Regulatory B-cells Limit CNS Inflammation and Infarct in Experimental Stroke

描述（由申请方提供）：最近的研究已经确定了一个小但强大的IL-10产生CD 1 dhiCD 5+调节性B淋巴细胞（B10）亚群，可以限制啮齿动物模型中的CNS炎症和神经系统疾病的临床体征。在这个多PI应用中，我们假设内源性调节性B细胞，包括B10亚群，通过控制局灶性卒中在CNS和外周免疫器官中触发的免疫介导的炎症来限制MCAO中的梗死面积。此外，我们预测，无论是选择性诱导或被动转移B10细胞，以增加B10细胞的频率将提供额外的调节作用，在野生型（WT）小鼠，并导致较小的梗死和改善功能性中风的结果。本申请的目的是：1）确定B淋巴细胞耗竭和调节性B10细胞恢复和治疗对MCAO后脾和脑半球中的梗死面积、免疫细胞组成和炎症特征的影响。假设是B细胞KO小鼠比WT小鼠承受更大的组织学损伤和较差的功能结果。我们将确定负责的B细胞亚群，包括分泌IL-10的B10亚群。2)评价PD-1/PD-L阴性共活化途径对脾和脑中梗死面积、免疫细胞组成和炎症特征的贡献。假设是B淋巴细胞，特别是CD 1 dhiCD 5 + B细胞中受体-配体途径的激活改变了MCAO的结果。该目的将评估B细胞上的PD-1、PD-L1和PD-L2表达，证实我们在PD-1 KO小鼠中梗死面积增加的初步发现，将分析扩展至PD-L1和PD-L2 KO小鼠，并通过用WT B10细胞重建PD-L KO小鼠来评估B10细胞对卒中后PD-1/PD-L阴性共激活的贡献。3)评估B细胞抑制小胶质细胞（MG）激活和神经毒性因子释放的机制。假设是B细胞，特别是CD 1 dhiCD 5 + B调节细胞，通过抑制小胶质细胞活化改善缺血后结果，通过分泌因子或直接细胞-细胞接触实现PD-1阴性共活化机制。具体而言，该目的将评价PD-L+ CD 19 + B细胞或产生IL-10的CD 1dhiCD 5 + B-reg细胞与活化的PD-1+ MG之间的相互作用。拟议的研究由于其以下特点对卒中领域产生了很大影响：1）完全新奇，2）关注选定B细胞亚群的炎症抑制调节能力，3）为急性卒中中的调节性B细胞治疗奠定转化基础的能力。 公共卫生相关性：缺血性中风是美国死亡和残疾的主要原因之一，但对中风期间对免疫系统的贡献和影响知之甚少。我们关注了中风期间外周免疫系统的早期和晚期事件，观察到脾脏的重要变化与脑损伤的进展同时发生，并且正在研究一种重要的免疫细胞类型B淋巴细胞，其可能具有在中风事件后调节脑损伤和脾脏炎症的能力。如果我们最初的发现得到证实，一类B淋巴细胞可能会对受伤的大脑提供保护。

DESCRIPTION (provided by applicant): Recent studies have identified a small but powerful subset of IL-10-producing CD1dhiCD5+ regulatory B lymphocytes (B10) that can limit CNS inflammation and clinical signs of neurological disease in rodent models. In this multi-PI application, we hypothesize that endogenous regulatory B-cells, including the B10 subset, limit infarct size in MCAO by controlling immune-mediated inflammation triggered both in the CNS and in peripheral immune organs by focal stroke. Further, we predict that either selective induction or passive transfer of B10 cells so as to augment B10 cell frequency will provide additional regulatory effects in wild type (WT) mice, and result in smaller infarct and improved functional stroke outcome. The aims of this application are: 1) Determine the effects of B lymphocyte depletion and regulatory B10 cell restoration and treatment on infarct size, immune cellular composition and inflammatory signature in both spleen and brain hemispheres after MCAO. The hypothesis is that B cell KO mice sustain larger histological damage and poor functional outcome than do WT mice. We will determine the responsible B-cell subpopulations, including the IL-10-secreting B10 subset. 2) Evaluate the contribution of the PD-1/PD-L negative co-activation pathway to infarct size, immune cell composition and inflammatory signature in spleen and brain. The hypothesis is that activation of this receptor-ligand pathway in B lymphocytes, particularly CD1dhiCD5+ B-cells, alters outcomes from MCAO. This aim will evaluate PD-1, PD-L1 and PD-L2 expression on B-cells, confirm our preliminary findings of increased infarct size in PD-1 KO mice, extend the analysis to PD-L1 and PD-L2 KO mice, and evaluate the contribution of B10- cells to the PD-1/PD-L negative co-activation after stroke by reconstituting PD-L KO mice with WT B10-cells. 3) Evaluate the mechanisms through which B-cells inhibit microglial (MG) activation and release of neurotoxic factors. The hypothesis is that B cells, particularly CD1dhiCD5+ B regulatory cells, improve post-ischemic outcomes by suppressing microglial activation, either via secreted factors or direct cell-cell contact that enables a PD-1 negative co-activation mechanism. Specifically, this aim will evaluate interaction between PD-L+ CD19+ B-cells or IL-10-producing CD1dhiCD5+ B-reg cells and activated PD-1+ MG. The proposed studies offer high impact for the stroke field by virtue of their: 1) complete novelty, 2) focus on the inflamma-suppressive regulatory capacity of select B-cell subsets and 3) ability to lay translational groundwork for regulatory B-cell therapy in acute stroke. PUBLIC HEALTH RELEVANCE: Ischemic stroke is one of the leading causes of death and disability in the United States yet relatively little is known about the contribution to and effects on the immune system during stroke. We have focused on both early and late events in the peripheral immune system during stroke, have observed important changes in the spleen that occur simultaneously with the evolving brain injury, and are studying an important type of immune cells, B lymphocytes, which may have the ability to modulate both brain injury and spleen inflammation after a stroke event. If our initial findings are borne out, a class of B lymphocytes may offer protection to the injured brain.