This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Relationship: 2017

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Stimulation of TLR7/8 leads to Increase of IL-23

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Stimulation of TLR7/8

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increase of IL-23

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Stimulation of TLR7/8 in dendric cells leading to Psoriatic skin disease	adjacent	High	High	Hiroyuki Komatsu (send email)	Under development: Not open for comment. Do not cite	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. 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 (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).

A distinct population of human blood DCs that are defined by the selective expression of the 6-sulfo LacNAc residue on the P-selectin glycoprotein ligand 1 membrane molecule was described previously. 6-Sulfo LacNAc DCs (slanDCs) stand out by a marked production of TNF-α, and they were recognized as the major source of IL-12p70 among blood leukocytes when stimulated with CD40 ligand or LPS of gramnegative bacteria (Hänsel et al. 2011).

According to the current concept, these inflammatory DCs are CD1c^–, CD11c⁺ cells locally expressing TNF-α and iNOS. They were also referred to as TNF and inducible nitric oxide synthase–expressing DCs (Tip-DCs) (Lowes et al. 2005) or inflammatory dermal DCs (Zaba et al. 2009). In contrast, resident dermal DCs express CD1c and CD11c and were shown to lack inflammatory markers. The phenotype of slanDCs (CD11c⁺ and CD1c^-) and their local production of IL-23p19, TNF-α, and iNOS identify slanDCs as being a population of inflammatory dermal DCs and so-called Tip-DCs in psoriasis (Hänsel et al. 2011).

Stimulation of blood DCs with self-RNA–LL37 complexes induced a robust TNF-α response (Hänsel et al. 2011). TNF-α affects Tip-DCs in an autocrine and/or paracrine manner (Zaba et al. 2007).

DC activation is known to be enhanced by IFN-α in the presence of TNF-α (Luft et al. 1998).

R848 induces IL-23 production from activated human monocyte-derived DCs (moDCs) by enhanced transcriptional activity (Schwarz et al. 2013).

IL-23 is a heterodimer, sharing a p40 subunit with IL-12 but having a distinct p19 subunit. IL-23 binds to IL-12Rβ1 but not IL-12Rβ2. The receptor for this cytokine is heterodimeric and uses a novel second subunit, IL-23R, which is a member of the hematopoietin receptor family (Lee et al. 2004).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The molecular structure and function of TLR7 and TLR8 are evident based on sufficient scientific findings as mentioned above. The known mechanisms for stimulation of TLR7/8 by each ligand are initiated by the formation of homodimer. TLR7-mediated signaling in pDC is mediated in a MyD88-dependent fashion, which initiates an IRF7, IRAK1, TRAF6, TRAF3, and IKKα-mediated response, secreting vast amounts of IFN type 1 (Kawai and Akira. 2011).

Similarly, upon engagement of ligands 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).

DC activation is known to be enhanced by IFN-α in the presence of TNF-α (Luft et al. 1998).

R848 induces IL-23 production from activated human monocyte-derived DCs (moDCs) by enhanced transcriptional activity (Schwarz et al. 2013).

TNF and inducible nitric oxide synthase–expressing DCs also known as Tip-DCs or inflammatory dermal DCs differentiates from moDCs by inflammation (Hänsel et al. 2011).

As mentioned above, stimulation of TLR7 in pDCs, and TLR8 in moDCs and Tip-DCs leads to activation of Tip-DCs, which leads to the overproduction of IL-23 from matured Tip-DCs.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Much experimental data is available that supports the stimulation of TLR7 in pDC induced by TLR7 agonist, which subsequently promote secretion of IFN-α in MyD88-dependent fashion. For example, three populations of cells were evaluated for type I IFN production following imidazoquinoline stimulation: human PBMC, pDC-depleted PBMC, and pDC-enriched cells. Human PBMC produce IFN-α following imiquimod (0.3–30 μM) or resiquimod (0.03–30 μM) treatment. Peak levels of IFN-α were reached with imiquimod and resiquimod at 3 μM. PBMC, depleted of pDC, did not produce detectable levels of IFN-α in response to imiquimod or resiquimod treatment.

The imidazoquinoline-treated pDC-enriched cultures produced 2–20 times more IFN-α than similarly treated PBMC as measured over the entire dose range. Peak levels of Resiquimod- and imiquimod-induced IFN-α production were reached with 0.3 μM and 30 μM, respectively (Gibson et al. 2002).

In addition, pDCs were stimulated with LL37 premixed with total human RNA extracted from U937 cells to confirm that LL37 can interact with self-RNA and convert it into a trigger of IFN-α production. U937-derived self-RNA induced dose-dependent IFN-α production when mixed with LL37, but not when given alone or mixed with the scrambled peptide GL37. Similar to self-DNA (Lande et al., 2007), pDCs activated by self-RNA mixed with LL37 produced high levels of IFN-α, but did not produce TNF-α or IL-6 or undergo maturation as assessed by measuring the expression of costimulatory molecules CD80 and CD86 (Ganguly et al. 2009).

Importantly, self-RNA isolated from a variety of cell types and tissue samples from various types of skin pathologies induced similar levels of IFN-α when mixed with LL37, indicating that cellular- or disease-dependent variations in RNA composition do not play a role in the responses to self-RNA. These data demonstrate that LL37 can convert otherwise nonstimulatory self-RNA into a trigger of pDC activation to produce IFN-α, and thus enable the RNA released during cell death to induce innate immune activation (Ganguly et al. 2009).

IFN-α induced in pDCs by self-RNA–LL37 complexes was inhibited in a dose-dependent manner by bafilomycin, which blocks endosomal acidification and TLR signaling. To specifically inhibit TLR7, we used the short oligonucleotide C661, which selectively blocks TLR7 (Barrat et al. 2005), as shown by the inhibition of IFN-α induction by the synthetic TLR7 agonist R837 but not the TLR9 agonist CpG2006. Pretreatment of pDCs with C661 specifically blocked the IFN-α induction by self-RNA–LL37 complexes, indicating that pDC activation by self-RNA–LL37 complexes occurs through TLR7 (Ganguly et al. 2009).

Self-RNA–LL37 complexes but not self-RNA alone activated mDCs to produce the proinflammatory cytokines TNF-α and IL-6, but not IFN-α (Ganguly et al. 2009). Self-RNA–LL37 complexes also activated mDCs to undergo maturation as shown by the up-regulation of CD80 and CD86 expression (Ganguly et al. 2009) . mDC activation by self-RNA–LL37 complexes was entirely dependent on self-RNA, given that these responses were abrogated by decreasing the amount of self-RNA in the complexes (unpublished data). In contrast to self-RNA–LL37 complexes, self-DNA–LL37 complexes were unable to activate mDCs (Ganguly et al. 2009) . In accordance with these findings, stimulation of mDCs with supernatants of apoptotic cells combined with LL37 induced the secretion of proinflammatory cytokines, and this secretion was entirely dependent on self-RNA because activity was abolished by depletion of self-RNA but not self-DNA (Ganguly et al. 2009).

Compared with stimulation with either supernatant of activated pDCs or self-RNA–LL37 alone, the combination of both significantly enhanced the activation of mDCs to secrete IL-6 and TNF-α and enhanced their differentiation into mature CD83+ DCs (Ganguly et al. 2009). This activity was completely blocked by antibodies against IFN-α, IFN-β and IFN-αβR (Ganguly et al. 2009). Thus, self-RNA–LL37 complexes can trigger mDC activation and maturation, and this process is enhanced by the concomitant activation of pDCs to produce IFN-α.

Self-RNA was also internalized by mDCs when complexed with LL37 but not when given alone. The cytokine production such as TNF-α and IL-6 of mDCs induced by self-RNA–LL37 complexes but not by the TLR4 agonist LPS was completely inhibited by bafilomycin in a dose-dependent manner, demonstrating that mDC activation by self-RNA–LL37 complexes involved endosomal TLR activation. Using 293T cells transfected with TLR8 and TLR3 expression vectors along with a NF-κB luciferase reporter plasmid, it was confirmed that self-RNA–LL37 complexes activated TLR8 but not TLR3. In support of this finding, synthetic short ssRNA sequences that activate TLR8 in human mDCs (Diebold et al. 2004, Heil et al. 2004) also activated mDCs when complexed with LL37 but not when given alone (Ganguly et al. 2009).

Dose-dependent DC maturation was observed with increasing concentrations from 10 IU/ml up to 1000 IU/ml of IFN-α2a or IFN-α8 added to cultures containing GM-CSF, IL-4, and TNF-α. Both of the IFNs had a similar capacity to up-regulate HLA-A, B, C, CD80, and CD86 and to down-regulate CD1a and CD11b expression on the cell population (Luft et al. 1998).

DC cultured in GM-CSF, TNF-α, and IL-4-containing medium until day 14, and type I IFNs were added daily between days 14 and 17. Proportions of positive cells for each markers were analyzed by FACS on day 17 (Luft et al. 1998).

When GM-CSF, TNF-α, and IL-4-containing cultures were washed on day 14 and continued until day 17 in serum-free medium containing GM-CSF and IL-4, without or with TNF-α (20 ng/ml, standard conditions), IFN-a (1000 IU/ml), or both, IFN-α alone did not enhance DC maturation as compared with standard conditions. When both of TNF-α and IFN-α exist, optimal maturation was observed than either TNF-α or IFN-α alone. Thus, the enhancement of DC activation by IFN-α under serum-free conditions required the presence of TNF-α (Luft et al. 1998).

LL37 is highly expressed in the inflamed skin of psoriasis but is undetectable in inflamed skin of atopic dermatitis or in healthy skin (Lande et al. 2007). To determine whether extracellular self-RNA–LL37 complexes form in vivo, Staining skin sections with Ribogreen and DAPI revealed that numerous extracellular Ribogreen⁺/ DAPI^- complexes in the dermal compartment of psoriatic skin lesions, but not in skin of atopic dermatitis or healthy skin (Ganguly et al. 2009). These tissue RNA complexes presented several features of self-RNA–LL37 complexes generated in vitro, including the size and bead-like branched structures resulting from the aggregation of smaller particles (Ganguly et al. 2009).

Skin sections of psoriatic tissues were stained with an anti-LL37 antibody and Ribogreen to determine whether the self-RNA complexes in the tissues contained LL37 and it was found that the majority of these complexes contained LL37 (Ganguly et al. 2009). Importantly, psoriatic skin also contained substantial numbers of particulate self-DNA–LL37 complexes.

Serial sections of lesional psoriatic skin were stained for RNA complexes and DC-LAMP, a lysosomal marker specific for mature mDCs to determine whether the presence of tissue self-RNA complexes is associated with the presence of activated DCs in psoriatic skin. Consistent with previous reports (Lowes et al. 2005), it was found that large clusters of DC-LAMP–positive mature mDCs (Ganguly et al. 2009). We also found tissue self-RNA–LL37 complexes within these clusters, and, occasionally, even inside the DCs as shown by the colocalization with endolysosomal compartments stained with DC-LAMP (Ganguly et al. 2009). The number of tissue self-RNA complexes significantly correlated with the numbers of DC-LAMP–positive mDCs in psoriatic skin (Ganguly et al. 2009). Together, these findings strongly support in vitro data that self-RNA complexes can activate mDCs and suggest that this pathway is operational in psoriasis.

When mRNA expression normalized to HARP for IL-23 subunits, such as p19 and p40 were quantified by RT-PCR in monocyte-derived DCs (moDCs) matured without and with etanercept, a dimeric human tumor necrosis factor recepter p75-Fc fusion protein made of 2 extra-cellular domains of the human 75kD TNFR linked by the constant Fc portion of human IgG1 (Haraoui and Bykerk. 2007), significant decrease in expression of IL-23 subunits p19 and p40 by etanercept were observed (Zaba et al. 2007). MoDCs cultured with etanercept decreased CD86 expression threefold and HLA-DR expression fivefold. In addition, moDCs cultured with etanercept were also an average of two to threefold less stimulatory than control DCs in a mixed leukocyte reaction. Gene array on control moDCs compared with those cultured with etanercept revealed that CD163, a macrophage scavenger receptor, was up-regulated 6.5-fold (Zaba et al. 2007).

In psoriatic dermis, mRNA expression normalized to HARP for multiple inflammatory products of Tip-DCs, including iNOS, TNF-α and IL23 p40 subunit, are reduced within 1–2 weeks after beginning etanercept, whereas the number of CD11c⁺ DCs in the tissue is minimally affected during this time, suggesting an initial blockade of cytokine production by these cells rather than cell reduction (Zaba et al. 2007). These facts suggest that TNF-α is an autocrine or paracrine inducer of IL-23 from Tip-DC (Zaba et al. 2007).

R848-treatment to moDCs, which were generated from monocytes isolated from buffy coats of healthy donors, resulted in concentration-dependent expression of IL-23. 2×10⁵ moDCs/ml were plated in DC medium and stimulated with 0-5 µg/ml R848. After 24 h of TLR stimulation, supernatants were harvested and cytokine expression was measured by ELISA. In addition, the combination of NOD1 and NOD2 agonists with R848 stimulated high levels of IL-23 secretion (Schwarz et al. 2013).

qRT-PCR for moDCs stimulated with TLR agonists in the absence or presence of NOD1 and NOD2 ligands at 8 h and 24 h post induction revealed that synergistic cytokine expression observed in NOD1/NOD2- and R848-stimulated cells is largely mediated by enhanced transcriptional activity (Schwarz et al. 2013).

In time kinetic studies, moDCs were stimulated with R848 in the absence or presence of MDP and iE-DAP which are ligands of NOD1/2, for 30 min, 2 h, 8 h or 24 h and mRNA levels of IL-23 as well as the cumulative cytokine release were measured by qRT-PCR and sandwich-ELISA, respectively. At the mRNA level, synergistic effects of both NOD ligands with R848 are already detectable after 8 h of stimulation. In agreement with IL-23 mRNA expression, synergistic effects are detectable by ELISA after 8 h; nevertheless, these effects are even more pronounced after 24 h of stimulation (Schwarz et al. 2013).

These findings show that dose responses and temporality of MIE and KE1 seem to be in sequence.

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Although unpublished, it has been reported that human slanDCs (Tip-DCs) lack the DNA-binding structureTLR9 but can express the endosomal RNA-binding receptorsTLR8 (slanDCs andCD1c⁺ DCs) and TLR7 (slanDCs but not CD1c⁺ DCs; Hänsel et al, unpublished data, June 2010) (Hänsel et al. 2011). There are not any other reports which mentioned TLR7 expression in Tip-DCs, so whether or not TLR7 exists in human Tip-DCs is still unknown.

In addition, freshly isolated human pDCs have been reported to 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. Therefore, it is still unknown that whether or not TLR7 exists in human mDCs, and how much it does contribute recognition of R848 or LL37-RNA in these cells (Iwasaki and Medzhitov. 2004).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

MIE:

Much experimental data is available that supports the stimulation of TLR7 in pDC induced by TLR7 agonist, which subsequently promote secretion of IFN-α in MyD88-dependent fashion. For example, 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 relative to medium control. As a result, dose-dependent increase in NF-κB-dependent luciferase activity in HEK293 transfected with hTLR7 was observed with increasing concentrations from 0.01 μM up to 10 μM of resiquimod, and 0.1 μM up to 15 μM of imiquimod. Maximal NF-κB activation with resiquimod is achieved with 10-30 μM, which yields an 18-fold increase in luciferase production. Maximal NF-κB activation with imiquimod requires 10-15 μM compound and induces a 5-6-fold increase in luciferase production (Gibson et al. 2002).

In addition, three populations of cells were evaluated for type I IFN production following imidazoquinoline stimulation: human PBMC, pDC-depleted PBMC, and pDC-enriched cells. Human PBMC produce IFN-α following imiquimod (0.3–30 μM) or resiquimod (0.03–30 μM) treatment. Peak levels of IFN-α were reached with imiquimod and resiquimod at 3 μM. PBMC, depleted of pDC, did not produce detectable levels of IFN-α in response to imiquimod or resiquimod treatment.

In different experiments, pDCs were stimulated with LL37 premixed with total human RNA extracted from U937 cells to confirm that LL37 can interact with self-RNA and convert it into a trigger of IFN-α production. U937-derived self-RNA induced dose-dependent IFN-α production when mixed with LL37, but not when given alone or mixed with the scrambled peptide GL37 (Ganguly et al. 2009).

R848 (0.001-10 µg/mL) induced NF-κB activation in HEK293 cells transfected with human TLR8 in a dose-dependent manner (Jurk et al. 2002). In addition, the production of TNF-α and IL-6, and the maturation

of mDCs induced by self-RNA–LL37 complexes but not by the TLR4 agonist LPS was completely inhibited by bafilomycin in a dose-dependent manner, demonstrating that mDC activation by self-RNA–LL37 complexes involved endosomal TLR activation (Ganguly et al. 2009).

In accordance with these findings, compared with stimulation with either supernatant of activated pDCs or self-RNA–LL37 alone, the combination of both significantly enhanced the activation of mDCs to secrete IL-6 and TNF-α and enhanced their differentiation into mature CD83+ DCs (Ganguly et al. 2009). This activity was completely blocked by antibodies against IFN-α, IFN-β and IFN-αβR (Ganguly et al. 2009). Thus, self-RNA–LL37 complexes can trigger mDC activation and maturation, and this process is enhanced by the concomitant activation of pDCs to produce IFN-α.

KE 1

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Human PBMC, pDC-deficient PBMC, and pDC -enriched from human PBMC (pDC-enriched) were cultured with various concentrations of resiquimod or imiquimod. After 24 h in culture, cell-free supernatants were collected and IFN-α was analyzed by ELISA (Gibson et al. 2002).

Suspensions containing RNA-LL37 or supernatants of dying cells were added to pDC and mDC cultures. After overnight culture, supernatants of pDCs and mDCs were collected and IFN-α, TNF-α and IL-6 were measured by ELISA (Ganguly et al. 2009). pDCs and mDCs were also stained with fluorochrome-labeled anti-CD80, anti-CD86, and anti-CD83 antibodies and analyzed by flow cytometry. mDCs were also cultured with supernatants of pDCs stimulated for 24 h with self-DNF-LL37 (Ganguly et al. 2009).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

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, CD4CD8DN DC and pDC express TLR7 in mice (Iwasaki and Medzhitov. 2004).

IFN-α, but not TNF-α and IL-6 production by human pDCs after stimulation with self-RNA-LL37 complex was detected (Ganguly et al. 2009). However, in mice, IFN-α production from splenic pDCs was induced by IMQ treatment. The production of TNF-α and IL-23 was also induced by IMQ treatment. Although 4–8% of mPDCA-1^- CD11c⁺ DCs were contaminated in prepared mPDCA-1⁺ pDC fraction, it was confirmed that splenic mPDCA-1^- CD11c⁺ DCs enriched to approximately 80% purity could not produce TNF-α and IL-23 by IMQ stimulation. In Tlr7^-/- splenic pDCs, these cytokines (IFN-α, TNF-α and IL-23) were not induced by IMQ treatment, although stimulation by CpG, a TLR9 ligand, resulted in induction of these cytokines at the same level as was produced by wild-type splenic pDCs. These data indicate that, in mice, IMQ application can induce the production via TLR7 of IFN-α, TNF-α and IL-23 from pDCs existing in the skin in vivo (Ueyama et al. 2014).

When BMDCs were generated by 10-day culture with GM-CSF and IL-4 and characterized their phenotypes, CD11c+ BMDCs showed MHC II^high, CD11b^high, B220^-, CD86^high, Mac-3⁺, and had the ability to produce high levels of TNF-α and NO/iNOS in response to LPS stimulation, which represents a similar phenotype to Tip-DCs (Xu et al. 2007, Ueyama et al. 2014).

In these BMDCs which represents a similar phenotype to Tip-DCs, IMQ weakly but significantly induced the production of IL-23. In addition, although IFN-α had no effect alone, co-stimulation with IFN-α and IMQ resulted in marked induction of IL-23 production. However, using BMDCs derived from Tlr7^-/- mice, these effects of IMQ and IFN-α was not observed, verifying that it is also mediated via TLR7 (Ueyama et al. 2014).

In mice, purified bone marrow dendritic cells (BMDCs) derived from wild-type mice stimulated with IFN-α showed increase in Tlr7 mRNA expression (Ueyama et al. 2014). In addition, TLR7 expression was also observed in the inflamed skin of IMQ-treated mice (Ueyama et al. 2014). These data suggest that the synergistic effect of IMQ and IFN-α on BMDCs was caused by induction of TLR7 expression by IFN-α (Ueyama et al. 2014).

Taken together, in mice, IFN-α produced by IMQ-primed pDCs may enhance the effects of IMQ to activate Tip-DC, resulting in the release of a large amount of IL-23 in IMQ-induced psoriasis-like skin lesion (Ueyama et al. 2014).

References

List of the literature that was cited for this KER description. More help

Akira, S., Uematsu, S. and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124(4): 783-801.
Barret, F.J., Meeker, T., Gregorio, J., Chan, J.H., Uematsu, S., Akira, S., Chang, B., Duramad, O. and Coffman, R.L. (2005). Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. Journal of experimental medicine, 202(8), 1131-1139.
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.
Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. and Reis e Sousa, C. (2004). Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science, 303(5663), 1529-1531.
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.
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.
Haraoui, B. and Bykerk, V. (2007). Etanercept in the treatment of rheumatoid arthritis. Therapeutics and clinical risk management 3(1), 99-105.
Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G., Wagner, H. and Bauer, S. (2004). Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 303(5663), 1526-1529.
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.
Lande, R., Gregorio, J., Facchinetti, V., Chatterjee, B., Wang, Y.H., Homey, B., Cao, W., Wang, Y.H., Su, B., Nestle, F.O., Zal, T., Mellman, I., Schrӧder, J.M., Liu, Y.J. and Gilliet, M. (2007). Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449(7162), 564-569.
Lee, E., Trepicchio, W.L., Oestreicher, J.L., Pittman, D., Wang, F., Chamian, F., Dhodapkar, M. and Krueger, J.G. (2004). Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris. Journal of experimental medicine 199(1), 125-130.
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.
Lowes, M.A., Chamian, F., Abello, M.V., Fuentes-Duculan, J., Lin, S.L., Nussbaum, R., Novitskaya, I., Carbonaro, H., Cardinale, I., Kikuchi, T., Gilleaudeau, P., Sullivan-Whalen, M., Wittkowski, K.M., Papp, K., Garovoy, M., Dummer, W., Steinman, R.M. and Krueger, J.G. (2005). Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a). Proceedings of the national academy of sciences of the United States of America 102(52), 19057-19062.
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.
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.
Schwarz, H., Posselt, G., Wurm, P., Ulbing, M., Duschl, A. and Horejs-Hoeck, J. (2013). TLR8 and NOD signaling synergistically induce the production of IL-1β and IL-23 in monocyte-derived DCs and enhance the expression of the feedback inhibitor SOCS2. Immunobiology 218(4), 533-42.
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.
Xu, Y., Zhan, Y., Lew, A.M., Naik, S.H. and Kershaw, M.H. (2007). Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking. Journal of immunology 179(11), 7577-7584.
Zaba, L.C., Cardinale, I., Gilleaudeau, P., Sullivan-Whalen, M., Suárez-Fariñas, M., Fuentes-Duculan, J., Novitskaya, I., Khatcherian, A., Bluth, M.J., Lowes, M.A. and Krueger, J.G. (2007). Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. Journal of experimental medicine 204(13), 3183-3194.
Zaba, L.C., Krueger, J.G. and Lowes, M.A. (2009). Resident and “inflammatory” dendritic cells in human skin. Journal of investigative dermatology 129(2), 302-308.
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.
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.