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

描述（由申请人提供）：类风湿性关节炎（RA）是一种自身免疫性疾病，其特征是滑膜内膜增生、炎症以及软骨和骨的破坏。在RA中，Bcl-2家族的抗凋亡和促凋亡成员之间的平衡可能转向生存。虽然我们证明抗凋亡蛋白，Bcl-2和Mcl-1在RA滑膜组织中与对照组相比增加，但没有研究检测拮抗Bcl-2抗凋亡成员在关节炎中的治疗潜力。Bcl-2家族抗凋亡成员的缺陷研究因胚胎致死或早期产后死亡而复杂化。相比之下，缺乏促凋亡Bcl-2成员如Bak、Bax和Bim的小鼠存活并进入成年期。由于Bim通过隔离Bcl-2/Mcl- 1和/或激活Bak和Bax而成为凋亡的关键激活因子，因此它是治疗RA的潜在靶点。我们证明，与对照组相比，RA滑膜组织中凋亡引发因子Bim的表达明显减少。此外，缺乏Bim而没有下游效应物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、促炎分子表达增加和pannus中巨噬细胞增多有关。在LPS刺激下，Bim-/-巨噬细胞表现出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。基于这些数据，我们假设Bim与Bcl-2和/或Mcl-1的比例是决定关节内巨噬细胞增生和活化程度的分子变阻器。我们将使用药理学方法、全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。这些研究将有可能为类风湿关节炎带来新的治疗方法。. 1。研究改变Bcl-2家族成员的关联在炎性关节炎发展中的作用。由信用证。确定Bak、Bax或Bcl-2功能丧失对Bim-/-小鼠炎症性关节炎发展的影响。出具。确定单核细胞和巨噬细胞靶向缺失Bim对炎性关节炎发展的影响。各。确定bim介导的巨噬细胞活化抑制机制。细胞死亡和生长的调节对维持人体的平衡至关重要。然而，在自身免疫性疾病，类风湿性关节炎（RA）的开始和/或进展过程中，这种平衡被破坏。在类风湿性关节炎中，细胞生长增加，同时细胞死亡减少，导致附着于软骨/骨连接处（滑膜衬里）的组织异常增加。在类风湿性关节炎中，滑膜衬里侵入并破坏相邻的软骨和骨。对RA患者关节组织的分析显示，巨噬细胞的数量与较差的预后相关。我们证明由死亡前蛋白Bim介导的死亡信号级联在RA患者巨噬细胞中功能失调。此外，我们已经证明，所有细胞类型中缺乏Bim的小鼠患关节炎的情况更严重，这些小鼠的巨噬细胞高度活化，这意味着它们会产生大量有害因子，从而加剧炎症。我们已经开发出一种潜在的治疗分子，由Bim的一部分组成，称为BH3结构域。我们现在已经证明，给小鼠全身递送BH3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肽作为治疗类风湿性关节炎的潜在分子的功效。我们的近期目标是开发BH3肽疗法作为RA和其他风湿病的一线治疗。

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.