Endogenous single-stranded RNA ligands for endosomal Toll-like receptors

内体 Toll 样受体的内源性单链 RNA 配体

• 批准号: 10666209
• 负责人: Yohei Kirino
• 金额: $ 23.4万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10666209
• 关键词: Bacteria; Biological Markers; Bovine Tuberculosis; Cell surface; Cells; Communicable Diseases; Data; Disease; Elements; Endosomes; Foundations; Future; Goals; Human; Immune; Immune response; Individual; Infection; Innate Immune Response; Innate Immune System; Invaded; Investigation; Knock-out; Knowledge; Ligands; Ligation; Lysosomes; Macrophage; MicroRNAs; Microbe; Molecular; Mycobacterium tuberculosis; NF-kappa B; Nature; Nucleic Acids; Pathway interactions; Patients; Pattern; Pattern recognition receptor; Periodicity; Plasma; Procedures; Production; RNA; Receptor Activation; Research; Ribosomal RNA; Sampling; Signal Transduction Pathway; Surface; TLR7 gene; TLR8 gene; Therapeutic; Tissues; Toll-Like Receptor Pathway; Toll-like receptors; Transcriptional Activation; Transfer RNA; Tuberculosis; Untranslated RNA; Virus; cell type; chemokine; cytokine; extracellular; extracellular vesicles; fungus; human disease; inorganic phosphate; insight; monocyte; novel; pathogen; pathogenic microbe; response; therapeutic target; tool; transcription factor; transcriptome; transcriptome sequencing

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. Ligand recognition by TLRs initiates signal transduction pathways that culminate in the activation of transcription factors such as NF-kappaB, resulting in production of cytokines and chemokines to protect the host. A subset of TLRs recognizes nucleic acids as their ligands, and nucleic acids from bacteria, viruses, and fungi have been extensively investigated as TLR stimulators to induce the innate immune responses. Endosomal TLR7 and -8 recognize single-stranded RNAs (ssRNAs) as their ligands. In addition to foreign ssRNAs from bacteria or viruses, endogenous self- ssRNAs, such as microRNAs (miRNAs) in host cells, have been shown to activate TLR7 and -8. However, in contrast to extensive research on foreign ssRNAs, those endogenous ssRNA ligands of ssRNA-sensing TLRs have not been fully elucidated, representing significant scientific knowledge gaps. We propose that many short non-coding RNAs (sncRNAs) remain to be discovered as endogenous ssRNA ligands for TLR7 and -8. The knowledge gaps partly result from technical limitations of standard RNA-seq that is unable to capture all RNA species expressed in the cells and tissues. For example, the RNAs containing a 2′,3′-cyclic phosphate (cP) at the 3′-ends cannot be ligated to a 3′-adapter and thus cannot be amplified and sequenced in standard RNA-seq procedure, forming hidden components of the transcriptome. Despite the “invisible” nature, functional significance of the cP-containing RNA (cP-RNAs) derived from tRNAs and rRNAs have been increasingly apparent in various human diseases including infectious diseases. Our lab has developed “cP-RNA-seq” which can specifically sequence cP-RNAs. In preliminary studies, our cP-RNA-seq identified numerous, abundant tRNA- or rRNA-derived sncRNAs accumulated upon infection of Mycobacterium bovis BCG and accompanying surface TLR activation in human monocyte-derived macrophages (HMDMs), allowing us to observe infection- induced sncRNAs that have never been recognized and characterized previously. Importantly, those sncRNAs are abundantly accumulated not only in HMDMs but also in their secreted extracellular vehicles (EVs), and some specific scnRNAs, such as a 5′-tRNAHisGUG half, have activity to stimulate endosomal TLR7 or -8 when delivered to endosomes of recipient cells. Our further preliminary results showed a large increase of the levels of these sncRNAs in plasma samples of patients infected with Mycobacterium tuberculosis (Mtb). These results have led us to hypothesize that endogenous sncRNAs function as integral elements in the innate immune response by activating ssRNA-sensing TLRs, and we propose to characterize infection-induced sncRNAs in plasma of Mtb- infected patients (Aim 1) and to investigate their activity in endosomal TLR stimulation (Aim 2).

先天免疫系统部署各种模式识别受体，包括toll样受体（TLRs），以识别微生物的入侵并启动保护性反应。tlr在各种细胞类型（如巨噬细胞）的细胞表面或细胞内区室（如内核体和溶酶体）表达，并识别病原体相关的分子模式。TLRs对配体的识别启动信号转导途径，最终激活NF-kappaB等转录因子，从而产生细胞因子和趋化因子来保护宿主。TLR的一个子集识别核酸作为它们的配体，来自细菌、病毒和真菌的核酸作为TLR刺激物诱导先天免疫反应已被广泛研究。内体TLR7和-8识别单链rna （ssrna）作为它们的配体。除了来自细菌或病毒的外源ssRNAs外，内源性自ssRNAs，如宿主细胞中的microRNAs (miRNAs)，已被证明可以激活TLR7和-8。然而，与外源ssRNA的广泛研究相比，ssRNA传感tlr的内源ssRNA配体尚未完全阐明，存在重大的科学知识空白。我们认为许多短链非编码rna （sncRNAs）仍有待发现，作为TLR7和-8的内源性ssRNA配体。知识差距部分是由于标准RNA-seq的技术限制，无法捕获细胞和组织中表达的所有RNA物种。例如，在3 ‘端含有2 ’，3 ' -环磷酸（cP）的rna不能连接到3 ' -适配器上，因此不能在标准rna测序程序中扩增和测序，从而形成转录组的隐藏成分。尽管具有“看不见”的性质，但源自trna和rnas的含cp RNA （cp -RNA）的功能意义在包括传染病在内的各种人类疾病中越来越明显。我们实验室开发了“cP-RNA-seq”，可以对cp - rna进行特异性测序。在初步研究中，我们的cP-RNA-seq鉴定出大量丰富的tRNA或rrna衍生的sncRNAs，这些sncRNAs是在牛分枝杆菌BCG感染后积累的，并伴随人类单核细胞源性巨噬细胞（HMDMs）的表面TLR激活，这使我们能够观察到以前从未被识别和表征过的感染诱导的sncRNAs。重要的是，这些sncrna不仅在HMDMs中大量积累，而且在其分泌的细胞外载体（ev）中也大量积累，并且一些特定的sncrna，如5 ' -tRNAHisGUG一半，在递送到受体细胞的核内体时具有刺激内体TLR7或-8的活性。我们进一步的初步结果显示，在感染结核分枝杆菌（Mtb）的患者血浆样本中，这些sncrna的水平大幅增加。这些结果使我们假设内源性sncRNAs通过激活ssrna传感的TLRs在先天免疫应答中起着不可或缺的作用，我们建议表征Mtb感染患者血浆中感染诱导的sncRNAs (Aim 1)，并研究它们在内体TLR刺激中的活性（Aim 2）。