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Event: 1706

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Stimulation, TLR7/8

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Stimulation of TLR7/8

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Skin disease by stimulation of TLR7/8 MolecularInitiatingEvent Evgeniia Kazymova (send email) Under development: Not open for comment. Do not cite Under Development

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

In general, quantification of TLR7/8 activation can be done by:

  • Reporter gene assay
  • ELISA

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
  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. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. Iwasaki, A. and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune responses. Nature immunology 5(10), 987-995.
  13. 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.
  14. 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.
  15. 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.
  16. 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.
  17. 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.
  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.