Inhibitory Effect And Structure-activity Relationship Of A Human Microsatellite DNA Mimicking Oligonucleotide

Innate immunity is the first line of host defense against invading pathogens. Bypattern recognition receptors (PRR) on the surface, innate immune cells canspecifically recognize microbial pathogen associated molecular patterns, and theninduce a strong inflammatory response via activated signaling pathways to remove theinvading pathogens. Toll-like receptor (TLR) is currently one of the most studiedfamilies of PRR. Under normal physiological conditions, TLR-mediated innateimmune response contributes to host defence against pathogen invasion. However, ifthe response is out of control, the innate immune cells could be over-activated andproduce excessive cytokines, causing inflammatory and autoimmune diseases.Obviously, it is required to develop agents to inhibit the activation of overwhelminginnate immune response for the treatment of such diseases.Inhibitory oligonucleotide (inhibitory ODN) is a synthetic single-stranded DNAmolecule with a specific sequence characteristics. It can inhibit the activation ofTLR-mediated innate immune and display good prospects for the application ontreating TLR activation-related diseases. In the previous work of the laboratory, wedesigned a human microsatellite DNA-mimicking ODN with CCT repeats, named asSAT05f (5′-CCTCCTCCTCCTCCTCCTCCTCCT-3′). It can inhibit PBMC IFN-αproduction induced by viral nucleic acid, protect mice against lethal shock caused byD-GalN/CpG ODN and reduce the generation of anti-double stranded DNAantibodies in mice with lupus-like syndrome. These results imply that SAT05f has agood potential for the treatment of excessive immune activation mediated disease.In this study, we further detected the inhibitory effects of SAT05f on CpGODN-induced proliferation of mouse splenocytes and activation of mousemacrophages (RAW264.7) and human pDC-like cells (CAL-1), and observe thetreatment of SAT05f on heat-killed bacteria induced septic shock mouse. In addition,we also studied the structure-activity relationship of SAT05f and preliminalydiscussed the mechanism of SAT05f. 1. Inhibitory effects of SAT05f in vitro1.1Effects of SAT05f on CpG ODN-induced proliferation of mouse splenocytesTo observe the inhibitory effect of SAT05f on the proliferation of immune cell,mouse splenocytes (5×105/well) were isolated and cultured with SAT05f (at a doserange of0.1-1μM) alone or in presence of CpG1826(0.3μM) for48h, and thensubjected to MTT assay. The results show that SAT05f inhibited splenocyteproliferation induced by CpG1826in a dose-dependent manner. While used alone,even if1μM, SAT05f does not induce or inhibit the proliferation of mousesplenocytes.1.2Analysis of cell population responsive to SAT05f in splenocytesTo determine the cell population responsive to SAT05f, mouse splenocytes werestimulated with CpG1826(0.3μM) alone or in presence of SAT05f (1μM) for48h,and then stained with FITC-labeled anti-CD19mAb or FITC-labeled anti-CD3mAb,followed by analysis on a FACScan. The results show that CpG1826activated Blymphocytes in the splenocytes are the cells responsive to SAT05f.1.3Effects of SAT05f on cell binding and uptake of CpG1826In order to observe whether inhibiting the binding and uptake of CpG1826by Bcell contribute to the inhibition of SAT05f on the CpG1826induced proliferation,0.3μM FITC-1826alone or in combination with1μM SAT05f was added to BALB/cmouse splenocytes. Then the cells were cultured at4oC for10min (bindingexperiment) or at37oC for1h (uptake experiment). After stained by PE-CD19mAb,the spleen cells were detected by the flow cytometry. The results show that SAT05fcan significantly inhibit the binding and uptake of CpG1826by B cells.1.4Effects of SAT05f on TNF-α production in RAW264.7induced by CpG1826Excessive TNF-α production has been associated with the pathogenesis of severaldiseases, including rheumatoid arthritis, Crohn's disease and sepsis. To observe theinhibitory effect of SAT05f on TNF-α production, we chose CpG1826as a stimulatorto induce the TNF-α production by RAW264.7, and added SAT05f at equal molarconcentration at the same time. After culture6h, we detected the levels of TNF-α inthe culture supernatant by measuring the biological activity of TNF-α. The resultsshow that SAT05f can inhibit the TNF-α production by RAW264.7(P=0.003). Whenused alone, SAT05f does not induce the TNF-α production by RAW264.7. 1.5Effects of SAT05f on the expression of TLR9mRNA induced by CpG1826In order to detect whether the inhibiton on the expression level of TLR9mRNAcontributes to the inhibition of SAT05f on IFN-α production in RAW264.7induced byCpG1826, we add CpG1826and SAT05f at equal molar concentration into culturemedia of RAW264.7for6h. After isolated RNA from cells as templates, we detectedthe expression level of TLR9mRNA by Real-time PCR. The results show thatSAT05f can inhibit the expression of TLR9mRNA induced by CpG1826(P=0.046).1.6Effects of SAT05f on TNF-α production in CAL-1induced by YW002Abnormal activation of plasma cell-like dendritic cells (pDC) plays an importantrole in the development and progression of some autoimmune diseases such assystemic lupus erythematosus. In order to expand the application of SAT05f in thesediseases, we must confirm the inhibition of SAT05f on the activation of pDC. Whilethe establishment of test platform based on the pDC in vitro is limited because theisolated process of pDC is tedious and pDC can not be passaged in vitro. Therefore,we selected CAL-1cells (pDC-like cell line) and established TNF-α productionplatform in CAL-1induced by CpG ODN (YW002). To observe the effect of SAT05f,CAL-1was stimulated by6μg/ml YW002and added1μM SAT05f at the same time.After culture48h,the supernatant of CAL-1was havasted. Then the levels of TNF-αin the culture supernatant was measured by the biological activity of TNF-α. Theresults show that1μM SAT05f can significantly inhibit the TNF-α production inCAL-1induced by YW002(P=0.005).2. Effects of SAT05f on septic shock induced by heat-killed bacteria in miceWe have found that SAT05f could inhibit TNF-α production in RAW264.7andCAL-1in vitro. These results suggested that SAT05f has a good prospect for treatingthe excess TNF-α-related diseases, such as sepsis. In order to observe the inhibitoryeffect of SAT05f in vivo, we have established a mouse model of septic shock inducedby D-galactosamine (D-GalN.) and heat-killed bacteria (HKB). The process is asfollows: female BALB/c mice were injected intraperitoneally with1g/kg D-GalN, andthen were injected intraperitoneally with8×108CFU heat-killed bacteria after1.5h.By the survival status of mice as an indicator, we observed the effects of SAT05f onthis mouse model of septic shock. The results show that SAT05f couldn't prolong thesurvival time or increase the survival rate of mouse at dose of10μg and50μg, whileSAT05f protected40%of the mice from the lethal shock at dose of250μg. 3. Study on structure-activity relationship of SAT05fTo develop more efficient inhibitory ODNs, we designed and synthesized a seriesof ODNs based on the sequence of SAT05f by changing repeat number of CCT unit,substituting CCT unit with AAG at3′end or5′end or in the middle and by forminghairpin at5′or3′end, and tested their inhibitory effect on the CpG ODN inducedproliferation and TNF-α production of murine immune cells. The results indicated that1) at least8CCT units were required for a CCT repeat ODN to display its inhibitoryactivity;2) CCT unit at3′end of SAT05f was necessary for its full inhibitory activity;3)5′end of SAT05f could be modified to design a more potent SAT05f derivedinhibitory ODN.In summary, SAT05f can significantly inhibit the TLR-mediated activation inimmune cell. And the sequence at5' end of SAT05f could be optimized to obtainmore potent inhibitory ODNs. Therefore, SAT05f and its derivatives could bedeveloped as a candidate medicament for the treatment of diseases associated withover-activated innate immune response.