B Cell Expressed COX2 and AID Dependent Sjogrens Syndrome Autoimmunity

描述（由申请方提供）：炎症通常促进三级淋巴组织的形成，三级淋巴组织含有B细胞灶，其引起自身免疫病理学，偶尔转化为恶性肿瘤。在这些位点，DNA修饰酶，激活诱导的胞嘧啶脱氨酶（AID），在产生致病性自身抗体（autoAb）和诱导致癌突变中起重要作用。该实验室最近在体外发现，体外复制的人B细胞内的环氧合酶（考克斯-2）轴显著增强AID表达/功能，为这些事件的机制提供了新的见解。该提议的长期目标是检验体内表达的B细胞考克斯-2在自身免疫环境中增强AID和下游致病性IgG自身抗体中起重要作用的假设。考克斯-2活性对于正常和自身免疫小鼠中IgG Ab的产生是重要的证据已经被报道。然而，目前还不清楚考克斯-2是否在B细胞或在邻近的非B细胞的功能参与。干燥综合征（SjS）是一种自身免疫性疾病，其特征在于唾液腺炎症、表达AIDS的B细胞、损害唾液分泌的致病性IgG自身抗体和高比率的B细胞淋巴瘤。此外，已知在人和/或小鼠模型中与SjS相关的刺激物是体外B细胞考克斯-2表达的诱导物。因此，SjS显然是一种与B细胞考克斯-2表达相关的疾病。本项目将测试B细胞表达的考克斯-2是否在促进IgG Ab驱动的SjS疾病表现中重要。对此至关重要的是（a）最近产生的SjS易感NOD. B10小鼠系，其floxed考克斯-2基因可以是Cre介导的失活的靶标，以及（B）用于产生含有生物活性分子的mAb结合的纳米颗粒（NP）的专有方法。含有rCre作为“有效载荷”的B细胞靶向的、可生物降解的NP将用电流体动力学方法产生，该方法将mAb掺入可生物降解的外壳中，并将Cre掺入内核中。将用在普遍存在的启动子和floxed终止信号的控制下表达荧光指示基因（GFP）的小鼠进行确认体外和体内NP特异性和活性的原理证明研究。随后，将在表达floxed或野生型考克斯-2基因的SjS易感NOD.B10小鼠中测试B细胞靶向Cre负载纳米颗粒。B细胞中选择性Cre介导的考克斯-2失活可防止SjS的唾液分泌不足特征，并损害针对M3 R毒蕈碱乙酰胆碱受体的致病性IgG抗体的形成，这一证据将是重要的，并将表明特异性NP有望在人类疾病中选择性靶向治疗分子。 公共卫生相关性：这项拟议中的研究与公共卫生有关，因为它将辨别B淋巴细胞中新发现的考克斯-2通路是否对自身免疫性疾病中产生致病性自身抗体很重要。虽然研究将在干燥综合征的小鼠模型中进行，这是一种主要针对50岁以上女性的衰弱性自身免疫性疾病，但这项工作的影响将扩展到其他疾病。该项目的结果应显示是否应靶向B细胞考克斯-2，并提供“原理证明”证据，支持选择性靶向治疗B细胞的创新方法。

DESCRIPTION (provided by applicant): Inflammation often promotes formation of tertiary lymphoid tissue, containing B cell foci which cause autoimmune pathology and occasionally transform into malignancy. In these sites, a DNA-modifying enzyme, activation-induced cytosine deaminase (AID), has an important role in generating pathogenic autoantibody (autoAb) and inducing oncogenic mutations. This laboratory's recent in vitro discovery that a cyclooxygenase (COX-2) axis within in vitro replicating human B cells significantly augments AID expression/function provide new insights into a mechanism for these events. This proposal's long term goal is to test the hypothesis that in vivo-expressed B cell COX-2 plays a significant role in augmenting AID and downstream pathogenic IgG autoAb in an autoimmune setting. Evidence that COX-2 activity is important for IgG Ab production in normal and autoimmune mice has been reported. Nevertheless, it remains unclear whether COX-2 function in B cells or in neighboring non-B cells is involved. Sjogren's Syndrome (SjS) is an autoimmune disease characterized by salivary gland inflammation, AID-expressing B cells, pathogenic IgG autoAbs that impair salivation, and a high rate of B cell lymphoma. Furthermore, stimuli linked to SjS in humans and/or mouse models are known to be inducers of B cell COX-2 expression in vitro. Thus, SjS is clearly a disease in which B cell COX-2 expression may be relevant. This project will test whether B cell-expressed COX-2 is important in promoting IgG Ab-driven disease manifestations in SjS. Crucial to this is (a) a recently generated SjS- susceptible NOD.B10 mouse line whose floxed COX-2 gene can be a target for Cre-mediated inactivation and (b) a proprietary method for generating mAb-bound nanoparticles (NP) containing biologically active molecules. B cell-targeted, biodegradable NP containing rCre as a "payload" will be generated with an electrohydrodynamic approach that incorporates mAb into the outer biodegradable shell and Cre into an inner core. Proof of principle studies to confirm in vitro and in vivo NP specificiy and activity will be performed with mice expressing the fluorescence indicator gene (GFP) under the control of a ubiquitous promoter and floxed stop signal. Subsequently, B cell-targeted, Cre-laden nanoparticles will be tested in SjS-susceptible NOD.B10 mice expressing floxed or wild-type COX-2 genes. Evidence that selective Cre-mediated COX-2 inactivation in B cells prevents the hyposalivation characteristic of SjS and impairs the formation of pathogenic IgG Abs to the M3R muscarinic acetylcholine receptor would be important and would suggest that specific NP hold promise in the selective targeting of therapeutic molecules in human disease. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will discern whether a newly discovered COX-2 pathway in B lymphocytes is important for generating pathogenic autoantibodies in autoimmune disease. While studies will be performed in a mouse model for Sjogren's Syndrome, a debilitating autoimmune disease which primarily targets women over the age of fifty, the implications of the work will extend to other conditions. Results from this project should show whether B cell COX-2 should be targeted and provide "proof of principle" evidence supporting an innovative means of targeting therapeutics selectively to B cells.