Identification of Specific Roles for Ets-1 in B Cell Tolerance

描述（由申请人提供）：抗体分泌细胞（ASC）产生抗体对于针对许多不同病原体的免疫反应至关重要。然而，在 B 细胞耐受性被破坏的自身免疫性疾病中，许多 ASC 会分泌自身抗体，并对器官病理学产生重要影响。 B 细胞向 ASC 的分化是由一组关键转录因子控制的，其中包括 Ets-1，Ets-1 是该过程的负调节因子。在缺乏 Ets-1 的情况下，B 细胞会加速分化为 ASC，其中许多 ASC 会分泌自身抗体，如血清中高滴度的 IgM 和 IgG 自身抗体所证明的那样。最近，Toll 样受体 (TLR) 信号传导，特别是通过 TLR7 和 TLR9，与自身反应性 B 细胞分化为 ASC 的激活有关。有趣的是，Ets-1缺陷的B细胞在体外表现出对TLR7和TLR9配体的增强的反应，而缺乏TLR接头蛋白Myd88的Ets-1敲除小鼠自身抗体的产生减少。 Toll 样受体信号传导是 Blimp-1 表达的有效诱导剂，Blimp-1 是驱动 ASC 分化的关键转录因子。 Ets-1 与 Blimp-1 发生物理相互作用，抑制 Blimp-1 结合靶 DNA 序列的能力。相反，密切相关的转录因子 Ets-2 无法阻断 Blimp-1 结合或抑制 ASC 分化。 Ets-1 也被认为可以调节控制 ASC 形成的关键靶基因的表达，但大多数这些靶基因的身份仍不清楚。在此应用中，我们提出了多种测定法来进一步确定 Ets-1 在调节靶基因表达、Blimp-1 活性和 ASC 分化中的作用。通过比较可阻止 ASC 分化的 Ets-1 与缺乏这种活性的密切相关的 Ets-2 蛋白的生物活性，将有助于这些研究。该提案的具体目标是（1）确定哪些细胞类型需要 Ets-1 活性来限制 ASC 形成、自身抗体分泌和自身免疫性疾病，（2）检查 TLR7 和 9 配体在激活 Ets-1 缺陷 B 细胞中的体内作用，（3）确定调节 ASC 形成的 Ets-1 依赖性基因表达途径，（4）确定 Ets-1 的结构特征，这些特征赋予调节 ASC 发育的独特能力。我们的研究将为 B 细胞分化的转录调控提供新的见解，特别是在自身抗体分泌的情况下。 公共卫生相关性：作为对病原体免疫反应的一部分，B 细胞分化为抗体分泌细胞 (ASC)，产生高水平的抗体来对抗感染。在自身免疫性疾病中，存在大量的 ASC 分泌与自身组织发生反应的抗体。从所提议的研究中收集的信息将有助于确定 B 细胞如何分化为 ASC 的分子细节，以及该过程在自身免疫性疾病中如何存在缺陷，从而有可能确定临床上刺激或抑制这一过程的新策略。

DESCRIPTION (provided by applicant): The production of antibodies by antibody-secreting cells (ASCs) is critically important for immune responses to many different pathogens. However, in autoimmune diseases where B cell tolerance is broken, many ASCs secrete autoantibodies and contribute in a major way to organ pathology. The differentiation of B cells into ASCs is controlled by a cohort of key transcription factors including Ets-1, which serves as a negative regulator of this process. In the absence of Ets-1, B cells undergo enhanced differentiation into ASCs many of which secrete autoantibodies as demonstrated by high titers of IgM and IgG autoantibodies in the serum. Recently Toll- like receptor (TLR) signaling, particularly via TLR7 and TLR9, has been implicated in the activation of autoreactive B cells to differentiate into ASCs. Interestingly, Ets-1 deficient B cells exhibit enhanced responses to TLR7 and TLR9 ligands in vitro and Ets-1 knockout mice lacking the TLR adaptor protein Myd88 have reduced autoantibody production. Toll-like receptor signaling is a potent inducer of the expression of Blimp-1, a key transcription factor that drives ASC differentiation. Ets-1 physically interacts with Blimp-1 to inhibit the ability of Blimp-1 to bind target DNA sequences. In contrast, a closely related transcription factor Ets-2 is unable to block Blimp-1 binding or to inhibit ASC differentiation. Ets-1 also is thought to regulate the expression of critical target genes controlling ASC formation, but the identity of most of these targets remains unclear. In this application we propose a variety of assays to further define the role of Ets-1 in regulating target gene expression, Blimp-1 activity and ASC differentiation. These studies will be aided by comparing biological activities of Ets-1, which can block ASC differentiation, with the closely related Ets-2 protein, which lacks this activity. The specific aims of the proposal are (1) to determine which cell types require Ets-1 activity to limit ASC formation, autoantibody secretion and autoimmune disease, (2) to examine the in vivo role of TLR7 and 9 ligands in activating Ets-1 deficient B cells, (3) to identify Ets-1 dependent gene expression pathways regulating ASC formation and (4) to identify structural features of Ets-1 that impart a unique ability to regulate ASC development. Together our studies will provide novel insights into the transcriptional regulation of B cell differentiation particularly in situations of autoantibody secretion. PUBLIC HEALTH RELEVANCE: As part of the immune response to pathogens, B cells differentiate into antibody-secreting cells (ASCs) that produce high levels of antibodies to fight infection. In autoimmune disease, there are large numbers of ASCs that secrete antibodies that react with self-tissues. Information gleaned from the studies proposed will help define the molecular details of how B cells differentiate into ASCs and how that process is defective in autoimmune disease, thereby potentially identifying new strategies to stimulate or inhibit this process clinically.