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)，来识别微生物的入侵并启动保护反应。 TLR 在各种细胞类型（例如巨噬细胞）的细胞表面或细胞内区室（例如内体和溶酶体）表达，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 NF-κB 等转录因子，从而产生细胞因子和趋化因子来保护宿主。 TLR 的一个子集将核酸识别为其配体，来自细菌、病毒和真菌的核酸已作为 TLR 刺激物被广泛研究，以诱导先天免疫反应。内体 TLR7 和 -8 将单链 RNA (ssRNA) 识别为其配体。除了来自细菌或病毒的外源 ssRNA 之外，内源性自身 ssRNA，例如宿主细胞中的 microRNA (miRNA)，已被证明可以激活 TLR7 和 -8。然而，与国外对 ssRNA 的广泛研究相比，ssRNA 传感 TLR 的内源 ssRNA 配体尚未完全阐明，代表了重大的科学知识差距。我们提出，许多短非编码 RNA (sncRNA) 作为 TLR7 和 -8 的内源 ssRNA 配体仍有待发现。知识差距部分是由于标准 RNA-seq 的技术限制造成的，它无法捕获细胞和组织中表达的所有 RNA 种类。例如，在 3' 末端含有 2',3'-环磷酸 (cP) 的 RNA 无法连接到 3'-接头，因此无法在标准 RNA-seq 程序中进行扩增和测序，从而形成转录组的隐藏成分。尽管具有“看不见”的性质，但源自 tRNA 和 rRNA 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日益明显。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 鉴定了牛分枝杆菌 BCG 感染以及人单核细胞衍生巨噬细胞 (HMDM) 中伴随的表面 TLR 激活时积累的大量丰富的 tRNA 或 rRNA 衍生的 sncRNA，使我们能够观察以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，而且在其分泌的细胞外载体 (EV) 中也大量积累，并且一些特定的 scnRNA，例如 5'-tRNAHisGUG half，在递送至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，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 sncRNA 通过激活 ssRNA 感应 TLR 在先天免疫反应中发挥不可或缺的作用，并且我们建议表征 Mtb 感染患者血浆中感染诱导的 sncRNA（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).