B Cell Expressed COX2 and AID Dependent Sjogrens Syndrome Autoimmunity

描述(申请人提供)：炎症通常促进第三级淋巴组织的形成，含有B细胞病灶，导致自身免疫病理，有时会转化为恶性肿瘤。在这些位点中，一种DNA修饰酶，激活诱导胞嘧啶脱氨酶(AID)，在产生致病自身抗体(AutoAb)和诱导致癌突变方面起着重要作用。该实验室最近在体外发现，在体外复制的人B细胞中的环氧合酶(COX-2)轴显著增强了AID的表达/功能，这为这些事件的机制提供了新的见解。这项提议的长期目标是检验这样一个假设，即在体内表达的B细胞COX-2在自身免疫环境中增强AID和下游致病的Ig G自身抗体方面发挥着重要作用。已有证据表明，COX-2活性对正常和自身免疫小鼠的免疫球蛋白抗体产生具有重要作用。然而，目前还不清楚COX-2是在B细胞还是在邻近的非B细胞中发挥作用。干燥综合征(Sjogren‘s综合征，SjS)是一种自身免疫性疾病，以唾液腺炎症、表达AID的B细胞、损害唾液的致病免疫球蛋白自身抗体和高发病率的B细胞淋巴瘤为特征。此外，在人类和/或小鼠模型中，与SjS相关的刺激在体外是B细胞COX-2表达的诱导剂。因此，SjS显然是一种与B细胞COX-2表达相关的疾病。该项目将测试B细胞表达的COX-2是否在促进SjS的免疫球蛋白抗体驱动的疾病表现方面起重要作用。关键是(A)最近产生的SjS敏感的NOD.B10小鼠系，其脱落的COX-2基因可以作为Cre介导的失活的目标，以及(B)产生含有生物活性分子的单抗结合纳米粒(NP)的专利方法。以B细胞为靶标、以rCre为“有效载荷”的可生物降解NP将通过电流体动力学方法产生，该方法将mAb加入到可生物降解的外层，将Cre加入到内核中。在体外和体内证实NP特异性和活性的原理研究的证据将在普遍存在的启动子和带花环的停止信号的控制下，在表达荧光指示器基因(GFP)的小鼠身上进行。随后，以B细胞为靶点的、携带Cre的纳米颗粒将在表达FLOXED或野生型COX-2基因的SjS敏感的NOD.B10小鼠身上进行测试。有证据表明，选择性Cre介导的COX-2在B细胞中的失活阻止了SjS的低活性特征，并损害了针对M3R M受体的致病抗体的形成，这将是重要的证据，并将提示特异性NP在人类疾病治疗分子的选择性靶向中具有前景。 公共卫生相关性：这项拟议的研究与公共健康相关，因为它将查明新发现的B淋巴细胞中的COX-2途径是否对自身免疫性疾病中产生致病自身抗体具有重要作用。虽然研究将在干燥综合征的小鼠模型上进行，这是一种主要针对50岁以上女性的衰弱自身免疫性疾病，但这项工作的影响将延伸到其他疾病。这个项目的结果应该表明B细胞COX-2是否应该成为靶点，并提供支持选择性靶向B细胞治疗药物的创新方法的“原则证据”。

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.