Synthesis and immunological activities of novel Toll-like receptor 7 and 8 agonists

Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.     

Synthesis and immunological activities of novel agonists of toll-like receptor 9

Novel agonists of TLR9 with two 5′-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-α production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-α and IP-10) in rhesus macaques after intra-muscular administration.     

Synthesis and immunoregulatory activities of conjugates of a Toll-like receptor 7 inert ligand

In the synthesis and modification of the analogs of an adenine type of Toll-like receptor (TLR) 7 agonists, we found a special compound, 9-propionyloxy-8-hydroxy-2-(2-methoxyethoxy)-adenine (6). It is a synthesized TLR7 inert ligand, which does not respond to TLR7 itself. However, it can be coupled with protein or peptide antigens via propionyloxy functional group to promote their immunogenicity significantly. The compound was covalently coupled to protein and peptide to get the conjugates. The inductivity of cytokine production by the conjugates was 872.4-fold compared with the unconjugated antigens in vitro by mouse splenocyte. These data show that the immunostimulatory activity of inert TLR7 ligand can be endowed, and the activity of antigens can be amplified by conjugation with various proteins and peptides, thus broadening the potential therapeutic application and reducing the risk of TLR7 agonists’ side effects.     

Synthetic oligoribonucleotides containing arabinonucleotides act as agonists of TLR7 and 8

In continuation of our studies with stabilized immune modulatory RNA (SIMRA) compounds, we have synthesized novel SIMRA compounds incorporating arabinonucleotides to study their effects on TLR7 and TLR8 activation. The SIMRA compounds containing ara-G, ara-C, ara-U or ara-A substitutions activated TLR8 in HEK293 cells. Interestingly, the SIMRA compound containing ara-C also activated TLR7 and stimulated immune responses in vivo in mice. In human PBMC and pDC assays, SIMRA compounds containing arabinonucleotides induced Th1-type cytokine profiles. These results suggest that SIMRA compounds containing arabinonucleotides act as agonists of TLR7 and TLR8.     

Polymorphism distribution and structural conservation in RNA-sensing Toll-like receptors 3, 7, and 8 in pigs

Background

Viral genomic RNA—both single-stranded (ss) and double-stranded (ds)—is recognized by RNA-sensing Toll-like receptors (TLRs), notably TLR3 (dsRNA), TLR7 (ssRNA), and TLR8 (ssRNA). However, our knowledge of the roles of porcine TLR3, 7, and 8 in antiviral immunity is inadequate.

Methods

From information on exon–intron boundaries obtained through comparisons of the genomic and cDNA sequences, polymorphisms in the coding sequences of each gene were detected in 84 male pigs of 11 breeds.

Results

Genomic structures are conserved between pigs and humans. The RNA-sensing TLR genes had fewer polymorphisms causing amino acid alterations than did the cell-surface TLR genes, but the alterations were distributed with a similar bias toward ectodomains.

Conclusions

The low level of diversity of substitutive polymorphisms in RNA-sensing TLRs than cell-surface ones implies that polymorphisms severely affecting function have been eliminated by selection pressure during longstanding pig breeding.

General significance

Recognition of virus-derived RNA is critical in host defense against infection. These results should provide a useful clue to analysis of the association between polymorphisms in RNA-sensing TLRs and disease resistance.     

Phylogenetic and expression analysis of amphibian Xenopus Toll-like receptors

An anuran amphibian, South African clawed frog (Xenopus laevis), is used to study the immune system, as it possesses a set of acquired immune system represented by T and B lymphocytes and the immunoglobulins. The acquired immune system is impaired throughout the larva and the metamorphosis stage in the amphibians. On the other hand, the role of innate immune system in the tadpole remains unclear. Recently, insect Toll protein homologues, namely, Toll-like receptors (TLRs), have been identified as sensors recognizing microbe-pattern molecules in vertebrates. Whole-genome analysis of Xenopus tropicalis supported the existence of the tlr genes in the frog. In this study, we annotated 20 frog tlr gene nucleotide sequences from the latest genome assembly version 4.1 on the basis of homology and identified cDNAs of the predicted frog TLR proteins. Phylogenetic analysis showed that the repertoire of the frog TLRs consisted of both fish- and mammalian-type TLRs. We showed that the frog TLRs are constitutively expressed in the tadpole as well as in the adult frog. Our results suggest that tadpoles are protected from microbes by the innate system that includes TLRs, despite impaired acquired immune system in tadpoles. This is the first report on the properties of TLRs in the most primitive terrestrial animals like amphibia. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of polymorphisms in bovine toll-like receptors 3, 7, 8, and 9

The toll-like receptors (TLRs) detect molecular signatures of invaders known as pathogen-associated molecular patterns (PAMPs). Ten members of the TLR family have been identified in cattle to date and 4 of these recognize PAMPs specific to viruses (TLRs 3, 7, 8, 9). The objective of this work was to detect polymorphisms in the genomic sequences of bovine TLRs 3, 7, 8, and 9. To achieve this objective, a panel of nine breeds representing Bos taurus and Bos indicus was assembled for sequencing and comparison with the Bovine Genome Project sequence. Comparative sequence analysis revealed a total of 139 polymorphisms, which include single-nucleotide polymorphisms and insertion-deletion polymorphisms. Of the 139 polymorphisms, 88% (N=123) are novel. In addition, the protein domain architecture of these four TLRs was examined between human, mouse, cow, and dog, which revealed several regions of conservation in the TLR variable leucine-rich-repeat patterning.     

Effect of the antimicrobial peptide LL-37 on Toll-like receptors 2-, 3- and 4-triggered expression of IL-6, IL-8 and CXCL10 in human gingival fibroblasts

The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IκBα and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam₃CSK₄, but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.     

mTOR Signaling pathway as a master regulator of memory CD8+ T-cells,Th17, and NK cells development and their functional properties

The mammalian target of rapamycin (mTOR) is a member of the evolutionary phosphatidylinositol kinase-related kinases (PIKKs). mTOR plays a pivotal role in the regulation of diverse aspects of cellular physiology such as body metabolism, cell growth, protein synthesis, cell size, autophagy, and cell differentiation. Immunologically, mTOR has a fundamental part in controlling and shaping diverse functions of innate and adaptive immune cells, in particular, T-cell subsets differentiation, survival, and metabolic reprogramming to ultimately regulate the fate of diverse immune cell types. Researchers report that rapamycin, a selective mTOR inhibitor, and immunosuppressive agent, has surprising immunostimulatory effects on inducing both quantitative and qualitative aspects of virus-specific memory CD8⁺ T-cells differentiation and homeostasis in a T-cell-intrinsic manner. The mTOR signaling pathway also plays a critical role in dictating the outcome of regulatory T cells (Treg), T helper 17 (Th17) cells, and natural killer (NK) cells proliferation and maturation, as well as the effector functions and cytotoxic properties of NK cells. Manipulation of mTOR activity is a critical therapeutic approach for pharmacological agents that seek to inhibit mTOR. This approach should enhance specific memory CD8 ⁺ T-cells responses and induce fully functional effector properties of NK cells to provoke their antitumor and antiviral activities.     

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.     

Local injections of OK432 can help the infiltration of adoptively transferred CD8+ T cells into the tumor sites and synergistically induce the local production of Th1-type cytokines and CXC3 chemokines

The effect of local injections with streptococcal preparation OK432 on the antitumor effect induced by adoptive immunotherapy (AIT) was investigated. Draining lymph node cells on day 14 after B7-P815 inoculation were used for AIT after in vitro stimulation. AIT on days 7 and 10 showed no effect on the growth of s.c. established P815 mastocytoma, but local injections with OK432 into the tumor sites on days 3, 6 and 9 resulted in a moderate antitumor effect. On the other hand, the combination therapy significantly suppressed tumor growth, and the tumor-bearing mice survived longer than those receiving only one of the treatment modalities. The significant infiltration of CD4⁺ or CD8⁺ T cells and multiple necrosis in the tumor sites were observed only when the tumor-bearing mice were treated with the combination therapy. In addition, a transfer experiment using labeled effector cells revealed these infiltrated CD8⁺ T cells and CD4⁺ T cells to be derived from the donor and the host respectively. More importantly, the combination therapy clearly led to higher expression of the mRNA for Th1-type cytokines and CXC3 chemokines in the tumor sites than resulted from each of the treatment modalities alone. Collectively, these results indicate that local injections with OK432 can help the infiltration of adoptively transferred CD8⁺ T cells into the tumor sites and synergistically induce the local production of the Th1-type cytokines and CXC3 chemokines. Received: 3 April 2000 / Accepted: 5 May 2000     

Cytokine decoy and scavenger receptors as key regulators of immunity and inflammation

IL-1R2 was the first decoy receptor to be described. Subsequently receptors which act as pure decoys or scavengers or trigger dampening of cytokine signaling have been described for cytokines and chemokines. Here we review the current understanding of the mode of action and significance in pathology of the chemokine atypical receptor ACKR2, the IL-1 decoy receptor IL-1R2 and the atypical IL-1 receptor family IL-1R8. Decoy and scavenger receptors with no or atypical signaling have emerged as a general strategy conserved in evolution to tune the action of cytokines, chemokines and growth factors.     

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.     

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E(2) (PGE(2)) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE(2) to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter.     

Toll/Interleukin-1 Receptor Domain Dimers as the Platform for Activation and Enhanced Inhibition of Toll-like Receptor Signaling

TIR (Toll/IL-1 receptor) domains mediate interactions between TLR (Toll-like) or IL-1 family receptors and signaling adapters. While homotypic TIR domain interactions mediate receptor activation they are also usurped by microbial TIR domain containing proteins for immunosuppression. Here we show the role of a dimerized TIR domain platform for the suppression as well as for the activation of MyD88 signaling pathway. Coiled-coil dimerization domain, present in many bacterial TCPs, potently augments suppression of TLR/IL-1R signaling. The addition of a strong coiled-coil dimerization domain conferred the superior inhibition against the wide spectrum of TLRs and prevented the constitutive activation by a dimeric TIR platform. We propose a molecular model of MyD88-mediated signaling based on the dimerization of TIR domains as the limiting step.     

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.     

Discovery of N-substituted 7-azaindoline derivatives as potent,orally available M1 and M4 muscarinic acetylcholine receptors selective agonists

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M₁ and M₄ mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.     

3-Ylidenephthalides as a new class of transient receptor potential channel TRPA1 and TRPM8 modulators

Following the recent identification of the naturally occurring 3-ylidene-4,5-dihydrophthalide ligustilide and its oxidation product dehydroligustilide as novel TRPA1 modulators, a series of seventeen 3-ylidenephthalides was synthesized and tested on TRPA1 and TRPM8 channels. Most of these compounds acted as strong modulators of the two channel types with EC₅₀ and/or IC₅₀ values distinctly lower than those of the reference compounds.     

Assessment of the effect of TLR7/8, TLR9 agonists and CD40 ligand on the transformation efficiency of Epstein-Barr virus in human B lymphocytes by limiting dilution assay

Infection of human B cells with Epstein-Barr virus (EBV) induces polyclonal activation in almost all infected cells, but a small proportion of infected cells are transformed to immortalized lymphoblastoid cell lines. Since B cells are activated also by CD40 ligand (CD40L) and Toll-like receptor (TLR) agonists via a similar signaling pathway, it is likely that costimulation through these molecules could result in synergistic enhancement of the transformation efficiency of EBV. In this study, the stimulatory effect of TLR7/8 (R848), TLR9 (CpG) agonists and/or CD40L on transformation efficiency of EBV in normal human B cells was assessed using the limiting dilution assay. Costimulation of peripheral blood mononuclear cells (PBMCs) with CpG and R848, but not CD40L, increased significantly the frequency of EBV transformed B cells (p < 0.001). Neither synergistic nor additive effects were observed between TLR agonists and CD40L and also TLR7/8 and TLR9 agonists. Costimulation with R848, CpG and CD40L enhanced the proliferative response of B cells infected with EBV. This effect was more evident when enriched B cells were employed, compared to PBMCs. The promoting effect of TLR agonists stimulation, implies that EBV may take advantage of the genes induced by the TLR stimulation pathway for viral latency and oncogenesis.