TLR9-mediated Immune Responses Research Articles

Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.

Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation.

Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

Stk38 protein kinase preferentially inhibits TLR9-activated inflammatory responses by promoting MEKK2 ubiquitination in macrophages.

NDR/LATS kinase family is highly conserved from yeast to human. It remains unknown whether the members of this family function in innate immune responses. Here we demonstrate that Stk38 negatively regulates TLR9-mediated immune responses in macrophages. Stk38 constitutively associates with ubiquitin E3 ligase Smurf1, and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation. MEKK2 is required for CpG-induced ERK1/2 activation, TNF-α and IL-6 production but not required for LPS-induced TNF-α and IL-6 production. Accordingly, Stk38 deficiency increases CpG-induced ERK1/2 activation, TNF-α and IL-6 production without significantly affecting LPS-induced TNF-α and IL-6 production. Stk38-deficient mice produce more TNF-α and IL-6, and display increased lethality than control wild-type mice upon E. coli infection. Stk38-deficient mice are also more susceptible to CLP-induced sepsis than control mice. Thus, Stk38 is important in limiting inflammatory cytokine production and necessary for protecting host from inflammatory injury during infection, possibly by negatively regulating TLR9 signalling.

Guanine-Modified Inhibitory Oligonucleotides Efficiently Impair TLR7- and TLR9-Mediated Immune Responses of Human Immune Cells

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX3–5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.

C7: A CpG oligodeoxynucleotide that induces protective immune response against megalocytivirus in Japanese flounder (Paralichthys olivaceus) via toll-like receptor 9-mediated signaling pathway

Megalocytivirus is the causative agent of severe disease outbreaks in farmed fish. Currently there is no effective control against megalocytivirus in aquaculture. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to possess marked immunostimulatory properties. In this study, we investigated the potentials of ten CpG ODNs as antiviral agents in a model of Japanese flounder (Paralichthys olivaceus). We found that, when administered into flounder, three of the ten CpG ODNs inhibited viral replication in kidney, spleen, and liver. ODN C7, which exhibited the strongest inhibitory activity, was able to promote proliferation of peripheral blood leukocytes and enhance activation of head kidney mononuclear adherent phagocytes. When the expression of toll-like receptor 9 (TLR9) was knocked down in vivo by small interfering RNA, C7-mediated immune response and antiviral activity were significantly blocked. Moreover, when C7 was co-administered with pCN86, a DNA vaccine against megalocytivirus, a significant increase in vaccine-induced protection was observed compared to administration with pCN86 alone. Further analysis showed that compared to fish immunized with pCN86, fish immunized with pCN86 plus C7 exhibited significantly upregulated expression of a wide range of genes involved in innate and adaptive immunity. Taken together, these results indicate that ODN C7 activates TLR9-mediated immune response and possesses antiviral and adjuvant potentials that may be exploited for the control of megalocytivirus infection in farmed flounder.

Immune-Stimulatory Dinucleotide at the 5'-End of Oligodeoxynucleotides Is Critical for TLR9-Mediated Immune Responses.

Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2'-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5'-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5'-end region and the other toward the 3'-end region, incorporated 2'-O-methylribonucleotides selectively preceding the 5'- or 3'-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5'-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents.

IMO-8400, a novel TLR7, TLR8 and TLR9 antagonist, inhibits disease development in lupus-prone NZBW/F1 mice (119.12)

Abstract Several studies have implicated Toll-like receptors (TLRs) 7, 8 and 9 in the development and progression of lupus. We have developed IMO-8400, a synthetic DNA-based antagonist that has shown potent inhibition of TLR7-, TLR8-, and TLR9-mediated immune responses in cell-based assays and in non-human primates. To assess the therapeutic potency of IMO-8400, we have tested this compound in lupus-prone NZBW/F1 mice. Mice were injected subcutaneously with IMO-8400 at 1.9, 3.8 or 7.5 mg/kg once a week or 7.5 mg/kg once every two weeks from the age of week 21 to 33, and sacrificed at week 34. NZBW/F1 mice treated with IMO-8400 at the weekly dose of 3.8 or 7.5 mg/kg or 7.5 mg/kg every two weeks showed a significant reduction in urine protein and blood urea nitrogen levels in compression to that of untreated mice. Additionally, anti-DNA, anti-RNP and anti-SM IgG levels were also lower in treated mice than untreated controls. No treatment-related side-effects were observed. Histological examination of kidney sections revealed improvement in nephritis (reduced glomerular sclerosis and decreased interstitial inflammation), accompanied by decreased glomerular deposits of IgG immune complex in mice treated with IMO-8400. These results indicate that IMO-8400 suppressed autoimmune antibody production and improved renal function by inhibiting immune responses in lupus-prone NZBW/F1 mice. Thus, IMO-8400 has the potential for the treatment of lupus and other autoimmune diseases.

IMO-8400, a novel TLR7, TLR8 and TLR9 antagonist, inhibits disease development in mouse models of psoriasis (119.8)

Abstract Toll-like receptors (TLR) are involved in the pathogenesis of numerous autoimmune diseases, including psoriasis. The role of TLR7 and TLR9 in psoriasis is well established and TLR8 may also contribute to this disease. Blocking the activation of these receptors through a TLR antagonist represents a novel approach to the treatment of psoriasis. We have developed IMO-8400, a synthetic DNA compound that inhibits TLR7-, TLR8-, and TLR9-mediated immune responses in cell-based assays. Additionally, following subcutaneous administration in non-human primates, it suppresses ex-vivo immune responses mediated through these receptors. IMO-8400 was evaluated in preclinical mouse models of psoriasis, induced by IL-23 cytokine or antimicrobial peptide LL-37. Psoriasis-like skin lesions were induced in the ear of C57BL/6 mice by intradermal injection of IL-23 or LL37. Injected mice were treated with subcutaneous administration of IMO-8400. Mice were euthanized at end of study and tissue samples were taken for histological examination. Increase in ear thickness was inhibited in a dose-dependent manner by treatment with IMO-8400. Reduction in epidermal hyperplasia and infiltrating leukocytes was observed in sample tissues. Treatment with IMO-8400 was associated with elevation of serum IL-10 levels. IMO-8400 can effectively suppress lesion development in these psoriasis models. IMO-8400 has potential as a novel agent for the treatment of psoriasis and other autoimmune diseases.

In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV)-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of GVHSV protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9–CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding GVHSV gene are due to GVHSV rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants.

Impact of Nature and Length of Linker Incorporated in Agonists on Toll-Like Receptor 9-Mediated Immune Responses

Oligodeoxynucleotides containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). We previously reported a novel class of TLR9 agonists, referred to as immune-modulatory oligonucleotides (IMOs), in which two 11-mers of the same sequence are attached via their 3'-ends through a 1,2,3-propanetriol linker and contain a synthetic immune-stimulatory motif, Cp7-deaza-dG. In the present study, we have examined the impact of length, nature, and stereochemistry of the linker incorporated in agonists for TLR9 activation. The new linkers studied include (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexanetriol, cis,trans-1,3,5-cyclohexanetriol, 1,3,5-tris(2-hydroxyethyl)isocyanurate, tetraethyleneglycol, and hexaethyleneglycol in place of 1,2,3-propanetriol linker. Agonists with various linkers are studied for TLR9-mediated immune responses in HEK293 cells, human cell-based assays, and in vivo in mice. Results of these studies suggest that C3-C5 linkers, 1,2,3-propanetriol, (S)-(-)-1,2,4-butanetriol, or 1,3,5-pentanetriol, are optimal for stimulation of TLR9-mediated immune responses. Rigid C3 linkers with different stereochemistry have little effect on immune stimulation, while linkers longer than C5 reduced TLR9-mediated immune stimulation.

Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity.

Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune-stimulatory effects of bacterial DNA and are recognized by Toll-like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9-dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9-mediated immune response, but this response is insufficient to induce optimal interferon-alpha-mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure-activity relationship for TLR9 agonist compounds with diverse immune effects.