Role of BH3-Domain Proteins in the Effector Phase of RA

描述(申请人提供)：类风湿性关节炎(RA)是一种自身免疫性疾病，以滑膜衬里增生、炎症和软骨和骨骼破坏为特征。在RA中，抗凋亡和促凋亡的Bcl2家族成员之间的平衡可能向生存转移。虽然我们证明了抗凋亡蛋白Bcl2和Mcl-1在RA滑膜组织中与对照组相比增加，但还没有研究探讨拮抗Bcl2抗凋亡成员在关节炎中的治疗潜力。由于胚胎死亡或出生后早期死亡，对Bcl-2家族抗凋亡成员缺陷的研究变得复杂起来。相比之下，Bak、Bax和Bim等缺乏促细胞凋亡的Bcl2成员的小鼠存活下来，并达到成年。由于Bim通过封闭Bcl2/Mcl-1和/或激活Bak和Bax而成为关键的细胞凋亡激活剂，因此它是治疗类风湿关节炎的潜在靶点。我们发现，与对照组相比，类风湿关节炎患者滑膜组织中凋亡启动子Bim的表达显著减少。此外，缺乏Bim但没有下游效应物Bak或Bax的小鼠会患上更严重的炎症性关节炎。在Bim-/-小鼠中，这种加重的疾病与细胞凋亡减少、促炎分子的表达增加以及血管内皮中更多的巨噬细胞有关。BIM-/-巨噬细胞在内毒素刺激下，IL-6、TNF1和IL-12水平升高，CD40和CD69表达增强，活性p38水平持续升高。根据这些数据，我们假设Bim与Bcl2和/或Mcl-1的比率作为分子变阻器，决定了关节内巨噬细胞的增殖和激活程度。我们将使用药理学方法、整体动物方法和细胞特异性方法来确定BIM缺乏如何加剧炎症性关节炎。这些研究可能会导致类风湿关节炎的新的治疗方法。A.1.研究改变Bcl2家族成员在炎症性关节炎发生发展中的作用。A.2.确定Bak、Bax或Bcl2功能丧失对Bim-/-小鼠炎性关节炎发展的影响。A.3.确定靶向缺失单核细胞和巨噬细胞中的Bim在炎性关节炎发生发展中的作用。A.4.确定Bim介导的抑制巨噬细胞活化的机制。细胞死亡和生长的调节对于维持人体内的平衡至关重要。然而，在自身免疫性疾病-类风湿性关节炎(RA)的启动和/或进展过程中，这种平衡被打破。在RA中，细胞生长增加，细胞死亡减少，导致附着在软骨/骨交界处的组织异常增加，滑膜衬里。在RA期间，滑膜衬里侵入并破坏邻近的软骨和骨。对RA患者关节组织的分析表明，巨噬细胞的数量与预后不良相关。我们证明，在RA患者的巨噬细胞中，由促死亡蛋白Bim介导的死亡信号级联是功能障碍的。此外，我们还表明，在所有细胞类型中都缺乏Bim的小鼠会患上更严重的关节炎，并且这些小鼠的巨噬细胞高度激活，这意味着它们会产生大量的有害因素，加剧炎症。我们已经开发出一种潜在的治疗分子，它由Bim的一部分组成，称为BH3结构域。我们现在已经证明，系统地给小鼠递送BH3多肽可以防止炎症性关节炎的发展，对小鼠没有毒性。这些研究首次显示BH3多肽作为治疗类风湿关节炎的潜在分子的有效性。我们的近期目标是开发BH3多肽疗法，作为RA和其他风湿性疾病的一线治疗。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. In RA, the balance between anti- and pro-apoptotic members of the Bcl-2 family may be shifted towards survival. While we demonstrated that the anti-apoptotic proteins, Bcl-2 and Mcl-1 are increased in RA synovial tissue as compared to controls, no study has examined the therapeutic potential of antagonizing Bcl-2 anti-apoptotic members in arthritis. The study of deficiencies in anti-apoptotic members of the Bcl-2 family is complicated by embryonic lethality or early post-natal death. In contrast, mice deficient in pro-apoptotic Bcl-2 members such as Bak, Bax, and Bim survive and reach adulthood. Since Bim is a critical activator of apoptosis by virtue of its sequestration of Bcl-2/Mcl- 1 and/or its activation of Bak and Bax, it is a potential target for treatment of RA. We demonstrated that expression of the apoptotic initiator Bim is markedly reduced in synovial tissue from RA compared to controls. Further, mice lacking Bim but not the downstream effectors Bak or Bax develop a more severe form of inflammatory arthritis. This exacerbated disease in Bim-/- mice is associated with decreased apoptosis, increased expression of pro-inflammatory molecules, and more macrophages in pannus. Bim-/- macrophages display elevated levels of IL-6, TNF1, and IL-12, enhanced expression of CD40 and CD69, and increased and sustained level of active p38 in response to stimulation with LPS. Based on these data, we hypothesize that the ratio of Bim to Bcl-2 and/or Mcl-1 serves as molecular rheostat that determines the extent of hyperplasia and activation of macrophages in the joint. We will use a pharmacological approach, a whole animal approach, and a cell-specific approach to identify how deficiency in Bim exacerbates inflammatory arthritis. These studies will potentially lead to novel therapeutic approaches to RA. A.1. Examine the effect of altering the association of Bcl-2 family members in the development of inflammatory arthritis. A.2. Determine the effect of loss of function in Bak, Bax, or Bcl-2 on the development of inflammatory arthritis in Bim-/- mice. A.3. Determine the effect of targeted deletion of Bim in monocytes and macrophages on the development of inflammatory arthritis. A.4. Determine the mechanism of Bim-mediated suppression of macrophage activation. The regulation of cell death and growth is vital for maintaining a balance in the human body. However, during the initiation and/or progression of the autoimmune disease, rheumatoid arthritis (RA), this balance is disrupted. In RA there is an increase in cellular growth and a concomitant decrease in cell death leading to an abnormal increase in the tissue that attaches to the cartilage/bone junction, the synovial lining. During RA, the synovial lining invades and destroys the adjacent cartilage and bone. Analysis of tissue from joints of patients with RA revealed that the number of macrophages, correlated with a worse prognosis. We demonstrated that the death signaling cascade mediated by the pro-death protein Bim is dysfunctional in macrophages from patients with RA. Additionally, we have shown that mice lacking Bim in all cell types develop a worse form of arthritis and that the macrophages from these mice are highly activated, meaning they produce significant amounts of deleterious factors that exacerbate the inflammation. We have developed a potential therapeutic molecule that consists of a portion of Bim, termed BH3 domain. We have now demonstrated that systemic delivery of BH3 peptides to mice prevents the development of inflammatory arthritis with no toxicity to the mice. These studies are the first to show the efficacy of BH3 peptides as a potential molecule for treatment of RA. Our immediate goal is to develop the BH3 peptide therapy as a front line treatment for RA and other rheumatic diseases.