| In homeostatic conditions,immune activation is protective in a host. Functioning as pattern recognition receptors,Toll-like receptors(TLRs),which are broadly distributed on immune cells,initiate innate immune activation in response to conserved microbial molecules including lipopolysaccharide(LPS),flagellin, unmethylated CpG DNA,viral RNA,etc,and result in the production of inflammatory cytokines,activation of DC and B cells and the induction of specific, adaptive immunity to the pathogen.However,if the innate immune system through the TLR dysregulated,the activation may implicated in various genetic diseases and a number of conditions including susceptibility to infections,lung diseases, autoimmune diseases such as systemic lupus erythematosus(SLE),inflammatory bowel disease(IBD) and atherosclerosis.Recent studies demonstrate that the use of suppressive oligodeoxynucleotide,an artificial single-stranded DNA molecule with length of dozens nucleotides,may potentially have significant clinical utility in those immune activation associated disease.In this study,among the ODN designated as MTODN we desigened,we describe here the identification of a candidate suppressive oligodeoxynucleotide (MT01).MT01 was subsequently analysed in detail in terms of inhibitory potency. kinetics,and mode of action.It was found that MT01 could down-regulate TLR7/9-dependent IFN production and proliferation in cultured human peripheral blood monouclear cells(PBMCs) and murine spleen cells,induced by CpG oligodeoxynucleotides(CpG ODN).imiquimod,inactivated herpessimplexvirus-1 (HSV-1),inactivated influenza A virus Puerto-Rico-Stamm 8(PR8),influenza A virus A/Fort Monmouth/1/1947 H1N1 strain(FM1) and sera from SLE patients.Moreover. MT01 was examined for its functionality in a hepatitis B surface antigen(HBsAg) plus CpG ODN immunized mouse model.1 Designing and screening of MT ODN1.1 Designing of MTODNTo develop immunosuppressive oligodeoxynucleotides with novel sequences as potential candidates for treating immune responses-associated diseases such as autoimmune diseases,serials of ODNs were designed and screened in our lab.It has been reported that there are certain DNA in mammalian genome which negatively regulate immune activations.According to sequence of mitochondrial DNA and basic sequence feature of suppressive ODN,we designed 13 sequences of MT ODN (MT00～12).Among them,repeat units were from 2 to 9 nucleotides.1.2 Screening of MT ODNThe inhibitory effects of the MT ODNs were tested in a VSV protection bioassay for determining IFN production from human PBMCs induced by CpG 2216 (1μg/mL),a typical A-type CpG ODN.The result showed that the supernatant of human PBMCs induced by CpG 2216 could protect Veto E6 cells from VSV challenge and MT01(8μg/mL) displayed significant inhibition but other MT ODNs couldn't.When used alone,none of MTODNs could stimulate human PBMCs to produced IFN evidently.Compared with all of other MTODNs,MT01 possessed the feature of C-rich sequence.1.3 Evaluating supernatant of human PBMCsIt was necessary to know whether the cytotoxicity of remaining MT01 in the supernatant of human PBMCs lead to the inhibition of MT01.Vero cells were cultured with the supernatant for 48 h and they all growed as well.Then we detect the culture supernatant of each group by 3%agarose gel electrophoresis.The result showed that there weren't remaining ODN in the supernatant of Medium group,MT01 group,CpG 2216 group and CpG 2216+MT01 group.It is implied that the cell death was due to the inhibition of MT01 on CpG 2216 induced immune activation.1.4 Confirming the inhibitory effect of MT01 Human PBMCs were cultured with CpG 2216(1μg/mL) and/or MT01(0.25, 0.5,1,2,4,8 and 16μg/mL) for 48 h or 72 h.The results showed that the inhibition of MT01 was enhanced by the increasing concentration.The time point of 48 h was chosen for the following experiments.2 Effect of MT01 on TLR9 induced human PBMC activationAs a common artificial TLR9 agnist,CpG ODN could stimulate pDC to produce a large amount of IFN-α(known as A-type CpG ODN,e.g.CpG 2216) and stimulate B cell proliferation(known as B-type CpG ODN,e.g.CpG 2006,CpG BW006).And C-type CpG ODN possess both A-and B-type characteristics. Meanwhile,we took heat inactivated DNA virus-herpessimplexvirus-1(HSV-1) as natural TLR9 agnist to investigate the inhibitory effect of MT01.2.1 Effect of MT01 on CpG ODN induced antiviral activity of human PBMCsHuman PBMCs were cultured with CpG 2216(1μg/mL) and/or MT01(0.25,0.5,1,2,4,8 16 and 32μg/mL) for 48 h.The doses of MT01 that began to act its suppressive effect were 1μg/mL,when the dose of MT01 increased the inhibitory effect enhanced.MT01 at 4μg/mL reached suppressive flat when human PBMCs were stimulated by CpG 2216 and CpG BW006.MT01 at 8μg/ml was selected as the optimal dosage in latter vitro experiments for testing its inhibitory effect with consistency.2.2 Effect of MT01 on human PBMC proliferation induced by CpG ODNHuman PBMCs were cultured with CpG 2006(1μg/mL),CpG BW006,CpG C274 and/or MT01(0.25,0.5,1,2,4,8 16 and 32μg/mL) for 48 h.From the result of VSV protection bioassay,MT01 was found to inhibit CpG ODN induced human PBMC proliferation in a dose-dependent manner.While used alone.MT01 couldn't stimulate human PBMC proliferation.As the parallel control,MS19 couldn't inhibit CpG ODN-induced IFN production and proliferation of human PBMCs.indicating that MT01 certainly possessed inhibitory role on immune responses without any toxicity.2.3 Kinetics of MT01 on CpG ODN induced IFN production of human PBMCsTo exclude the possibility whether the MT01 displayed inhibition was due to its extracellular interference with CpG ODN,the kinetics of the inhibition of MT01 was studied when using CpG 2216 as the stimulatory CpG ODN.MT01(8μg/mL) was added to human PBMCs at various times(0.25h,0.5h,1h,2h,3h,4h,5h,6h) after or before the addition of CpG 2216(1μg/mL).The result showed that significant inhibition was observed when MT01 were co-administered with CpG 2216 or added before CpG 2216(p＜0.01).MT01 was noticeably inhibitory even when added less then 2 h after addition of CpG 2216(p＜0.01) and could also inhibit CpG 2216 induced IFN production when added at the time point of 3 h and 6 h after addition of CpG 2216(p＜0.05).It was indicating that the inhibition of MT01 on TLR9 activation was consistent and was not because of the extracellular interference with CpG ODN.2.4 Structure-function analysis of MT01Structurally,MT01 was entirely composed of three repeats of a C-rich motif.To determine whether the repeat numbers of the motif contributed to the suppressive effect,ODNs that contained one and two of the motif were synthesized,named MT01a and MT01b respectively.We used VSV protection bioassay and proliferation assay to compare the inhibitory effect of them.The result showed that the inhibitory effect of MT01 a was much weaker than MT01b,while that of MT01 b was also weak than MT01.The inhibitory effect of MT01 on CpG ODN induced IFN production and proliferation was at the maximum so we didn't observe the inhibitory effect of ODNs with four or more motifs.2.5 Effect of MT01 on antiviral activity of human PBMCs induced by heat inactive DNA virusAct as natural TLR9 agonist.DNA virus could also result in acute or chronic diseases.Human PBMCs were cultured with heat inactive HSV-1 and/or MT01(8 g/mL) for 3.6,12,24 h.The result of VSV protection assay showed that MT01 could inhibit heat inactive HSV-1 induced antiviral activity of human PBMCs once the antiviral activity induced by the heat inactive HSV-1 was detected.3 Effect of MT01 on TLR7 induced human PBMC activationIt was reported that TLR7 activation could be a therapeutic target in autoimmune diseases.It was necessary to know whether MTO1 could inhibit the immune activation though TLR7.3.1 Effect of MT01 on antiviral activity of human PBMCs induced by IMQ Human PBMCs were cultured with IMQ(0.25μg/mL) and/or MT01(8μg/mL) for 48 h.The result of VSV protection bioassay showed that MT01 inhibited imiquimod(p＜0.01) induced IFN production.3.2 Effect of MT01 on antiviral activity of human PBMCs induced by heat inactive RNA virusRNA virus,natural TLR7 agonist,could induce human PBMCs to produce IFN. Human PBMCs were cultured with heat inactive influenza A virus Puerto-Rico-Stamm 8(PR8),influenza A virus A/Fort Monmouth/1/1947 H1N1 strain (FM1) and/or MT01(8μg/mL) for 48 h.The antiviral activity was detected by VSV protection assay.The result of showed that supernatant of human PBMCs induced by heat inactive PR8 or FM1 could protect Veto cells against VSV attack,and MT01 could inhibit the antiviral activity induced by the heat inactive RNA virus.4 Effect of MT01 on antiviral activity induced by serum from SLE patientsHuman PBMCs were treated with serum from 16 SLE patients for 48 h. Compared with normal serum group,the supernatant of SLE-1,SLE-9,SLE-14 and SLE-15 could protect the Vero E6 against VSV attacking.Then human PBMCs were cultured with serum from the above 4 SLE patients or in the present of MT01 for 48 h. In VSV protection assay,MT01 remarkably inhibited the antiviral activity induced by serum from SLE patients.The data indicated that MT01 could inhibit endogenous ligands of SLE patients,providing a therapeutic point for SLE or other autoimmune diseases.5 Suppressive effect of MT01 on cultured mouse cellsIn the above experiments used human PBMCs as the cell model.MT01 displayed its inhibitory effect on TLR7/9 activation.Some ODNs were granted in a species-specific manner.To determine whether MT01 was also species dependent and the feasibility of evaluating its in vivo effect in mice,we established two platforms for detecting mouse reactive inhibitory ODN. 5.1 Effect of MT01 on antiviral activity of mouse splenocytes induced by CpG ODNAfter screening the concentration(0,0.25,0.5,1,2,4,8,16 and 32μg/mL) and the incubation time(3,6,12,24,48,72 and 96 h) of CpG 2216,the dilution of the supernatant induced by CpG 2216,the concentration(1,2,4,8,16,32 or 64μg/mL) of A151,the method established was as followed:mouse splenocytes(cell density 5×106·mL-1) were cultured with 2μg/mL CpG 2216 and/or 16μg/mL suppressive ODN for 24 h.The supernatants were collected and added to L929 cells by 1:4 dilution.After 18 h incubation,the cells were cultured with VSV for 48 h and A578 was detected to screen suppressive ODN.Using CpG 2216 as the stimulator and A151 as the positive suppressive ODN,the conditions of screening experiment were established successfully and could be used for identifying mouse reactive suppressive ODN.It was demonstrated that MT01could inhibit the antiviral activity of mouse splenocytes induced by CpG 2216 by this method(P＜0.01),and the inhibitory effect was not due to cytotoxicity by 3%agarose gel electrophoresis.5.2 Effect of MT01 on proliferation of spleen cells induced by CpG ODNNext,we established a MTT method to detect mouse splenocytes proliferation. The method established was as followed:mouse splenocytes(cell density 5×106·mL-1) were cultured with 4μg/mL CpG 2006 and/or 16gg/mL suppressive ODN for 24 h and then the cell proliferation was detected with routine MTT.Using this method, MT01was detected to inhibit mouse splenocyte proliferation induced by CpG 2006(P＜0.01).Mouse splenocytes were cultured with MT01(8μg/mL) and/or CpG ODN(B type CpG ODN,CpG 2006 and CpG BW006)(C type CpG ODN,CpG C274)(1μg/mL) for 48 h to confirm above results.The result of 3H-thymidine incorporation assay also showed that MT01 could inhibit mouse splenocytes proliferation by above CpG ODN(P＜0.01).5.3 Effect of MT01 on antiviral activity of mouse splenocytes induced by heat inactive virusTo investigate whether MT01 could inhibit immune activation by natural TLR7/9 agonist in mouse splenocyte model,mouse splenocytes were cultured with heat inactive virus(HSV-1 or FM1) and/or MT01 for 48 h.The result showed that the antiviral activity of mouse splenocytes induced by HSV-1 and FM1MT01 could be inhibited by MT01(P＜0.01),but not by MS19.5.4 Effect of MT01 on proliferation of mouse splenocyte induced by LPSWe chose LPS as TLR4 agonist to investigate whether MT01 could inhibit LPS induced proliferation.The result of 3H-thymidine incorporation assay showed neither MT01 nor MS19 could inhibit LPS induced proliferation of mouse splenocytes,indicating that MT01 couldn't inhibit activation indeced by TLR4 agonist.6 Effect of MT01 on antibody production in mice and its mechanismFrom above in vitro experiments,it was demonstrated that MT01 could work in both human PBMC model and mouse splenocyte model.Next,we investigate the in vivo effect of MT01.6.1 Effect of MT01 on anti-HBs production in HBsAg immunized miceMice immunized with HBsAg or HBsAg plus CpG BW006were used as an animal model to test whether MT01 could inhibit antibody production in vivo.Female BALB/c mice(n=6) were immunized with HBV vaccine(containing 1μg HBsAg/mouse) or plus CpG BW006(5μg/mouse) on days 1,29 by injection the agents into the tibialis posterior muscle,and were injected with MT01(50μg/mouse) on day 0,7,14,21,28,35,42 and 49 to investigate whether MT01 could inhibit the production of anti-HBs antibodies in mice.The mice were bled on days -2,13,20,27, 34,41,48,and 55 to collect sera for detecting anti-HBs antibodies by ELISA.MT01, in mice immunized with HBV vaccine plus CpG BW006.inhibited the production of anti-HBs antibodies on day 35,42.49 and 56(p＜0.01).But in mice immunized with HBV vaccine alone.MT01 didn't inhibit the production,indicating that MT01 inhibited only the CpG ODN enhanced antibody response.6.2 The possible mechanism of MT01 mediated inhibition on CpG ODN induced antibody productionTo determine the cells on which the MT01 exerted its inhibition on antibody production induced by antigens,splenocytes from BALB/c mouse were stimulated by CpG BW006 in the presence of MT01 or MS19 as a negative control ODN.Cells were then doubly stained with PE-labeled anti-CD19 mAb and FITC-labeled anti-CD69 mAb at 12 h,with PE-labeled anti-CD19 mAb and FITC-labeled anti-CD80 mAb at 72 h and followed by the analysis on a FACS Calibur.The result showed that CpG BW006 obviously up-regulate CD69 and CD80 expression of the gated CD19+ cells.The up-regulation of the CD69 was almost completely inhibited by MT01,and the up-regulation of the CD80 was inhibited by half.The results demonstrate that the inhibition of MT01 on antibody production could be through the TLR9 pathways in B cells.To sum up,the C-rich oligodeoxynucleotide designated as MT01 designed by our laboratory could inhibit the immune activation of human PBMC or mouse splenocyte induced by various stimuli,and could inhibit over-production of antibody in mice.As a novel class of immunosuppressive ODN,MT01 could be of therapeutic use in immune activation associated diseases.
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