Distinct Functional Outcomes of BCR/TLR7 and BCR/TLR9 Co-engagement

BCR/TLR7 和 BCR/TLR9 共同参与的不同功能结果

• 批准号: 9033830
• 负责人: Ann Marshak-Rothstein
• 金额: $ 51.36万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-12 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9033830
• 关键词: Accounting; Adverse effects; American; Animal Disease Models; Antibodies; Antigen-Antibody Complex; Autoantibodies; Autoantigens; Autoimmune Process; Autophagosome; B cell repertoire; B-Cell Activation; B-Lymphocytes; Biochemical; Biological Assay; Bone Marrow; CRISPR/Cas technology; Cells; Cessation of life; ChIP-seq; Chimera organism; Clinical; Computer Analysis; Confocal Microscopy; DNA; Data; Defect; Dendritic Cells; Dependence; Development; Diagnosis; Disease; Disease Progression; Etiology; Feedback; Future; Gene Expression Profile; Gene Targeting; Genetic; Genomics; Goals; Health; IRF4 gene; Immunosuppressive Agents; In Vitro; Inbred BALB C Mice; Inbred MRL lpr Mice; Indium; Lead; Life; Ligands; Longevity; Lupus; Mediating; MicroRNAs; Modeling; Molecular; Monitor; Mus; Mutation; Nature; Nucleic Acid Binding; Onset of illness; PTPRC gene; Pathway interactions; Patients; Pattern; Pattern recognition receptor; Phenotype; Phosphorylation; Phosphorylation Site; Plasma Cells; Plasmablast; Play; Population; Post-Translational Protein Processing; Pristane; Process; Production; RNA; Regimen; Regulation; Regulator Genes; Risk Factors; Role; Scheme; Source; Staging; Stem cells; Surface; Symptoms; System; Systemic Lupus Erythematosus; TLR7 gene; Techniques; Technology; Testing; Therapeutic; Toll-like receptors; Transgenic Organisms; Variant; anti-IgM; autoreactive B cell; base; cell type; cohort; design; ds-DNA; functional outcomes; in vivo; in vivo Model; insight; interest; macrophage; mouse model; neutrophil; novel; protective effect; purge; receptor; reconstitution; response; small molecule; systemic autoimmune disease; trafficking; transcriptome sequencing

 DESCRIPTION (provided by applicant): SLE is a devastating systemic autoimmune disease of unknown etiology that presents with a diverse array of clinical symptoms and afflicts over 1.5 million Americans. Numerous risk factors have been identified, and it is generally accepted that SLE can result from a spectrum of immunoregulatory defects. Current treatments can involve immunosuppressive regimens associated with debilitating adverse side effects. Given the heterogeneous nature of the disease, it is remarkable that nucleic acid binding Toll-like receptors TLR7 and TLR9 has been found to play a critical role in the production of autoantibodies and disease development in all animal models of disease, and may prove to be critical targets for future therapies. Intriguingly, TLR9 appears to play both a protective and disease promoting role in the disease onset and progression - TLR9 is required for the production of autoantibodies against DNA-associated autoantigens but, quite inexplicably, TLR9-deficient autoimmune-prone mice develop much more severe clinical disease than their TLR9- sufficient counterparts. Our recent studies have now demonstrated a unique role for TLR9 in purging the autoreactive B cell repertoire - B cells activated by BCR/TLR9 engagement first undergo several rounds of division and then undergo a quite profound post-proliferative death. By contrast, BCR/TLR7 activated B cells survive and readily convert to plasmablasts. The goals of this project are to gain a better understanding of: (1) where in the course of disease TLR9 can mediate this protective effect; (2) when in the course of disease TLRs play a key role in B cell activation; (3) whether TLR9 mediates a suppressive role in any other cell types; (4) what unique features of the transcriptional networks downstream of BCR/TLR9 vs BCR/TLR7 co- engagement account for the distinct functional outcomes of these activation schemes, (5) how TLR9 and TLR7 contribute to autophagosome formation, and (6) how specific Unc93b post translational modifications preferentially promote TLR9 vs TLR7 activation in primary B cells, dendritic cells and macrophages. Our studies are based on a unique experimental system in which B cells are activated by physiologically relevant autoantigen immune complexes (IC), by using either IgG2a-specific transgenic B cells or by using a novel anti-IgM/IC dual variable domain polyclonal targeting antibody. ICs that incorporate both DNA and RNA will be used to parse the unique features of BCR/TLR9 and BCR/TLR7 activation pathways by varying the genetic background (TLR7- or TLR9-deficient) of the responder B cell populations. Advanced genomic and computational analyses will be used to elucidate gene regulatory networks. A highly efficient lentiviral retrogenesis strategy will be used to rapidly screen Unc93b variants for their capacity to support TLR9 vs TLR7 driven responses in specific cell types. These studies should provide important insights regarding the best strategies for designing effect TLR-based therapeutics for the treatment of patients diagnosed with SLE and other related systemic autoimmune diseases.

 描述（由申请人提供）：SLE 是一种病因不明的破坏性系统性自身免疫性疾病，具有多种临床症状，困扰着超过 150 万美国人。已经确定了许多危险因素，并且普遍认为系统性红斑狼疮可能是由一系列免疫调节缺陷引起的。目前的治疗可能涉及与使人衰弱的不良副作用相关的免疫抑制疗法。鉴于该疾病的异质性，值得注意的是，核酸结合Toll样受体TLR7和TLR9被发现在所有疾病动物模型中自身抗体的产生和疾病发展中发挥着关键作用，并且可能被证明是未来治疗的关键靶点。有趣的是，TLR9 似乎在疾病的发生和进展中发挥着保护性和疾病促进作用——TLR9 是产生针对 DNA 相关自身抗原的自身抗体所必需的，但令人费解的是，TLR9 缺陷的易患自身免疫的小鼠比 TLR9 充足的小鼠患上更严重的临床疾病。我们最近的研究证明了 TLR9 在清除自身反应性 B 细胞库方面的独特作用 - 由 BCR/TLR9 参与激活的 B 细胞首先经历几轮分裂，然后经历相当严重的增殖后死亡。相比之下，BCR/TLR7 激活的 B 细胞存活并容易转化为浆母细胞。该项目的目标是更好地了解：（1）在疾病过程中TLR9可以在何处介导这种保护作用； (2) 在疾病过程中，TLRs在B细胞激活中发挥关键作用； (3) TLR9是否在任何其他细胞类型中介导抑制作用； (4) BCR/TLR9 与 BCR/TLR7 共参与下游转录网络的哪些独特特征解释了这些激活方案的不同功能结果，(5) TLR9 和 TLR7 如何促进自噬体形成，以及 (6) 特定的 Unc93b 翻译后修饰如何优先促进原代 B 细胞中 TLR9 与 TLR7 的激活， 树突状细胞和巨噬细胞。我们的研究基于独特的实验系统，其中通过使用 IgG2a 特异性转基因 B 细胞或使用新型抗 IgM/IC 双可变域多克隆靶向抗体，B 细胞被生理相关的自身抗原免疫复合物 (IC) 激活。包含 DNA 和 RNA 的 IC 将用于通过改变应答 B 细胞群的遗传背景（TLR7 或 TLR9 缺陷）来解析 BCR/TLR9 和 BCR/TLR7 激活途径的独特特征。先进的基因组和计算分析将用于阐明基因调控网络。高效的慢病毒逆转录策略将用于快速筛选 Unc93b 变体，以确定其支持特定细胞类型中 TLR9 与 TLR7 驱动反应的能力。这些研究应该为设计基于 TLR 的有效疗法的最佳策略提供重要见解，以治疗诊断为 SLE 和其他相关系统性自身免疫性疾病的患者。