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

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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).