Human TLR8 is activated upon recognition ofBorrelia burgdorferiRNA in the phagosome of human monocytes

Abstract

Phagocytosed Borrelia burgdorferi (Bb), the Lyme disease spirochete, induces a robust and complex innate immune response in human monocytes, in which TLR8 cooperates with TLR2 in the induction of NF-κB-mediated cytokine production, whereas TLR8 is solely responsible for transcription of IFN-β through IRF7. We now establish the role of Bb RNA in TLR8-mediated induction of IFN-β. First, using TLR2-transfected HEK.293 cells, which were unable to phagocytose intact Bb, we observed TLR2 activation by lipoprotein-rich borrelial lysates and TLR2 synthetic ligands but not in response to live spirochetes. Purified Bb RNA, but not borrelial DNA, triggered TLR8 activation. Neither of these 2 ligands induced activation of TLR7. Using purified human monocytes we then show that phagocytosed live Bb, as well as equivalent amounts of borrelial RNA delivered into the phagosome by polyethylenimine (PEI), induces transcription of IFN-β and secretion of TNF-α. The cytokine response to purified Bb RNA was markedly impaired in human monocytes naturally deficient in IRAK-4 and in cells with knockdown TLR8 expression by small interfering RNA. Using confocal microscopy we provide evidence that TLR8 colocalizes with internalized Bb RNA in both early (EEA1) and late endosomes (LAMP1). Live bacterial RNA staining indicates that spirochetal RNA does not transfer from the phagosome into the cytosol. Using fluorescent dextran particles we show that phagosomal integrity in Bb-infected monocytes is not affected. We demonstrate, for the first time, that Bb RNA is a TLR8 ligand in human monocytes and that transcription of IFN-β in response to the spirochete is induced from within the phagosomal vacuole through the TLR8-MyD88 pathway.

TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-β Production via a TAK1–IKKβ–IRF5 Signaling Pathway

“…Human blood monocytes also sense Borrelia burgdorferi RNA via TLR8, resulting in IFN-b production (43,44). Although this finding is in agreement with our present study on S. aureus, the proposed signaling mechanisms are different, as they suggested IRF7 to be the central transcription factor involved.…”

Bacterial RNA: An Underestimated Stimulus for Innate Immune Responses

“…Similarly, both human PBMCs and murine bone marrow-derived macrophages (BMDMs) responded to intracellular delivery of total bacterial RNA with TNF production (27)(28)(29)(30). In addition to inducing classical NF-kB-dependent cytokines, bacterial RNA has been identified as a strong trigger of IFN-a in plasmacytoid dendritic cells (pDCs) and of IFN-b in myeloid dendritic cells (mDCs) in an IRF-dependent manner (28,(30)(31)(32)(33)(34). Depending on the model system, the involvement of IRF1, IRF3, IRF5, or IRF7 has been implicated (30,31,33,35).…”

“…Early studies using HEK293 cells stably expressing human TLR3, TLR7, and TLR8 indicated that bacterial RNA can trigger IL-8 secretion by all three TLRs (29), and a potential contribution of TLR8 in transcriptional regulation of IFN-b production in human monocytes was suggested by Cervantes et al (31). Yet, the current evidence for a role of TLR8 or other TLRs in bacterial RNA sensing in primary human immune cells is weak or, in the case of NF-kB-dependent cytokines, still lacking.…”

“…Two important points to consider are the release of RNA from bacteria and, thus, the mechanisms of how to get access to any innate immune receptor and second, a point that is often overlooked, the concentration necessary to trigger an immune response. From the studies using purified bacterial RNA, it seems obvious that bacterial RNA, in general, can activate innate immune cells, because Gram-positive and Gram-negative RNA preparations induced cytokine secretion (28,30,31,36,38,39,64). Yet the contribution of RNA to whole bacteria recognition has been less well analyzed.…”

“…Cervantes et al (31,70) showed that B. burgdorferi induced IFN-b in a TLR8-dependent manner and that whole bacteria recognition required TLR2 and TLR8 cooperativity. Along the same lines, B. burgdorferi extracts depleted enzymatically for nucleic acids showed dependency on TLR7 with respect to IFNs, yet they also induced NF-kB-dependent genes (37).…”