| A variety of complex mechanisms on the pathogenesis of autoimmune diseases still remain poorly understood.Currently,the use of clinical drugs cannot eliminate autoimmune diseases(AID)effectively,and even may cause severely side effects.A growing number of studies show that innate immune disorders are associated with AID.The pattern recognition receptors(PRRs)in the innate immune system can specifically recognize pathogen-associated molecular patterns(PAMPs),which initiate relevant signal transmission releasing inflammatory cytokines.However,excessive inflammation is able to initiate AID,such as systemic lupus erythematosus(SLE),scleroderma,psoriasis,and rheumatoid arthritis(RA).So,to improve autoimmune pathogenesis by identification of new therapeutic targets has become a research priority.Toll-like receptors(TLRs)expressed in dendritic cells(DCs)and macrophages are a family of proteins,and these receptors constitute the first line of immune system against related pathogens.TLRs can recognize specific PAMPs,such as TLR4 can combine with LPS,TLR3 and TLR7/8 are respectively able to recognize double-stranded(ds)and single-stranded(ss)RNA,while TLR9 can recognize unmethylated Cp G-DNA originated from microorganisms.These receptors take advantage of different downstream signaling pathways: TLR3 relys on the TRIF pathway and TLR7 and 9 completely depends on the MyD88 pathway.Even so,different TLRs activation leads to similar inflammatory responses and induces release of cytokines from immune cells.It was also reported that some TLRs activation can lead to the formation and activation of the Nod-like receptor 3(NLRP3)inflammasome,and increase the release of caspase-1,IL-18 and I L-1β,which are closely related to AID.The TLRs and their related signaling pathways constitute a complicated network,which is difficult to identify key therapeutic targets.Furthermore,excessive inflammationis usually caused by multiple TLRs activation.Consequently,the co-activation of different TLRs makes it more complex.Clinical drugs non-selectively target the terminal process and control the pro-inflammatory reponses.Some antibodies against IL-6,TNF-?,IL-23 and IL-17 have been used to relieve AID.Although this clinical therapeutic way has shown some promise,it is also related to a higher risk of serious infections.In contrast to targeting the terminal process,blocking upstream pathway could reduce side effects and would serve as an optimal therapeutic way.However,rational drug target has not been identified so far.CpG-c41,previously known only as a TLR9 antagonist,is composed of nucleotide sequences of 20bp(5’-TGGCGCGCACCCACGGCCTG-3’).In this study,we futher investigate the immune characteristics of CpG-c41 and its immunoeffects on different TLRs-mediated inflammation in vivo and in vitro used a variety of related experimental techniques,such as ELISA,Western blot and immunofluorescence microscopy.Moreover,we also evaluate the safety profile of Cp G-c41 by acute toxicity testing and CCK-8.This study presents multiple immunosuppressive effects of CpG-c41 on intracellular TLR-mediated inflammation.And CpG-c41 has a very good therapeutic effect on psoriasis.These results suggest that it may be possible to develop drugs.Object:To investigate the immune characteristics of non-stimulatory CpG-c41 and its immunoeffects on TLRs-mediated inflammation in vivo and in vitro.First,Evaluation of the safety profile of the Cp G-c41 molecule is preliminarily observed.Then we investigate the effects of CpG-c41 on the activation of multiple TLRs,and research the mechanism of action of this molecule.Finally,we establish the psoriasis-like mouse model induced by Imiquimod cream and research on the mechanism of disease treatment.Methods:1.Safety profile of CpG-c41Preliminarily research on animal acute harm induced by Cp G-c41 used 10 times dosage(80mg/kg)in the animal acute toxicity testing;and research on CpG-c41 under the high concentration(2-64μM)to L929 cells damage degree used CCK-8.2.Multiple immunoeffects of CpG-c41 on TLRs-mediated inflammation(1)Immunoeffects of CpG-c41 on cytokine secretion induced by TLRs activationWe researched the immunoeffects of Cp G-c41 on the levels of related cytokineinduced by TLRs activation in BMDMs and BMDCs and human THP-1 cells,and two TLRs activation in RAW264.7 cells.All cells were seeded into 96-well tissue culture plates and stimulated as indicated: TLR2 agonist(zymosan,200μg/ml),TLR3 agonist(Poly(I:C),100μg/ml),TLR4 agonist(LPS,100ng/ml),TLR7 agonist(imiquimod,2μg/ml),TLR7/8agonist(ssRNA120,30μg/ml)mixed with DOTAP,TLR7 agonist(R848,0.2μg/ml),and TLR9 agonist(CpG-1826,2μM)in the presence or absence of CpG-c41(4μM).After 24 hours,the levels of cytokines in cell-free culture supernatants were determined by ELISA.WT mice(18-20g)received intraperitoneal injection as indicated: TLR3 agonist(Poly(I:C),40μg/20g),TLR7 agonist(R848,10μg/20g)or TLR9 agonist(Cp G-1826,160μg/20g).Meanwhile,these mice received CpG-c41(320μg/20g)or normal saline(NS)by tail vein injection.The levels of TNF-α,IL-6,IFN-α,and IL-12/23p40 in sera from the different time points were measured by ELISA.(2)Effects of Cp G-c41 on NLRP3 inflammasome formation and activationFormation of the NLRP3 inflammasome is also promoted by TLR activation.We investigated the immunoeffects of CpG-c41 on the basic elements and related effector molecules of the NLRP3 inflammasome.RAW264.7 cells and BMDMs were seeded into culture plates,and then were stimulated with TLR3 agonist(Poly(I:C),100μg/ml),TLR4 agonist(LPS,100ng/ml),TLR7 agonist(R848,1μg/ml),or TLR9 agonist(CpG-1826,3μM)in the absence or presence of CpG-c41(8μM)for 4 hours.Cells were then stimulated with ATP(5mM)for30 min.NLRP3 and caspase-1 protein expression in RAW264.7 cells were detected by Western blot.The levels of IL-1β from BMDMs cultures were measured by ELISA.(3)Research on relationship between multiple immunoeffects of Cp G-c41 and TLR9TLR9-/-BMDMs were seeded into 96-well culture plates at 5×105/200 μl/well.Cells were stimulated with TLR3 agonist(Poly(I:C),100 μg/ml),TLR7 agonist(Imiquimod,2μg/ml),TLR7 agonist(R848,0.2μg/ml),and TLR9 agonist(CpG-1826,2μM)in the absence or presence of CpG-c41(4μM)for 24 hours.The levels of TNF-α,IL-6 in cell-free culture supernatants were determined by ELISA.3.Pharmacodynamic study of CpG-c41 on IMQ-induced psoriasis-like disease(1)CpG-c41 improved damage from IMQ-induced psoriasis-like diseaseMouse model of psoriasis-like skin inflammation with commercially available Aldaracream(5% imiquimod,IMQ)was established.Female BALB/c mice(8-10 weeks,18-20g/mouse)were divided into placebo and treatment groups.The placebo group was administered with normal saline(NS,100μl/mouse)and the treatment group with Cp G-c41(320μg/mouse)by subcutaneous injection(100μl/mouse).Both groups were topically applied 45 mg of IMQ cream on the shaved back skin once per day.The process was performed for 6 consecutive days.Meanwhile,the psoriasis area and severity index(PASI)was recorded daily.Three parameters,including scaling,erythema,and thickness,were measured and scored independently on a scale from 0 to 4(0,none;1,slight;2,moderate;3,marked;4,very marked).The cumulative score was the sum of the three parameters,ranging from 0 to 12.For histological assessment,dorsal skin from the mice model were fixed in 10%formalin for 24 h at 22 ℃ on day 7.Dorsal skin was embedded in paraffin and eparaffinized 5-μm sections were stained with hematoxylin erythrosine saffron,and last evaluated by light microscopy.(2)CpG-c41 attenuates IMQ-induced psoriasis-like inflammation in vivoTo investagate the mechanism of CpG-c41 attenuating IMQ-induced psoriasis-like inflammation,so we used histological immunofluorescence assessment.After disease induction,5-μm frozen sections of dorsal skin from 24 hours,72 hours,and 7 days were prepared.Primary antibodies to CD3(1:150),IL-23p19(1:200)and antibody to F4/80(1:150,Alexa Fluor 488),and Cy3-labeled goat anti-rabbit secondary antibody(1:1000)were used according to the manufacturer’s instructions.Finally,immune cells and cytokine infiltrating into IMQ-damaged skin was assessed by immunofluorescence microscopy.Results1.Safety profile of CpG-c41Both test and control group showed mice were no obvious symptoms after given a high dose of Cp G-c41.CCK-8 test showed that there were no differences in the activity of L929 cells between in absence and in presence of Cp G-c41(64μM)(p>0.05).2.Multiple immunosuppressive effects of Cp G-c41 on intracellular TLRsmediated inflammation(1)CpG-c41 suppresses intracellular TLR-induced inflammationThe immunoeffects of non-stimulatory Cp G-c41 on the activation of TLRs wasinvestigated in murine BMDMs and BMDCs.CpG-c41 respectively decreased the levels of TNF-α,IL-6 induced by TLR3 agonists(poly I:C),TLR7 agonists(R848)and TLR9agonists(CpG-1826)(P<0.01),but not those induced by TLR2 or 4 agonists(zymosan or LPS).The immunoeffects of CpG-c41 on TLR8 activation was also observed in the human monocytic cell line THP-1.The results showed that CpG-c41 also effectively inhibited TLR8 activation induced by ssRNA120(TLR7/8 agonist)(P<0.01).TLR3,7/8,and 9 are intracellular receptors,and TLR2 and 4 are cell membrane receptors.Therefore,these data suggested that Cp G-c41 selectively suppresses intracellular,but not cell membrane,TLRs.Furthermore,the immunoeffects of CpG-c41 on RAW264.7 cells was investigated in which two intracellular TLRs were stimulated simultaneously.CpG-c41 significantly reduced cytokines release(P<0.01).Thus,CpG-c41 showed an immunosuppressive effect on intracellular TLR co-activation.Then we studied the immunoeffects of Cp G-c41 in vivo.Mice serum TNF-α,IL-6,IFN-α levels were elevated an hour after treatment with TLR agonists,and were significantly decreased 3 hours after treatment.Serum IL-12/23 total p40 levels were elevated in the third hour,and then significantly decreased in the sixth hour after treatment.Treatment with CpG-c41 effectively suppressed production of serum cytokines at each time point(P<0.01).(2)Cp G-c41 inhibits intracellular TLR-mediated inflammasome formation and activationLPS,R848,and Cp G-1826 could increase the expression of NLRP3,induced the cleavage of caspase-1,and promoted the secretion of IL-1β.However,CpG-c41 could inhibit the inflammasome activation induced by R848 and CpG-1826.It reduced the levels of NLRP3 and caspase-1,and significantly suppressed IL-1β production(P<0.01).Unfortunately,in contrast to previous reports,we could not detect Poly(I:C)-induced NLRP3 inflammasome activation,(3)TLR9-independent immunosuppressive effects of CpG-c41The in vitro experiments were repeated using TLR9-/-BMDMs.Cp G-1826,a TLR9 agonist,lost its immunostimulatory effect in TLR9-/-BMDMs;however,TLR3 and 7 couldbe activated normally by their ligands.Interestingly,non-stimulatory CpG-c41 was still able to significantly suppress the releases of TNF-? and IL-6 induced by TLR3 and 7activation in the TLR9-/-BMDMs(P<0.01).3.Pharmacodynamic study of CpG-c41 on psoriasis(1)CpG-c41 reduces damage from IMQ-induced psoriasis-like diseaseThe PASI scores showed that CpG-c41 treatment significantly decreased IMQ-induced skin injury(P<0.01).The treated skin was relatively smooth and reduced thickness compared with the skin of the placebo group at day 6.Pathological analysis showed that acute skin inflammation wasd improved in the treatment group.(2)CpG-c41 attenuates IMQ-induced psoriasis-like inflammation in vivoPeak F4/80+macrophage infiltration was on day 3 in the placebo group,with significantly lower infiltration on day 7.IL-23p19 increased expression in the epidermal layer on day 7,and T cell infiltration increased in the epidermis and dermis on day 7.In contrast to the placebo group,CpG-c41 remarkably decreased macrophage infiltration(P<0.01),reduced IL-23p19 release(P<0.01),and significantly inhibited T cell infiltration(P<0.01).Conclusions(1)These results suggested that Cp G-c41 had a good safety profile.It had no toxicology in vitro and in vivo.(2)TLR3,7/8,and 9 are intracellular receptors,and TLR2 and 4 are cell membrane receptors.Therefore,these data indicated that CpG-c41 selectively suppresseed intracellular,but not cell membrane,TLRs-mediated inflamattion.Moreover,Cp G-c41 significantly inhibited intracellular TLR-mediated NLRP3 inflammasome formation and activation.However,the multiple immunosuppressive effects of Cp G-c41 did not require interaction with TLR9.(3)These findings indicated that Cp G-c41 can attenuate IMQ-induced psoriasis-like inflammation by suppressing macrophage activation,cytokine release and T cell infiltration. |
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