Relationship: 2017: Stimulation of TLR7/8 leads to Increase of IL-23

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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Although unpublished, it has been reported that human slanDCs (Tip-DCs) lack the DNA-binding structureTLR9 but can express the endosomal RNA-binding receptorsTLR8 (slanDCs andCD1c⁺ DCs) and TLR7 (slanDCs but not CD1c⁺ DCs; Hänsel et al, unpublished data, June 2010) (Hänsel et al. 2011).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 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).

Key Event Relationship Description

Toll-like receptors (TLRs) are members of interleukin-1 (IL-1) receptor/TLR superfamily, as they share the intracellularToll-IL-1 receptor (TIR) domain with the IL-1 receptor.

Toll-like receptor (TLR) 7 and TLR8 is known to mediate the recognition of guanosine- and uridine-rich single-stranded RNA (ssRNA) derived from ssRNA viruses and synthetic antiviral imidazoquinoline components (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 Supporting this KER

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.

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.

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).

Quantitative Understanding of the Linkage

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).

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).

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Relationship: 2018: Increase of IL-23 leads to Th17 cell migration and inflammation induction

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

In mice, application of IL-23 causes psoriatic-like epidermal hyperplasia, but this effect does not occur in IL-17A and IL-22KO mice. Therefore, it is thought that IL-17A and IL-22 play an important role downstream of IL-23（Rizzo HL. Et al. 2011）.

Recombinant mIL-23 (rmIL-23) injected into the ear of WT mice induced IL-17A and IL-22 expression, and showed ear swelling and epidermal hyperplasia. When rmIL-23 was injected into IL-22 KO mice, IL-22 was induced, but ear swelling and epidermal hyperplasia were less than in WT mice. When rmIL-23 was injected into IL-17A KO mice, IL-22 was induced, but ear swelling and epidermal hyperplasia hardly occurred. WT mice after administration of IL-22 or IL-17A inhibitor completely inhibited IL-23-induced epidermal hyperplasia. These results indicate that two cytokines, IL-22 and IL-17A, are downstream mediators of IL-23-induced changes in mouse skin and are required for the generation of IL-23-mediated skin lesions. (Hansel et al. 2011)

Key Event Relationship Description

IL-23 is important for differentiation and proliferation of Th17 cells. As a major source of IL-23, Tip-DC is present in the skin lesions of psoriatic patients and works to activate the Th17 pathway (Hansel et al. 2011).

Signaling through the heterodimeric IL-23 receptor (subunits of p19 and p40) of Th17 cells stimulates the production of proinflammatory keratinocyte cytokines that mediate the psoriatic response and induces the production of IL-17. Th17 cells are increased in the peripheral blood and lesion skin of psoriatic patients, and IL-17 and IL-22 produced from Th17 act on epidermal keratinocytes to cause inflammatory chemokines and hyperproliferation (Michelle A. et al. 2005).

IL-17A, which is highly expressed by Th17 cells, has a direct effect on the regulation of genes expressed by keratinocytes that are involved in innate immune defense, including defensins,8, 9 S100 family proteins, lipocalin, and LL37/cathelicidin, as well as a range of CXCL chemokines that regulate neutrophil trafficking (Gilliet et al. 2004). IL-22, which is expressed by Th22 and Th17 cells, and related IL-20 family members promote keratinocyte hyperproliferation and abnormal differentiation (Krueger et al. 2012).

Evidence Supporting this KER

IL-17A, which is highly expressed by TH17 cells, has a direct effect on the regulation of genes expressed by keratinocytes that are involved in innate immune defense, thorough expressions of defensins,8, 9, S100 family proteins, lipocalin and LL37/cathelicidin, as well as a range of CXCL chemokines that regulate neutrophil trafficking. IL-22, which is expressed by TH22 and TH17 cells, and related IL-20 family members promote keratinocyte hyperproliferation and abnormal differentiation （Gilliet et al.2012）.

In vitro Reconstituted Human Epidermis (RHE) model stimulated for 48 hours with medium containing IL-17, IL-22 and TNFα mix (concentration 3 ng / mL) as psoriasis-specific cytokines. Controls were cultured in normal medium. After fixing RHE and embedding in paraffin, 4 μm sections were stained with hematoxylin-eosin or immunolabeled with anti-filaggrin, anti-S100A7, anti-hBD-2 mAb.

RHE stimulated with cytokine mix showed dramatic expression of these protein. In the RHE with normal medium, antibacterial peptide S100A7 was expressed locally, but BD-2 protein was not detected. This is due to the synergistic effect of IL-17 added to the IL-22 / TNFα combination. Filaggrin, S100A7 and BD-2 mRNA expression by RT-qPCR analysis increased 20-fold (S100A7) or -50-fold (BD-2) compared to controls. This is a downstream event that can be modeled using keratinocytes and cytokines and relies on upstream mechanisms of recruitment and activation of other innate adaptive immune cells.（Bernard et al. 2012.）.

Quantitative Understanding of the Linkage

KE1:

IL-23, which maintains Th17 cells, is released from TNF-a and inducible nitric oxide synthase (iNOS) -producing dendritic cells (TIP-DC). TIP-DC activates IL-17, IL-22, IL-23, and TNF-a mRNA expression in psoriatic skin. Cytokine staining analysis of peripheral blood mononuclear cell (PBMC) in patients with psoriasis showed a three-fold increase in Th17 cells compared to normal PBMC. Thl7 cells produce IL-22 and stimulate keratinocyte proliferation. IL-22 activates STAT3 and induces the production of cytokine (such as IL-8), chemokines and the synthesis of antimicrobial peptides (Zaba et al. 2005).

KE 2

The epidermis of psoriasis patients did not have many T cells, but the analysis was similar to peripheral blood and dermis. The proportion of Th17 cells in the dermis was significantly higher than that in normal skin, and TNF and IFN-g were produced from Th17 cells. Skin and peripheral blood contained a subset of Th17 cells producing IFN-g / TNF.

Keratin 16, IL-17, IFN-g, and IL-22 mRNA expression increased in psoriatic skin, but cyclosporine therapy returned these mRNA to normal levels. The average expression of IL-17 / human acidic ribosomal protein (hARP) in non-lesional skin was 0.4 compared to 10.8 in lesional skin, and cyclosporine administration returned to non-lesional levels. That IL-17 mRNA return to baseline, effective treatment supports that Th17 in psoriasis is a central pathogenic.(Lowes et al.2008)

References

1. Anja Hänsel,Claudia Günther, Jens Ingwersen, Josephine Starke, Marc Schmitz, MichaelBachmann,Michael Meurer, Ernst Peter Rieber, Knut Schäkel. Human slan (6-sulfo LacNAc) dendritic cells are inflammatory dermal dendritic cells in psoriasis and drive strong TH17/TH1 T-cell responses.J.Allergy. Clin. Immunol. 2011, 127, 787-794.
2. Michelle A. Lowes, Francesca Chamian, Maria Veronica Abello, Judilyn Fuentes-Duculan,Shao-Lee Lin, Rachel Nussbaum, Inna Novitskaya, Henrietta Carbonaro, Irma Cardinale, Toyoko Kikuchi, Patricia Gilleaudeau, Mary Sullivan-Whalen, Knut M. Wittkowski, Kim Papp, Marvin Garovoy, Wolfgang Dummer, Ralph M. Steinman, James G. Kruege. Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a). Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 19057–19062.
3. James G Krueger, Scott Fretzin, Mayte Suarez-Farinas, Patrick A Haslett, Krista M Phipps, Gregory S Cameron, Juliet Mccolm, Artemis Katcherian, Inna Cueto, Traci White, Subhashis Banerjee, and Robert W Hoffman. IL-17A is essential for cell activation and inflammatory gene ciorcuits in subjects with psoriasis. Jourmal of Allergy and Clinical Immunology 2012, 130(1): 145-154
4. Michel Gilliet, Curdin Conrad, Michael Geiges, Antonio Cozzio, Wolfgang Thürlimann, Günter Burg, Frank O. Nestle, Reinhard Dummer. Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.Arch.Dermatol.2004, 140, 1490-1495.・Bernard 2012
5. Lisa C. Zaba, Irma Cardinale, Patricia Gilleaudeau, Mary Sullivan-Whalen, Mayte Suárez-Fariñas, Judilyn Fuentes-Duculan, Inna Novitskaya, Artemis Khatcherian, Mark J. Bluth, Michelle A. Lowes, James G. Krueger. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. J. Exp. Med. 2007, 204, 3183-3194.
6. Michelle A. Lowes, Toyoko Kikuchi, Judilyn Fuentes-Duculan, Irma Cardinale, Lisa C. Zaba, Asifa S. Haider, Edward P. Bowman, and James G. Krueger. Psoriasis Vulgaris Lesions Contain Discrete Populations of Th1 and Th17 T Cells. Journal of Investigative Dermatology. 2008, 128, 1207-1211.
7. Rizzo HL, Kagami S, Phillips KG, Kurtz SE, Jacques SL, Blauvelt A. IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A. J Immunol. 2011 Feb 1; 186(3): 1495-502.

Relationship: 2019: Th17 cell migration and inflammation induction leads to Skin disease

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Initiation of plaque formation in the Aldara psoriasis mouse model is dependent on RORγt +, skin infiltrating γδ T cells, and innate lymphocyte cells (ILC). Vγ4 + γδT cells and innate lymphoid cells (ILC) are the dominant and important sources of IL-17A, IL-17F, and IL-22 in the formation of acute psoriatic lesions, rather than Th cells (Pantelyushin et al. 2012).

Amyloid A: SAA, an inflammatory marker, is high in the serum of patients with psoriasis. When C57B6 mice were given SAA protein subcutaneously on the back, epidermal thickening and inflammatory cell wetting were frequent on days 5-7. Skin inflammation was significantly suppressed when IL-12 / IL-23p40 protein, a target molecule of psoriasis biologics, was administered in advance. By SAA administration, a similar reaction to psoriatic eruption was formed in the immunological reaction, and a psoriatic eruption model mouse was established. (J Dermatolog Trest. 2013; 24 (6): 477-80)

Key Event Relationship Description

Th17 cells produce the cytokines IL-17 and IL-22. IL-17 is inflammatory, promotes the migration of neutrophils to psoriatic lesions, contributes to the formation of Munro's micro-abscess, and through DCL and memory T cells, including Th17 cells and CCR6, via CCL20 Incorporate into the affected area. IL-22 causes abnormal keratinocyte proliferation by down-regulating genes that control terminal differentiation, leading to altered differentiation and keratinization. Both IL-17 and IL-22 induce keratinocyte expression of the antibacterial S100A7 (psoriacin).（Nograles et al. 2008）

STAT3 is important for Th17 differentiation. Cytokine signaling SOCS3-deficient mice show increased IL-17 expression by increasing STAT3 activity in response to IL-23 binding to IL-17. Associated with increased activity of STAT3 in response to IL-23 capable of binding to IL-17 and IL-17F promoters. STAT3 overexpression promotes Th17 differentiation, whereas STAT3 deficiency inhibits Th17 differentiation. STAT3 signaling from IL-6, IL-21, IL-23 regulates the expression of lineage specific master transcription factors RORγt22, 63, 66 and RORα67. It has been found that patients with psoriasis with mutations in STAT3 cannot generate a Th17 response.（Martinez et al. 2008）

Evidence Supporting this KER

The biological activity of the combination of cytokines was investigated. The combination of IL-17A and IFN-γ or IL-17A and TNF-α has a synergistic effect on CXCL8 production by keratinocytes. IL-17A and IL-22 exert a synergistic effect in upregulation of β-defensin 2: BD-2 and S100A9 production] IL-1α, IL-17, IL-22, Oncostatin M: OSM, and TNFα binding are associated with increased expression of inflammatory molecules such as soriacin / S100A7 or BD-2, IL-8 in vitro by NHEK Although very potent synergistic, removal of IL-22 from the cytokine mixture reduces CXCL8 and BD-2 expression by 30% and removal of IL-17 reduces it by 70%.

Ex vivo studies on human skin explants showed upregulation of BD-2, S100A7, and CXCL8 expression in response to the same combination of cytokines, and intradermal injection of cytokines into mice resulted in neutrophil infiltration and early epidermis CXCL1, CXCL2, CXCL3, S100A9, and BD-3 expression related to epidermal thickening was increased. (Bernard et aaal. 2012)

Resident memory tissue T cells (TRM cells) confer both resistance and immunity depending on the local microenvironment, and CD8 TRM can be tracked by phenotypic markers CD49a and CD103. Circulating effector T cells infiltrate the site of skin inflammation and turn into long-lived epidermal TRM cells when the skin inflammation is resolved. Epidermal TRM cells are thought to form pathological site-specific disease memory at the site of recurrent psoriasis.(Cheuk et al. 2014)

Single cell suspensions of epidermis and dermis were analyzed by flow cytometry within 30 hours of sampling. In active psoriasis, CD8 T cells increased about 100-fold in the epidermis compared to normal skin, whereas CD4 T cells increased 10-fold in the dermis. In healthy skin, 20-30% of epidermal CD8 T cells co-expressed integrin CD103 and CD49a, which are phenotypic markers of TRM cells. In active psoriasis, approximately half of epidermal CD8 T cells co-expressed these TRM phenotypic markers, a 100-fold increase compared to healthy skin.（Cheuk et al. 2014）

Cytokines cannot be specified for genes associated with abnormalities in psoriatic skin. Many genes that are up-regulated in psoriatic lesions can be attributed to IFN-γ, including IL-17 and IL-22. Cytokines synthesized by Th1 / Th17 cells regulate different gene expression pathways in epidermal keratinocytes and other skin resident cells. The psoriatic transcriptome may result from activation of multiple independent pathways.（Nograles et al. 2008）

After daily topical application of Aldara containing imiquimod (IMQ) to humans, significant skin thickening, redness and scaling were observed 3 days later (doi: 10.1172 / JCI61862DS1). The clinical course of plaque formation on the ear and back skin and histopathology were similar. Aldara treatment resulted in impaired keratinocyte hyperproliferation and epidermal differentiation, as indicated by epidermal thickening and hyperkeratosis. There was a terminal neutrophil accumulation in the stratum corneum reminiscent of a Munro micro-abscess in psoriasis. Extensive leukocyte infiltration was observed in the dermis.(Pantelyushin et al. 2012)

Quantitative Understanding of the Linkage

KE2

High levels of Th17 cytokines were observed in psoriatic skin induced by CD4 + T cells. IL-23 p40 subunit or IL-22 significantly prevented the development of skin lesions.

IL-22-induced acantosis and inflammation were reduced in IL-22-deficient mice compared to WT mice. Blocking IL-22 increases IL-1α, IL-1β, IL-6, IL-17, IL-17F, and TNF-α. (K. A. et al. 2013)

AO

Anti-IL-17 antibody administration results in decreased keratinocyte proliferation and differentiation, leukocyte infiltration, and keratinocyte release of inflammatory cytokines. In psoriatic lesioned keratinocytes, changes in mRNA and protein expression of IL-17 regulatory products occurred. Within 2 weeks of antibody administration, the expression of LL37 (cathelicidin), β-defensin 2, S100A7, and S100A8 proteins was markedly decreased in keratinocytes, and the expression reached normal levels after 6 weeks.(Krueger et al. 2012)

Epidermal keratinocyte expression genes that were elevated in psoriatic lesions of patients with psoriasis with stage-type skin eruption: mRNA expression level of keratin6a and 16, s100A7A, S100A12, DEFB4, IL-1F6, CCL20, IL-17C, etc. was rapidly reduced by 700㎎  single intravenous dose of brodalumab and decreased to non-lesional skin level 2 weeks after administration. On the other hand, leukocyte expression genes with increased expression in psoriatic lesion skin: IL-17A, IL-17F, IL-23F, IL-12B, IL-22, IFN-γ and other mRNA expression levels decreased with brodalumab administration. However, at 2 weeks after administration, the level did not decrease to the level of the non-lesional skin. Since the expression of pathophysiology-related genes is reduced prior to the decrease in the expression of leukocyte expression genes is reduced prior to the  decrease in the expression of leukocyte expression genes and the decrease in the PASI score, Brodalumab is reduced expression of  pathophysiology-related genes by blocking IL-17 signaling in the epidermal keratinocytes of psoriatic lesions It is possible to improve the skin eruption promptly. (Kyowa Hakko Kirin Co., Ltd.)

References

1. K. E. Nograles, L.C. Zaba, E. Guttman, J. Fuentes-Duculan, M. Suarez-Farinas, I. Cardinale, A. Khatcherian, J. Gonzalez, K. C. Pierson, T. R. White, C. Pensabene, I. Novitskaya, M. A. Lowes, and J. G. Krueger. Th17 cytokines interleukin(IL)-17 and IL-22 modulate distinct inflammatory and kerationocyte-response pathways. Br J Dermatol 2008 Nov.; 159(5): 1092-1102.
2. Gustavo J. Martinez, Roza I. Nurieva, Xuexian O. Yang, and Chen Dong. Regulation and Function of Proinflammatory TH17 Cells. Ann N. Y. Acead Sci. 2008 Nov; 1143: 188-211.
3. Francois-Xaver Bernard, Franck Morel, Magalie Camus, Nathalie Pedretti, Christine Barrault, Julien Garnier, and Jean-Claude Lecron. Keratinocytes under Fire of Proinflammatory Cytokines: Bone Fide Innate Immune Cells Involved in the Physiopathology of Chronic Atopic Dermatitis and Psoriasis. Journal of Allergy. 2012. Article ID 718725, 10.
4. Stanley Cheuk, Maria Wiken, Lennart Blomqvist, Susanne Nylen, Toomas Taqlme, Mona Stahle and Liv Eidsmo. Epidermal Th22 and Tc17 Cells From a Localized Disease Memory in Clinically Healed Psoriasis. J Immunol. 2014 Apr 1; 192(7): 3111-3120. 2014
5. Stanislav Pantelyushin, Stefan Haak, Barbara Ingold, Paulina Kulig, Frank L. Hepponer, Alexander A. Navarini, and Burkhand Becher. Roryt innate lymphocytes and yo T cells intiate psoriasiform plaque foemation in mice. J Clin Invest. 2012 Jun 1; 122(6): 2252-2256.
6. K.A. Papp, R.G. Langley, B. Sigurgeirsson, M. Abe,D.R. Baker, P. Konno, S. Haemmerle, H.J. Thurston, C. Papavassilis, H.B. Richards. Efficacy and safety of secukinumab in the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind,placebo-controlled phase II dose-ranging study. Br. J. Dermatol. 2013, 168, 412-421.
7. James G Krueger, Scott Fretzin, Mayte Suarez-Farinas, Patrick A Haslett, Krista M Phipps, Gregory S Cameron, Juliet Mccolm, Artemis Katcherian, Inna Cueto, Traci White, Subhashis Banerjee, and Robert W Hoffman. IL-17A is essential for cell activation and inflammatory gene ciorcuits in subjects with psoriasis. Jourmal of Allergy and Clinical Immunology 2012, 130(1): 145-154
8. Kyowa Hakko Kirin Co., Ltd. Clinical pharmacology study, internal data
9. Tanizaki H, Nakahigashi K, Miyachi Y, and Kabashima K. Comparison of the efficacy of fexofenadine 120 and 240 mg/day on chronic idiopathic urticarial and histamine-induced skin responses in Japanese populations. J Dermatolog Treat. 2013; Dec; 24(6): 477-80.