Cell cycle checkpoint defects lead to chronic inflammation in RA

描述（由申请人提供）：一般认为，对病原体的适应性免疫应答的启动分为两部分，通过先天免疫系统识别“危险”和通过适应性免疫系统识别外来抗原。广泛接受的是，适应性免疫应答中耐受性的破坏导致有助于自身免疫的自身抗原的识别。在这个模型中，“危险”的来源 从未被完全定义。我们提出了一个新的模型，即“危险”的来源实际上是内部的，而不是外部的，我们在这个应用程序中探索。在我们的模型中，由于环境暴露，例如吸烟，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，DNA损伤每天在个体中积累。细胞周期检查点的激活和DNA修复机制修复DNA损伤。然而，在类风湿性关节炎中，通过由HLA-DRB 1 *04等位基因的存在或其他内在途径调节的内在机制，这些修复机制ATR、DNA-PKcs和ATM以及细胞周期检查点JNK 2、p53、p21、p27、CHEK 2、RANGAP 1是有缺陷的，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导致慢性NF- B活化和促炎细胞因子的诱导。由于促炎细胞因子也激活NF- B，这产生了一个连续的循环，导致慢性炎症.在细胞模型和类风湿性关节炎受试者的淋巴细胞中，甲氨蝶呤纠正JNK 2、p53、p21和p27的表达水平，但不纠正CHEK 2和RANGAP 1的表达水平。因此，为了进一步验证我们的假设，我们建议对开始甲氨蝶呤治疗的患者进行纵向研究，以评估这些蛋白质表达的恢复、细胞周期检查点的恢复、基因组完整性的恢复以及慢性NF-kB激活恢复到基础水平。 另一种模型是慢性炎症驱动过量的细胞因子产生和NF-kB活化，而过量的NF-kB活化对抗细胞周期检查点和DNA修复机制，从而产生在类风湿性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Enbrel治疗的患者进行纵向研究，该治疗将降低过量的TNF-α水平， 由过量水平的TNF-α驱动的NF-κ B活化。我们将确定TNF-α水平恢复到基线水平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。 我们的假设的第二个主要测试是，细胞周期检查点缺陷，DNA修复机制的损失，基因组完整性的损失和过度的NF-κ B激活存在于RA淋巴细胞是遗传决定的，部分是由RA的主要遗传风险因素，HLA-DRB 1 *04等位基因。为了验证这一假设，我们将确定这种分子和细胞表型在HLA-DRB 1 *04阳性和阴性受试者中存在和不存在类风湿性关节炎的程度。

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.