Activation of Innate Immunity by Small Ribonucleoprotein

描述（由申请人提供）： 许多患有慢性自身免疫性疾病（例如系统性红斑狼疮 (SLE)）的患者会产生针对普遍存在的宿主细胞分子的自身抗体，例如含有小 RNA U1、U2、U4-6 和 U5 RNA 的 Sm 小核糖核蛋白 (snRNP)。这些小 RNA 是高度结构化的分子，含有广泛的双链（双链）RNA 区域。 RNA病毒复制产生的高浓度dsRNA通过与TLR3和/或dsRNA依赖性蛋白激酶(PKR)等dsRNA结合蛋白结合来激活先天免疫，导致I型干扰素(IFN)的产生增加，从而产生抗病毒反应。该项目的目的是阐明 snRNP 激活系统性红斑狼疮（SLE）先天免疫的机制。 抗 snRNP 自身抗体增加的 SLE 患者的单核细胞表达较高水平的基因，通常由 I 型 IFN 诱导，I 型 IFN 是一种在 SLE 中发挥关键作用的先天细胞因子。 其他初步数据表明，来自 snRNP 的双链 (ds) U1 RNA 刺激单核细胞激活 dsRNA 诱导的蛋白激酶 (PKR)，这是 IFN 反应的标志。 据推测，snRNP 通过 U1 RNA 与先天免疫细胞中的 Toll 样受体 3 (TLR3) 结合，从而触发 IFN 产生。 初步研究表明，具有针对小核糖核蛋白 nRNP/Sm 和 Ro/La 的自身抗体的患者子集的外周血单核细胞表达的 IFN 诱导基因水平高于健康对照或这些自身抗体呈阴性的狼疮患者子集。此外，还发现纯化的真实 U1 RNA 和合成的 U1 茎环 II 是 IFN 诱导基因表达和 PKR 自磷酸化的有效刺激剂。有趣的是，I 型干扰素也与 SLE 的发病机制有关。我们假设与 U1 snRNP 相关的小 RNA 通过刺激 TLR3 和/或 PKR 信号传导，促进对 UI snRNP 蛋白质成分的自身免疫反应。通过刺激 I 型 IFN 的产生，细胞来源的高度结构化小 RNA 可以促进树突状细胞成熟和自身反应性 T 细胞的激活和/或 snRNP 特异性的自身反应性 B 细胞的激活和成熟。提出了三个具体目标。目的 I 是通过研究 IFN 反应是否由 TLR3 信号传导、U1 RNA 内化随后结合和激活 PKR 或其他途径介导，来检查 U1 RNA 如何刺激抗病毒 (IFN) 反应。将鉴定 U1 RNA 中负责免疫刺激作用的部分，并检查免疫抑制性 dsRNA 也存在的可能性。目标 2 是确定通常是什么阻止内源性 U1 RNA 激活 IFN 系统。据推测，与 RNA 结构区域结合的蛋白质会阻止其与 TLR3 或 PKR 结合。将检查一系列部分破坏的 U1 snRNP 片段刺激 IFN 反应或 PKR 激活的能力。目标 3 是确定响应 U1 RNA 而被激活的细胞类型。将确定 U1 RNA 对树突状细胞成熟、巨噬细胞活化和 B 细胞发育的影响。这些研究可能有助于更好地理解为什么 SLE 中的自身免疫反应选择性地针对 Sm（U 系列）和其他 snRNP。通过触发先天免疫，这些分子中包含的高度结构化的 RNA 可以通过促进树突细胞成熟和/或自身反应性 B 或 T 淋巴细胞的激活来促进自身免疫。

DESCRIPTION (provided by applicant): Many patients with chronic autoimmune diseases such as systemic lupus erythematosus (SLE) develop autoantibodies to ubiquitous host cellular molecules, such as Sm small ribonucleoproteins (snRNPs) containing the small RNAs U1, U2, U4-6, and U5 RNAs. These small RNAs are highly structured molecules containing extensive regions of duplex (double-stranded, ds) RNA. High concentrations of dsRNA produced by replicating RNA viruses activate innate immunity by binding to dsRNA binding proteins such as TLR3 and/or the dsRNA dependent protein kinase (PKR), causing increased production of type I interferon (IFN) leading to an antiviral response. The purpose of this project is to elucidate the mechanism by which snRNP activates innate immunity in systemic lupus erythematosus (SLE). Mononuclear cells from SLE patients with increased anti-snRNPs autoantibodies express higher levels of genes usually induced by type-I IFN, an innate cytokine that plays a key role in SLE. Additional preliminary data show that double-stranded (ds) U1 RNA from snRNP stimulates mononuclear cells to activate dsRNA-induced protein kinase (PKR), a hallmark of the IFN response. It is hypothesized that snRNP triggers IFN production by engaging Toll-like receptor 3 (TLR3) in innate immune cells via U1 RNA. Preliminary studies indicated that peripheral blood mononuclear cells from a subset of patients with autoantibodies against the small ribonucleoproteins nRNP/Sm and Ro/La express higher levels of IFN inducible genes than healthy controls or the subset of lupus patients who are negative for these autoantibodies. Moreover, it was found that purified authentic U1 RNA and synthetic stem-loop II of U1 are potent stimulators of IFN-inducible gene expression and PKR autophosphorylation. Interestingly, type I IFNs also have been implicated in the pathogenesis of SLE. We hypothesize that the small RNAs associated with U1 snRNPs promote autoimmune responses to the protein components of UI snRNPs by stimulating signaling via TLR3 and/or PKR. By stimulating type I IFN production, highly structured small RNAs of cellular origin may promote dendritic cell maturation and activation of autoreactive T cells and/or the activation and maturation of autoreactive B cells specific for snRNPs. Three Specific Aims are proposed. Aim I is to examine how U1 RNA stimulates an antiviral (IFN) response by investigating whether the IFN response is mediated by TLR3 signaling, internalization of the U1 RNA followed by binding and activation of PKR, or some other pathway. The portion(s) of U1 RNA responsible for the immunostimulatory effect will be identified and the possibility that immunoinhibitory dsRNAs also exist will be examined. Aim 2 is to determine what normally prevents endogenous U1 RNA from activating the IFN system. It is hypothesized that proteins bound to structured regions of the RNA prevent it from engaging TLR3 or PKR. The ability of series of partially disrupted U1 snRNP fractions to stimulate IFN responses or PKR activation will be examined. Aim 3 is to identify the cell type that becomes activated in response to U1 RNA. The effects of U1 RNA on dendritic cell maturation, macrophage activation, and B cell development will be determined. These studies may lead to a better understanding of why autoimmune responses in SLE selectively target the Sm (U series) and other snRNPs. By triggering innate immunity, the highly structured, RNAs contained in these molecules could promote autoimmunity by promoting dendritic cell maturation and/or the activation of autoreactive B or T lymphocytes.