Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo.

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria.

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.

Ligand-induced conformational changes allosterically activate Toll-like receptor 9.

Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.

Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9.

Hemozoin (HZ) is an insoluble crystal formed in the food vacuole of malaria parasites. HZ has been reported to induce inflammation by directly engaging Toll-like receptor (TLR) 9, an endosomal receptor. "Synthetic" HZ (beta-hematin), typically generated from partially purified extracts of bovine hemin, is structurally identical to natural HZ. When HPLC-purified hemin was used to synthesize the crystal, beta-hematin had no inflammatory activity. In contrast, natural HZ from Plasmodium falciparum cultures was a potent TLR9 inducer. Natural HZ bound recombinant TLR9 ectodomain, but not TLR2. Both TLR9 stimulation and TLR9 binding of HZ were abolished by nuclease treatment. PCR analysis demonstrated that natural HZ is coated with malarial but not human DNA. Purified malarial DNA activated TLR9 but only when DNA was targeted directly to the endosome with a transfection reagent. Stimulatory quantities of natural HZ contain <1 microg of malarial DNA; its potency in activating immune responses was even greater than transfecting malarial DNA. Thus, although the malarial genome is extremely AT-rich, its DNA is highly proinflammatory, with the potential to induce cytokinemia and fever during disease. However, its activity depends on being bound to HZ, which we propose amplifies the biological responses to malaria DNA by targeting it to a TLR9(+) intracellular compartment.

TLR9 and the recognition of self and non-self nucleic acids.

Toll-like receptors (TLRs) are involved in the innate recognition of foreign material and their activation leads to both innate and adaptive immune responses directed against invading pathogens. TLR9 is intracellularly expressed in the endo-lysosomal compartments of specialized immune cells. TLR9 is activated in response to DNA, in particular DNA containing unmethylated CpG motifs that are more prevalent in microbial than mammalian DNA. By detecting foreign DNA signatures TLR9 can sense the presence of certain viruses or bacteria inside the cell and mount an immune response. However, under certain conditions, TLR9 can also recognize self-DNA and this may promote immune pathologies with uncontrolled chronic inflammation. The autoimmune disease systemic lupus erythematosis (SLE) is characterized by the presence of immune stimulatory complexes containing autoantibodies against endogenous DNA and DNA- and RNA-associated proteins. Recent evidence indicates that the autoimmune response to these complexes involves TLR9 and the related single-stranded RNA-responsive TLRs 7 and 8, and therefore some breakdown in the normal ability of these TLRs to distinguish self and foreign DNA. Evidence suggests that immune cells use several mechanisms to discriminate between stimulatory and nonstimulatory DNA; however, it appears that TLR9 itself binds rather indiscriminately to a broad range of DNAs. We therefore propose that there is an additional recognition step by which TLR9 senses differences in the structures of bound DNA.

mRNA is an endogenous ligand for Toll-like receptor 3.

Toll-like receptors (TLRs) are the basic signaling receptors of the innate immune system. They are activated by molecules associated with pathogens or injured host cells and tissue. TLR3 has been shown to respond to double stranded (ds) RNA, a replication intermediary for many viruses. Here we present evidence that heterologous RNA released from or associated with necrotic cells or generated by in vitro transcription also stimulates TLR3 and induces immune activation. To assess RNA-mediated TLR3 activation, human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-kappaB-dependent luciferase reporter were generated. Exposing these cells to in vitro transcribed RNA resulted in a TLR3-dependent induction of luciferase activity and interleukin-8 secretion. Treatment with in vitro transcribed mRNA activated nuclear factor-kappaB via TLR3 through a process that was dose-dependent and involved tyrosine phosphorylation. Furthermore, in vitro transcribed natural or 2'-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-alpha, and interleukin-1 receptor-associated kinase-M mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-alpha secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses.

Induction of Macrocyst Formation by Factors Secreted by Giant Cells in Dictyostelium discoideum: (D. discoideum/sexual cycle/induction of macrocyst formation).

The fusion of cells of complementary mating types to produce giant cells has been shown to be the critical event to induce macrocyst formation in Dictyostelium discoideum. We have examined the way in which giant cells use diffusible factors to influence the developmental mode of nearby cells using an experimental design in which NC4 cells are allowed to develop on a dialysis membrane above a suspension of giant cells. We have observed that giant cells are able to inhibit independent aggregation and stream formation in the upper cells and become the dominant aggregation centers. In addition giant cells are able to redirect local amoeba away from the fruiting-body and toward the macrocyst mode of development. We show that these effects are mediated by diffusible factors of under 2,000 MW. and discuss possible mechanisms of action.