Effects Of CpG Oligodeoxynucleotides Research Articles

CpG ODN‐induced matrix metalloproteinase‐13 expression is mediated via activation of the ERK and NF‐κB signalling pathways in odontoblast cells

To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.

Effect of CpG-ODNs belonging to different classes on resistance of olive flounder ( Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus (Ciliata; Scuticociliatia) infections

The objective of this study was to investigate the effects of different classes of CpG oligodeoxynucleotides (CpG-ODNs) on resistance of olive flounder ( Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish administered CpG-ODN 2216 (A-class ODN) showed the highest resistance against VHSV infection and the strongest expression of Mx and ISG15 genes. Fish administered CpG-ODN 2395 (C-class ODN) showed intermediate resistance against VHSV between ODN 1668 and ODN 2216. Whereas, olive flounder intraperitoneally (i.p.) injected with CpG-ODN 1668 (B-class ODN) showed no enhanced resistance against VHSV challenge and no significant increase in expression of Mx and ISG15 genes. In contrast, in the M. avidus challenge experiment, fish injected with CpG-ODN 1668 showed the highest survival rate and the strongest scuticocidal activity of serum. Similar to the results in the VHSV infection experiment, administration of CpG-ODN 2395 elicited secondly higher survival rate against M. avidus challenge. Although fish administered CpG-ODN 2216 eventually showed 100% mortality by M. avidus challenge, the cumulative mortality until 2 weeks post-challenge was similar to the group of fish injected with CpG-ODN 1668. The present results suggest that CpG-ODNs belonging to different classes have different abilities to increase resistance against different pathogens in olive flounder. Therefore, selection of a CpG-ODN appropriate to the characteristics of a certain pathogen is crucial to induce immune responses that are optimal for defense against the pathogen.

The Mechanism for the Ameliorative Effect of CpG-Oligodeoxynucleotides on Bone Marrow Hemopoiesis Radiation Injury

Bone marrow is a major site of radiation injury. The extreme sensitivity of bone marrow cells to genotoxic stress largely determines the adverse side effects of radiation. CpG-oligodeoxynucleotide (ODN) is known to be radioprotective in extramedullary hemopoiesis, but its effect on bone marrow hemopoiesis remains unknown. In this study, we investigated whether CpG-ODN ameliorated hemopoiesis radiation injury when administered after total-body irradiation (TBI). Mice were treated with 50 μg of CpG-ODN via intraperitoneal injection (i.p) 30 min., 24 and 48 hr after TBI. Our results show that CpG-ODN was able to mediate the activation of nuclear factor κB (NF-κB) via degradation of inhibitor NF-κB (IκB-α), and some oxidative stress parameters (malondialdehyde, glutathione and superoxide dismutase) showed significant differences between the radiation control group and the radiation and administration of CpG-ODN group. White blood cell count, bone marrow cell count and bone marrow histological examination indicated that CpG-ODN minimized bone marrow damage induced by radiation. Exogenous colony-forming unit-spleen count indicated that CpG-ODN reduced primitive hemopoietic stem cell damage and reconstituted the hemopoietic system after TBI. The survival of mice was also enhanced after various levels of TBI. The calculated dose reduction factor was 1.2. Thus, we conclude that CpG-ODN may contribute to the amelioration of hemopoiesis radiation injury.

CpG Oligodeoxynucleotide 1826 Enhances the Lewis Lung Cancer Response to Radiotherapy in Murine Tumor

CpG oligodeoxynucleotides (ODNs) are synthetic DNA sequences containing unmethylated cytosine-guanine motifs with potent immune modulatory effects. Via Toll-like receptor 9 agonists of dendritic cells and B cells, CpG ODNs induce cytokines, activate natural killer cells, and elicit vigorous T-cell responses that lead to significant antitumor effects. On the basis of these properties of CpG ODNs, a previous study has tested that they could enhance tumor response to single-dose radiotherapy. The present study extended this finding to the fractionated radiotherapy of the Lewis lung cancer and assessed the ability of CpG ODN 1826 to increase the immune function of mice and the effect of CpG ODN 1826 on the apoptosis of Lewis lung cancer. First, tumor growth delay was observed, and the enhancement ratio of CpG ODN 1826 was found to be 2.4; decreased tumor weight was found after combined treatments with CpG ODN 1826 and X-ray radiation compared with either treatment alone (P < 0.01). Second, enhanced cell apoptotic index was found after combined treatments with CpG ODN 1826 and X-ray radiation compared with either treatment alone (p < 0.01). Increased tumor necrosis factor-α and decreased interleukin-10 concentration in serum and enhanced spleen exponent were observed after combined treatments with CpG ODN 1826 and X-ray radiation compared with X-ray radiation treatment alone (p < 0.01). Results suggest that CpG-ODN1826 can increase the radiosensitivity of Lewis lung cancer, which may be associated with stimulation of immune system and enhanced cell apoptosis.

Effect of CpG oligodeoxynucleotide on the immune response of 28 kDa outer membrane protein of Brucella

In order to develop an effective new generation vaccine against brucellosis, it is important to identify and evaluate the immune response of protein antigens of Brucella. It has been demonstrated that 28 kDa outer membrane protein (OMP28) of Brucella induced Th2 type of immune response with predominant IgG1 and suppressed IL-2 production. However, coadministration of CpG oligodeoxynucleotide caused a shift in the immune response of antigen towards Th1 type with predominant IgG2a and upregulated IL-2 production. This suggested that the OMP28 of Brucella may act as a B-cell antigen and may induce Th1 type of immune response on addition of suitable adjuvant.

Potential Immunological Adjuvant of 'K'-Type CpG-Oligodeoxynucleotides Enhanced the Cell Proliferation and IL-6 mRNA Transcription in Canine B Cells

CpG oligodeoxynucleotides (CpG-ODNs) are ligands for toll-like receptor 9 (TLR9), signaling of which plays a role in innate immunity by inducing T helper 1 (TH1)-cell responses and pro-inflammatory cytokine production. The activation of TLR9 signaling is considered to be effective for the therapy of cancer, infectious diseases, and allergies and preclinical studies using CpG-ODNs have been performed in dogs and humans. In order to investigate the precise mechanisms responsible for the effect of CpG-ODNs in dogs, we examined their role in cell proliferation and cytokine gene expression in canine B cells. Canine B cells were collected by a magnetic cell isolation method using anti-CD21 antibody. Flow cytometric analysis for the intracellular CD79α revealed the purity of canine B cells to be as high as 90.2 ± 2.1%. Transcription of TLR2, TLR4, and TLR9 mRNA on canine CD21(+) cells was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). CpG-ODNs induced dose-dependent proliferation of canine CD21(+) cells (P<0.05 compared with control-ODNs) detected by BrdU incorporation. Quantification of IL-6, IL-10, and IL-12p40 mRNA transcription on canine CD21(+) cells revealed that CpG-ODNs enhanced IL-6 mRNA transcription but not IL-10 and IL-12p40 mRNA transcription (P<0.05 compared with control-ODNs). These responses to CpG-ODNs in the canine B cells indicated that CpG-ODNs would be useful as an immunological adjuvant for vaccine in dogs.

Synthetic innate defence regulator peptide enhances in vivo immunostimulatory effects of CpG-ODN in newborn piglets

The in vivo immunoadjuvant effects of CpG oligodeoxynucleotides (CpG-ODN) have been studied extensively in mice and relatively fewer studies have been done in piglets. But so far, the innate immunostimulatory effects of CpG-ODN combination with innate defense-regulator peptides (IDRs) have not been demonstrated. The purpose of this study is to determine the potential effects of CpG-ODN with IDR in newborn piglets. The immunostimulatory abilities of four selected IDRs were compared, among them HH2 showed best immunostimulatory effects in newborn piglets. Hereafter, the abilities of CpG-ODN combined with HH2 to enhance innate immune responses were examined in newborn piglets. The complex of HH2 and CpG-ODN could induce much stronger Th1 cytokine and chemokine responses than HH2 or CpG-ODN alone. HH2-CpG-ODN immunized piglets showed higher B cell percentage in PBMCs than CpG-ODN alone. These in vivo data demonstrated for the first time that subcutaneously (SC) administration of CpG-ODN combined with HH2 is efficient to stimulate innate immune system in newborn piglets.

Effect of CpG oligodeoxynucleotides on the growth of aggressive human B-cell lymphomas.

8076 Background: Immunostimulatory CpG oligodeoxynucleotides (CpG ODN) are potent activators of T cell immunity and ADCC, and under study as immunotherapeutic agents for a variety of cancers, including B cell lymphomas. Recently, anti-CD20 antibody-CpG conjugates have been shown to eradicate rituximab-resistant B cell lymphoma in a syngeneic murine lymphoma model (D. Betting et al, ASCO 2009). CpGs strongly stimulate the proliferation of normal B cells. Paradoxically, CpG markedly inhibits the in vitro growth of the murine B-cell lymphoma A20, prompting us to investigate the direct effects of CpGs on the growth of human B cell lymphomas. Methods: A panel of 12 human lymphoma cell lines (DLBCL, Burkitt's, mantle cell) were cultured in the presence or absence of varying concentrations of CpGs of A, B, or C classes (50, 10, or 2 ug/ml) or control ODN. Proliferation was measured by 3H-thymidine incorporation in quadruplicate 72-hour cultures, and apoptosis measured by annexin V flow cytometry. Results: CpG ODNs strongly stimulated the proliferation of normal peripheral blood B cells (stimulation index for class B 27.5 at 5 ug/ml). In contrast, the proliferation of all 12 lymphoma lines were inhibited by CpGs. The strongest inhibitory effects were seen with CpG 7909, a class B CpG under clinical development for cancer therapy (Pfizer, PF-3512676). Raji cells were inhibited by 77.9%, 40.7%, and 8.8% at CpG concentrations of 50, 10, and 2 ug/ml, respectively (p≤0.01 for all comparisons vs. media alone). Among the 12 tested cell lines, the percentage growth inhibition using 50 ug/ml CpG 7909 was 61.2-80.4% for germinal center-type DLBCL (SUDHL-4, SUDHL-6, OCI-Ly19), 50-59.5% for activated B cell- type DLBCL (SUDHL-2, OCI-Ly3, OCI-Ly10), 56.4-79.3% for Burkitt's lymphomas (Raji, Ramos, Daudi, BJAB), and 69.6-69.9% for mantle cell lymphomas (Jeko-1, Granta-519). CpG 7909 also induced significant levels of apoptosis in Raji and Jeko-1 cells. Conclusions: We report here for the first time on the ability of CpGs to directly inhibit the proliferation of a large panel of human B-cell lymphomas representing the majority of aggressive histologies. These results provide a novel mechanism of action for CpGs as therapeutic agents for B-cell lymphomas. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pfizer

CpG‐DNA Protects Against UV‐Induced Apoptosis Via Enhancement of AKT and mTORC1 in Keratinocytes And Dendritic Cells

CpG‐DNA and its related synthetic CpG oligodeoxynucleotides (CpG‐ODNs) play an important role in immune cell survival and activation. Existing data have shown that AKT activation is involved in this process. However, the potential role of CpG‐ODN in protection against UV‐induced skin damage has thus far not been tested. In this study, we found that CpG‐ODN protects against UV‐induced cell death and apoptosis in both skin keratinocytes (HaCaT cells) and cultured dendritic cells (XS106, or DCs). In mechanism research, we found that CpG‐ODN pre‐treatment enhances UV induced activation of AKT (ser473, thr308 phosphorylation) as well as downstream signal TORC1 (S6K and 4E‐BP1 phosphorylation) in both HaCaT cells and DCs. AKT deficiency (by using AKT1/2 double knockout Mouse Embryonic Fibroblasts as well as LY 294002, a pharmacological inhibitors of AKT) or mTORC1 inhibition (by using rapamycin, a pharmacological inhibitor of mTORC1) largely neutralize the protective effects of CpG‐ODN, indicating that AKT and downstream signaling mTORC1 activation are necessary for CpG‐ODN induced protective effects against UV induced cell death. Our findings suggest that CpG‐ODN may be utilized to prevent from UV‐induced skin aging and provide a novel mechanism of protective effects against UV radiation.

Toll-like receptor 9-mediated inhibition of apoptosis occurs through suppression of FoxO3a activity and induction of FLIP expression

Synthetic oligodeoxynucleotides (ODN) with a CpG-motif are recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Stimulation of macrophages with TLR9 agonist prevented apoptosis induced by serum deprivation through increased expression of FLICE-like inhibitory protein (FLIP). CpG ODN-mediated anti-apoptosis depended on the TLR9-Akt-FoxO3a signaling pathway. Inhibition of TLR9 by small interfering (si) RNA or an inhibitor suppressed CpG ODN-mediated anti-apoptosis. Analysis of signaling pathways revealed that the anti-apoptotic effect of CpG ODN required phosphorylation of FoxO3a and its translocation from the nucleus to the cytosol. Overexpression of FoxO3a increased apoptosis induced by serum deprivation and CpG ODN blocked these effects through FLIP expression. In contrast, siRNA knock-down of FoxO3a decreased apoptosis by serum deprivation. In addition, Akt activation was involved in CpG ODN-induced phosphorylation of FoxO3a, expression of FLIP, and anti-apoptosis. Taken together, these results demonstrate the involvement of Akt-FoxO3a in TLR9-mediated anti-apoptosis and indicate that FoxO3a is a distinct regulator for FLIP expression.

The thymus-independent immunity conferred by a pneumococcal polysaccharide is mediated by long-lived plasma cells

AbstractIt was recently shown that bacterial thymus-independent (TI) antigens confer long-lasting immunity and generate memory B lymphocytes. However, reactivation of TI memory B cells is repressed in immunocompetent mice, thus raising the issue of the mechanism whereby TI vaccines confer immune protection. Here, we propose an explanation to this apparent paradox by showing that a Streptococcus pneumoniae capsular polysaccharide (PS) generates long-lived bone marrow (BM) plasma cells which frequency can be increased by CpG oligodeoxynucleotides (ODNs). The adjuvant effect of CpG ODNs on the PS3 Ab response is directly targeted to B cells and does not involve B-1a cells. We also demonstrated that BM plasma cells generated in response to the thymus-dependent (TD) form of the PS vaccine have a higher secretion capacity than those produced after immunization with the CpG-adjuvanted PS vaccine. Finally, we show that the PS-specific BM plasma cell compartment is sufficient to confer full protection of vaccinated mice against S pneumoniae infection. Altogether, our results show that TI antigens like their TD counterparts can generate both the lymphoid and the plasma cell component of B-cell memory. They also provide a framework for the improvement and widespread usage of TI vaccines.

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.

PyNTTTTGT Prototype Oligonucleotide IMT504, a Novel Effective Adjuvant of the FMDV DNA Vaccine

Synthetic oligodeoxynucleotides (ODNs) such as CpG can stimulate B and plasmacytoid dendritic cells in vertebrate immune systems. Several studies showed that non-CpG ODNs could also induce strong stimulation of B and T cells. PyNTTTTGT ODNs, non-CpG ODNs, can activate and cause immunoglobulin secretion by B cells and proliferation of T cells in vivo. By using PyNTTTTGT ODNs as an adjuvant for a FMDV DNA vaccine, we found that levels of antibody production, T-cell proliferation, and CTL activity were significantly increased compared with the DNA vaccine alone. Compared with the adjuvant effects of CpG ODNs on DNA vaccination, similar levels of antibody production and T-cell proliferation, and higher levels of CTL activity and IFN-gamma expression in CD8 T cells were induced by the IMT504 ODNs. On the other hand, RT-PCR results show that IMT504 ODN may activate the DNA sensor of DAI (DNA-dependent activator of IFN regulatory factors) and partially stimulate TLR9. At this point, the PyNTTTTGT prototype IMT504 ODN can reasonably be predicted to be a good adjuvant for FMDV DNA vaccine in small animals, but its efficacy in larger animals remains to be explored.

The Effect of CpG-Oligodeoxynucleotides with Different Backbone Structures and 3' Hexameric Deoxyriboguanosine Run Conjugation on the Treatment of Asthma in Mice

CpG-Oligodeoxynucleotide (ODN) has two backbones. Phosphorothioate backbone (PS) shows a strong immunostimulating effect while phosphodiester (PE) shows little in vivo. 3' hexameric deoxyriboguanosine-run (3' dG6-run) conjugation to PE CpG-ODN has been reported to enhance immunostimulation and to protect against asthma when injected at the time of sensitization in mice. We evaluated the treatment effects of PE and PS CpG-ODN with or without 3' dG6-run on asthma in presensitized mice. BALB/c mice sensitized with ovalbumin and alum were challenged with 1% ovalbumin on three days. CpG-ODNs (100 µg) or PBS were injected 4 times; 27 hr before challenge and 3 hr before each challenge (CpG-dG6: CpG-ODN with 3' dG6-run, PE*-CpG-dG6: PE-CpG-dG6 with two PS backbones at the 5' terminus). PE-CpG showed no treatment effect. PE-CpG-dG6 only increased ovalbumin-specific IgG2a. PE*-CpG-dG6 increased ovalbumin-specific IgG2a but also reduced BAL fluid eosinophils and airway hyperresponsiveness. PS-CpG increased ovalbumin-specific IgG2a, reduced airway inflammation and airway hyperresponsiveness. PS-CpG-dG6 was less effective than PS-CpG on airway inflammation and airway hyperresponsiveness. In pre-sensitized mice, PE-CpG required not only 3' dG6-run but also the modification of two PS linkages at 5' terminus to inhibit features of asthma. PS-CpG was strong enough to inhibit asthma but PS-CpG-dG6 was less effective.

Immunotherapeutic Potential of CpG Oligonucleotides in Chickens

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs activate innate and adaptive immune responses in numerous vertebrate species. The protective effects of CpG ODN against viral, bacterial and protozoal pathogens have been well documented in various mouse models of disease. CpG ODN are also being evaluated in humans as an immunotherapeutic agent against infectious diseases, cancer, allergy and as a vaccine adjuvant. In species of veterinary importance where the immune activity of CpG ODN has been investigated, CpG ODN has shown the greatest potential in chickens, as indicated by its protective effects against experimental bacterial infections. Surprisingly, chicken do not appear to express Toll-like receptor 9 (TLR9), the receptor that is involved in CpG-mediated immune activation in humans and many animal species. We will review progress on CpG research with particular emphasis on avian species.

Treatment with LL-37 Peptide Enhances Antitumor Effects Induced by CpG Oligodeoxynucleotides Against Ovarian Cancer

There is an urgent need for innovative therapies against ovarian cancer, one of the leading causes of death from gynecological cancers in the United States. Immunotherapy employing Toll-like receptor (TLR) ligands, such as CpG oligodeoxynucleotides (CpG-ODN), may serve as a potentially promising approach in the control of ovarian tumors. The CpG-ODN requires intracellular delivery into the endosomal compartment, where it can bind to TLR9 in order to activate the immune system. In the current study, we aim to investigate whether the antimicrobial polypeptide from the cathelicidin family, LL-37, could enhance the immunostimulatory effects of CpG-ODN by increasing the uptake of CpG-ODN into the immune cells, thus enhancing the antitumor effects against ovarian cancer. We found that treatment with the combination of CpG-ODN and LL-37 generated significantly better therapeutic antitumor effects and enhanced survival in murine ovarian tumor-bearing mice compared with treatment with CpG-ODN or LL-37 alone. We also observed that treatment with the combination of CpG-ODN and LL-37 enhanced proliferation and activation of natural killer (NK) cells, but not CD4(+) or CD8(+) T cells, in the peritoneal cavity. Furthermore, in vivo antibody depletion experiments indicated that peritoneal NK cells played a critical role in the observed antitumor effects. Thus, our data suggest that the combination of CpG-ODN with LL-37 peptide may lead to the control of ovarian tumors through the activation of innate immunity.

Comparative Proteomics Study Reveals That Bacterial CpG Motifs Induce Tumor Cell Autophagy in Vitro and in Vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.

Anti‐tumor activity of an anti‐endoglin monoclonal antibody is enhanced in immunocompetent mice

In the present study, we investigated the mechanisms by which anti-endoglin (EDG; CD105) monoclonal antibodies (mAbs) suppress angiogenesis and tumor growth. Antihuman EDG mAb SN6j specifically bound to murine endothelial cells and was internalized into the cells in vitro. SN6j effectively suppressed angiogenesis in mice in the Matrigel plug assay. We found that SN6j is more effective for tumor suppression in immunocompetent mice than in SCID mice. We hypothesized that T cell immunity is important for effective antitumor efficacy of SN6j in vivo. To test this hypothesis, we investigated effects of CpG oligodeoxynucleotides (ODN) and depletion of CD4(+) T cells and/or CD8(+) T cells on antitumor efficacy of SN6j in mice. Systemic (i.v.) administration of a relatively small dose (0.6 mug/g body weight/dose) of SN6j suppressed growth of established s.c. tumors of colon-26 in BALB/c mice and improved survival of the tumor-bearing mice. Addition of CpG ODN to SN6j synergistically enhanced antitumor efficacy of SN6j. In contrast, such enhancing effects of CpG ODN were not detected in SCID mice. Antitumor efficacy of SN6j in BALB/c mice was abrogated when CD4(+) T cells and/or CD8(+) T cells were depleted; effect of CD8(+) T cell depletion was stronger. Interestingly, CD4-depletion decreased tumor growth while CD8-depletion enhanced tumor growth in the absence of SN6j. SN6j induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner which indicates an additional mechanism of antiangiogenesis by SN6j. (c) 2008 Wiley-Liss, Inc.

CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen-specific Th2-cell migration into lung

Allergic airway inflammation is one of the most typical characteristic features of bronchial asthma. T(h)2 cells, which produce IL-4, IL-5 and IL-13, are well known as major effector lymphocytes of the inflammation. In the present work, we found that subcutaneous injection of Toll-like receptor-9-ligand, CpG-oligodeoxynucleotides (CpG-ODN), remarkably suppressed eosinophilia and mucus hyper-production in T(h)2 cell-dependent airway inflammation model at the effector phase. The injection of CpG-ODN significantly blocked T(h)2 cell migration into lung. The inhibitory effects of CpG-ODN were observed even when IFN-gamma-deficient T(h)2 cells were transferred into IFN-gamma(-/-) mice. In contrast, the administration of neutralizing mAbs against type I cytokines such as IFN-alpha, IFN-beta and IL-12 significantly suppressed the inhibitory effect of CpG-ODN on airway inflammation and T(h)2 cell migration into the lung. We further demonstrated that the production of T(h)2 chemokines, thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), was significantly reduced by the CpG-ODN. The reduction of both TARC and MDC was also inhibited by the blockade of IFN-alpha, IFN-beta and IL-12 with mAbs. Thus, we revealed here that IFN-alpha, IFN-beta and IL-12, but not IFN-gamma, were required for the inhibitory effect of CpG-ODN in T(h)2 cell-mediated allergic airway inflammation. The present evidence strongly suggest that induction of type I cytokines would be promising therapeutic targets in T(h)2-dependent allergic diseases such as bronchial asthma.

Systemic Administration of Olygodeoxynucleotides with CpG Motifs at Priming Phase Reduces Local Th2 Response and Late Allergic Rhinitis in BALB/c Mice

Oligodeoxynucleotides (ODN) with CpG motifs (CpG ODN) induce T helper (Th)1-type reaction. We aimed to evaluate the therapeutic effect of CpG ODN in the development of late allergic rhinitis induced by ovalbumin (OVA), which is one of Th2 diseaes, in BALB/c mice. Effects of a single dose of synthetic CpG-ODN (50 microg) intraperitoneally (i.p.) at the priming phase (on day 0) by OVA on the development of late eosinophilic rhinitis at respiratory areas were compared to the control mice treated with its vehicle (ODN without CpG motifs; 50 microg). Animals were again sensitized by OVA (on day 10) i.p., and 4 days after second sensitization animals were challenged by OVA intranasally (on day 14). Four days after challenge, eosinophilic reactions, nasal lesions and local cytokine values were examined. Compared to the control group, the CpG ODN-administration increased production of OVA-specific Th1 cytokine (interferon-gamma) and decreased productions of ovalubmin-specific Th2 cytokines [interleukin (IL)-5 and IL-13] in nasal cavity fluids, supernatants of splenocytes and/or sera. Also, eosinophilia and increased total IgE values were decreased in mice treated with the CpG ODN compared to the control group. Moreover, nasal lesions with infiltration of eosinophils were prominently reduced by the CpG ODN-treatment compared to the control mice. The present study suggests that the systemic administration of CpG ODN at the priming phase may reduce local OVA-specific Th2 responses, resulting in decreased nasal pathology in the late allergic eosinophilic rhinitis.