Epigenetic downregulation of the antibody and autoantibody response

描述（由申请人提供）：抗体的表观遗传下调和自身抗体反应表观遗传标记包括DNA甲基化、组蛋白修饰和microRNA。正如我们所说，这些与调节 B 细胞功能的遗传程序“相互作用”，例如类别转换 DNA 重组 (CSR)、体细胞超突变 (SHM) 和浆细胞分化，从而通知抗体反应。表观遗传失调可导致抗体对外源抗原（例如病毒和细菌抗原）或自身抗原（例如狼疮中的染色质、组蛋白和 dsDNA）产生异常反应。 我们假设表观遗传调节剂组蛋白脱乙酰酶 (HDAC) 抑制剂 (HDI) 抑制 B 细胞内在功能 CSR/SHM 和浆细胞分化，从而减弱抗体和自身抗体反应。我们进一步认为，HDI 通过上调选定的 microRNA 来抑制这些 B 细胞功能，包括 miR-155、miR-181b 和 miR-361 来下调 AID（Aicda 基因，对 CSR/SHM 至关重要），以及 miR-23b、miR-30a 和 miR-125 来下调 Blimp1（Prdm1 基因，对浆细胞分化至关重要）。我们的假设特别关注 B 细胞，并得到我们令人信服的数据的支持，这些数据使用纯化的人和小鼠 B 细胞在体外、在正常小鼠体内对 T 依赖性和 T 非依赖性抗原作出反应，以及通过 HDI 预防疾病和“治疗”易患狼疮的小鼠。 我们在 B 细胞生物学、分子 CSR/SHM 机制和自身免疫方面的优势，以及我们尖端的表观遗传工具和方法，使我们能够以独特的方式检验我们的假设。我们将：（目标 1）分析 HDIs（丙戊酸、丁酸和 SAHA）对 CSR/SHM 和浆细胞分化的影响，以及对正常 C57BL/6 和 Balb/c 小鼠中的特异性 T 依赖性和 T 非依赖性抗体反应以及狼疮倾向 MRL/Faslpr/lpr 和 B6.NZM/Sle1.Sle2.Sle3 小鼠中自身抗体反应的影响； （目标 2）使用分子生物学、生物化学和高通量/生物信息学工具通过增强组蛋白乙酰化和 microRNA“宿主基因”转录来分析 B 细胞中 HDI 介导的 miR-155、miR-181b、miR-361、miR-23b、miR-30a 和 miR-125b 上调，解决这些 microRNA 对 AID 和 Blimp1 的下调，并构建使用 Ago HITS-CLIP 绘制 Aicda 和 Prdm1 3'UTR 的 microRNA 体内靶向图谱； （目标 3）证明 B 细胞 microRNA 在介导 HDI 抑制抗体和自身抗体反应中的关键作用，使用三管齐下的体内集成方法，包括构建在 Aicda 或 Prdm1 3'UTR 中缺乏特定 microRNA 靶向位点的新敲入小鼠、B 细胞条件 Dicer 或 Drosha KO 小鼠，以及表达 miR-155、miR-181b 和 miR-361 特异性的“海绵”抑制剂的小鼠（针对 Aicda）和/或 B 细胞中的 miR-23b、miR-30a 和 miR-125b（针对 Prdm1）。 我们的提案具有高度创新性和出色的转化性。它将提供表观遗传学和免疫调节的机制见解和未来方向，包括 B 细胞 microRNA 在抗体/自身抗体反应中的关键作用，以及表观遗传学工具作为自身免疫新疗法的作用。

DESCRIPTION (provided by applicant): Epigenetic downregulation of the antibody and autoantibody response Epigenetic marks include DNA methylation, histone modifications and microRNAs. As we have contended, these "interact" with genetic programs to regulate B cell functions, such as class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation, thereby informing the antibody response. Epigenetic dysregulation can result in aberrant antibody responses to exogenous antigens, such as those on viruses and bacteria, or self-antigens, such as chromatin, histones and dsDNA in lupus. We hypothesize that the epigenetic modulators histone deacetylase (HDAC) inhibitors (HDIs) inhibit the B cell intrinsic functions CSR/SHM and plasma cell differentiation, thereby blunting antibody and autoantibody responses. We further argue that HDIs inhibit these B cell functions by upregulating selected microRNAs, including miR-155, miR-181b and miR-361 to downregulate AID (Aicda gene, critical for CSR/SHM), as well as miR-23b, miR-30a and miR-125 to downregulate Blimp1 (Prdm1 gene, critical for plasma cell differentiation). Our hypotheses uniquely focus on B cells and are supported by our compelling data using purified human and mouse B cells in vitro, and in vivo in normal mice responding to T-dependent and T- independent antigens, and prevention of disease and "treatment" of lupus-prone mice by HDI. Our strengths in B cell biology, molecular CSR/SHM mechanisms and autoimmunity, and our cutting-edge epigenetic tools and approaches make us uniquely poised to test our hypotheses. We will: (Aim 1) analyze the impact of HDIs (valproic acid, butyrate and SAHA) on CSR/SHM and plasma cell differentiation, and on specific T-dependent and T-independent antibody responses in normal C57BL/6 and Balb/c mice and autoantibody responses in lupus-prone MRL/Faslpr/lpr and B6.NZM/Sle1.Sle2.Sle3 mice; (Aim 2) use molecular biology, biochemistry and high-throughput/bioinformatics tools to analyze HDI-mediated upregulation of miR-155, miR-181b, miR-361, miR-23b, miR-30a and miR-125b in B cells through enhanced histone acetylation and transcription of the microRNA "host genes", address downregulation of AID and Blimp1 by these microRNAs, and construct microRNA in vivo targeting maps of Aicda and Prdm1 3'UTRs using Ago HITS-CLIP; (Aim 3) prove the critical role of B cell microRNAs in mediating HDI suppression of antibody and autoantibody responses using a three-prong integrated in vivo approach involving construction of new knockin mice lacking specific microRNA targeting sites in Aicda or Prdm1 3'UTR, B cell conditional Dicer or Drosha KO mice, and mice expressing "sponge" inhibitors specific for miR-155, miR-181b and miR-361 (targeting Aicda) and/or for miR-23b, miR-30a and miR-125b (targeting Prdm1) in B cells. Our proposal is highly innovative and exquisitely translational. It will provide mechanistic insights and future directions in epigenetics and immunoregulation, including the critical role of B cell microRNAs in antibody/autoantibody responses and epigenetic tools as new therapeutics in autoimmunity.