B Cell Expressed COX2 and AID Dependent Sjogrens Syndrome Autoimmunity

描述（由申请人提供）：炎症通常促进三级淋巴组织的形成，含有B细胞灶，引起自身免疫病理，偶尔转化为恶性肿瘤。在这些位点上，一种dna修饰酶，激活诱导胞嘧啶脱氨酶（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，体外复制人B细胞的环氧化酶（COX-2）轴显著增强了AID的表达/功能，为这些事件的机制提供了新的见解。本研究的长期目标是验证体内表达的B细胞COX-2在自身免疫环境下增强AID和下游致病性IgG自身抗体中发挥重要作用的假设。有证据表明，COX-2活性对正常小鼠和自身免疫小鼠的IgG Ab产生很重要。然而，COX-2是否在B细胞或邻近的非B细胞中起作用尚不清楚。干燥综合征（SjS）是一种自身免疫性疾病，其特征是唾液腺炎症、表达艾滋病的B细胞、致病性IgG自身抗体损害唾液分泌，以及B细胞淋巴瘤的高发率。此外，已知在人类和/或小鼠模型中与SjS相关的刺激是B细胞COX-2体外表达的诱导剂。因此，SjS显然是一种与B细胞COX-2表达有关的疾病。本项目将检验B细胞表达的COX-2是否在SjS中促进IgG抗体驱动的疾病表现中起重要作用。对此至关重要的是(a)最近产生的SjS易感NOD。B10小鼠系，其封闭的COX-2基因可以成为cre介导的失活靶标；(b)一种专有方法，用于生成含有生物活性分子的mab结合纳米颗粒（NP）。以B细胞为靶点的、可生物降解的含有Cre作为“有效载荷”的NP将通过电流体动力学方法生成，该方法将mAb整合到外部可生物降解的外壳中，将Cre整合到内核中。为了证实体外和体内NP的特异性和活性，将在普遍存在的启动子和封闭停止信号的控制下，用表达荧光指示基因（GFP）的小鼠进行原理证明研究。随后，将在sjs易感NOD中测试B细胞靶向、含cree的纳米颗粒。B10小鼠表达固定型或野生型COX-2基因。B细胞中选择性cre介导的COX-2失活可阻止SjS的低活化特征，并损害致病性IgG抗体对M3R毒毒碱乙酰胆碱受体的形成，这一证据将是重要的，并表明特异性NP在人类疾病治疗分子的选择性靶向方面具有希望。

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.