Inhibitory Effect Of Human Microsatellite DNA Mimicking Oligonucleotides On The Activation Of Human Immune Cells

DNA is a kind of biomacromolecule which is composed of nucleotides, almost all the genetic information determining biological features is stored in DNA molecule. The sequence character and modified style of DNA varies significantly between different species, such as prokaryotic and eukaryotic organism, animal and plant, microbe and vertebrate. Recent studies demonstrate that DNA has some special biological activities in addition to carrying and transmitting genetic information, for example, certain DNA with specific sequence and modification has stimulatory or inhibitory effect on the immune system. The investigation on the immunoregulatory activity of DNA molecule not only helps to recognize the discipline of organic evolution, some DNA which has specific sequence and modification can be used for the prevention and treatment of diseases.Mammalian immune system consists of innate immune and adaptive immune. The innate immune system senses diverse pathogen-associated molecular patterns (PAMP) derived from microbes through pattern recognition receptors (PPR). TLR family is an important group of PRR. TLR3, TLR7, TLR8 and TLR9 detect bacterial and viral nucleic acid (DNA and RNA). Recognition of PAMPs by TLRs culminates in the production of inflammatory cytokines, chemokines, IFNs and upregulation of co-stimulatory molecules and subsequently activates adaptive immune. However, TLR-mediated activation might also result in deleterious inflammation and autoimmune. It has been demonstrated that self DNA and RNA might be endogenous ligands for TLRs. After stimulated by microbial and self DNA, pDC and auto-reactive B cells expressing TLR9 product high level of IFN-αand autoantibodies, resulting in autoimmune diseases, such as SLE.In physiological settings, however, self DNA released due to cell apoptosis does not promote immune activations, resulting in autoimmune. It has been reported that there are certain DNA in mammalian genome which negatively regulate immune activations. Microsatellite (MS) DNA is short tandemly repeated DNA composed of 1~6 bp units and distributes randomly in coding and non-coding regions. Because of its polymorphic property, MS DNA has been used as ideal molecular markers. Certain MS DNA is associated with cancers and neurological disorders. Up to now, very little is known about the immune regulatory property of human MS DNA.From the evolutionary point of view, it has been established that microbial DNA are sensed as"exogenous danger signals"through TLR9-dependent and–independent pathways, resulting in immune activation. Immune system might sense self DNA released during cell apoptosis as"endogenous danger signals", resulting in inactivation of excessive immune responses. We hypothesize that human MS DNA released from cells may block the excessive immune response for maintaining a homeostasis. To investigate this, we synthesized a series of ODNs with the sequences of human MS DNA (designated as MS ODN) and used them to mimic human MS DNA. We test whether MS ODNs could inhibit the activation of human PBMCs through TLR9-dependent and -independent pathways. The results demonstrate that MS ODNs can down-regulate the immune activation of human PBMCs induced by CpG ODN, HSV, Flu virus, PHA, PMA and alloantigens. The inhibition of MS ODN on CpG ODN correlates with their ability to block CpG ODN's entry into PBMCs. MS ODNs are expected to be developed as novel immunoregulatory medicine and used for the treatment of diseases associcated with imbalance of immune response, such as autoimmune diseases, graft rejection and hypersensitivity.1 Preliminary screening of suppressive ODN1.1 Establishment of screening platform for suppressive ODN B-type and C-type CpG ODN (BW006 and C274) were used as stimulators respectively and A151 reported previously was used as positive suppressive ODN. Human PBMCs were incubated with CpG ODN alone or in the presence of A151. The cell proliferation was examined by thymidine incorporation. The result showed that BW006 and C274-induced cell proliferations were both inhibited by A151 significantly. Time point of A151 adding (-30~60 min) did not affect its inhibitory effect. To obtain strong inhibitory effect of A151 and shorten the experimental cycle, PBMCs should be incorporated with 3H-TdR after culturing for 48 h.1.2 Design and screening of suppressive ODNTotal 68 suppressive ODNs from 3 groups were designed, synthesized and screened by"PBMC proliferation assay". 667B transformed from CpG ODN and SAT05 derived from microsatellite DNA were more active than others. 667B contains G-rich sequence and SAT05 is composed of CT repeats, their inhibition was dose-dependent. We next optimized the sequence of SAT05 and found that the inhibitory effect of SAT05f comprised with CCT repeats was higher than A151 and SAT05.1.3 Optimization of screening platform for suppressive ODNTo exclude the possibility of nonspecific affect caused by high concentration of ODN, the working concentration of CpG ODN and suppressive ODN were both optimized in"PBMC proliferation assay". The stimulation of CpG ODN reached the platform at 1μg/mL and the suppression of SAT05f began to be obvious at 4μg/mL and got stronger with its dose increasing.1.4 Effect of SAT05f on the cell cycle and morphology of PBMCFACS analysis showed that the cell cycle status was not interfered with CpG ODN, but the percentage of cells in S phase increased with the presence of SAT05f, indicating that SAT05f arrests cell cycle in S phase. The micrograph of PBMCs showed that many cell proliferative colonies caused by CpG ODN were observed, while the number of cell colonies decreased significantly in the presence of SAT05f.1.5 Establishment of VSV protection assayA-type and C-type CpG ODN can stimulate plasmacytoid dendritic cells (pDC) to produce IFN-αmediating anti-viral acitivity. To investigate whether CpG ODN-induced anti-viral acitivity can be inhibited by candidate suppressive ODN,"VSV protection assay"was established and optimized. The supernatant of PBMCs stimulated with 1μg/mL CpG ODN can protect Vero cells from VSV challenge, and the dilution of supernatant to be examined should be 1/20~1/80.1.6 Inhibition of SAT05f on CpG ODN-induced anti-viral activityVSV protection assay was conducted to investigate whether SAT05f could inhibit the anti-viral activity of human PBMCs induced by CpG ODN. The result showed that SAT05f inhibited the anti-VSV acitivity induced by A-type and C-type CpG ODN (2216 and C274) in both dose-dependent and sequence-specific manner.2 Immunosuppressive effect of human microsatellite DNA mimicking ODNWith the screening of preliminary 3 groups of suppressive ODNs, we found that human microsatellite (MS) DNA derived C and T repeats, SAT05 and SAT05f, have significant suppressive activity. To study the Immunosuppressive effect of human MS DNA systemically, we synthesized a series of ODNs with the sequences of human MS DNA (designated as MS ODN) and used them to mimic human MS DNA.2.1 Effect of MS ODN on the proliferation of human PBMC induced by CpG ODNPBMC proliferation assay was conducted to test the suppressive effect of MS ODNs on CpG ODN. The result showed that most of the T and C containing repeats could inhibit CpG BW006-induced PBMCs proliferation remarkably and MS08 which composed of TC repeats was the most active MS ODN. In addition, when used alone, MS ODNs comprised with T and C containing repeats had stimulatory effect on PBMCs proliferation to some extent. MS08 was selected as a prototype of MS ODN to conduct further studies, while MS19 with little inhibitory activity was used as control ODN. We incubated human PBMCs with BW006 and increasing amounts of MS08, the result showed that the suppressive effect of MS08 got stronger with its dose increasing, but not MS19, suggesting that the inhibition of MS08 was dose-dependent and sequence-specific.2.2 Inhibition of MS08 on the proliferation of human PBMC induced by B-type and C-type CpG ODNTo investigate whether MS08 could also inhibit the stimulation of C-type and other B-type CpG ODN, CpG C274 (C-type) and CpG 2006 (B-type) were used as stimulators respectively. The result showed that MS08 significantly inhibited the proliferation of human PBMCs induced by C274 and 2006 in a dose-dependent manner, but not MS19.2.3 Stimulatory effect of MS08 at low dose on PBMC proliferationTo investigate the stimulatory effect of MS08 on PBMC proliferation, we incubated human PBMCs with increasing amounts of MS08. The result showed that, when used alone at low concentrations (0~0.5μg/mL), the stimulatory ability of MS08 got stronger with its dose increasing; while when used alone at higher concentrations (0.5~16μg/mL), the stimulation of MS08 declined with its dose increasing. The stimulatory effect of MS08 on cell proliferation might be associated with the backbone phophorothioate-modification because that the Po-MS08 with phosphodiester linkage showed little stimulatiory activity.2.4 Inhibition of MS08 on the anti-viral activity induced by A-type and C-type CpG ODNVSV protection assay was conducted to investigate whether MS08 could inhibit the anti-viral activity of human PBMC induced by A-type and C-type CpG ODN. CpG 2216 (A-type) and C274 (C-type) were used as stimulators respectively. The result showed that MS08 inhibited the anti-VSV activities induced by 2216 and C274 in both dose-dependent and sequence-specific manner. 2.5 Inhibition of MS08 on the upregulation of co-stimulatory molecules induced by CpG ODNCpG ODN has been shown to trigger the up-regulation of co-stimulatory molecules (CD80 and CD86) on antigen presenting cells (APC). We tested whether MS08 might inhibit the adaptive immune response by affecting APCs functions. The FACS analysis showed that C274-induced upregulation of CD80 and CD86 on human PBMCs was significantly inhibited by MS08, but not by MS19.2.6 Inhibition of MS08 on the anti-viral activity induced by HSV-1 and PR8-FluTo test whether MS08 could inhibit the activation of immune cells induced by natural virus, we used HSV-1 and PR8-Flu virus as stimulators respectively. The result of VSV protection assay showed that the anti-VSV activities induced by HSV-1 and PR8-Flu were blocked by MS08 significantly, demonstrating the suppressive activity of MS08 on natural DNA and RNA virus in addition to their inhibition on synthesized CpG ODN.2.7 Inhibition of MS08 on the proliferation of human PBMC induced by PHA and PMATo investigate whether MS08 can also inhibit the proliferation of human PBMC triggered by stimulators other than TLR9 agonists, we tested the effect of MS08 on the proliferation of human PBMC stimulated by mitogens (PHA and PMA). MS08 resulted in significant suppression on the proliferation of human PBMCs induced by PHA and PMA and the inhibition is dose-dependent, but MS19 was not inhibitory. This result suggests that the immune suppression of MS08 was not specific to TLR9.2.8 Inhibition of MS08 on the two-way mixed lymphocyte reaction (MLR) of human PBMCWhen lymphocytes from two different donors were mixed together, T cells were stimulated to proliferate by the other alloantigen (HLAⅡ). We assessed the effect of MS08 on alloantigen-induced cell proliferation in a two-way MLR of human PBMCs. The result showed that MS08 at high concentration could inhibit the proliferation of mixed PBMCs, but not MS19, suggesting that the MS08 could inhibit alloantigen-induced activation of immune cells and could be used for the treatment of graft rejection.3 Mechanism and characters of MS08's inhibition on CpG ODN3.1 MS08 inhibits CpG ODN binding and uptake by human PBMCsCpG ODN must be bound and internalized by immune cells so that it can interact with TLR9 and induce stimulatory signals. To provide insight into how MS08 causes the observed inhibitory effects on CpG ODN, we cultured human PBMCs with FITC-labeled BW006 (FITC-BW006) and/or MS08. The FACS analysis showed that MS08 reduced the binding and uptake of FITC-BW006 by PBMCs in a dose-dependent manner, but not MS19. Next, we cultured human PBMCs with FITC-BW006 and/or Cy3-labeled MS08 (Cy3-MS08). The FACS analysis showed that, when used together at equal concentration (1μg/mL), FITC-BW006 and Cy3-MS08 could be bound and internalized by PBMCs simultaneously, when used at at 8μg/mL, Cy3-MS08 dramatically reduced the FITC-BW006 (1μg/mL) binding and internalization, indicating a competition between MS08 and BW006. The inhibition occurred in monocytes, neutrophils and a group of lymphocytes which have been identified to be parts of T cells and B cells. Confocal micrograph showed that the green fluorescence of FITC-BW006 and red fluorescence of Cy3-MS08 merged very well, suggesting that they might bind to the same cells. 3.2 Competition of MS08 and CpG ODN for shared receptor on cell surfaceWe pre-incubated human PBMCs with CpG BW006, MS08 or MS19, followed with Cy3-MS08. FACS analysis showed that MS08 or BW006, not MS19, dramatically reduced the binding of Cy3-MS08 by PBMCs in a dose-dependent manner, and that the MS08 was more efficient than BW006, suggesting that MS08 and CpG BW006 might bind to the same site on the cell surface. We next incubated PBMCs with increasing amounts of Cy3-MS08 and the FACS analysis showed that the Cy3-MS08 binding by PBMCs is saturable, revealing the existence of a shared receptor on cell surface for both MS08 and CpG ODN.3.3 Chronergy analysis of MS08's inhibitionTo exclude the possibility that the binding of MS08 with CpG ODN outside of cells, we conducted PBMC proliferation assay in which MS08 was added before or after (-2~6 h) the CpG ODN (C274 and BW006) administration. The result showed that the significant suppression was observed when MS08 was added at each time point, suggesting that the inhibitory effect of MS08 was not derived from their binding with CpG ODN. In another experiment, to investigate the effect of MS08 on the activity of immune cells, we pretreated human PBMCs with MS08 or C274 for overnight and then each group of cells were washed and cultured with C274 and/or MS08 and tested by PBMC proliferation assay. The result showed that the proliferation of PBMCs pretreated with MS08 could still be promoted by C274, indicating MS08 is nontoxic to PBMCs at least. MS08 inhibited C274-induced additional proliferation of PBMCs pretreated with either MS08 or C274 demonstrating that the MS08 has inhibitory effect on CpG ODN if has enough action time.3.4 Structure-function analysis of MS08We analyzed the structure features of MS08 by PBMC proliferation assay. The results showed that (TC)12, (CCT)8 and (CT)12 could inhibit PBMC proliferation induced by CpG C274, suggesting that T and C containing tandem repeats are responsible for the suppressive activity. C274-induced cell proliferation could be inhibited by (TC)12 and (TC)9, but not (TC)6 and (TC)3 at equal molar concentration, suggesting that the optimal number of TC repeat is required for the inhibition of MS08. Recombinant ODNs coming from integrating CpG ODNs with TCTC to their 5' ends showed similar stimulatory potencies as their original ones, revealing the TC repeats could not interfere with the stimulatory activities of CpG motif in a cis manner. Similar inhibitions were observed when three types of MS08 (phosphorothioate-modified, semi-phosphorothioate modified and unmodified) were used in C274-induced PBMC proliferation, indicating that backbone modification has no effect on the inhibitory activity of MS08.3.5 Species-specificity analysis of MS08We replaced human PBMCs with BALB/c spleen cells and investigated whether MS08 is still inhibitory on the activation of mouse immune cells stimulated by CpG ODN. The results showed that, in cell proliferation assay, MS08 slightly inhibited BW006 and C274-induced proliferation of spleen cells, but not MS19; however, in VSV protection assay, MS08 did not reduced the anti-VSV acitivity of spleen cells induced by C274. These results indicate there is species-specificity for MS08 function in some experiments.In summary, our study is the first to investigate the biologic activity of human microsatellite (MS) DNA from the immunological point of view. In human MS DNA, we found a class of ODNs (MS ODN) which are composed of T and C tandem repeats and have strong immunosuppressive activity. These MS ODNs can down-regulate the TLR9-dependent and -independent activation of human immune cells. These findings indicate that human MS DNA released from cells may inhibit the excessive immune response for maintaining a homeostasis and preventing tissue cells from damage. This class of MS ODNs therefore represents a novel immunoregulatory drugs that could be of prophylactic and therapeutic use in diseases associated with immune imbalance, such as autoimmune disease, organ-graft rejection and hypersensitivity.