Role of rRNA-derived short non-coding RNAs in innate immune response

项目概要和摘要 微生物感染会引起多种症状和疾病。持续努力了解 针对微生物感染的细胞防御系统机制对于制定有效的预防措施至关重要 方法和治疗应用。先天免疫系统部署各种模式识别受体， 包括 Toll 样受体 (TLR)，用于识别微生物的入侵并启动保护反应。 TLR 在细胞表面或细胞内区室（例如内体和溶酶体）以多种形式表达。 细胞类型，例如巨噬细胞，并识别病原体相关分子模式 (PAMP)，例如 脂多糖（LPS）和肽聚糖（PGN）。 PAMP 识别启动信号转导 最终激活转录因子（例如 NF-κB）的途径，从而产生 细胞因子和趋化因子来保护宿主。 TLR 下游的转录调控 途径涉及多个层面的调控，包括转录后步骤。在此背景下，短非 编码 RNA (ncRNA) 已发展成为关键的转录后调节因子。各种短ncRNA中 来自核糖体 RNA (rRNA) 的物种，称为 rRNA 片段 (rRF)，是最丰富的一类 短 ncRNA，最近其表达和功能意义受到越来越多的关注。 rRF 在包括人类在内的多种生物体中组成型表达，其表达参与 细胞周期和增殖、细胞凋亡以及脂质和葡萄糖代谢的调节。在人类中，他们的 表达以性别和人群特异性方式调节，并与 2 型糖尿病和其他疾病相关 疾病。我们认为 rRF 在先天免疫反应中发挥重要作用。在初步研究中，我们 发现人单核细胞来源的表面 TLR 的激活可上调 rRF 的表达 巨噬细胞（HMDM）。重要的是，短的 ncRNA 测序数据证明了丰富的积累 rRF 不仅存在于 HMDM 中，而且存在于其分泌的细胞外载体 (EV) 中。进一步的实验 结果表明，特定的细胞外 rRF 激活受体 HMDM 中的内体 TLR，从而诱导细胞因子 分泌，表明 rRF 不仅作为 rRNA 降解副产物积累，而且 积累为促进免疫反应的功能分子。在拟议的研究中，我们将确定 HMDM 及其 EV 中 TLR 途径诱导的 rRF 的表达谱（目标 1）。我们将进一步阐明 细胞外 rRF 在内体 TLR 激活中的分子功能（目标 2）。最后，我们将描述 健康受试者和结核分枝杆菌感染患者血浆样本中的 rRF 表达 （目标 3）。通过定义一类新的 TLR 通路诱导的 rRF 并进一步确定它们的作用，我们的研究 将揭示先天免疫反应中一种新的 rRNA 参与的 ncRNA 途径，并可能支持未来 探索微生物感染后针对 ncRNA 的生物标志物和有效的治疗应用。

Project Summary and Abstract Infection of microbes causes a wide range of symptoms and diseases. Sustained efforts to understand the mechanism of cellular defense systems against microbe infections are essential to develop effective prevention methods and therapeutic applications. The innate immune system deploys various pattern-recognition receptors, including Toll-like receptors (TLRs), to recognize the invasion of microbes and initiate protective responses. TLRs are expressed at the cell surface or intracellular compartments (e.g., endosome and lysosome) in various cell types, such as macrophages, and recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and peptidoglycan (PGN). The PAMP recognition initiates signal transduction pathways that culminate in the activation of transcription factors such as NF-B, resulting in production of cytokines and chemokines to protect the host. Following transcriptional regulation, the downstream of TLR pathways involves regulation at multiple levels including post-transcription steps. In this context, short non- coding RNAs (ncRNAs) have evolved as key post-transcriptional regulators. Among various short ncRNA species, those from ribosomal RNAs (rRNAs), termed rRNA fragments (rRFs), are the most abundant class of short ncRNAs and have recently gained increasing attentions on their expression and functional significance. rRFs are constitutively expressed in multiple organisms including humans, and their expression is involved in regulation of cell cycle and proliferation, apoptosis, and lipid and glucose metabolism. In humans, their expression is modulated in a sex- and population-specific manner, and associated with type 2 diabetes and other diseases. We propose that rRFs play important roles in innate immune response. In preliminary studies, we found that the expression of rRFs is upregulated by the activation of surface TLR in human monocyte-derived macrophages (HMDMs). Importantly, short ncRNA sequencing data demonstrated the abundant accumulation of the rRFs not only in HMDMs but also in their secreted extracellular vehicles (EVs). Further experiments showed that a specific extracellular rRF activates endosomal TLR in the recipient HMDMs to induce cytokine secretion, suggesting that rRFs are not just accumulated as degradation by-products of rRNAs, but are accumulated as functional molecules promoting immune response. In the proposed studies, we will identify the expression profiles of TLR pathway-induced rRFs in HMDMs and their EVs (Aim 1). We will further elucidate the molecular function of extracellular rRFs in endosomal TLR activation (Aim 2). Finally, we will characterize the rRF expression in plasma samples from healthy subjects and patients infected with Mycobacterium tuberculosis (Aim 3). By defining a novel class of TLR pathway-induced rRFs and further establishing their roles, our study will reveal a novel rRNA-engaged ncRNA pathway in the innate immune response and may support the future exploration of biomarkers and efficacious therapeutic applications targeting ncRNAs upon microbe infection.