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

先天免疫系统部署各种模式识别受体，包括Toll样受体(TLRs)，识别微生物的入侵并启动保护性反应。TLRs在不同类型的细胞(如巨噬细胞)中表达在细胞表面或细胞内隔间(如内小体和溶酶体)，并识别病原体相关的分子模式。TLRs的配体识别启动信号转导通路，最终激活转录因子，如核因子-kappaB，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其配体，来自细菌、病毒和真菌的核酸被广泛研究为TLR的刺激物，以诱导先天免疫反应。内体TLR7和TLR8识别单链RNA作为其配体。除了来自细菌或病毒的外来单链RNA外，内源性自身单链RNA，如宿主细胞中的microRNAs(MiRNAs)，已被证明可激活TLR7和-8。然而，与国外对单链RNA的广泛研究相比，对单链RNA敏感的TLRs的内源单链RNA配体还没有完全阐明，存在着重大的科学知识空白。我们认为，许多短的非编码RNA(SNcRNAs)作为TLR7和-8的内源性单链RNA配体仍有待发现。知识差距的部分原因是标准RNA-seq的技术限制，无法捕获在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’-接头上，因此不能在标准的RNA-SEQ程序中进行扩增和测序，从而形成转录组的隐藏成分。尽管含有CP的RNA(CP-RNAs)具有“看不见”的性质，但由tRNAs和rRNAs衍生而来的CP-RNAs在包括传染病在内的各种人类疾病中的功能意义已经越来越明显。本实验室已研制出能对CP-RNAs进行特异性测序的“CP-RNA-seq”。在初步研究中，我们的CP-RNA-SEQ发现了大量丰富的tRNA或rRNA衍生的SncRNAs，这些SncRNAs是在牛分枝杆菌BCG感染后积累的，并伴随着人单核细胞衍生巨噬细胞(HMDM)表面TLR的激活，使我们能够观察到以前从未发现和表征的感染诱导的SncRNAs。重要的是，这些SncRNAs不仅在HMDM中大量积聚，而且在它们分泌的细胞外载体(EV)中也大量积累，一些特定的scnRNAs，如5‘-tRNAHisGUG一半，当被运送到受体细胞的内体时，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)的患者的血浆样本中，这些SncRNAs的水平显著增加。这些结果使我们假设内源性SncRNAs通过激活单链RNA敏感的TLR在先天性免疫反应中发挥不可或缺的作用，我们建议表征感染诱导的结核分枝杆菌感染患者血浆中的SncRNAs(目标1)，并研究它们在内体TLR刺激中的活性(目标2)。

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).