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Relationship: 2017
Title
Stimulation of TLR7/8 leads to Increase of IL-23
Upstream event
Downstream event
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 | Evgeniia Kazymova (send email) | Under development: Not open for comment. Do not cite | Under Development |
Taxonomic Applicability
Sex Applicability
Life Stage Applicability
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).
| ID | Experimental Design | Species | Upstream Observation | Downstream Observation | Citation (first author, year) | Notes |
|---|
| Title | First Author | Biological Plausibility |
Dose Concordance |
Temporal Concordance |
Incidence Concordance |
|---|
Biological Plausibility
Dose Concordance Evidence
Temporal Concordance Evidence
Incidence Concordance Evidence
Uncertainties and Inconsistencies
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).
Response-response Relationship
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.
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 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).
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).
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
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).
Time-scale
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).
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).
Known Feedforward/Feedback loops influencing this KER
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).
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.
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 IIhigh, CD11bhigh, B220-, CD86high, 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).