Inhibitory Effect Of Oligonucleotides Containing CCT On TLR7/9 Mediated Innate Immune Response

Inhibitory oligodeoxynucleotides (ODNs), an artificial single-stranded DNA molecule typically with a 15-30 nucleotides length, could regulate innate immunity activation. Recent studies demonstrate that inhibitory ODNs show a promise to develop an agent for the treatment of diseases associated with Toll-like receptor activation.As part of the innate immune system, TLRs recognize microbe derived molecular structures (pathogen-associated molecular patterns, PAMPs) and therefore sense the microbe invasion and consequently initiate a rapid response that is characterized by the secretion of type I interferon (IFN) and pro-inflammatory cytokines [IL-1, IL-6, tumor-necrosis factor-α(TNF-α)], depending on the nature of the microbes. In mammals, TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11, expressed at the cell surface, respond to extra-cellular microbes, and TLR3, TLR7, TLR8 and TLR9, confined to intracellular compartments, respond to nucleic acids. Preferentially, TLR7 senses single-stranded RNA [ssRNA], and TLR9 senses DNA molecules including single-stranded CpG containing oligonucleotides (CpG ODN). In homeostatic conditions, TLR activation leads to a protective immune response against pathogens through multiple inflammatory pathways. On the other hand, if unwanted, TLR activation is associated with the development of a profile of diseases, designated as Toll like receptor activation associated diseases, including SLE and sepsis.We synthesized a series of ODNs with the sequences of human MS DNA (designated as IMO-ODN) and used them to mimic human microsatellite DNA. We test whether IMO-ODNs could inhibit the activation of human PBMCs through TLR7/9-dependent. We found the screening platform by the heat-inactivated HSV-1 and PR8-flu virus as stimulus for the development of novel ODN with immunoregulatory activity. The results demonstrate that an ODN containing CCT motif, designated as IMO03, can down-regulate the immune activation of human PBMCs induced by HSV-1, flu virus,CpG ODN and imiquimod. IMO03 has been studied for its capacity of negatively regulating innate immunity induced by TLR7/TLR9 agonists in vitro and in mice.1 Preliminary screening of inhibitory ODN1.1 Establishment of screening platform for inhibitory ODN by inactivated virus as stimulusConsidering the virus infection causes frequently an excessively innate immune response and DNA and RNA are as nature TLR7/9 ligands. In some extent, inactivated virus can simulate the real immune response. Heat-inactivated HSV-1 and PR8-flu virus were used as stimulus respectively and the well studied inhibitory ODN, IRS954, was selected as positive inhibitory ODN. The human PBMCs were stimulated by the inactivated virus and/or IRS954, then the anti-viral activity of the culture supernatant was tested by the anti-VSV bioassay. The experimental conditions were also well optimized. The result showed that heat-inactivated HSV-1 and PR8-flu virus induced anti-VSV bioassay were inhibited by IRS954 significantly. Time point of IRS954 adding (-30~30 min) did not affect its inhibitory effect. To obtain shorten the experimental time, the supernatant were harvest after culturing PBMC with inactivated virus and ODN for 48h.1.2 Screening of IMO-ODNsSix IMO-ODNs were screened by"anti-VSV bioassay". The results indicated that IMO06, IMO04 and IMO03 inhibited the anti-viral activity of the culture supernatant of human PBMCs. Their inhibition was in a dose-dependent manner. IMO01 was found almost inert. Because the inhibitory effect of IMO03 containing CCT motif was higher than IMO04 and IMO06, so IMO03 was as a more promise ODN for following experiments, and IMO01 was as a control ODN.1.3 Inhibition of IMO03 on CpG ODN induced anti-viral activityVSV protection assay was conducted to investigate whether IMO03 could inhibit the anti-viral activity of human PBMCs induced by CpG ODN and imiqumod. The result showed that IMO03 inhibited the anti-VSV activity induced by CpG ODN and imiquimod, but not IMO01. The results showed that the inhibitory effect of IMO03 was base on a sequence-specific manner.1.4 Species-specificity analysis of IMO03We replaced human PBMCs with BALB/c spleen cells and investigated whether IMO03 is still inhibitory on the activation of mouse immune cells stimulated by CpG ODN. The results showed that, in cell proliferation assay, IMO03 remarkable inhibited CpG 1826-induced proliferation of spleen cells, but not IMO01; however, in VSV protection assay, IMO03 reduced the anti-VSV activity of spleen cells induced by CpG 1585. These results indicate that there is not species-specificity for IMO03 function.1.5 IMO03 inhibited cytokine production through TLR9 on human pDCThe effect of IMO03 on cytokine production from pDCs induced by CpG ODN was also observed. The purified pDCs were incubated with CpG 2216 or in the presence of IMO03, A151 or IMO01 for 22 h and the supernatants were collected and assayed by ELISA. When used together, IMO03 significantly inhibited IFN-αproduction from the pDCs triggered by CpG 2216. Comparatively, the inhibition displayed by IMO03 was more profound than that displayed by A151. IMO03 and IMO01 alone did not induce the IFN-α, TNF-αand IL-6 production. Obviously, IMO03, displayed a tendency to, couldn't induce significant inhibition on the production of TNF-αand IL-6 from the pDCs treated by CpG 2216. The result indicates that IMO03 targets human pDCs, cells expressing TLR7/TLR9, to control the production of IFN-α, possibly other cytokines induced via TLR9 activation.2. The application of IMO03 on platform of Toll-like receptor associated disease2.1 The inhibitory effect of IMO03 on IFN production from human PBMCs stimulated with serum of SLE patients and the CpG motif-containing DNA fragment from SLE patients.Human PBMCs were treated with or without serum from eight anti-dsDNA–positive SLE patients for 48 h in the present and absent of IMO03, and the supernatants were harvested for anti-VSV assay. Three healthy donors serum are as negative control. The results indicated that the supernatant of serum from eight anti-dsDNA–positive SLE patients incubated with PBMC protect the Vero E6 against VSV attacking,but not normal serum. IMO03 inhibited the IFN production from human PBMCs stimulated with the serum from SLE patient.We next synthesized a CpG motif-containing fragment from a SLE patient serum, KJ-6 was complexed to Lipofectamine 2000 with human PBMCs for 48 h in the present and absent of IMO03. In anti-VSV activity bioassay, we found that KJ-6 alone also stimulates highly IFN production from human PBMCs with the help of Lipofectamine 2000. The results show that IMO03 induced a dramatic inhibition of IFN secretion. In cell proliferation assay, IMO03 remarkable inhibited KJ-6-induced proliferation of human PBMCs. Together, the data indicated that IMO03 could inhibit endogenous ligands of SLE patients by inhibiting TLR7/9 signaling.2.2 Effect of IMO03 on cytokine-mediated lethal shock induced by TLR9 agonist in mice.Upon the in vitro analysis, we then evaluated the in vivo effect of IMO03 in a mouse model of excessive cytokine-mediated lethal shock induced CpG ODN. To create the model, BALB/c mice were pre-sensitized with D-GalN for 1.5 h and then injected i.p. with CpG 1826. The survivals of the mice were recorded. All mice in CpG 1826 group were dead in 24 h. In contrast, 100% of mice in CpG 1826 plus IMO03 group, 40% of mice in CpG 1826 plus IMO01 group and 20% of mice in CpG 1826 plus chloroquine group were still alive in 168 h, demonstrating that IMO03 could rescue mice from excessive cytokine-mediated lethal shock induced by TLR9 activation.3 Mechanism and characters of IMO03's inhibition 3.1 The action of IMO03 is in a selected fashion.To investigate whether IMO03 could also inhibit activation of other TLRs, LPS (TLR4-ligand) and Poly I:C (TLR3-ligand) were used as stimulators to induce excessive cytokine-mediated lethal shock in D-GalN-treated mice. The result showed that both Poly I:C (50μg/mouse) and LPS (1μg/mouse) could induce death of D-GalN-treated mice in 24 h. However, IMO03 couldn't rescue D-GalN-treated mice from LPS- and Poly I:C-induced excessive cytokine-mediated lethal shock even at 250μg. This may hint that IMO03 displays the inhibitory role on innate immune response by selecting TLR9 pathway but not TLR3 and TLR4 pathways.3.2 The action of IMO03 is in a sequence-specificity and length–dependent manner.To analyzing the structure features of IMO03 by model mouse of cytokine-mediated lethal shock, we synthesized IMO04 containing CT motif and IMO06 containing CT motif with 20 nucleotides length. IMO06, IMO04, IMO03 are injected into sepsis model mouse in vivo separately. The result showed that IMO06 has not demonstrated any inhibitory function. But IMO04 had demonstrated certain the inhibitory function for rescuing the mice from excessive cytokine-mediated lethal shock in D-GalN-treated mice. The results indicate that the action of IMO03 is in a sequence-specificity and length -dependence manner.3.3 Inhibitory action of IMO03 is in a time and dose-dependent manner.We tested the suitable time for administration of IMO03 in D-GalN/CpG 1826-treated mice. IMO03 was separately injected i.p. into D-GalN sensitized mice at -1 h and -0.5 h before CpG 1826 injection, simultaneously with CpG 1826 injection or 0.5 h, 1 h and 2 h after CpG 1826 injection. The survivals of the mice were recorded. The result showed that IMO03 could effectively protect mice from CpG 1826-induced excessive cytokine-mediated lethal shock when injected at -1 h, -0.5 h or simultaneously with CpG 1826 injection but failed to display the protection role when injected in 0.5 h or later after CpG 1826 injection. This may hint that IMO03 competed with CpG 1826 at the same site on target cells. Subsequently, dose-effect of IMO03 on resisting CpG 1826-induced lethal shock was observed in D-GalN-treated mice. All mice in groups of CpG 1826 alone or plus 6.25μg of IMO03 were dead in 24 h, whereas 30%, 50% and 80% of mice in groups of CpG 1826 plus IMO03 at 12.5μg, 25μg and other three doses of 50, 75 and 100μg were still alive in 168 h. This result reveals that 50μg of IMO03 may be an optimal dosage for rescuing mice from TLR9 activation-induced excessive cytokine-mediated lethal shock.3.4 Inhibitory action of IMO03 isn't due to its extracellular interaction with TLR agonists.To observe the influence of the ways of administrating IMO03 on its inhibitory effect, inactivated flu virus PR8 or HSV-1 was premixed with IMO03 or IMO01 for 2 h at 37°C or used separately to act on human PBMCs for 48 h. The supernatants were assayed in VSV protection assays. The results show that in two different ways of administration, IMO03 inhibit IFN production induced by inactivated virus. IMO03 or IMO01, premixed or added simultaneously with inactivated flu virus PR8 or HSV-1, displayed the similar inhibition on IFN production from human PBMCs. The data indicate that IMO03 mediated inhibition isn't due to its extracellular interaction with TLR agonists like flu virus PR8 and HSV-1.3.5 Inhibitory action of IMO03 is possible related with target cell activation. We next tested whether IMO03 targeted pDCs to display its immunesuppression. Afterward, CpG 2216 was selected as stimulator for pDCs. The pDCs were stimulated with CpG 2216 alone or in the presence of IMO03, A151 or IMO01 for 22 h and then analyzed on a FACS Canto for its expression of surface molecules after staining with fluorescence-labeled mAbs. The result showed that the expression of CD86, CD80 and HLA-DR on the surface of pDCs was up-regulated not only by CpG 2216 (P<0.05) or A151 (P<0.05) but also by IMO03 or CpG 2216 plus IMO03. The activities of IMO03 on up-regulating the surface molecules of pDCs were similar as that of A151 but much higher than that of IMO01.⑴The results indicated that IMO03 inhibited TLR9 activation at the same time of inducing pDC's maturation. Between the two sides does not have the contradiction.⑵Since pDCs are the major cells to produce IFN-αin response to TLR9 activation and A151, as a reported inhibitory ODN, could up-regulate the expression of CD86, CD80 and HLA-DR on the surface of pDCs, we suppose that inhibitory action of IMO03 was dependent target cell activation.In present study, an IMO-ODN containing CCT motif, designated as IMO03, was studied for its inhibitory effect on TLR7/9 activation. It was found that IMO03 could down-regulate TLR7/9-dependent IFN-αproduction induced by viral nucleic acids in vitro, protect mice from D-GalN/CpG ODN induced lethal shock but not TLR3/4 activation-mediated death, and suppress IFN-αproduction from human pDCs induced by CpG ODN. Interestingly, IMO03 could also activate pDCs, manifested by up-regulating their MHC class II and co-stimulatory molecules. The data suggest that IMO03 could be used as a potential candidate for developing a medicament for the treatment of TLR9/TLR7 activation associated diseases by blocking the TLR7/9 signaling. Although the mechanism of inhibitory ODN is unclear, we prove that the action of an oligonucleotides containing CCT motif had been related with three primary factors including the sequence specificity, certain length and PS-modification skeleton.