Immunoregulatory Effect Of Suppressive Oligodeoxynucleotides Through DNA Sensors And The Mechanism In Treatment Of Septic Peritonitis

The immunoregulatory oligodeoxynucleotides(rODNs)are oligodeoxynucleotides with immunoregulatory or inhibitory activity,whose motifs are similar to their own cell DNA and can inhibit the innate immune responses stimulated by PAMPs and/or DAMPs to reduce their own tissue damage.There are several DNA sensors in the cytoplasm,which can sense intracellular DNA to initiate innate immune responses.It is different from viral DNA produced in cells that rODNs contact intracellular DNA sensor only when they enter into cells.How the extracellular DNA is delivered into cells in which intracellular DNA sensor can sense it remains unclear.The septic peritonitis is the host's systemic inflammatory response to the bacteria,initiated by PAMPs including LPS and lipid A from ?-negative bacteria and LTA and peptidoglycan from ?-positive bacteria.The pathogenesis of sepsis includes countless disturbances of the host immune system starting with a harmful infection-triggered exaggerated inflammatory cascade.Numerous exogenous(endoand exotoxin)and endogenous mediators are capable of activating monocytes and macrophages,which synthesize cytokines.In this process,macrophages produce a great amount of pro-inflammatory cytokines that lead to host mortality.In order to maintain body health and immune homeostasis in the fight against pathogenic microorganisms and tissue injury,it is necessary to develop drugs that inhibit the excessive activation of innate immune system.In this study,the immunoregulatory effects of SAT05 f and MS19 on the expression of cytoplasmic DNA sensors in U937 cells was investigated.In addition,the role and possible mechanism of MS19 in inflammatory response were studied by using septic peritonitis model in mice infected with E.coli.These results will help to provide valuable experimental basis,and develop an inhibitory oligonucleotide drug for the treatment of autoimmune inflammatory diseases or autoimmune diseases.1.rODNs can regulate the mRNA expression of cytoplasmic DNA sensors in U937 cellsrODNs can inhibit the innate immune response stimulated by PAMPs and/or DAMPs and reduce the damage of their own tissue cells.Monocyte-macrophages are the main effector cells that cleave apoptotic or dead cells in vivo.The regulation of monocyte-macrophages in their own DNA in vivo may be the main reason for inducing innate immune tolerance.In order to verify whether rODNs can induce the activation of U937 cells,real-time PCR was used to detect the expression of a large number of known cytoplasmic DNA sensors in U937 cells.The results showed that the mRNA expression of many DNA sensors in the cytoplasm was regulated by ODNs at different times.rODNs had the most obvious regulation of DNA sensors after 2 hours' treatment.2.The relationship between SAT05f/MS19 and DEC-205 into U937 cellsA recent report demonstrated that DEC-205 could act as a cell surface receptor for Cp G ODN in human p DCs.To survey the possible role of DEC-205 for delivering SAT05 f and MS19 into U937 cells,the cells were labeled with anti-DEC-205 antibody followed by flow cytometry and indirect immunofluorescence analysis.The results showed that:(1)At 4oC culture condition,SAT05 f and MS19 didn't affect the level of DEC-205 on the cells.When the cells were cultured at 37 oC,SAT05f and MS19 decreased the level of DEC-205 on the surface of the cells.(2)DEC-205 expressed on the surface of the cells when cultured at 4oC,whereas moved into the cells when cultured at 37 oC.Indicate that DEC-205 can internalize into the cells along with SAT05 f and MS19.3.The role of IFI16 in the production of IFN-? induced by SAT05 f and MS19It is reported that IFI16 in the cytoplasm can be combined with endoplasmic reticulum intrinsic protein STING,and activate the type I interferon signaling pathway.Therefore,the effects of SAT05 f and MS19 on IFN-? expression was investigated by means of q RT-PCR,Western Blot,RNAi,ELISA and indirect immunofluorescence assay.The results showed that:(1)Up-regulated expression of IFN-? was observed at both mRNA and protein levels in U937 cells treated with SAT05 f and MS19.(2)The effect of SAT05 f and MS19 on the production of IFN-? in U937 cells was abolished by IFI16 knockout.(3)In the cytoplasm of U937 cells,the co-localization of IFI16 with SAT05 f and MS19 was observed.Suggesting that in U937 cells,SAT05 f and MS19 can bind to IFI16 and induce IFN-? production.4.Induction of septic peritonitis in mice by intraperitoneal injection of E.coliAccording to the common infection sites of sepsis,E.coli was choosen as pathogens,through the way of intraperitoneal injection to establish a primary infection in the abdominal cavity of septic peritonitis mice model,through clinical symptoms score,survival and inflammatory response of PLCs,to determine whether the model was established successfully.The results showed that:(1)Intraperitoneal injection of E.coli can cause some systemic symptoms of severe infection in mice,including lethargy,tremor,vertical hair,periorbital secretions and respiratory distress.(2)Autopsy found that the liver of the mice had diffuse bleeding and intestinal edema.(3)Intraperitoneal injection of E.coli could cause mice to die,death peak concentrated in 18-28 h after infection.The 72 h mortality rate is 65%.(4)The levels of i NOS,TNF-? and IL-6 in the PLCs were significantly increased after the bacterial infection.Thus,intraperitoneal injection of E.coli can induce septic peritonitis in mice.5.The effects of SAT05 f and MS19 on the survival of septic peritonitis mice and the regulation of inflammatory factors expression in PLCsrODNs have the effect of reducing and/or inhibiting inflammation.Excessive inflammatory response is the main reason for sepsis death.Therefore,we study the effects of SAT05 f and MS19 on the survival of E.coli induced septic peritonitis mice and the regulation of inflammatory factors expression in PLCs.The results showed that:(1)MS19 could prolong the survival in model mice;SAT05f was weaker.(2)MS19 could down-regulate the expression of i NOS,TNF-? and IL-6 in PLCs of peritonitis mice;SAT05f had a slight decrease in the expression of these genes,but there was no significant difference compared with the control group.The results suggest that MS19 saved the lives of model mice by inhibiting the inflammatory response.6.The possible mechanism of MS19 inhibits the expression of inflammatory factors in monocyte-macrophagesIRF5 was reported as a reliable marker for M1 macrophages at sites of inflammation.IRF5 expression is induced during differentiation of human monocytes and murine bone marrow–derived macrophages into M1 macrophages.In turn,IRF5 directly induces the production of pro-inflammatory cytokines such as IL-6 and TNF-?.The amino-terminal DBD of IRF5 is involved in recognition of target DNA sequences,with the putative binding motif(G/C)(A)AAA(N)2-3AAA(G/C)(T/C).The motif is in consensus with MS19.Based on this,we used LPS-treated RAW264.7 cells to mimic the macrophages in the inflammatory site and LPS-treated U937 cells to mimic human bacterial inflammatory cell model.Through the study of the two cell models,we found that:(1)MS19 can down-regulate the production of i NOS,TNF-?,IL-6 and IRF5 from LPS-treated cells.(2)MS19 prevents the excessive activation of downstream inflammatory signals possibly by interfering nuclear transportation of IRF5.In summary,the research attempted to explore the signaling pathway of rODNs enter human monocytes.The experimental findings revealed that SAT05 f and MS19 were predominantly sensed by IFI16 along with DEC-205 internalization in U937 cells,and thereby triggered IFN-? production.Interference with IFI16 si RNA can partially inhibit the IFN-? production induced by SAT05 f and MS19.In addition,the model of septic peritonitis in mice by E.coli was established,and research found that MS19 could prolong the survival in model mice,down-regulate the expression of i NOS and IRF5,interfere nuclear transportation of IRF5,inhibit the polarization of M1 macrophages,and prevent the excessive activation of downstream inflammatory signals.These results will help to elucidate the mechanism of MS19 and provide valuable experimental evidence for the development of an inhibitory oligonucleotide drug for the treatment of autoimmune inflammatory diseases or autoimmune diseases.