMA）已经被广泛应用于治疗和预防疾病。 在3期临床试验中显示可降低MS复发的频率。然而，这些药物都不是 对所有患者都有效，没有一种是有效的。粒细胞-巨噬细胞集落刺激因子（GM-CSF），a 骨髓细胞生长因子，已经成为MS的一个有前途的治疗靶点。 在未经治疗的复发缓解型MS患者中以相对较高的频率发生，并已在 多发性硬化症脑组织。GM-CSF的缺乏或中和赋予对实验性自身免疫的抵抗力 GM-CSF在自身免疫性脑脊髓炎（EAE）中的作用机制 脱髓鞘还有待明确证实。在初步研究中，我们发现GM-CSF信号传导 是过继免疫诱导的EAE小鼠慢性神经功能缺损发展所必需的。 髓鞘特异性Th 17细胞的转移。GM-CSF阻断或缺乏导致数量减少， 和CNS浸润的供体T细胞和粒细胞的百分比，而CD 88（C5 a受体）表达 骨髓细胞富集。我们发现CNS CD 88+单核细胞和髓样树突状细胞（mDC）表达水平较低， 髓鞘反应性T细胞的抗原呈递细胞。在癌症和哮喘的动物模型中，CD 88+髓系 细胞具有免疫调节特性。基于这些观察，我们建议调查两个潜在的 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缺乏可加速EAE浸润中促炎性iNOS+髓样细胞向 免疫抑制性CD 88 + β-淀粉酶-1+表型。我们将比较细胞表面标志物的表达， GM-CSF受体缺陷的抗原呈递能力、细胞因子谱和免疫调节特性 （GM-CSFR-/-）与从GM-CSFR-/-/ WT和WT混合骨髓的CNS分离的WT髓样细胞 EAE峰值时的嵌合体小鼠。我们还将确定是否治疗GM-CSFR-/-过继转移 接受CD 88拮抗剂的患者加重EAE并促进慢性残疾。在目标2中， 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破坏， 到CNS。我们将确定CXCL 1（一种中性粒细胞吸引趋化因子）的强制CNS表达，或 用重组粒细胞集落刺激因子（G-CSF）治疗，可预防GM-1型EAE缓解。 CSFR-/-主机。这项拟议的研究可以增加我们对EAE免疫发病机制的了解 和MS，并最终导致用于治疗MS的新型骨髓细胞调节药物的开发。 对淋巴细胞靶向DMA无反应的MS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.