Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Skin disease by stimulation of TLR7/8||MolecularInitiatingEvent|
Key Event Description
Toll-like receptors (TLRs) are members of interleukin-1 (IL-1) receptor/TLR superfamily, as they share the intracellularToll-IL-1 receptor (TIR) domain with the IL-1 receptor.
Toll-like receptor (TLR) 7 and TLR8 is known to mediate the recognition of guanosine- and uridine-rich single-stranded RNA (ssRNA) derived from ssRNA viruses and synthetic antiviral imidazoquinoline components specifically that lead to activation of sequential signalling pathway (Akira et al. 2006, Blasius and Beutler. 2010). They also mediate the recognition of self RNA that is released from dead or dying cellsand activation of Myeloid differentiation primary response 88 (MyD88)-dependent signals can occur that leads to inflammation process as well as ssRNA derived from viruses.
TLR7 are exclusively expressed in plasmacytoid DCs (pDCs), which have the capacity to secrete vast amounts of type I interferon (IFN) in rapid response to viral infection (Gilliet et al. 2008, Reizis et al. 2011). TLR8 is express in various tissues, with its highest expression in monocytes. Myeloid DCs (mDCs) also express TLR8 in human (Iwasaki and Medzhitov. 2004). Thus, TLR8 ligands can directly activate mDCs via TLR8. TLR8 signalling activates mDCs to secrete TNF-α and IL-6 (Ganguly et al. 2009). TLR7 and TLR8 are localize in the endoplasmic reticulum of expressing cells (Lai et al. 2017).
Human TLR7 (hTLR7) and human TLR8 (hTLR8) contain 1049 and 1041 amino acid residues, respectively with molecular weight of 120.9 kDa and 119.8 kDa, respectively (Chuang and Ulvitch. 2000). The full-length hTLR7 protein includes a signal peptide of 26 amino acids (1–26 aa). The mature hTLR7 protein ectodomain, trans-membrane, and TIR domain are composite structure of 27–839, 840–860, and 889–1,036 amino acids, respectively (Gupta et al. 2016).
hTLR7 and hTLR8 form a subfamily of proteins that each contain an extracellular domain of >800 residues and share functional and structural features. hTLR7 and hTLR8 contains 27 and 26 leucine-rich repeats (LRRs), which is the largest number of LRRs among TLRs whose structures have been reported (Tanji et al. 2013).
As mentioned above, TLR7 and TLR8 are localize in the endoplasmic reticulum of expressing cells. They are deliver to the endosomes by interacting UNC93B1, which is a 12 membrane-spanning protein (Kawai and Akira. 2011, Itoh et al. 2011). After the trafficking, they initiate cellular responses upon their activation by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Lai et al. 2017).
Structural characterization was conducte with recombinant TLR7 from monkey (Macaca mulatta; 96.8% sequence identity with human TLR7) expressed in Drosophila S2 cells (Zhang et al. 2016). Rhesus TLR7 exists as a monomer in the absence of ligands. This TLR7 is activate by dimerization triggered by guanosine and uridine-containing ssRNA, which are degradation products of ssRNA, synergistically. Specifically, this TLR7 molecule has two ligand-binding sites. The first site conserved in TLR7 and TLR8 is use for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 is use for ssRNA-binding that enhances the affinity of the first-site ligands. The first site preferentially recognize guanosine and the second site specifically bound to non-terminal uridine in ssRNA which have more than 3 bases. Rhesus TLR7 is also activate by dimerization induced by resiquimod alone, which is bound to only the first site (Zhang et al. 2016).
In contrast, hTLR8 exists as preformed dimer before ligand recognition. hTLR8 molecule has two ligand-binding sites as well as TLR7. The first site preferentially recognize uridine and second site recognize short-oligonucleotide. hTLR8 transforms into activated form upon binding of these two degradation products of ssRNA. hTLR8 is also activated by transformation induced by resiquimod alone, which is bound to only the first site (Tanji et al. 2015).
The key residues involved in TLR7 dimerization are LYS410, ASN503, SER504, GLY526, ASN527, SER530, THR532, ARG553, and TYR579 (Gupta et al. 2016).
Cellular signalling initiated by TLR7 activation with ssRNA in pDC is mediated in a Myeloid differentiation primary response 88 (MyD88)-dependent fashion, and activates NF-κB and IRF7, which results in induction of inflammatory cytokines and type I interferon, respectively (Kawai and Akira. 2011).
MyD88-dependent IRF7 activation in pDCs is mediate by activation of IRAK1, TRAF6, TRAF3, and IKKα and is facilitate by IFN-inducible Viperin expressed in the lipid body (Kawai and Akira. 2011).
IRF7, which is constitutively expresse by pDCs, binds MyD88 and forms a multiprotein signalling complex with IRAK4, TRAF6, TRAF3, IRAK1 and IKKα (Kawai and Akira. 2008). In this complex, IRF7 becomes phosphorylate by IRAK1 and/or IKKα, dissociates from the complex and translocates into the nucleus to induce transcription of type I IFN by binding to its promoter proximal region (Kawai and Akira. 2008, Génin et al. 2009).
Signalling initiated by TLR8 engagement with ssRNAs in endosomes is also mediated the MyD88-dependent pathway culminating in synthesis and release of proinflammatory mediators, such as TNF-α via NF-κB activation (Tanji et al. 2015).
How It Is Measured or Detected
In general, quantification of TLR7/8 activation can be done by:
- Reporter gene assay
Measurement of transcriptional activation of human TLR and NF-κB-luciferase co-transfected cells
HEK293 cells were transiently co-transfected with human TLR7 and a NF-κB-luciferase reporter. The cells were incubated for 48 hours following transfection and then stimulated with various concentrations of resiquimod or imiquimod. Luciferase activity was measured 48h post-stimulation and the results are reported as fold-increase in luciferase production relative to medium control (Gibson et al. 2002). Likewise, resiquimod (0.001-10 µg/mL) induced NF-κB activation in HEK293 cells transfected with human TLR7 or human TLR8 and a NF-κB-luciferase reporter is detected in the same manner (Jurk et al. 2002).
Measuring of cytokine levels in supernatants
IFN-α in cell-free supernatants collected after stimulation of human PBMC and/or pDC-enriched cells by imidazoquinoline derivatives is detected by ELISA (Gibson et al. 2002).
TNF-α and IL-6 in cell-free supernatants collected after stimulation of mDCs by RNA-LL37 are measured by ELISA (Ganguly et al. 2009).
Domain of Applicability
TLR7 and TLR8 are conserved among humans and mice (Gupta et al. 2016). In addition, these molecules are also conserved among humans and rhesus monkeys.
Alignment of amino acid residues between human toll-like receptor 7 (NP_057646.1) and murine toll-like receptor 7 (NP_573474.1, NP_001277684.1, NP_001277685.1, NP_001277686.1, NP_001277687.1, XP_006528776.1, XP_011246087.1 and XP_011246088.1) was 80.74-80.76% identification. Murine TLR7 proteins have 1050 or 1053 amino acids. Human and rhesus toll-like receptor 7 (NP_001123898.1) was 98.00% identification. Rhesus TLR7 protein has 1049 amino acid residues.
In addition, alignment of amino acid residues between human toll-like receptor 8 (NP_619542.1) and murine toll-like receptor 8 (NP_001300689.1) was 70.97% identification. Likewise, human and rhesus toll-like receptor 8 (NP_001123899.1) was 96.73% identification. Murine and rhesus TLR8 referred here have 1029 and 1039 amino acids residues, respectively.
Studies of DC subsets isolated from humans and mice have revealed that TLRs have distinct expression patterns. TLR7 is expressed in freshly isolated human pDCs, whereas TLR8 is expressed in CD11c+ human myeloid DCs (mDCs). In some studies, TLR7 expression was detected on both pDCs and mDCs, whereas other reports showed that TLR7 was exclusively expressed in pDCs (Iwasaki and Medzhitov. 2004).
In mice, CD4+ dendritic cells (DC), CD4/CD8 double negative DC and pDC express TLR7. All splenic DC subsets express TLR8. Moreover, mouse CD8α+ DCs lack TLR7 expression and fail to respond to TLR7 agonists. (Iwasaki and Medzhitov. 2004).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
- Akira, S., Uematsu, S. and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124(4): 783-801.
- Blasius, A.L. and Beutler, B. (2010). Intracellular toll-like receptors. Immunity 32(3), 305-315.
- Chuang, T.H. and Ulevitch R.J. (2000). Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. European cytokine network 11(3), 372-378.
- Diaz, M.O., Bohlander, S. and Allen, G. (1993). Nomenclature of the human interferon genes. Journal of interferon research 13(3), 243-244.
- Ganguly, D., Chamilos, G., Lande, R., Gregorio, J., Meller, S., Facchinetti, V., Homey, B., Barrat, F.J., Zal, T. and Gilliet, M. (2009). Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8. Journal of experimental medicine 206(9), 1983-1994.
- Génin, P., Lin, R., Hiscott, J. and Civas, A. (2009). Differential regulation of human interferon A gene expression by interferon regulatory factor 3 and 7. Molecular and cellular biology 29(12), 3435-3450.
- Gibson, S.J., Lindh, J.M., Riter, T.R., Gleason, R.M., Rogers, L.M., Fuller, A.E., Oesterich, J.L., Gorden, K.B., Qiu, X., McKane, S.W., Noelle, R.J., Kedl, R.M., Fitzgerald-Bocarsly, P. Tomai, M.A. and Vasilakos, J.P. (2002). Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cellular immunology 218(1-2), 74-86.
- Gilliet, M., Cao, W. and Liu, Y.J. (2008). Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nature reviews immunology 8(8), 594-606.
- Gupta, C.L., Akhtar, S., Sayyed, U., Pathak, N. and Bajpai P. (2016). In silico analysis of human toll-like receptor 7 ligand binding domain. Biotechnology and applied biochemistry 63(3), 441-450.
- Hänsel, A., Günther, C., Ingwersen, J., Starke, J., Schmitz, M., Bechmann, M., Meurer, M., Rieber, E.P. and Schäkel, K. (2011). Human slan (6-sulfoLacNAc) dendritic cells are inflammatory dermal dendritic cells in psoriasis and drive strong TH17/TH1 T-cell responses. Journal of allergy and clinical immunology 127(3), 787-794.
- Itoh, H., Tatematsu, M., Watanabe, A., Iwano, K., Funami, K., Seya, T. and Matsumoto, M. (2011). UNC93B1 physically associates with human TLR8 and regulates TLR8-mediated signaling. PLoS One 6(12), e28500.
- Iwasaki, A. and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune responses. Nature immunology 5(10), 987-995.
- Jurk, M., Heil, F., Vollmer, J., Schetter, C., Krieg, AM., Wagner, H., Lipford, G. and Bauer, S. (2002). Human TLR7 and TLR8 independently confer responsiveness to the antiviral compound R848. Nature immunology 3(6), 499.
- Kawai, T. and Akira, S. (2008). Toll-like receptor and RIG-I-like receptor signaling. Annals of the New York academy of sciences 1143, 1-20.
- Kawai, T. and Akira, S. (2010). The role of pattern-recognition receptors in innate immunity:update on toll-like receptors. Nature immunology 11(5), 373-384.
- Kawai, T. and Akira, S. (2011). Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34(5), 637-650.
- Lai, C.Y., Su, Y.W., Lin, K.I., Hsu, L.C. and Chuang, T.H. (2017). Natural modulators of endosomal toll-like receptor-mediated psoriatic skin inflammation. Journal of immunology research 7807313, 15 pages.
- Reizis, B., Bunin, A., Ghosh, H.S., Lewis, K.L. and Sisirak, V. (2011). Plasmacytoid dendritic cells: recent progress and open questions. Annual reviews of immunology 29, 163-183.
- Tanji, H., Ohto, U., Shibata, T., Miyake, K. and Shimizu, T. (2013). Structural reorganization of the toll-like receptor 8 dimer induced by agonistic ligands. Science 339(6126), 1426-1429.
- Tanji, H., Ohto, U., Shibata, T., Taoka, M., Yamauchi, Y., Isobe, T., Miyake, K. and Shimizu, T. (2015). Toll-like receptor 8 senses degradation products of single-stranded RNA. Nature structural and molecular biology 22(2), 109-115.
- Zhang, Z., Ohto, U., Shibata, T., Krayukhina, E., Taoka, M., Yamauchi, Y., Tanji, H., Isobe, T., Uchiyama, S., Miyake, K. and Shimizu, T. (2016). Structural analysis reveals that toll-like receptor 7 is a dual receptor for guanosine and single-stranded RNA. Immunity 45(4), 737-748.