B cells alter adaptive autoimmunity to protect from ischemic injury after stroke

 描述(申请人提供)：在美国，每年将有超过80万人罹患中风，但只有一种FDA批准的急性干预措施。减少中风后炎症的临床试验失败，突显了更好地了解中枢神经系统(CNS)内的炎症和神经保护机制的必要性。重复低氧预适应(RHP)在治疗结束后的几个月内自然保护中风，最大限度地减少梗塞体积、血脑屏障(BBB)破坏以及单核细胞、T细胞和中性粒细胞渗入缺血脑中。相反，RHP特异性地诱导了一种免疫抑制的B细胞表型，这种表型在中风后预适应小鼠受损的中枢神经系统中得到增强。与PBS或野生型(WT)B细胞处理的对照组相比，过继转移RHP处理的B细胞(B(RHP)细胞)在雄性小鼠短暂性卒中后6小时减少了梗塞体积并改善了功能恢复。B(RHP)细胞治疗还抑制了中风后对中枢神经系统衍生的神经元和髓鞘抗原(Ag)的自身免疫反应，这是适应性自身免疫的一个新发现，仅在继发感染背景下的中风临床前模型中进行研究。这项新研究人员拨款的目标是确定B(Rhp)细胞作为轻度缺氧可转移适应性免疫的一部分，是否通过产生白介素10(IL10)和干扰素-γ(干扰素-TERG)来介导内源性保护。白介素10是一种促进神经元修复的细胞因子，干扰素是一种细胞因子，可以将效应T细胞(即促炎T细胞)转化为抑制其他神经病理免疫细胞的调节性T细胞(Treg)。具体目标1将测试B细胞通过产生IL10和直接神经元相互作用来提供急性神经保护的假设。我们将使用最先进的3-D系列双光子断层扫描进行全脑成像、MRI和行为分析，以证实IL10增强的B(RHP)细胞与缺血半球中更多存活神经元共存，以促进长期功能恢复。我们还将利用从IL10缺失和WT小鼠获得的B(RHP)细胞，在体外证实B(RHP)细胞来源的IL10在缺氧-葡萄糖剥夺(OGD)后对神经元存活和轴突生长的作用。特异性目标2将验证这样的假设，即来自WT而不是干扰素-γ缺失小鼠的B(Rhp)细胞将诱导一种新的Treg群体来抑制来自Th1细胞的促炎自身反应。将体外暴露的Tregs转移到B(RHP)细胞除了抑制中枢神经系统衍生的自身反应外，还将促进长期的神经血管和功能恢复(Aim 1A方法)。最后，Glatiramer Acetate或Copaxone(R)是FDA批准的药物，可上调多发性硬化症患者B细胞来源的IL10和Treg的转化。具体目标3将验证这样的假设，即经Copaxone处理的WT B细胞将上调IL10和干扰素-γ，以实现表型B细胞介导的保护。我们将测试经Copaxone处理的B细胞在体外对OGD后神经元活性的影响(Aim 1B方法)。在卒中后，用Copaxone或对照倍他松治疗的B细胞将被过继转移，以确定长期恢复的效果(Aim 1A方法)。

 DESCRIPTION (provided by applicant): More than 800,000 people in the U.S. will suffer from stroke each year, yet there remains only one FDA- approved acute intervention. Clinical trials to minimize post-stroke inflammation failed, highlighting the need to better understand inflammatory and neuroprotective mechanisms within the central nervous system (CNS). Repetitive hypoxic preconditioning (RHP) naturally protects from stroke for months beyond treatment, minimizing infarct volumes, blood-brain barrier (BBB) disruption, and monocyte, T cell, and neutrophil diapedesis into the ischemic brain. In contrast, RHP specifically induces an immunosuppressed B cell phenotype that is enhanced in the injured CNS of preconditioned mice following stroke. The adoptive transfer of RHP-treated B cells (B(RHP)cells) 6h after transient stroke in male mice reduced infarct volumes and improved functional recovery compared to PBS- or wild type (WT) B cell-treated controls. B(RHP)cell therapy also suppressed a post-stroke autoimmune response to CNS-derived neuronal and myelin antigen (Ag), a novel finding for adaptive autoimmunity that has only been studied in preclinical models of stroke in the context of secondary infection. The goal of this New Investigator grant is to determine if B(RHP)cells, as part of a transferrable adaptive immunity to mild hypoxia, mediate endogenous protection through the production of interleukin 10 (IL10), a cytokine that promotes neuronal repair, as well as interferon (IFN)-γ, a cytokine that converts effector (i.e. pro-inflammatory, Th1) T cells to regulatory T cells (Tregs) that suppress other neuropathological immune cells. Specific Aim 1 will test the hypothesis that B cells confer acute neuroprotection through the production of IL10 and direct neuronal interaction. We will confirm, using state-of-the-art 3-D serial two-photon tomography for whole brain imaging, MRI, and behavioral assays, that IL10- enhanced B(RHP)cells colocalize to a higher number of surviving neurons in the ischemic hemisphere to improve long-term functional recovery. We will also confirm the role of B(RHP)cell-derived IL10 on neuronal viability and axon outgrowth in vitro following oxygen-glucose deprivation (OGD), with B(RHP)cells harvested from IL10-null and WT mice. Specific Aim 2 will test the hypothesis that B(RHP)cells from WT but not IFN-γ-null mice will induce a novel Treg population to suppress pro-inflammatory autoreactive responses from Th1 cells. The transfer of Tregs exposed in vitro to B(RHP)cells will also promote long-term neurovascular and functional recovery (Aim 1A methods), in addition to suppressing CNS-derived autoreactivity. Finally, Glatiramer Acetate, or Copaxone(r), is an FDA-approved drug that upregulates B cell-derived IL10 and Treg conversion in patients with multiple sclerosis. Specific Aim 3 will test the hypothesis that WT B cells treated with Copaxone will upregulate IL10 and IFN-γ to phenocopy B(RHP)cell-mediated protection. We will test the efficacy of Copaxone-treated B cells on neuronal viability in vitro following OGD (Aim 1B methods). Copaxone- or control Betaseron-treated B cells will be adoptively transferred post-stroke to determine efficacy on long-term recovery (Aim 1A methods).