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（TLR），以识别微生物的入侵并启动保护性反应。TLR在细胞表面或细胞内区室（例如，核内体和溶酶体），并识别病原体相关的分子模式。TLR的配体识别启动信号转导途径，最终激活转录因子如NF-κ B，导致产生细胞因子和趋化因子以保护宿主。TLR的一个子集识别核酸作为它们的配体，并且来自细菌、病毒和真菌的核酸已经被广泛研究作为TLR刺激物以诱导先天性免疫应答。内体TLR 7和TLR 8识别单链RNA（ssRNA）作为其配体。除了来自细菌或病毒的外源ssRNA之外，内源性自身ssRNA，如宿主细胞中的微小RNA（miRNA），已显示激活TLR 7和TLR 8。然而，与对外源ssRNA的广泛研究相反，ssRNA敏感TLR的那些内源性ssRNA配体尚未完全阐明，这代表了重大的科学知识空白。我们提出，许多短的非编码RNA（sncRNA）仍然被发现作为TLR 7和TLR 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种类。例如，在3′端含有2′，3 ′-环磷酸（cP）的RNA不能连接到3′-衔接子上，因此不能在标准RNA-seq程序中扩增和测序，形成转录组的隐藏组件。尽管具有“不可见”的性质，但源自tRNA和rRNA的含cP-RNA（cP-RNA）的功能性意义在包括感染性疾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鉴定了在牛分枝杆菌BCG感染后积累的大量丰富的tRNA或rRNA衍生的sncRNA以及伴随的人单核细胞衍生的巨噬细胞（HMDM）中的表面TLR活化，使我们能够观察到先前从未被识别和表征的感染诱导的sncRNA。重要的是，这些sncRNA不仅在HMDM中大量积累，而且在它们分泌的细胞外载体（EV）中也大量积累，并且一些特异性scnRNA（例如5′-tRNAHisGUG半体）在递送至受体细胞的内体时具有刺激内体TLR 7或-8的活性。我们进一步的初步结果显示，这些sncRNA的水平在感染结核分枝杆菌（Mtb）的患者的血浆样品中有很大的增加。这些结果使我们假设内源性sncRNA通过激活ssRNA敏感TLR在先天性免疫应答中作为组成部分起作用，并且我们提出表征Mtb感染患者血浆中感染诱导的sncRNA（目的1）并研究它们在内体TLR刺激中的活性（目的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).