API

Event: 1706

Key Event Title

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Stimulation, TLR7/8 in dendritic cells

Short name

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Stimulation of TLR7/8

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Cell term
dendritic cell


Organ term

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Organ term
immune system


Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Skin disease by stimulation of TLR7/8 MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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Life Stages

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Sex Applicability

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Key Event Description

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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 (Akira et al. 2006, Blasius and Beutler. 2010). They also mediate the recognition of self RNA that is released from dead or dying cells.

Human TLR7 (hTLR7) and human TLR8 (hTLR8) contains 1049, 1041 amino acid residues with a calculated 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. TLR8 contains 26 leucine-rich repeats (LRRs), which is the largest number of LRRs among TLRs whose structures have been reported (Tanji et al. 2013).

Monkey TLR7 exists as a monomer in the absence of ligands, and TLR7 dimerization is induced by R848 alone, but not by poly U or guanosine alone, although these two ligands synergistically triggered TLR7 dimerization (Zhang et al. 2016). In contrast, hTLR8 exists as preformed dimer before ligand recognition. TLR8 is activated by R848 alone, or both uridine and ssRNA synergistically (Tanji et al. 2013).

The key residues interacting two TLR7 molecules into dimer confirmation are LYS410, ASN503, SER504, GLY526, ASN527, SER530, THR532, ARG553, and TYR579 (Gupta et al. 2016).

TLR3, TLR7, TLR8, and TLR9 localize to the endoplasmic reticulum and are trafficked to the endosomal compartment where they initiate cellular responses upon their activation by PAMPs and DAMPs (Lai et al. 2017).

TLR7 are exclusively expressed in plasmacytoid DCs (pDCs), which have the capacity to secrete vast amounts of type I IFN rapidly in response to viral infection (Gilliet et al. 2008, Reizis et al. 2011).

TLR8 is expressed 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.

TLR7-mediated signaling in pDC is mediated in a MyD88-dependent fashion, which initiates an IRF7-mediated response, secreting vast amounts of IFN type 1 (Kawai and Akira. 2011).

MyD88-dependent IRF7 activation in pDCs is mediated by activation of IRAK1, TRAF6, TRAF3, and IKKα and is facilitated by IFN-inducible Viperin expressed in the lipid body (Kawai and Akira. 2011).

IRF7, which is constitutively expressed by pDCs, binds MyD88 and forms a multiprotein signaling complex with IRAK4, TRAF6, TRAF3, IRAK1 and IKKα (Kawai and Akira. 2008). In this complex, IRF7 becomes phosphorylated by IRAK1 and/or IKKα, dissociates from the complex and translocates into the nucleus.

The interferons (IFNs) are a primary defense against pathogens because of the strong antiviral activities they induce. Three types of IFNs, types I, II and III, have been classified based on of their genetic, structural, and functional characteristics and their cell-surface receptors (Zhou et al. 2014). IFN-α belongs to the type I IFNs, the largest group which includes IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ and IFN-ζ.

The IFN-α of type I IFN family in humans is composed of 12 subtypes encoded by 14 nonallelic genes including one pseudogene and two genes that encode the same protein. The various IFN-α subtypes have many common points. For example, all are clustered on chromosome 9 (Diaz et al. 1993). IFN-αs, which are composed of 165 to 166 aa, have 80% amino acid sequence identities (Li et al. 2018).

Upon engagement of ssRNAs in endosomes, TLR8 initiate the MyD88-dependent pathway culminating in synthesis and release of proinflammatory mediators, such as TNF-α via NF-κB activation (Tanji et al. 2015).

Stimulation of blood DCs with self-RNA–LL37 complexes induces a robust TNF-α response (Hänsel et al. 2011). DC activation is known to be enhanced by IFN-α in the presence of TNF-α (Luft et al. 1998).


How It Is Measured or Detected

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HEK293 cells were transiently co-transfected with human TLR7 and NF-κB-luciferase reporter. After 48 hours, the cells were 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). R848 (0.001-10 µg/mL) induced NF-κB activation in HEK293 cells transfected with human TLR8 is detected in the same manner (Jurk et al. 2002).

IFN-α in cell-free supernatants collected after imidazoquinoline stimulation to human PBMC and/or pDC-enriched cells is detected by ELISA (Gibson et al. 2002).

TNF-α and IL-6 in cell-free supernatants collected after RNA-LL37 stimulation to mDCs were measured by ELISA (Ganguly et al. 2009). mDCs were also stained with fluorochrome-labeled anti-CD80, anti-CD86, and anti-CD83 antibodies and analyzed by flow cytometry (Ganguly et al. 2009).

 


Domain of Applicability

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Thirteen mammalian TLR members (10 in humans and 13 in mice) have been identified so far, of which TLR1, 2, 4, 5, and 6 are membrane bound and catalytic site for pathogenic structural components, whereas TLR3, 7, 8, and 9 expressed within the endosomal compartment

are dedicated to nucleic acids. TLRs 1–9 are conserved among humans and mice, yet TLR10 is present only in humans and TLR11 strictly restricted to rodents (Gupta et al. 2016).

Mouse TLR10 is not functional because of a retrovirus insertion, and TLR11, TLR12 and TLR13 have been lost from the human genome. (Kawai and Akira. 2010).

In addition, alignment of amino acid residues between human toll-like receptor 7 (AAF60188.1) and murine toll-like receptor 7 (AGX25544.1) was 80.74% identification. Both proteins have 1049 amino acid residues.

Structural characterization was conducted with recombinant TLR7 from monkey (Macaca mulatta; 96.8% sequence identify with human TLR7) expressed in Drosophila S2 cells (Zhang et al. 2016).

Studies of DC subsets isolated from humans and mice have revealed that TLRs have distinct expression patterns. Freshly isolated human pDCs express TLR7 and TLR9, whereas CD11c+ human myeloid DCs (mDCs) express TLR1, TLR2, TLR3, TLR5, TLR6 and TLR8. In some studies, TLR7 expression was detected on both pDCs and mDCs, whereas others found TLR7 was exclusively expressed in pDCs (Iwasaki and Medzhitov. 2004).

In mice, all splenic DC subsets express TLRs 1, 2, 4, 6, 8 and 9. However, mouse pDCs do not express TLR3. Moreover, mouse CD8α+ DCs lack TLR5 and TLR7 expression and fail to respond to TLR7 agonists. In short, CD4+ DC, CD4/CD8DN DC and pDC express TLR7 in mice (Iwasaki and Medzhitov. 2004).


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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References

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  1. Akira, S., Uematsu, S. and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124(4): 783-801.
  2. Blasius, A.L. and Beutler, B. (2010). Intracellular toll-like receptors. Immunity 32(3), 305-315.
  3. 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.
  4. Diaz, M.O., Bohlander, S. and Allen, G. (1993). Nomenclature of the human interferon genes. Journal of interferon research 13(3), 243-244.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. Iwasaki, A. and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune responses. Nature immunology 5(10), 987-995.
  11. 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.
  12. 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.
  13. 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.
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  15. 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.
  16. Li, S.F., Gong, M.J., Zhao, F.R., Shao, J.J., Xie, Y.L., Zhang, Y.G. and Chang, H.Y. (2018). Type I interferons: Distinct biological activities and current applications for viral infection. Cell physiology and biochemistry 51(5), 2377-2396.
  17. Luft, T., Pang, K.C. Thomas, E., Hertzog, P., Hart, D.N., Trapani, J. and Cebon, J. (1998). Type I IFNs enhance the terminal differentiation of dendritic cells. Journal of immunology 161(4), 1947-1953.
  18. 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.
  19. 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.
  20. 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.
  21. 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.
  22. Zhou, H., Chen, S., Wang, M. and Cheng, A (2014). Interferons and their receptors in birds: a comparison of gene structure, phylogenetic analysis, and cross modulation. International journal of molecular sciences 15(11), 21045-21068.