Cell cycle checkpoint defects lead to chronic inflammation in RA

描述(申请人提供)：一个普遍的观点是，对病原体的适应性免疫反应的启动分为两个部分，即通过先天免疫系统识别‘危险’和由适应性免疫系统识别外来抗原。人们普遍认为，适应性免疫反应的耐受性被打破会导致对自身抗原的认可，这些自身抗原有助于自身免疫。在这个模型中，“危险”的来源是 从来没有被完全定义过。我们提出了一个新的模型，在这个模型中，“危险”的来源实际上是内部的，而不是外部的，我们在本申请中探索了这一点。在我们的模型中，由于环境暴露，如吸烟、紫外线或电离辐射、氧化应激、化学暴露、细胞复制、炎症应激或正常代谢活动，个体的DNA损伤每天都在积累。激活细胞周期检查点和DNA修复机制可以修复DNA损伤。然而，在类风湿关节炎中，这些修复机制(ATR、DNA-PKcs和ATM)以及细胞周期检查点JNK2、P53、p21、p27、CHEK2、RANGAP1存在缺陷，导致DNA修复失败和基因组完整性丧失。基因组完整性的丧失会导致慢性核因子-B的激活和促炎细胞因子的诱导。由于促炎细胞因子也激活核因子-B，这会产生一个连续的循环，导致慢性炎症。在类风湿关节炎患者的细胞模型和淋巴细胞中，甲氨蝶呤可以纠正JNK2、p53、p21和p27的表达水平，但不能纠正CHEK2和RANGAP1的表达水平。因此，为了进一步验证我们的假设，我们提议对开始接受甲氨蝶呤治疗的患者进行纵向研究，以评估这些蛋白的表达、细胞周期检查点、基因组完整性的恢复以及慢性核因子-kB的激活恢复到基础水平。另一种模型是，慢性炎症导致细胞因子的过度产生和核因子-kB的激活，而核因子-kB的过度激活与细胞周期检查点和DNA修复机制相反，从而产生在类风湿关节炎中实际观察到的上述表型。为了验证第二个假设，我们建议对开始接受Enbrel治疗的患者进行纵向研究，该治疗将减少过量的TNF-a水平和 由过高水平的肿瘤坏死因子-α驱动的核因子-kB激活。我们将确定将肿瘤坏死因子水平恢复到基线是否也能纠正有缺陷的细胞周期检查点和DNA修复机制，从而恢复基因组的完整性。我们假设的第二个主要检验是，存在于RA淋巴细胞中的细胞周期检查点缺陷、DNA修复机制丧失、基因组完整性丧失和过量的NF-kB激活在一定程度上是由RA的主要遗传危险因素--HLA-DRB1*04等位基因决定的。为了验证这一假设，我们将确定这种分子和细胞表型在患有和不患有类风湿性关节炎的HLA-DRB1*04阳性和阴性受试者中的存在程度。

DESCRIPTION (provided by applicant): A general view is that initiation of adaptive immune responses to pathogens is divided into two parts, the recognition of 'danger' via the innate immune system and the recognition of foreign antigen by the adaptive immune system. It is widely accepted that a breach in tolerance in the adaptive immune response leads to recognition of self-antigen contributing to autoimmunity. In this model, the source of 'danger' has never been completely defined. We propose a new model whereby the source of 'danger' is actually internal and not external, which we explore in this application. In our model, DNA damage accumulates every day in individuals as a result of environmental exposures, e.g. smoking, UV or ionizing radiation, oxidative stress, chemical exposures, cell replication, inflammatory stress, or normal metabolic activities. Activation of cell cycle checkpoints and the DNA repair machinery repairs DNA damage. However, in rheumatoid arthritis, via intrinsic mechanisms regulated by the presence of HLA-DRB1*04 alleles or other intrinsic pathways, these repair mechanisms; ATR, DNA-PKcs, and ATM, and cell cycle checkpoints, JNK2, p53, p21, p27, CHEK2, RANGAP1 are defective resulting in failure of DNA repair and loss of genomic integrity. Loss of genomic integrity results in chronic NF- B activation and induction of pro-inflammatory cytokines. Since pro- inflammatory cytokines also activate NF- B, this produces a continuous cycle resulting in chronic inflammation. Methotrexate, in cell models and in lymphocytes from subjects with rheumatoid arthritis, corrects expression levels of JNK2, p53, p21, and p27, but not CHEK2 and RANGAP1. Therefore, to further test our hypothesis, we propose longitudinal studies of patients initiating methotrexate therapy to assess restoration of expression of these proteins, of cell cycle checkpoints, of genomic integrity, and restoration of chronic NF-kB activation to basal levels. An alternative model is that chronic inflammation drives excess cytokine production and NF-kB activation and excessive NF-kB activation opposes cell cycle checkpoints and DNA repair machinery producing the above phenotype actually observed in rheumatoid arthritis. To test this second hypothesis, we propose to perform longitudinal studies of patients initiating Enbrel therapy that will reduce excess TNF-a levels and NF- kB activation driven by excessive levels of TNF-a. We will determine if restoration of TNF-¿ levels to baseline also corrects defective cell cycle checkpoint and DNA repair machinery, thus restoring genomic integrity. The second major test of our hypothesis is that cell cycle checkpoint defects, loss of DNA repair machinery, loss of genomic integrity and excessive NF-kB activation existing in RA lymphocytes are genetically determined, in part, by the major genetic risk factor for RA, HLA-DRB1*04 alleles. To test this hypothesis, we will determine the extent to which this molecular and cellular phenotype is present in HLA-DRB1*04 positive and negative subjects with and without rheumatoid arthritis.