Inhibition Effect Of Microsatellite Oligonucleotides On Pathogen-Associated Molecular Patterns Induced Inflammation Immune Response In Innate Immune Cells

The innate immune system constitutes the first line of defense against invading microbial pathogens and relies on a large family of pattern recognition receptors(PRRs),which detect distinct evolutionarily conserved structures on pathogens,termed pathogen-associated molecular patterns(PAMPs).Upon PAMP engagement,PRRs trigger intracellular signaling cascades ultimately culminating in the expression of a variety of cytokines and prionflammatory molecules,which together orchestrate the host immune response against bacteria,viruses,fungi,and parasites.However,these damage-associated molecular patterns(DAMPs)released from damaged or necrotic cells are recognized by certain PRRs and subsequent over-activate the innate immune response and produce a large number of cytokines.The over-produced cytokines,as found in basic and clinical studies,contribute to immune pathological damage,even leading to a fatal outcome.Obviously,it is required to develop agents to curb the over-reaction and the over-produced cytokines for maintain immune balance.Inhibitory oligodeoxynucleotides(ODNs),an artificially synthesized single-stranded DNA molecule,could regulate innate immunity activation.In the previous study,SAT05f and MS19,the MS ODNs(microsatellite DNA mimicking oligonucleotides)designed with reference of the sequence of human microsatellite DNA,are capable of improve the survival rate in mouse model of immune over-activated disease,such as sepsis,systemic lupus erythematosus,and arthritis.However,which types of innate immune cell involved in the role of MS ODNs on controlling the immune over-activated disease are not entirely clear.In this study,we isolated and cultured various innate immune cells from mouse bone marrow.Then,we detected the inhibition role of MS ODNs and MS19 mutant on the production of pro-inflammatory cytokines form bone marrow-derived cells.stimu-lated by various PRRs.In vivo experiments,we observed the effect of MS ODNs on acute lung injury induced by LPS in mice and study the mechanism.1.The inhibitory effect of MS ODNs on secretion of inflammatory cytokines and interferon(IFN)in bone marrow-derived macrophages(BMDMs),bone marrow-derived dendritic cells(BMDCs)and bone marrow-derived plasmacytoid dendritic cells(BM-pDCs)stimulated by DNA virus,RNA virus and bacteria in vitro.BM mononucleocytes were isolated from mouse and added the L929-conditioned medium,recombinant murine granulocyte-macrophage colony stimulating factor(rmGM-CSF)and recombinant murine FMS-like tyrosine kinase 3ligand(rmFLT3L)to obtain BMDMs,BMDCs and FLT3L~+cells(including 10-15%BM-pDCs),respectively.We choose the DNA viruses(Adenovirus(ADV),Herpes simplex virus(HSV)and DNA virus mimics VACV-70),RNA virus(Influenza virus,Reovirus(Reov)and RNA virus metabolism products dsRNA long poly(I:C),(LPIC)and bacterial components(lipopolysaccharide(LPS),flagellin and Gram-positive bacteria cell wall component Curdlan)as stimulus.MS19(1mM)or SAT05F(1mM)transfected with Lipofectamine 3000(Lipo 3000)into cells and then incubated with different stimulus for 16 h.Cells with transfect Lipo 3000 were as medium control.The concentration of TNF-aand IL-6 in the supernatant was detected by ELISA.The results showed that MS19 and SAT05F could significantly inhibit the TNF-?and IL-6 production from BMDMs triggered by DNA virus(VACV-70,HSV,ADV),RNA virus(PR8,Reov,LPIC)and LPS(P<0.01).Notably,MS19 and SAT05F failed to inhibit the IL-6 production from BMDMs stimulated by curdlan,a bacterial cell wall component(P>0.05).Additionally,MS19 and SAT05f were capable of inhibiting IFN-asecretion of BMDCs induced by influenza virus.Like BMDMs and BMDCs,MS19 and SAT05F can reduce the production of IFN-aand TNF-ain FLT3L~+cells in response to CpG A ODN and CpG B ODN and influenza virus.Based on these results,we found that MS ODNs(MS19 and SAT05F)displayed a strong inhibition role on the secretion of cytokines from BMDMs,BMDCs and FLT3L~+cells stimulated by various microbial pathogens,except for curdlan.2.The inhibitory effect of MS ODN and MS ODN mutant on production of inflammatory cytokines.from RAW264.7 cells stimulated by LPS.Single site mutants(G to C)were employed to measure sequence-dependent mechanism properties of AAAG-rich ODN(MS19)on inhibiting the production of inflammatory cytokines from innate immune cells.In the mouse RAW264.7macrophage cell line,the mRNA expression level of IL-6 and TNF-aof RAW264.7stimulated by LPS with or with not MS19 or MS19 mutant(MS19-M1)by RT-PCR.The results showed that MS19 could inhibit the production of IL-6 and TNF-afrom RAW 264.7 cells induced by LPS,while MS19-M1 could not.It suggested that G base is a key site in AAAG motif,which is associated with biological functions of MS19.3.Regulatory effect of MS ODN and MS ODN mutant on activation of p65 in NF-kB signaling pathway in RAW264.7 cells.Activation of p65 in response to a variety of stimuli is most typical process of NF-kB activated in RAW264.7 cells.To find the initial mechanism of MS ODN on inhibiting the production of pro-inflammatory cytokines in innate immune cells,we detected the expression of p65 phosphorylation in RAW264.7 cells stimulated by LPS with or with not MS19 or MS19 mutant(MS19-M1)by western blot.In contrast to the RAW264.7 cells treated with various LPS or VACV70 or influenza virus stimulation alone,the phosphorylation of p65 intensity was dramatically decreased in RAW264.7 cells treated simultaneously with MS19 and various stimulus.We speculated that the function of MS19 on inhibition of IL-6 and TNF-aproduction in innate immune cells relies on inhibiting the phosphorylation of p65 in the NF-?B signaling pathway.4.Inhibitory effect of MS19 on LPS-induced acute lung injury in miceUpon these,MS 19 could block the production of IL-6 and TNF-aby blocking the phosphorylation of p65 in macrophages and dendritic cells.Based on the published articles,a large number of innate immune cells like macrophages and dendritic cells harbor into the lung epithelial cells.When the immune system was over-activated,the localized macrophages and dendritic cells in the lung firstly secreted a large number of inflammatory factors that constitute to the acute inflamma-tory response.Here,we want to observe the effect of MS19 on LPS-induced acute lung injury in mice.On day 0,mice were inoculated intranasally with LPS(20 mg/kg)to establish LPS-induced acute lung injury model mice.The mice(n=8)sham-inoculated with sterile PBS were used as health control.On days 1 before infection,the model mice(n=8)were translocate with PBS or MS19 by mixing the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP).We observed the effect of MS19 on the morbidity,mortality,and lung inflammatory injury of mice induced by LPS.The results showed that the model mice received PBS displayed 13%mean weight loss on day 4 post infection,while model mice treated with MS19 only displayed 10%mean weight loss,indicated that MS19 significantly inhibit the weight loss of mice treated with LPS.Compared with the model mice treated with PBS,the mortality rates of the mice treated with MS19 successively increased from 62.5%to 87.5%.The lung of the mice treated with MS19 manifested thinner alveolar walls,lacking intra-alveolar edema fluid and infiltrated leukocytes.Furthermore,the pathological score of the lung tissue of MS19-treated mice was lower than that in the mice treated with PBS.Besides,MS19 significantly inhibit the mRNA expression levels of TNF-a,IL-6,IL-1bin the Bronchoalveolar lavage fluid(BALF)of model mice.In present study,we found that MS19 and SAT05f could significantly inhibit the production of IFN and inflammatory cytokine by blocking the activation of p65 in NF-kB signaling pathway in macrophages and dendritic cells induced by various stimulis in vitro.Meanwhile,in mice model of LPS-induced acute lung injury,we observed that MS19 inhibited the weight loss of mice,increase the survival rate of the mice and displayed dramatic effect on lessening the acute lung injury by decreasing the cytokines level in BALF and reducing consolidation,intra-alveolar edema and leukocyte infiltration in lungs of the mice.The data suggested that MS19 might be developed a candidate ODN for the treatment of over activated immune disease.