Effects Of CpG Oligodeoxynucleotides Research Articles

The immune enhancement effect of CpG-ODNs on the vaccine of inactivated Vibrio harveyi in tiger puffer (Takifugu rubripes)

Vibrio harveyi is an opportunistic pathogen among aquatic organisms, which can cause disease and even death of the cultured tiger puffer (Takifugu rubripes). Vaccination is one of the most promising strategies to prevent infection, and potent adjuvant systems are essential to enhance the immunogenicity of inactivated vaccines. CpG-oligodeoxynucleotides (CpG-ODNs) are novel immunopotentiators to induce strong humoral and cellular immunity. In the present study, the survival rate and immune response in the vaccinated individuals of T. rubripes were examined after the treatment with CpG-ODNs to verify its immune enhancement effect as an adjuvant against V. harveyi. After the V. harveyi challenge, the survival rate of puffers in the vaccine group (vaccinated with inactivated Vibrio harveyi), CpG + vaccine group (vaccinated inactivated Vibrio harveyi and treated with CpG), CpG group (treated with CpG), and control group (treated with PBS) was 53.33%, 60%, 36.67%, and 16.67%, respectively. The proliferation index of spleen lymphocytes in the vaccine group (2.13 ± 0.26), CpG + vaccine group (2.62 ± 0.67) and the CpG group (1.74 ± 0.16) were significantly higher than that in the PBS group (1.27 ± 0.18, p < 0.05), respectively. The concentration of cytokines (IL-1β, TNF-α, IL-6 and IL-8), IgM antibodies and non-specific enzymes (SOD and LZM) in the serum of puffers from vaccine group all increased significantly (p < 0.05) after the vaccination with inactivated V. harveyi. More noticeably, the concentration of IL-1β, TNF-α and IgM antibodies in the serum of CpG + vaccine group increased further (p < 0.05), which was 1.28-, 1.31-, and 1.18-fold of that in the vaccine group at 21 d post-vaccination. The results collectively indicated that the inactivated V. harveyi was able to induce both humoral and cellular immune responses of puffer to defend against infection, and CpG-ODNs could further enhance the immune protection of vaccine as a potential immunopotentiator.

A CpG-Oligodeoxynucleotide Suppresses Th2/Th17 Inflammation by Inhibiting IL-33/ST2 Signaling in Mice from a Model of Adoptive Dendritic Cell Transfer of Smoke-Induced Asthma.

Tobacco smoke exposure is a major environmental risk factor that facilitates the development and progression of asthma. Our previous study showed that CpG oligodeoxynucleotide (CpG-ODN) inhibits thymic stromal lymphopoietin (TSLP)-dendritic cells (DCs) to reduce Th2/Th17-related inflammatory response in smoke-related asthma. However, the mechanism underlying CpG-ODN -downregulated TSLP remains unclear. A combined house dust mite (HDM)/cigarette smoke extract (CSE) model was used to assess the effects of CpG-ODN on airway inflammation, Th2/Th17 immune response, and amount of IL-33/ST2 and TSLP in mice with smoke-related asthma induced by adoptive transfer of bone-marrow-derived dendritic cells (BMDCs) and in the cultured human bronchial epithelium (HBE) cells administered anti-ST2, HDM, and/or CSE. In vivo, compared to the HDM alone model, the combined HDM/CSE model had aggravated inflammatory responses, while CpG-ODN attenuated airway inflammation, airway collagen deposition, and goblet cell hyperplasia and reduced the levels of IL-33/ST2, TSLP, and Th2/Th17-cytokines in the combined model. In vitro, IL-33/ST2 pathway activation promoted TSLP production in HBE cells, which could be inhibited by CpG-ODN. CpG-ODN administration alleviated Th2/Th17 inflammatory response, decreased the infiltration of inflammatory cells into the airway, and improved the remodeling of smoke-related asthma. The underlying mechanism may be that CpG-ODN inhibits the TSLP-DCs pathway by downregulating the IL-33/ST2 axis.

CpG oligodeoxynucleotides attenuate ROR\u03b3t-mediated Th17 response by restoring histone deacetylase-2 in cigarette smoke-exposure asthma

BackgroundCigarette smoke (CS) exposure increases corticosteroid insensitive asthma related to increased Th17 phenotype, and new treatment strategies are needed for CS-associated asthma. Histone deacetylase 2 (HDAC2), found in the airway epithelium, is critical for ameliorating glucocorticoids insensitivity. We recently demonstrated the anti-inflammatory effects of CpG oligodeoxynucleotides (CpG-ODNs) on CS-exposure asthma. However, the effects of CpG-ODNs on HDAC2 expression and enzymatic activity remain unclear. This study aimed to assess whether CpG-ODNs protect against excessive Th17 immune responses in CS-induced asthma through HDAC2-dependent mechanisms and compared their effects with those of corticosteroids.MethodsThe effects of CpG-ODNs alone and in combination with budesonide (BUD) on airway inflammation and Th2/Th17-related airway immune responses were determined using an in vivo model of CS-induced asthma and in cultured bronchial epithelial (HBE) cells administered ovalbumin (OVA) and/or cigarette smoke extract (CSE). HDAC2 and retinoid-related orphan nuclear receptor γt (RORγt) expression were also assessed in mouse lung specimens and HBE cells.ResultsCpG-ODNs and BUD synergistically attenuated CS exposure asthmatic responses in vivo by modulating the influx of eosinophils and neutrophils, airway remodeling, Th2/Th17 associated cytokine and chemokine production, and airway hyperresponsiveness and blocking RORγt-mediated Th17 inflammation through induced HDAC2 expression/activity. In vitro, CpG-ODNs synergized with BUD to inhibit Th17 cytokine production in OVA- and CSE-challenged HBE cells while suppressing RORγt and increasing epithelial HDAC2 expression/activity.ConclusionsCpG-ODNs reversed CS-induced HDAC2 downregulation and enhanced the sensitivity of CS-exposed asthmatic mice and CSE-induced HBE cells to glucocorticoid treatment. This effect may be associated with HDAC2 restoration via RORγt/IL-17 pathway regulation, suggesting that CpG-ODNs are potential corticosteroid-sparing agents for use in CS-induced asthma with Th17-biased immune conditions.

Effects of CpG oligodeoxynucleotides on the differentiation of Treg/Th17 cells

AimThe balance between Th17 cells and T regulatory (Treg) cells has emerged as a prominent factor in regulating cancer development. However, the effect of CpG oligodeoxynucleotides (ODNs) on the differentiation of Treg/Th17 cells has not been well studied. We sought here to explore the function of CpG ODNs in the differentiation of Tregs and Th17 cells in vitro and in vivo. MethodsMouse spleen cells were cultured with anti-CD3 monoclonal antibodies in vitro. Tregs and Th17 cell differentiation was induced by transforming growth factor (TGF)-β and interleukin (IL)-2, or TGF-β, IL-6, and IL-23, respectively. Then cells were treated with two CpG ODNs, CpG 1982, or CpG 1826. FBL-3-inoculated C57Bl/6 mice were treated with CpG 1826, tumor vaccine, or combination of CpG 1826 and tumor vaccine. After treatment, spleen cells and serum were isolated, and Tregs/Th17 cells were detected by flow cytometry. The expression of forkhead box P3 (Foxp3), retinoid-related orphan receptor gamma-t (RORγt), IL-10, and IL-17 mRNA was measured by real-time PCR, and protein levels were measured by Western blot and enzyme-linked immunosorbent assay. ResultsThe frequency of Treg cells increased significantly (p < 0.05) in the FBL-3-inoculated leukemia mouse model compared with control mice, whereas the frequency of Th17 cells did not change. Median survival of mice after treatment with CpG 1826 and tumor vaccine was significantly prolonged compared with that of control mice (p < 0.05). The frequency of induced Treg cells decreased after treatment with CpG 1826, whereas the frequency of Th17 cells induced by cytokines in vitro and in the murine leukemia model increased following treatment with CpG 1826. Furthermore, after treatment with CpG 1826, the mRNA and protein levels of Foxp3 and IL-10 decreased significantly both in vitro and in vivo (p < 0.05), whereas those of RORγt and IL-17 increased significantly (p < 0.05). ConclusionCpG 1826 may inhibit the differentiation of Treg cells induced by cytokines, promote the differentiation of Th17 cells in vitro and in murine leukemia models, and prolong the median survival of mice with leukemia.

CpG‐ODN 2007 protects zebrafish ( Danio rerio ) against Vibrio vulnificus infection

Vibrio vulnificus is an aquatic pathogen that can cause primary sepsis and soft tissue infection. CpG oligodeoxynucleotides (CpG-ODN) are a type of essential immunomodulators, which can trigger or enhance immune responses in mammals, fish and humans. In this study, we evaluated the effect of CpG-ODN 2007 as a potential immunostimulant for zebrafish (Danio rerio) infected with V. vulnificus strain FJ03-X2. Fish injected with the CpG-ODN 2007 showed a lower mortality rate compared with the fish that did not receive treatment; the survival rates of the CpG-ODN 2007-treated group and PBS-treated group were 85% and 57.9% respectively. Also, our in vitro results demonstrated that CpG-ODN 2007 could effectively reduce the toxicity of V. vulnificus strain FJ03-X2 to zebrafish embryonic fibroblast (ZF4) cells. Furthermore, we assessed innate immune-related genes (TNF, IL-1β, IFNg1-2, MyD88, lysozyme, IL-10, IL-22, IL-26 and TLRs) in expression patterns of V. vulnificus strain FJ03-X2 infected zebrafish or ZF4 cells with and without CpG-ODN 2007 treatment. We found that CpG-ODN 2007 could reduce the toxicity of V. vulnificus to ZF4 cells and regulate the immune system to control the overexpression of specific genes. To sum up, our data indicated that CpG-ODN 2007 protects zebrafish against Vibrio vulnificus infection.

Phosphate-modif ied CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells.

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D- SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

Ameliorating effect of CpG-ODN (oligodeoxynucleotide) against radiation-induced lung injury in mice.

While radiation-induced lung injury (RILI) is known to be progressed by Th2 skewed, pro-inflammatory immune response, there have been few therapeutic attempts through Th1 immune modulation. We investigated whether the immunostimulant CpG-oligodeoxynucleotide (CpG-ODN) would be effective against RILI by way of measuring reactive oxygen species (ROS) and nitric oxides (NO), histopathology, micro-three-dimensional computer tomography (CT), and cytokine profiling. We found that KSK CpG-ODN (K-CpG) significantly reduced histopathological fibrosis when compared to the positive control (PC) group (p < 0.01). The levels of ROS production in serum and splenocyte of PC group were significantly higher than that of K-CpG group (p < 0.01). The production of nitric oxide (NO) in CpG-ODNs group was higher than that of PC group. Last, cytokine profiling illustrated that the protein concentrations of Th1-type cytokines such as IL-12 and TNF-α as well as Th2-type cytokine IL-5 in K-CpG group inclined to be significantly (p < 0.001 or p < 0.01) higher than those of in PC group. Collectively, our study clearly indicates that K-CpG is effective against RILI in mice by modulating the innate immune response. To our knowledge, this is the first note on anti-RILI effect of human type, K-CpG, clinically implying the potential of immunotherapy for RILI control.

Co-delivery of antigen and dual adjuvants by aluminum hydroxide nanoparticles for enhanced immune responses

Adjuvants that contain pathogen-associated molecular patterns (PAMPs) can enhance vaccination efficacy by binding to pattern recognition receptors (PRRs), thereby stimulating immune responses. Particularly effective may be the combination of multiple PAMPs that activate different PRRs, which occurs with natural pathogens. Here we hypothesized the enhanced effects would occur in two adjuvants that stimulate different PRRs: CpG oligodeoxynucleotide (CpG-ODN), which is Toll-like receptor 9 agonist; and 5′-triphosphate, short, double-stranded RNA (3pRNA), which activates retinoic acid-inducible gene I (RIG-I). The model antigen ovalbumin (OVA) was loaded and adjuvants were surface-adsorbed to aluminum hydroxide nanoparticles (hereafter NP-3pRNA-CpG) by electrostatic interaction with an average size of 120 nm and a negative surface charge for targeting lymph nodes. These nanoparticles were efficiently internalized by antigen-presenting cells (APCs) in the lymph nodes, and the resulting APC activation and antigen cross-presentation generated strong humoral immunity and cytotoxic T lymphocyte responses and IFN-γ secretion. NP-3pRNA-CpG significantly suppressed B16-OVA tumor growth and prolonged survival of tumor-bearing mice in therapeutic and prophylactic models, illustrating the enhanced effects of CpG-ODN and 3pRNA. Our study highlights the potential of combining multiple adjuvants for effective vaccine design.

Role of Kupffer cells in liver injury induced by CpG oligodeoxynucleotide and flucloxacillin in mice.

CpG oligodeoxynucleotide (CpG-ODN) is a Toll-like receptor 9 (TLR9) agonist that can induce innate immune responses. In a previous study, flucloxacillin (FLUX; 100 mg/kg, gavage)-induced liver injury in mice was enhanced by co-administration of CpG-ODN (40 μg/mouse, intraperitoneally). In this study, the mechanism of CpG-ODN sensitization to FLUX-induced liver injury was further investigated in mice inhibited of Kupffer cells (KCs) function by gadolinium chloride (GdCl3; 10 mg/kg, intravenously). GdCl3-treated mice administrated with CpG-ODN and FLUX showed lower liver injury than wild-type (WT) mice treated with CpG-ODN and FLUX. Upregulation of Fas and FasL by CpG-ODN was also inhibited in GdCl3-treated mice and mitochondrial swelling in response to FLUX failed to occur regardless of pre-treatment with CpG-ODN. When FasL-mutant gld/gld mice were treated with CpG-ODN, mitochondrial swelling in response to FLUX was also inhibited. These results suggest that KCs play an essential role in liver injury induced by CpG-ODN and FLUX. CpG-ODN may activate KCs, resulting in induction of Fas/FasL-mediated apoptosis of hepatocytes. The Fas/FasL pathway may also be an upstream regulator of CpG-ODN- and FLUX-induced changes in mitochondrial permeability transition. These results enhance our understanding of the mechanism of the adjuvant effect of CpG-ODN in this mouse model of liver injury.

PSVI-3 The effect of oligodeoxynucleotides on Toll-like receptor pathway genes in goat blood

Abstract The objective of this study was to investigate the effect of two classes of oligodeoxynucleotides (ODNs) on the expression of genes involved in Toll-like receptor signaling in goat blood. Toll-like receptors play a critical role in initiating innate immune responses. Select synthetic oligodeoxynucleotides containing methylated CpG motifs (CpG ODN) have immunologic effects similar to those seen with bacterial DNA. These short synthetic single-stranded DNA molecules are being explored as adjuvants to enhance animal health. Blood samples were collected from the jugular vein of BoerXSpanish goats (n = 3) into tubes containing an anticoagulant. Blood was treated with the 10 µg/ml CpG ODN (2216, 2006) class A and B, or 200µl of PBS which served as control. Cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 minutes. Total RNA was isolated using Trizol and then converted to cDNA using RETROscript kit (Qiagen). The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array was evaluated using real-time PCR. Fold change in gene expression was calculated using the 2−ΔΔCt method. The housekeeping genes GAPDH, ACTB, HPRT1, TBP, and YWHAZ was used to normalize the data. Of 84 genes tested, 74 genes were expressed in the control group. Following treatment with ODN 2006, 50 genes were expressed, 29 were up-regulated while 17 genes were down-regulated. The genes HSPD1, IFNG, MAPK8, and PTGS2 were induced by ODN 2006 treatment. Following treatment with ODN 2216, 52 genes were expressed. Treatment with ODN 2216, upregulated 26 genes and down-regulated the expression of 19 genes. The genes CLEC4E, HSPD1, IFNG, IRF1, MAPK8, PTGS2, and UBE2V1 were induced by ODN 2216. Thus specific patterns of TLR signaling may be involved in response to the immunostimulatory effect of CpG ODN in goats with implicates for their use as adjuvants to enhance animal health.

Melatonin biosynthesis restored by CpG oligodeoxynucleotides attenuates allergic airway inflammation via regulating NLRP3 inflammasome

AimsBoth CpG oligodeoxynucleotide (CpG-ODN) and melatonin have been reported to induce Th1 response and contribute to allergic asthma resistance. Here, we aimed to reveal how they confer such effect as well as whether they crosstalk with each other. Main methodsSix-week-old Female C57BL/6 mice were challenged by OVA to induce allergic airway inflammation, and were treated with CpG-ODN, CpG-ODN plus Luzindole or melatonin respectively. Bronchoalveolar lavage fluid (BALF) cellularity was classified and counted by Wright's-Giemsa staining. HE and PAS staining were used to analyze airway inflammation. The levels of IL-4, IL-5, IL-13,GM-CSF and IFN-γ, as well as IL-1β and IL-18 were analyzed by ELISA. Protein expressions of ASMT, AANAT, NLRP3, IL-1β and caspase-1 in lung tissue were detected by Western blotting, expression of ASMT and AANAT were further observed by immunohistochemistry. Key findingsWe found that CpG-ODN considerably suppressed OVA-induced airway leukocytes infiltration, goblet cell hyperplasia and Th2 cytokines production. Furthermore, the resolution effect of CpG-ODN on OVA-induced allergic airway inflammation occurred in parallel with decreased-activation of NLRP3 inflammasome and increased biosynthesis of melatonin. Blocking the effect of endogenous melatonin by Luzindole abolished the suppressive effect of CpG-ODN on OVA-induced airway inflammation and activation of NLRP3 inflammasome, suggesting such effect was mediated by endogenous melatonin. Moreover, exogenous melatonin pronouncedly ameliorated airway inflammation and decreased the activation of NLRP3 inflammasome. SignificanceThese results proven that CpG-ODN protects against allergic airway inflammation via suppressing the activation of NLRP3 inflammasome, and such effect may be resulted from the restored-production of melatonin.

Targeting Delivery of Oligodeoxynucleotides to Macrophages by Mannosylated Cationic Albumin for Immune Stimulation in Cancer Treatment.

To efficiently deliver CpG oligodeoxynucleotides (ODNs) to macrophages for the reversal of cancer-induced immunosuppression, nanoparticles ODN@MCBSA with mannosylated cationic albumin (MCBSA) as a macrophage targeting vector were constructed. Compared with ODN@CBSA with cationic albumin (CBSA) as a vector, ODN@MCBSA exhibited significantly improved cellular uptake mediated by mannose moieties, resulting in significantly enhanced secretion of proflammatory cytokines including IL-12, IL-6, TNF-α, and iNOS. The modulation of macrophages toward the favorable M1 phenotype was confirmed by the upregulated CD80 expression after being treated by ODN delivery systems. In addition to immune cells, the effects of the ODN delivery system on cancerous HeLa cells were also investigated. The results showed that ODN@MCBSA did not affect the overall tumor cell viability. However, enhanced NF-κB, p-Akt, PIK3R3, Fas, and FasL, as well as upregulated caspases were observed in tumor cells, implying the pleiotropic effects on tumor cells. Our study provides a more in-depth understanding on the immunotherapeutic effects of CpG ODNs and highlights the importance of macrophage targeting delivery to minimize the effects on tumor cells. These results indicate that MCBSA could serve as a promising delivery vector of CpG ODNs to macrophages for cancer immunotherapy.

Immunological Efficacy and Safety of Synthesized CpG Oligodeoxynucleotides

The technology for synthesis is described, and the adjuvant properties of CpG oligodeoxynucleotides (CpG-ODNs) are assessed. CpG-ODN sequences were generated according to the available sequences on an automatic synthesizer. The adjuvant activity was evaluated with CpG-ODNs in combination with a recombinant protective antigen and EA1, an S-layer protein of the anthrax agent. It was established that the use of the synthesized adjuvant CpG 2006, along with immunogenic antigens, leads to the development of long-term, high-level immunity in test animals. The synthetic CpG 2006 antigenic product was shown to have an advantage over alhydrogel in terms of adjuvant activity. Experiments on biomodels provided data confirming the absence of toxic and damaging effects of CpG-ODNs on cells and tissues of the macroorganism. Comparison of the cell-mediated immunity (content of CD4+ and CD8+) after immunization by the B. anthracis STI-1 strain or a recombinant anthrax vaccine prototype with CpG 2006 or alhydrogel as an adjuvant is evidence of the activation of the cellular component of the immune system in all of the compared groups.

CpG-ODNs and Budesonide Act Synergistically to Improve Allergic Responses in Combined Allergic Rhinitis and Asthma Syndrome Induced by Chronic Exposure to Ovalbumin by Modulating the TSLP-DC-OX40L Axis.

The experimental model of combined allergic rhinitis and asthma syndrome (CARAS) has shown that CpG oligodeoxynucleotides (CpG-ODNs) are potential inhibitors of type 2 helper cell-driven inflammatory responses. Currently available CpG-ODNs modestly inhibit allergic responses in CARAS, while a combination strategy for upper airway treatment by co-administration of CpG-ODNs and glucocorticoids may show good efficacy. This study aimed to assess the therapeutic effects of CpG-ODNs combined with budesonide (BUD) on upper and lower-airway inflammation and remodeling in mice with CARAS induced by chronic exposure to ovalbumin (OVA), exploring the possible underlying molecular mechanisms. A BALB/c mouse model of chronic CARAS was established by systemic sensitization and repeated challenge with OVA. Treatment with CpG-ODNs or BUD by intranasal administration was started 1h after OVA challenge. Then, nasal mucosa and lung tissues were fixed and stained for pathologic analysis. The resulting immunologic variables and TSLP-DC-OX40L axis parameters were evaluated. Both CpG-ODNs and BUD intranasal administration are effective on reducing Th2-type airway inflammation and tissue remodeling. Co-administration of CpG-ODNs and BUD was more effective than each monotherapy in attenuating upper and lower-airway inflammation as well as airway remodeling in chronic CARAS. Notably, combination of CpG-ODNs with BUD modulated the TSLP-DC-OX40L axis, as demonstrated by decreased TSLP production in the nose and lung, alongside decreased TSLPR and OX40L in DC. Intranasal co-administration of CpG-ODNs and BUD synergistically alleviates airway inflammation and tissue remodeling in experimental chronic CARAS, through shared cellular pathways, as a potent antagonist of the TSLP-DC-OX40L axis.

CpG oligodeoxynucleotide preconditioning improves cardiac function after myocardial infarction via modulation of energy metabolism and angiogenesis.

Unmethylated CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to protect against myocardial ischemia/reperfusion injury. However, the potential effects of CpG-ODN on myocardial infarction (MI) induced by persistent ischemia remains unclear. Here, we investigated whether and how CpG-ODN preconditioning protects against MI in mice. C57BL/6 mice were treated with CpG-ODN by i.p. injection 2 hr prior to MI induction, and cardiac function, and histology were analyzed 2 weeks after MI. Both 1826-CpG and KSK-CpG preconditioning significantly improved the left ventricular (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS) when compared with non-CpG controls. Histological analysis further confirmed the cardioprotection of CpG-ODN preconditioning. In vitro studies further demonstrated that CpG-ODN preconditioning increases cardiomyocyte survival under hypoxic/ischemic conditions by enhancing stress tolerance through TLR9-mediated inhibition of the SERCA2/ATP and activation of AMPK pathways. Moreover, CpG-ODN preconditioning significantly increased angiogenesis in the infarcted myocardium compared with non-CpG. However, persistent TLR9 activation mediated by lentiviral infection failed to improve cardiac function after MI. Although CpG-ODN preconditioning increased angiogenesis in vitro, both the persistent stimulation of CpG-ODN and stable overexpression of TLR9 suppressed the tube formation of cardiac microvascular endothelial cells. CpG-ODN preconditioning significantly protects cardiac function against MI by suppressing the energy metabolism of cardiomyocytes and promoting angiogenesis. Our data also indicate that CpG-ODN preconditioning may be useful in MI therapy.

The effect of the TLR9 ligand CpG-oligodeoxynucleotide on the protective immune response to radiation-induced lung fibrosis in mice.

CpG-oligodeoxynucleotide (CpG-ODN) is not only reported to protect against airway hyper responsiveness but is also known as a potent vaccine adjuvant for anti-tumor therapy. Little is known about the effect of CpG-ODN in mice with radiation-induced lung fibrosis (RILF), a common late stage form of tissue damage that occurs after thorax radiotherapy (RT). Here, we evaluated the immunomodulatory effects of CpG-ODN on the development of RILF. Mice were divided into four groups: (1) RT, single dose of 12Gy to the whole thorax; (2) CpG, only intraperitoneal injection of CpG-ODN for total 5 weeks; (3) RT+CpG, irradiation plus CpG-ODN treatment before and after irradiation for total 5 weeks; and (4) control (CTL): No RT or CpG-ODN treatment. In this study, we found that CpG-ODN treatment attenuated lung fibrosis and collagen deposition by increasing the number of M1 macrophagocytes, levels of Type-2 cytokines and TGF-β. CpG-ODN administration up-regulated the expression of TLR9 and STAT1 phosphorylation and reversed the expression of Type-2 immune response key transcription factor GATA-3. Activation of the JAK-STAT1 signaling pathway further enhanced M1 macrophage differentiation and Type-1 cytokine production. This study reveals the mitigating effect of early exposure to CpG-ODN on lung injury caused by irradiation in mice. The potential mechanism of action may be related to enhancement of Type-1 immunity. In conclusion, CpG-ODN may be a potential therapeutic target to treat RILF.

Polyethyleneimine-functionalized boron nitride nanospheres as an efficient carrier for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Polyethyleneimine-functionalized boron nitride nanospheres as an efficient carrier for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Functionalization of boron nitride nanosphere for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Functionalization of boron nitride nanosphere for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

Postconditioning with a CpG containing Oligodeoxynucleotide ameliorates myocardial infarction in a murine closed-chest model

AimsToll-like receptor (TLR)9 ligand CpG-oligodeoxynucleotide (CpG-ODN) exerts preconditioning in myocardial ischemia/reperfusion. We hypothesized a postconditioning effect of CpG-ODN in a murine closed-chest model of myocardial infarction. Materials and MethodsC57BL/6 (12weeks, male, WT) mice were instrumented at the left anterior descending artery, then allowed 5d of recovery before 30min ischemia. Treatments comprised: 1) PBS: 250μl phosphate buffer solution intraperitoneally 5min before reperfusion and 2) IPC (ischemic postconditioning): 3 twenty-second reperfusion and occlusion episodes at the end of ischemia 3) CpG-ODN: 1668 thioate 0.2μmol/kg BW intraperitoneally 5min before reperfusion. Infarct size was assessed via triphenyltetrazolium chloride (TTC) staining after 2 and 24h reperfusion. Myocardial mRNA-expression of cytokines was measured using real-time PCR after 2h reperfusion. Phosphatidylinositol-3 kinase (PI3K)-inhibitor wortmannin was injected intraperitoneally in WT 15min before postconditioning and PBS in each group. Cardiac function in WT was assessed with a left-ventricular pressure-volume catheter at 24h reperfusion. Key findingsFollowing 30min ischemia and 2h reperfusion, infarct size was diminished by 90% in WT postconditioned with CpG-ODN (2.4±1.55 IS/AAR%) and IPC (1.98±1.03 IS/AAR%) compared to PBS mice (23.2±3.97 IS/AAR%). Infarct size increased following 24h reperfusion but the differences remained robust. Expression of TNF-α and IL-10 was increased in CpG-ODN. Wortmannin abolished the postconditioning effect of CpG-ODN and IPC. Ejection fraction and preload-recruitable stroke work were significantly greater in CpG-ODN mice. SignificanceCpG-ODN confers postconditioning via activation of TLR9. Cardiac function is preserved following CpG-ODN postconditioning. The PI3K -inhibitor wortmannin attenuates CpG-ODN postconditioning.

CpG oligodeoxynucleotides enhance chemosensitivity of 5-fluorouracil in HepG2 human hepatoma cells via downregulation of the antiapoptotic factors survivin and livin

BackgroundRecent studies indicated that a synthetic oligonucleotide containing un-methylated CpG oligodeoxynucleotides (CpG-ODN) has a potential function for cancer therapy. In this study, we evaluated the chemosensitizing effects of CpG-ODN in 5-fluorouracil (5-FU)-treated HepG2 human hepatoma cells.MethodsCell viability assay were utilized to evaluate the direct cytotoxicity of CpG-ODN in the presence or absence of 5-FU in HepG2 cells, and apoptosis as well as cell-cycle was examined by flow cytometry analysis. The mRNA expression of Bcl-2, Livin and Survivin within HepG2 cells treated with CpG-ODN and/or 5-FU were analyzed by Real Time PCR assay in vitro.ResultsCpG-ODN in combination with 5-FU could decrease cell viability, increase apoptosis and further induce HepG2 cells cycle arrest at S phase when compared with CpG-ODN or 5-FU. CpG-ODN or 5-FU could down-regulate the mRNA expression of Bcl-2 within HepG2 cells. The mRNA expression of Livin and Survivin decreased in cells treated with CpG-ODN alone but increased in cells treated with 5-FU alone. However, CpG-ODN in combination with 5-FU could down-regulate the mRNA expression of Livin and Survivin within HepG2 cells.ConclusionsOur finding demonstrated that CpG-ODN enhanced the chemosentivity of 5-FU in HepG2 human hepatoma cells at least in part by down-regulating the expression of Livin and Survivin, leading to apoptosis and further inducing cell cycle arrest at S phase. Therefore, CpG-ODN may be a potential candidate as chemosensitizer for human hepatocellular carcinoma.