|
1
|
Ribes S, Zacke L, Nessler S, Saiepour N, Avendaño-Guzmán E, Ballüer M, Hanisch UK, Nau R. Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host. J Neuroinflammation 2021; 18:39. [PMID: 33531028 PMCID: PMC7852218 DOI: 10.1186/s12974-021-02077-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Accepted: 01/05/2021] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae. METHODS Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)-/- mice received an intraperitoneal injection of 100 μg CpG ODN or vehicle. To render mice neutropenic, anti-Ly-6G monoclonal antibody was daily administrated starting 4 days before infection with a total of 7 injections. Kaplan-Meier survival analyses and bacteriological studies, in which mice were sacrificed 24 h and 36 h after infection, were performed. RESULTS Pre-treatment with 100 μg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9-/- mice. There was a trend towards lower mortality in CpG ODN-treated immunocompetent and neutropenic wild-type mice. CpG ODN caused an increase of IL-12/IL-23p40 levels in the spleen and serum in uninfected animals. The effects of CpG ODN on bacterial concentrations and development of clinical symptoms were associated with an increased number of microglia in the CNS during the early phase of infection. Elevated concentrations of IL-12/IL-23p40 and MIP-1α correlated with lower bacterial concentrations in the blood and spleen during infection. CONCLUSIONS Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain.
Collapse
Affiliation(s)
- S Ribes
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany.
| | - L Zacke
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany
| | - S Nessler
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany
| | - N Saiepour
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany
| | - E Avendaño-Guzmán
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany
| | - M Ballüer
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany.,Department of Geriatrics, Protestant Hospital Göttingen-Weende, Göttingen, Germany
| | - U K Hanisch
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany
| | - R Nau
- Institute of Neuropathology, University Medical Center, Georg August University Göttingen, Robert-Koch-Straße 40, D-37075, Göttingen, Germany.,Department of Geriatrics, Protestant Hospital Göttingen-Weende, Göttingen, Germany
| |
Collapse
|
|
2
|
CpG enhances the immunogenicity of heterologous DNA-prime/protein-boost vaccination with the heavy chain myosin of Brugia malayi in BALB/c mice. Parasitol Res 2019; 118:1943-1952. [PMID: 31069533 DOI: 10.1007/s00436-019-06318-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2018] [Accepted: 04/09/2019] [Indexed: 10/26/2022]
Abstract
The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5 ± 0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6 ± 1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN-γ leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF-α in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.
Collapse
|
|
3
|
Hiramatsu Y, Satho T, Hyakutake M, Irie K, Mishima K, Miake F, Kashige N. The anti-inflammatory effects of a high-frequency oligodeoxynucleotide from the genomic DNA of Lactobacillus casei. Int Immunopharmacol 2014; 23:139-47. [PMID: 25193776 DOI: 10.1016/j.intimp.2014.08.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Revised: 08/08/2014] [Accepted: 08/13/2014] [Indexed: 12/16/2022]
Abstract
Genomic DNA has been identified as an anti-inflammatory component of Lactobacillus species, the effects of which are mediated through toll-like receptor (TLR) 9. In this study, we identified 14 novel anti-inflammatory oligodeoxynucleotide (ODN) from the genomic DNA of Lactobacillus casei by measuring their effects on the secretion of interleukin (IL)-8 (CXCL8) in the human epithelial colorectal adenocarcinoma cell line Caco-2 cells. The ODN TTTTGCCG strongly decreased IL-8 secretion. In the genomic DNA of Lactobacillus species, the frequency of TTTTGCCG was highest in the genomic DNA of L. casei and similar among strains of L. casei. Decreases in inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions in macrophage-like differentiated THP-1 cells confirmed the anti-inflammatory effect of TTTTGCCG. Furthermore, oral administration of TTTTGCCG ameliorated dextran sodium sulfate (DSS)-induced murine colitis and DSS-induced increased expression of inflammatory factor mRNAs, such as macrophage inflammatory protein (MIP)-2 (CXCL2), iNOS, and COX-2. The anti-inflammatory effect of TTTTGCCG was mainly regulated by an increase in heat shock protein (Hsp) 70 expression in the epithelium. TLR9 and Hsp90 may primarily mediate the anti-inflammatory effect of TTTTGCCG on Hsp70 signaling.
Collapse
Affiliation(s)
- Yukihiro Hiramatsu
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Tomomitsu Satho
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
| | - Mika Hyakutake
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Keiichi Irie
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Kenichi Mishima
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Fumio Miake
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| | - Nobuhiro Kashige
- Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
| |
Collapse
|
|
4
|
Zhuang X, Qiao T, Yuan S, Chen W, Zha L, Yan L. Dose-effect relationship of CpG oligodeoxyribonucleotide 1826 in murine Lewis lung cancer treated with irradiation. Onco Targets Ther 2013; 6:549-54. [PMID: 23723711 PMCID: PMC3666880 DOI: 10.2147/ott.s42485] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Background Cytosine-phosphate-guanine (CpG) oligodeoxyribonucleotides (ODNs), which induce signaling via Toll-like receptor 9, have recently been suggested to enhance sensitivity to traditional therapies, including chemotherapy, in certain cancer cell lines. This study aimed to define the dose-effect relationship for CpG ODN 1826 in increasing radiosensitivity and its impact on immune function in a mouse model of Lewis lung cancer. Methods The tumor-bearing mouse model was induced by injecting Lewis lung cancer cells into the right anterior leg subcutaneously. Sixty-four C57BL/6 J mice were evenly randomized into eight groups, comprising: a control group; an irradiation group; a CpG ODN 0.15 group; a CpG ODN 0.3 group; a CpG ODN 0.45 group; a CpG 0.15 + irradiation group; a CpG 0.3 + irradiation group; and a CpG 0.45 + irradiation group. Tumor growth, serum tumor necrosis factor-alpha and interleukin-12 concentrations, spleen and thymus exponents, and effect of CpG on the secondary immune response were measured, and apoptosis of tumor cells was investigated using TdT-mediated dUTP nick end labeling (TUNEL) after treatment. Results Tumor volumes in the treated groups were smaller than in the control group, with those of the CpG 0.45 + irradiation group being the smallest. TUNEL showed that the apoptosis rate in all the active treatment groups was higher than in the control group. CpG ODN apoptosis rate, serum tumor necrosis factor-alpha and interleukin-12 levels, and the spleen and thymus exponent showed greater improvement in the groups receiving combination therapy of CpG ODN and irradiation than the control group or the group receiving irradiation alone. With the increasing concentration of CpG ODN 1826, its effect became more and more significant, meanwhile, inoculation of Lewis lung cancer cells failed in those CpG ODN-cured mice. Conclusion CpG ODNs dramatically increased the radiosensitivity of Lewis lung cancer and enhanced immune function in mice in a dose-related manner.
Collapse
Affiliation(s)
- Xibing Zhuang
- Department of Oncology, Jinshan Hospital, Medical Center of Fudan University, Shanghai, People's Republic of China
| | | | | | | | | | | |
Collapse
|
|
5
|
Zhao T, Wu X, Song D, Fang M, Guo S, Zhang P, Wang L, Wang L, Yu Y. Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3. Int Immunopharmacol 2012; 14:665-73. [PMID: 23063973 DOI: 10.1016/j.intimp.2012.09.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Revised: 09/22/2012] [Accepted: 09/27/2012] [Indexed: 01/07/2023]
Abstract
Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 μg YW002 per mouse. When adjusted the amount at 10 μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.
Collapse
Affiliation(s)
- Tiesuo Zhao
- Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
6
|
Sommariva M, De Cecco L, De Cesare M, Sfondrini L, Ménard S, Melani C, Delia D, Zaffaroni N, Pratesi G, Uva V, Tagliabue E, Balsari A. TLR9 agonists oppositely modulate DNA repair genes in tumor versus immune cells and enhance chemotherapy effects. Cancer Res 2011; 71:6382-90. [PMID: 21878529 DOI: 10.1158/0008-5472.can-11-1285] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.
Collapse
Affiliation(s)
- Michele Sommariva
- Department of Human Morphology and Biomedical Sciences Città Studi, Università degli Studi di Milano, Italy
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
7
|
Caipang CMA, Gallage S, Lazado CC, Brinchmann MF, Kiron V. Unmethylated CpG oligodeoxynucleotides activate head kidney leukocytes of Atlantic cod, Gadus morhua. FISH PHYSIOLOGY AND BIOCHEMISTRY 2010; 36:1151-1158. [PMID: 20349339 DOI: 10.1007/s10695-010-9393-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Accepted: 03/15/2010] [Indexed: 05/29/2023]
Abstract
Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 μM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.
Collapse
|
|
8
|
Liu CS, Sun Y, Hu YH, Sun L. Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity. Vaccine 2010; 28:4153-61. [PMID: 20416262 DOI: 10.1016/j.vaccine.2010.04.016] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2009] [Revised: 02/26/2010] [Accepted: 04/05/2010] [Indexed: 11/19/2022]
Abstract
Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class IIalpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity.
Collapse
Affiliation(s)
- Chun-sheng Liu
- Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China
| | | | | | | |
Collapse
|
|
9
|
CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia. Cell Immunol 2010; 260:113-8. [PMID: 19883904 DOI: 10.1016/j.cellimm.2009.10.001] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2009] [Revised: 10/01/2009] [Accepted: 10/06/2009] [Indexed: 11/24/2022]
Abstract
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.
Collapse
|
|
10
|
Klaschik S, Tross D, Shirota H, Klinman DM. Short- and long-term changes in gene expression mediated by the activation of TLR9. Mol Immunol 2009; 47:1317-24. [PMID: 20005572 DOI: 10.1016/j.molimm.2009.11.014] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2009] [Revised: 11/10/2009] [Accepted: 11/16/2009] [Indexed: 01/26/2023]
Abstract
CpG DNA binds to Toll-like receptor 9 to stimulate a strong innate immune response. The magnitude, duration and scope of CpG-induced changes in gene expression are incompletely understood despite extensive studies of TLR9 mediated signal transduction pathways. In particular, the prolonged effects of CpG DNA on gene activation have not been investigated despite evidence that a single dose of CpG DNA alters immune reactivity for several weeks. This study used gene expression analysis to monitor changes in mRNA levels for 14 days, and identified the genes, pathways and functional groups triggered in vivo following CpG DNA administration. Two discrete peaks of gene activation (at 3h and 5 days) were observed after CpG injection. Both the behavior and function of genes activated during the second peak differed from those triggered shortly after CpG administration. Initial gene up-regulation corresponded to a period when TLR9 ligation stimulated genes functionally associated with the generation of innate and adaptive immune responses (e.g. the NF-kappaB and B-cell receptor pathways). The second peak reflected processes associated with cell division (e.g. cell cycle and DNA replication and repair). The complex bimodal pattern of gene expression elicited by CpG DNA administration provides novel insights into the long-term effects of TLR9 engagement on genes associated with immunity and cell proliferation.
Collapse
Affiliation(s)
- Sven Klaschik
- Laboratory of Experimental Immunology, Cancer and Inflammation Program, National Cancer Institute, NIH, Frederick, MD 21702, USA
| | | | | | | |
Collapse
|
|
11
|
Gregory SH, Mott S, Phung J, Lee J, Moise L, McMurry JA, Martin W, De Groot AS. Epitope-based vaccination against pneumonic tularemia. Vaccine 2009; 27:5299-306. [PMID: 19616492 DOI: 10.1016/j.vaccine.2009.06.101] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2008] [Revised: 06/23/2009] [Accepted: 06/30/2009] [Indexed: 10/20/2022]
Abstract
Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacterial pathogens known. No vaccine is currently approved for public use. Previously, we identified epitopes recognized specifically by T cells obtained from individuals following infection with F. tularensis. Here, we report that a subunit vaccine constructed based upon these epitopes elicited protective immunity in "humanized" HLA class II (DRB1*0401) transgenic mice. Vaccinated mice challenged intratracheally with a lethal dose of F. tularensis (Live Vaccine Strain) exhibited a rapid increase in pro-inflammatory cytokine production and diminished number of organisms in the lungs, and a concurrent increased rate of survival. These results demonstrate the efficacy of an epitope-based tularemia vaccine and suggest that such an approach might be widely applicable to the development of vaccines specific for intracellular bacterial pathogens.
Collapse
Affiliation(s)
- Stephen H Gregory
- Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI, United States.
| | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Klaschik S, Tross D, Klinman DM. Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo. J Leukoc Biol 2009; 85:788-95. [PMID: 19179452 DOI: 10.1189/jlb.1008671] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG-mediated signal transduction has been studied, the scope of TLR9-dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these experimental data, this study is the first to describe the complex regulatory networks responsible for TLR9-mediated gene expression. Current results are the first to establish that CpG-induced stimulation of the innate immune system proceeds in multiple waves over time, and gene up-regulation is mediated by a small number of temporally activated "major inducers" and "minor inducers". An additional study of TNF knockout mice supports the conclusion that the regulatory networks identified by our bioinformatic analysis accurately identified CpG ODN-driven gene-gene interactions in vivo. Equally important, this work identifies the counter-regulatory mechanisms embedded within the signaling cascade that suppresses the proinflammatory response triggered in vivo by CpG DNA stimulation. Identifying these network interactions provides novel and global insights into the regulation of TLR9-mediated gene activation, improves our understanding of TLR-mediated host defense, and facilitates the development of interventions designed to optimize the nature and duration of the ensuing response.
Collapse
Affiliation(s)
- Sven Klaschik
- Laboratory of Experimental Immunology, Cancer and Inflammation Program, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
| | | | | |
Collapse
|
|
13
|
Henry SP, Levin AA, White K, Mennear JH. Assessment of the Effects of ISIS 2302, an Anti-Sense Inhibitor of Human ICAM-1, on Cellular and Humoral Immunity in Mice. J Immunotoxicol 2008; 3:199-211. [DOI: 10.1080/15476910601046538] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
|
|
14
|
Patel BA, Gomis S, Dar A, Willson PJ, Babiuk LA, Potter A, Mutwiri G, Tikoo SK. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) predominantly induce Th1-type immune response in neonatal chicks. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2008; 32:1041-1049. [PMID: 18395255 DOI: 10.1016/j.dci.2008.02.007] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/19/2007] [Revised: 02/07/2008] [Accepted: 02/07/2008] [Indexed: 05/26/2023]
Abstract
Earlier, we demonstrated that intramuscular administration of oligodeoxynucleotides containing CpG motifs (CpG-ODN) induces protection in neonatal chicks against a lethal challenge of Escherichia coli. However, the mechanism of induction of the protection was not clear. In an attempt to elucidate the mechanism of induced protection, we determined the kinetics of expression of cytokines/chemokines in the spleen and bursa of Fabricius of newly hatched chicks that had received intramuscular administration of CpG-ODN or non-CpG ODN compared to saline-treated controls. SyBr green, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of the RNA demonstrated increased expression of IL-1beta, IL-6, IL-8, IL-10, IL-18, IFN-gamma and MIP-3alpha mRNAs in the spleen and; IL-10 and IFN-alpha in bursa of Fabricious of chicks that had received CpG-ODN. However, non-CpG ODN failed to induce any of the cytokine. The increased level of IL-18 and IFN-gamma but not IL-4 mRNA suggests that the administration of CpG-ODN elicits a Th1 biased immune response, which may be important in inducing protection against infections in neonatal chicks. To our knowledge, this is the first report evaluating the induction of cytokines/chemokines in neonatal chicks following administration of CpG-ODN.
Collapse
Affiliation(s)
- Bhavini A Patel
- Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Koyama S, Ishii KJ, Kumar H, Tanimoto T, Coban C, Uematsu S, Kawai T, Akira S. Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination. THE JOURNAL OF IMMUNOLOGY 2007; 179:4711-20. [PMID: 17878370 DOI: 10.4049/jimmunol.179.7.4711] [Citation(s) in RCA: 237] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-beta promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.
Collapse
Affiliation(s)
- Shohei Koyama
- Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan
| | | | | | | | | | | | | | | |
Collapse
|
|
16
|
Anderson RB, Cianciolo GJ, Kennedy MN, Pizzo SV. Alpha 2-macroglobulin binds CpG oligodeoxynucleotides and enhances their immunostimulatory properties by a receptor-dependent mechanism. J Leukoc Biol 2007; 83:381-92. [PMID: 17967834 DOI: 10.1189/jlb.0407236] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
CpG oligodeoxynucleotides (ODN) stimulate the immune system and are under evaluation as treatments and vaccine adjuvants for infectious diseases, cancer, and immune system disorders. Although they have shown promising results in numerous clinical trials, the ultimate use of CpG ODN-based therapeutics may hinge on improved pharmacokinetics and reduced systemic side-effects. CpG ODN efficacy and potency might be enhanced greatly by packaging them into particles that protect them from degradation and specifically target them for uptake by immune-competent cells. The plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) binds numerous biologically active macromolecules, including cytokines, chemokines, and growth factors, and can modulate their activity. Molecules bound to alpha 2M are protected from interactions with neighboring macromolecules and are targeted for receptor-mediated uptake by immune-competent cells. Here, we report that activated alpha 2M (alpha 2M*) binds CpG ODN and enhances their immunostimulatory properties significantly. Murine macrophages treated with alpha 2M*-ODN complexes respond more rapidly and produce a greater cytokine response than induced by free CpG ODN. Using human PBMC, alpha 2M*-ODN complexes exhibit fourfold enhanced potency and 15-fold greater efficacy for stimulating production of inflammatory cytokines. alpha 2M* targets delivery of CpG ODN specifically to immune-competent cells, which endocytose the complexes sixfold more rapidly than free CpG ODN. CpG ODN bound to alpha 2M* are also protected from degradation by nucleases. This novel targeting technology may improve CpG ODN-based therapeutics by increasing efficacy at reduced doses, thus reducing side-effects and cost.
Collapse
Affiliation(s)
- Ryan B Anderson
- Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA
| | | | | | | |
Collapse
|
|
17
|
Carrington AC, Secombes CJ. CpG oligodeoxynucleotides up-regulate antibacterial systems and induce protection against bacterial challenge in rainbow trout (Oncorhynchus mykiss). FISH & SHELLFISH IMMUNOLOGY 2007; 23:781-92. [PMID: 17459727 DOI: 10.1016/j.fsi.2007.02.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2006] [Revised: 02/23/2007] [Accepted: 02/23/2007] [Indexed: 05/15/2023]
Abstract
The effects of unmethylated CpG oligodeoxynucleotides (ODN) on the mammalian immune system are relatively well studied but much less is known of their effects on the immune systems of different fish species. Here we show that CpG ODNs significantly enhance the survival of rainbow trout (Oncorhynchus mykiss) following bacterial challenge when used both as stand-alone prophylactic agents, or as adjuvants to a commercially available vaccine. They are also capable of increasing serum lysozyme activity in vivo and stimulating the production of chemoattractant factors for rainbow trout head kidney (HK) leucocytes in vitro.
Collapse
Affiliation(s)
- Allison C Carrington
- Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, UK
| | | |
Collapse
|
|
18
|
Liu JW, Cheng J. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice. World J Gastroenterol 2007; 13:3847-54. [PMID: 17657840 PMCID: PMC4611218 DOI: 10.3748/wjg.v13.i28.3847] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.
METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.
RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes, including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule, proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.
CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.
Collapse
Affiliation(s)
- Jian-Wen Liu
- Institute of Infectious Diseases, Beijing Ditan Hospital, Beijing 100011, China.
| | | |
Collapse
|
|
19
|
Inoue J, Aramaki Y. Suppression of skin lesions by transdermal application of CpG-oligodeoxynucleotides in NC/Nga mice, a model of human atopic dermatitis. THE JOURNAL OF IMMUNOLOGY 2007; 178:584-91. [PMID: 17182599 DOI: 10.4049/jimmunol.178.1.584] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by an elevation of the total IgE level in plasma, the infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 cells. NC/Nga mice kept in conventional conditions are known to develop skin lesions resembling human AD. We examined in this study the alterations of immune response in NC/Nga mice kept in conventional conditions, following transdermal application of CpG-oligodeoxynucleotides (ODN), which plays a critical role in immunity via the augmentation of Th1-type and suppression of Th2-type responses. CpG-ODN remarkably changed the immune response from type Th2 to Th1 as determined from cytokine mRNA and Ab levels. The serum IgE level was decreased and the expression of IgG2a was up-regulated. The application of CpG-ODN to the skin also decreased inflammatory infiltration of mast cells, and suppression in the skin lesions was observed. Furthermore, the generation of regulatory T cells, which are considered immune suppressive T cells, was observed in the skin on treatment with CpG-ODN. These results suggested CpG-ODN is effective for immunotherapy in patients with AD, which is characterized by Th2-dominated inflammation.
Collapse
Affiliation(s)
- Joe Inoue
- School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan
| | | |
Collapse
|
|
20
|
Cerullo V, Seiler MP, Mane V, Brunetti-Pierri N, Clarke C, Bertin TK, Rodgers JR, Lee B. Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors. Mol Ther 2007; 15:378-85. [PMID: 17235317 DOI: 10.1038/sj.mt.6300031] [Citation(s) in RCA: 113] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
A major obstacle to the clinical application of systemic adenoviral gene replacement therapy is the host innate immune response. Although recent studies have attempted to characterize the cellular basis for this response to systemically administered helper-dependent adenoviral vector (HD-Ad), the underlying molecular components of the innate immune repertoire required to recognize the viral vector have yet to be identified. Here, we show that primary macrophages can sense HD-Ad vectors via the Toll-like Receptor 9 (TLR9) and respond by increasing pro-inflammatory cytokine secretion. Moreover, TLR9 sensing is involved in the rapid innate immune response to HD-Ad in vivo. TLR9 deficiency attenuates the innate immune response to HD-Ad, whereas TLR9 blockade reduces the acute inflammatory response after intravenous injection of the vector. Moreover, HD-Ad upregulates TLR9 gene expression independent of TLR9 function, suggesting that additional innate signaling pathways work cooperatively with TLR9. The identification of the components of the innate immune response to adenovirus will facilitate the development of combinatorial therapy directed at increasing the maximal tolerated dose of systemically delivered adenoviral vectors.
Collapse
Affiliation(s)
- Vincenzo Cerullo
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
21
|
Zhang L, Tian X, Zhou F. CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo. Res Vet Sci 2007; 83:171-81. [PMID: 17223146 DOI: 10.1016/j.rvsc.2006.11.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2006] [Revised: 11/06/2006] [Accepted: 11/21/2006] [Indexed: 01/27/2023]
Abstract
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.
Collapse
Affiliation(s)
- Linghua Zhang
- Microbiological Staff Room, College of Life Sciences, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou, GuangDong 510642,
| | | | | |
Collapse
|
|
22
|
Linghua Z, Xingshan T, Fengzhen Z. Vaccination with Newcastle disease vaccine and CpG oligodeoxynucleotides induces specific immunity and protection against Newcastle disease virus in SPF chicken. Vet Immunol Immunopathol 2006; 115:216-22. [PMID: 17157392 DOI: 10.1016/j.vetimm.2006.10.017] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2006] [Revised: 10/16/2006] [Accepted: 10/23/2006] [Indexed: 10/24/2022]
Abstract
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) have been proven to be immunoprotective in mouse models. However, little work has been conducted on in vivo immune responses in chicken with CpG ODN. The objective of this study was to investigate the immunoadjuvant effects of CpG ODN to Newcastle disease (ND) vaccine and its protective effects against ND virus in SPF chicken. In this report, the titre of serum IgG to ND vaccine and the proliferation of lymphocytes were monitored in SPF chickens. The results demonstrated that the above-mentioned immune responses were significantly stronger in chickens that received CpG ODN than in the birds that received only ND vaccine. Furthermore, ND vaccine plus CpG ODN protected SPF chicken from challenge with an otherwise lethal dose of ND virus. These data suggest that CpG ODN holds considerable promise as an adjuvant for future vaccines against ND virus.
Collapse
Affiliation(s)
- Zhang Linghua
- College of Life Sciences, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou, GuangDong 510642, China. lhzhang73212000yahoo.com.cn
| | | | | |
Collapse
|
|
23
|
Klaschik S, Gursel I, Klinman DM. CpG-mediated changes in gene expression in murine spleen cells identified by microarray analysis. Mol Immunol 2006; 44:1095-104. [PMID: 16930709 DOI: 10.1016/j.molimm.2006.07.283] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2006] [Revised: 07/07/2006] [Accepted: 07/10/2006] [Indexed: 11/21/2022]
Abstract
Unmethylated CpG motifs interact with Toll-like receptor 9 (TLR9), triggering an innate immune response characterized by the production of cytokines, chemokines and immunoglobulins. Microarray analysis of cDNA from murine spleen cells stimulated with CpG oligodeoxynucleotides (ODN) identified reproducible changes in gene expression over time. Eight genes are significantly up-regulated 2h post CpG ODN stimulation, most of which contribute to the induction of innate or adaptive immune responses. Network analysis indicates that TNF and NFKB1 are key regulators of gene expression at this early time point. At 4h, IL1B in addition to TNF and NFKB1 play dominant roles in the up-regulation of immune gene expression, whereas by 8h this function is mediated by TNF, IFNG, and MYC. Genes responsible for down-regulating CpG-induced responses were also identified, dampening what would otherwise be a continuous positive feedback loop. This work provides novel insights into the regulatory process embedded in the gene expression profile induced by CpG ODN, identifies novel genes associated with CpG-induced immune stimulation, and clarifies the breadth of the immune response elicited via TLR9.
Collapse
Affiliation(s)
- Sven Klaschik
- Section of Retroviral Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29A, Room 3D10, Bethesda, MD 20892, USA
| | | | | |
Collapse
|
|
24
|
Frost RA, Nystrom GJ, Lang CH. Multiple Toll-like receptor ligands induce an IL-6 transcriptional response in skeletal myocytes. Am J Physiol Regul Integr Comp Physiol 2006; 290:R773-84. [PMID: 16254126 DOI: 10.1152/ajpregu.00490.2005] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Toll-like receptors (TLRs) comprise a critical sentinel that monitors body compartments for the presence of pathogens. Skeletal muscle expresses TLRs and responds to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by mounting an innate immune response. In the present study, we used C2C12myocytes as a model system for skeletal muscle during infection. C2C12cells responded to LPS in a time frame and with a pattern of gene expression that faithfully mimicked the response of skeletal muscle to LPS in vivo. LPS from a variety of Escherichia coli serotypes stimulated IL-6 synthesis. C2C12cells expressed TLR1–7, but not TLR8 or TLR9, mRNA by RT-PCR. A synthetic tripalmitoylated cysteine-, serine-, and lysine-containing peptide (Pam) and LPS from Porphyromonas gingivalis, two TLR2 ligands, also stimulated IL-6 expression. LPS and Pam stimulated luciferase activity driven from NF-κB and IL-6 promoter-containing plasmids, and this response was blunted when the NF-κB binding site was mutated. LPS- and Pam-stimulated IL-6 expression was inhibited by the proteasome inhibitor MG-132 and the IκB kinase-2 (IKK2) inhibitor 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). Pam-stimulated NF-κB and IL-6 promoter activities were disrupted by a dominant-negative form of TLR2, but not TLR4. Local injection of LPS or Pam into the gastrocnemius muscle stimulated IL-6 mRNA expression in the injected, but not the contralateral, muscle. The LPS- but not Pam-stimulated expression of IL-6 mRNA was blunted in skeletal muscle of mice carrying an inactivating mutation in TLR4. The data suggest that skeletal muscle and muscle cells recognize pathogen-associated molecules with specific TLRs to initiate an IL-6 transcriptional response.
Collapse
Affiliation(s)
- Robert A Frost
- Department of Cellular and Molecular Physiology (H166), Pennsylvania State University College of Medicine, 500 University Dr., Hershey, Pennsylvania 17033, USA.
| | | | | |
Collapse
|
|
25
|
Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, Ludwig H, Sutter G, Suzuki K, Hemmi H, Sato S, Yamamoto M, Uematsu S, Kawai T, Takeuchi O, Akira S. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. Nat Immunol 2005; 7:40-8. [PMID: 16286919 DOI: 10.1038/ni1282] [Citation(s) in RCA: 620] [Impact Index Per Article: 31.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2005] [Accepted: 10/01/2005] [Indexed: 11/09/2022]
Abstract
The innate immune system recognizes nucleic acids during infection or tissue damage; however, the mechanisms of intracellular recognition of DNA have not been fully elucidated. Here we show that intracellular administration of double-stranded B-form DNA (B-DNA) triggered antiviral responses including production of type I interferons and chemokines independently of Toll-like receptors or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding interferon-beta through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kappaB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of interferon-beta and NF-kappaB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders.
Collapse
Affiliation(s)
- Ken J Ishii
- Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
26
|
Grønevik E, Mathiesen I, Lømo T. Early events of electroporation-mediated intramuscular DNA vaccination potentiate Th1-directed immune responses. J Gene Med 2005; 7:1246-54. [PMID: 15822067 DOI: 10.1002/jgm.760] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND Application of electrical pulses after DNA injection into muscle increases expression of the encoded genes, and is shown to improve antigen-specific immune responses when used for DNA vaccination. In addition, electroporation causes tissue injury and inflammatory reactions. Together with immune stimulatory motifs in the injected DNA these factors may potentiate the immune response by acting as adjuvants for the antigen. Here, we have examined the role of these factors in promoting the efficiency of DNA vaccination. METHODS We injected a plasmid DNA vector containing the gene Ag85B from M. tuberculosis into mouse quadriceps muscles followed by electroporation. Ag85B was under control of a Tet-responsive promoter, and was expressed either immediately or up to 28 days later by administrating doxycycline to the mice. Delayed expression was combined with injection of non-coding DNA or saline with or without electroporation to examine the ability of these factors to enhance the Ag85B-specific antibody response in the blood and cellular responses in the spleen. Blood samples were analysed with ELISA, while the number of Ag85B-specific IFN-gamma- and IL-4-producing spleenocytes was analysed with ELISpot. RESULTS Delaying Ag85B expression by 5 or 28 days caused lower anti-Ag85B-specific IgG2a levels. In contrast, the IgG1 antibody response was not significantly affected. Injection of non-coding DNA followed by electroporation moderately increased the IgG2a response. Delaying the Ag85B expression by 28 days reduced the average number of Ag85B-specific IFN-gamma-producing spleenocytes by over 60%. No significant change in the number of IL-4-producing Ag85B-specific spleenocytes was observed. CONCLUSIONS These results suggest that DNA and electroporation per se may act as good adjuvants in promoting efficient Th1-directed responses during DNA vaccination.
Collapse
Affiliation(s)
- Eirik Grønevik
- Department of Physiology, University of Oslo, Box 1103 Blindern, 0316 Oslo, Norway.
| | | | | |
Collapse
|
|
27
|
Tassakka ACMAR, Savan R, Watanuki H, Sakai M. The in vitro effects of CpG oligodeoxynucleotides on the expression of cytokine genes in the common carp (Cyprinus carpio L.) head kidney cells. Vet Immunol Immunopathol 2005; 110:79-85. [PMID: 16229898 DOI: 10.1016/j.vetimm.2005.09.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2005] [Revised: 08/23/2005] [Accepted: 09/08/2005] [Indexed: 10/25/2022]
Abstract
Synthetic oligodeoxynucleotides with CpG motifs (CpG-ODNs) have gained attention because it elicits strong innate immune responses. CpG-ODNs promote the production of T-helper 1(T(H)1) and pro-inflammatory cytokines. In fish, knowledge of the effects of CpG-ODNs on the expression of cytokine genes is scarce. In this study, we report that CpG-ODNs induce the expression of pro-inflammatory cytokines in common carp head kidney leucocytes in vitro. Evidence of a T(H)1 type immune stimulation was observed as the fish homologs of IP-10 (interferon gamma-inducible protein 10; CXCL10) and MCP (monocyte chemotactic protein a CC-chemokine) were induced by CpG. Interleukin 10, a cytokine inhibitory factor, failed to be induced by CpG. These results suggest a robust immune stimulation can be achieved by CpGs and has a potential as an immunostimulant in aquaculture.
Collapse
|
|
28
|
Wang J, Alvarez R, Roderiquez G, Guan E, Caldwell Q, Wang J, Phelan M, Norcross MA. CpG-independent synergistic induction of beta-chemokines and a dendritic cell phenotype by orthophosphorothioate oligodeoxynucleotides and granulocyte-macrophage colony-stimulating factor in elutriated human primary monocytes. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2005; 174:6113-21. [PMID: 15879106 DOI: 10.4049/jimmunol.174.10.6113] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Chemokines attract leukocytes bearing the relevant chemokine receptors and regulate innate immune responses. CpG oligodeoxynucleotides (ODN) and GM-CSF are potent vaccine adjuvants and in combination induce enhanced Th1 responses by mechanisms yet to be determined. We have examined combinations of CpG- or non-CpG-ODN and GM-CSF for effects on the production of chemokines and the differentiation of monocytes to dendritic cells. High levels of the Th1-attracting, HIV-1-inhibitory chemokines, CCL3/MIP-1alpha and CCL4/MIP-1beta, were induced in human primary monocytes when CpG- or non-CpG-ODN was combined with GM-CSF, but not with IL-4 or IFN-gamma. The synergistic induction of beta-chemokines by non-CpG-ODN was phosphorothioate (PS) chemistry dependent and inhibited by blocking endosome maturation/acidification and ERK1/2 activation. Chemokine and TLR9 mRNAs were induced by PS-ODN. Cells treated with non-CpG PS-ODN and GM-CSF expressed dendritic cell marker CD83 and high levels of HLA-DR and costimulatory molecules, and were CD14(-) or CD14(dim), consistent with monocyte differentiation into a dendritic cell phenotype. The induction of CD83 and beta-chemokines was tyrosine phosphorylation dependent. Secreted CCL3 and CCL4 were detected as a heterodimer. Our results indicate the CpG-independent synergy between PS-ODN and GM-CSF mediated through chemokine and dendritic cell induction. In addition, our observations suggest that PS-ODN plus GM-CSF may be useful as potent ex vivo dendritic cell differentiation/maturation agents for dendritic cell therapy and as vaccine adjuvants for tumor and infectious microorganisms, including HIV-1.
Collapse
Affiliation(s)
- Jinhai Wang
- Laboratory of Immunology, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
29
|
Sharma S, Karakousis CP, Takita H, Shin K, Brooks SP. Cytokines and chemokines are expressed at different levels in small and large murine colon-26 tumors following intratumoral injections of CpG ODN. Neoplasia 2005; 6:523-8. [PMID: 15548360 PMCID: PMC1531655 DOI: 10.1593/neo.04166] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Direct tumor injections of (CpG ODN) into murine colon tumor 26 (CT-26) tumors can induce a potent antitumor response. Tumor size at the beginning of treatment determines the final therapeutic outcome, with smaller tumors responding favorably to CpG ODN therapy whereas large tumors do not. CpG ODN injections in small tumors resulted in tumor necrosis and extensive inflammatory cell infiltration, with average survival that is significantly higher (48.1 +/- 34 days) when compared to control ODN-treated mice (16.1 +/- 3.5 days). Cytokines and chemokines are expressed at different levels in small and large CT-26 tumors following intratumoral injections of CpG ODN. We observed that granulocyte-macrophage colony-stimulating factor and interleukin (IL) 6 are the major cytokines that were overexpressed in CpG ODN-treated small tumors but not in large tumors. Similarly, several chemokines (CXCL1, CCL2, and CCL3) were also significantly higher in CpG ODN-treated small tumors compared to control ODN-treated tumors.
Collapse
Affiliation(s)
- Sanjay Sharma
- Department of Surgery, SUNY Buffalo and Kaleida Health, Buffalo General Hospital, Buffalo, NY 14203, USA
| | | | | | | | | |
Collapse
|
|
30
|
Xu HB, Xu W, Chu YW, Wang Y, Xiong S. Single B or T-cell epitope-based DNA vaccine using modified vector induces specific immune response against hepadnavirus. Immunol Lett 2005; 99:186-92. [PMID: 16009269 DOI: 10.1016/j.imlet.2005.02.014] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2004] [Revised: 12/16/2004] [Accepted: 02/15/2005] [Indexed: 10/25/2022]
Abstract
Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.
Collapse
MESH Headings
- Animals
- Cell Line
- Cloning, Molecular
- Ducks
- Epitopes, B-Lymphocyte/biosynthesis
- Epitopes, B-Lymphocyte/immunology
- Epitopes, T-Lymphocyte/biosynthesis
- Epitopes, T-Lymphocyte/immunology
- Hepadnaviridae Infections/immunology
- Hepadnaviridae Infections/prevention & control
- Hepatitis B Antibodies/biosynthesis
- Hepatitis B Core Antigens/biosynthesis
- Hepatitis B Core Antigens/genetics
- Hepatitis B Core Antigens/immunology
- Hepatitis B Surface Antigens/biosynthesis
- Hepatitis B Surface Antigens/genetics
- Hepatitis B Surface Antigens/immunology
- Hepatitis B Virus, Duck/genetics
- Hepatitis B Virus, Duck/immunology
- Mice
- Mice, Inbred C57BL
- Plasmids
- Transfection
- Vaccination
- Vaccines, DNA/administration & dosage
- Vaccines, DNA/immunology
Collapse
Affiliation(s)
- Huan-Bin Xu
- Department of Immunology and Key Laboratory of Molecular Medicine of the Ministry of Education, Shanghai Medical college of Fudan University, P.R. China
| | | | | | | | | |
Collapse
|
|
31
|
Matheu V, Treschow A, Teige I, Navikas V, Issazadeh-Navikas S. Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-beta knock-out mice. Respir Res 2005; 6:25. [PMID: 15748290 PMCID: PMC555575 DOI: 10.1186/1465-9921-6-25] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2004] [Accepted: 03/05/2005] [Indexed: 12/02/2022] Open
Abstract
Background CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-β in the immunomodulatory effects of CpG-ODN is not known. Objective Here, we aimed to elucidate the role of IFN-β in the anti-allergic effect of CpG motifs. Methods We assessed the immune response in OVA-primed/OVA-challenged IFN-β knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment with synthetic CpG motifs. Results Vaccination with CpG-ODN reduced the number of cells in airways of OVA-sensitized WT but not IFN-β-/- mice. Although airway eosinophilia was reduced in both treated groups, they were significantly higher in IFN-β-/- mice. Other inflammatory cells, such as lymphocytes and macrophages were enhanced in airways by CpG treatment in IFN-β-/- mice. The ratio of IFN-γ/IL-4 cytokines in airways was significantly skewed to a Th1 response in WT compared to IFN-β-/- group. In contrast, IL-4 and IgE were reduced with no differences between groups. Ag-specific T-cell proliferation, Th1-cytokines such as IFN-γ, IL-2 and also IL-12 were significantly lower in IFN-β-/- mice. Surprisingly, we discovered that intranasal treatment of mice with CpG-ODN results in mild synovitis particularly in IFN-β-/- mice. Conclusion Our results indicate that induction of Th1 response by therapy with CpG-ODN is only slightly and partially dependent on IFN-β, while IFN-β is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination.
Collapse
Affiliation(s)
- Victor Matheu
- Section of Medical Inflammation Research, Department of Cell & Molecular Biology; Lund University; Sweden
- Fundación Rafael Clavijo de Investigación Biomédica, Tenerife, Spain
| | - Alexandra Treschow
- Section of Medical Inflammation Research, Department of Cell & Molecular Biology; Lund University; Sweden
| | - Ingrid Teige
- Section of Medical Inflammation Research, Department of Cell & Molecular Biology; Lund University; Sweden
| | - Vaidrius Navikas
- Section of Medical Inflammation Research, Department of Cell & Molecular Biology; Lund University; Sweden
| | - Shohreh Issazadeh-Navikas
- Section of Medical Inflammation Research, Department of Cell & Molecular Biology; Lund University; Sweden
| |
Collapse
|
|
32
|
Yamada H, Ishii KJ, Klinman DM. Suppressive oligodeoxynucleotides inhibit CpG-induced inflammation of the mouse lung. Crit Care Med 2004; 32:2045-9. [PMID: 15483413 DOI: 10.1097/01.ccm.0000142397.38134.ef] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
OBJECTIVE To examine the effect of suppressive oligodeoxynucleotides (ODNs) on the pulmonary inflammation induced by immunostimulatory CpG DNA. DESIGN Prospective, randomized, controlled study. SETTING Research laboratories. SUBJECTS RAW 264.7 murine macrophage-like cell line and BALB/c mice. INTERVENTIONS RAW 264.7 cells were incubated with bacterial DNA or CpG ODN, alone or combined with suppressive ODN. The in vivo effect of suppressive ODN was determined using an acute lung injury model. CpG ODN alone or combined with suppressive ODN was instilled into the mouse lung. MEASUREMENTS AND MAIN RESULTS Production of tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 by RAW 264.7 cells were measured by enzyme-linked immunosorbent assay (ELISA), whereas their messenger RNA levels were determined by reverse transcriptase-polymerase chain reaction. Synthetic ODN containing CpG motifs (CpG ODN) mimicked the ability of bacterial DNA to stimulate the production of TNF-alpha and MIP-2. Suppressive ODN significantly inhibited the activation of RAW 264.7 cells by both bacterial DNA and CpG ODN. In the lung injury model, production of proinflammatory cytokines (TNF-alpha and IL-6) and chemokines (MIP-2 and KC) in bronchoalveolar lavage (BAL) fluids was measured by ELISA. Neutrophil accumulation in the alveolar spaces was also evaluated. Instillation of CpG ODN into the lungs of normal mice triggered the synthesis of TNF-alpha, IL-6, MIP-2, and KC. Suppressive ODN significantly blocked the production of these proinflammatory cytokines and chemokines and also reduced neutrophil mobilization into the alveolar spaces by CpG DNA. CONCLUSIONS Proinflammatory cytokines and chemokines are up-regulated by CpG motifs in bacterial DNA. Suppressive ODN significantly inhibits the inflammatory response induced by CpG DNA in murine macrophages and the lung. This study supports the use of suppressive ODN to reduce the deleterious inflammatory responses induced by bacterial DNA.
Collapse
Affiliation(s)
- Hiroshi Yamada
- Department of Anesthesiology, Yokohama City University, School of Medicine, Japan.
| | | | | |
Collapse
|
|
33
|
Carrington AC, Collet B, Holland JW, Secombes CJ. CpG oligodeoxynucleotides stimulate immune cell proliferation but not specific antibody production in rainbow trout (Oncorhynchus mykiss). Vet Immunol Immunopathol 2004; 101:211-22. [PMID: 15350751 DOI: 10.1016/j.vetimm.2004.04.022] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2003] [Revised: 03/26/2004] [Accepted: 04/30/2004] [Indexed: 10/26/2022]
Abstract
Bacterial DNA and CpG ODN have both been shown to have immunostimulatory effects in mammals, activating APCs and inducing a potent Th1 type immune response. They have also been shown to have a strong adjuvant effect and up-regulate MHC class 2 expression in murine cells, augment human and murine NK cell lytic activity, activate human B cells and induce murine B cell proliferation. However, little work has been carried out with regard to their effects on the piscine immune system. Here it is shown that various CpG ODN induce proliferation of peripheral blood leucocytes, spleen and head kidney cells from rainbow trout although, at the range of concentrations tested CpG ODN 2133 lacked the ability to induce specific antibody production to a protein antigen.
Collapse
Affiliation(s)
- Allison C Carrington
- Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
| | | | | | | |
Collapse
|
|
34
|
Deng JC, Moore TA, Newstead MW, Zeng X, Krieg AM, Standiford TJ. CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection. THE JOURNAL OF IMMUNOLOGY 2004; 173:5148-55. [PMID: 15470059 PMCID: PMC3001228 DOI: 10.4049/jimmunol.173.8.5148] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Bacterial pneumonia is a leading cause of mortality in the United States. Innate immune responses, including type-1 cytokine production, are critical to the effective clearance of bacterial pathogens from the lung. Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs (CpG ODN), which mimic the effects of bacterial DNA, have been shown to enhance type-1 cytokine responses during infection due to intracellular pathogens, resulting in enhanced microbial clearance. The role of CpG ODN in modulating protective innate immunity against extracellular pathogens is unknown. Using a murine model of Gram-negative pneumonia, we found that CpG ODN administration stimulated protective immunity against Klebsiella pneumoniae. Specifically, intratracheal (i.t.) administration of CpG ODN (30 microg) 48 h before i.t. K. pneumoniae challenge resulted in increased survival, compared with animals pretreated with control ODN or saline. Pretreatment with CpG ODN resulted in enhanced bacterial clearance in lung and blood, and higher numbers of pulmonary neutrophils, NKT cells, gammadelta-T cells, and activated NK1.1+ cells and gammadelta-T lymphocytes during infection. Furthermore, pretreatment with CpG ODN enhanced the production of TNF-alpha, and type-1 cytokines, including IL-12, IFN-gamma, and the IFN-gamma-dependent ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma in response to Klebsiella challenge, compared with control mice. These findings indicate that i.t. administration of CpG ODN can stimulate multiple components of innate immunity in the lung, and may form the basis for novel therapies directed at enhancing protective immune responses to severe bacterial infections of the lung.
Collapse
Affiliation(s)
- Jane C. Deng
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
| | - Thomas A. Moore
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
| | - Michael W. Newstead
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
| | - Xianying Zeng
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
| | | | - Theodore J. Standiford
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109
- Address correspondence and reprint requests to Dr. Theodore J. Standiford, University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, 1150 West Medical Center Drive, Medical Science Research Building III 6301, Ann Arbor, MI 48109-0642.
| |
Collapse
|
|
35
|
Bjersing JL, Eriksson K, Tarkowski A, Collins LV. The arthritogenic and immunostimulatory properties of phosphorothioate oligodeoxynucleotides rely on synergy between the activities of the nuclease-resistant backbone and CpG motifs. Inflammation 2004; 28:39-51. [PMID: 15072229 DOI: 10.1023/b:ifla.0000014710.44475.94] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Experiments with immunostimulatory unmethylated CpG-containing DNA are usually conducted with nuclease-protected phosphorothioate oligodeoxynucleotides (S-ODNs), rather than phosphodiester oligodeoxynucleotides (O-ODNs). We compared the murine immune responses to S-ODNs and O-ODNs that either contained or lacked CpG motifs. Both CpG and non-CpG S-ODNs induced synovitis, as did sequence-matched CpG O-ODN, but not GpC O-ODN. There was a minimum length requirement for arthritogenic S-ODNs since a CpC dinucleotide S-ODN did not induce arthritis. There were both sequence- (CpG > non-CpG) and backbone-dependent (S-ODN > O-ODN) differences in the levels of DNA-induced arthritis upon intra-articular injection with the ODNs. However, CpG O-ODN being an exception, induced more severe arthritis than the GpC S-ODN. The levels of in vitro proliferation and production of IL-6, TNF-alpha, IL-12, and RANTES by splenocytes following exposure to CpG S-ODN were significantly higher than those induced by CpG O-ODN. In addition, both proliferative responses and cytokine production induced by S-ODN-stimulated splenocytes increased significantly when the S-ODN contained a CpG motif. Transcription factor NFkappaB was activated by both CpG S-ODN and CpG O-ODN but interestingly not by GpC S-ODN. This indicates that the NFkappaB signal pathway modulates CpG-mediated immunostimulation, while sequence-independent immune activation by the phosphorothioate backbone is probably signalled via a different pathway.
Collapse
Affiliation(s)
- Jan L Bjersing
- Department of Rheumatology and Inflammation Research, University of Göteborg, Sweden.
| | | | | | | |
Collapse
|
|
36
|
Kato A, Ogasawara T, Homma T, Batchelor J, Imai S, Wakiguchi H, Saito H, Matsumoto K. CpG oligodeoxynucleotides directly induce CXCR3 chemokines in human B cells. Biochem Biophys Res Commun 2004; 320:1139-47. [PMID: 15249208 DOI: 10.1016/j.bbrc.2004.06.059] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2004] [Indexed: 11/30/2022]
Abstract
CpG oligodeoxynucleotides (CpG ODN) are known to elicit Th1 immune responses via TLR9. However, the precise mechanisms through which B cells are involved in this phenomenon are not fully understood. We investigated the effect of CpG ODN on the induction of Th1-chemoattractant CXCR3 chemokines, IP-10, Mig, and I-TAC, in B cells. Cells from the RPMI 8226 human B cell line and human peripheral B cells were stimulated with three distinct classes of CpG ODN. As a result, CXCR3 chemokines were strongly up-regulated by CpG-B and CpG-C, but only weakly by CpG-A. Though CXCR3 chemokines are known to be induced by IFNs, blocking mAbs against IFN receptors did not inhibit their induction by CpG-B. Induction of CXCR3 chemokines was blocked by two NF-kappaB inhibitors and a p38 inhibitor. These results strongly suggest that CXCR3 chemokines are directly induced by CpG ODN via NF-kappaB- and p38-dependent pathways in human B cells.
Collapse
Affiliation(s)
- Atsushi Kato
- Department of Allergy and Immunology, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan
| | | | | | | | | | | | | | | |
Collapse
|
|
37
|
Takeshita F, Suzuki K, Sasaki S, Ishii N, Klinman DM, Ishii KJ. Transcriptional Regulation of the Human TLR9 Gene. THE JOURNAL OF IMMUNOLOGY 2004; 173:2552-61. [PMID: 15294971 DOI: 10.4049/jimmunol.173.4.2552] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5'-PU box, 3'-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPalpha transcription factors. Other members of the C/EBP family, such as C/EBPbeta, C/EBPdelta, and C/EBPepsilon, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-kappaB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPalpha culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.
Collapse
MESH Headings
- Animals
- Base Sequence
- Blotting, Western
- CCAAT-Enhancer-Binding Proteins/immunology
- CCAAT-Enhancer-Binding Proteins/metabolism
- Cell Line, Tumor
- Cloning, Molecular
- CpG Islands/genetics
- CpG Islands/immunology
- Cyclic AMP Response Element-Binding Protein/immunology
- Cyclic AMP Response Element-Binding Protein/metabolism
- Gene Expression Regulation/immunology
- Genes, Reporter/genetics
- Genes, Reporter/immunology
- Genes, jun/immunology
- Humans
- Membrane Glycoproteins/genetics
- Membrane Glycoproteins/immunology
- Mice
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- NF-kappa B/immunology
- NF-kappa B/metabolism
- Promoter Regions, Genetic/genetics
- Promoter Regions, Genetic/immunology
- Receptors, Cell Surface/genetics
- Receptors, Cell Surface/immunology
- Reverse Transcriptase Polymerase Chain Reaction
- Sequence Homology, Nucleic Acid
- Toll-Like Receptor 9
- Toll-Like Receptors
- Transcription Factors/immunology
- Transcription Factors/metabolism
- Transcription, Genetic/genetics
- Transcriptional Activation/immunology
- Transfection
Collapse
Affiliation(s)
- Fumihiko Takeshita
- Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
| | | | | | | | | | | |
Collapse
|
|
38
|
Katakura T, Miyazaki M, Kobayashi M, Herndon DN, Suzuki F. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. THE JOURNAL OF IMMUNOLOGY 2004; 172:1407-13. [PMID: 14734716 DOI: 10.4049/jimmunol.172.3.1407] [Citation(s) in RCA: 146] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Classically activated macrophages (CAMphi) have been described as a major effector cell on the host's innate immunities. However, CAMphi are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMphi) predominate. In this study, the mechanism by which AAMphi suppress the ability of resident macrophages (RMphi) to generate CAMphi was investigated. AAMphi were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMphi were generated from RMphi (peritoneal Mphi from normal mice) through stimulation with CpG DNA, a typical CAMphi inducer. RMphi did not polarize to CAMphi when they were cultured with AAMphi in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMphi stimulated with CpG DNA did not convert to CAMphi when they were cultured with the culture fluids of AAMphi (AAMphi Culture-Sup). AAMphi Culture-Sup contained IL-6, IL-10, CCL17, PGE(2), and TGF-beta. Among these, CCL17 and IL-10 inhibited CAMphi generation. The ability of AAMphi Culture-Sup to inhibit CAMphi generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMphi inhibit CAMphi generation from RMphi stimulated with CpG DNA.
Collapse
Affiliation(s)
- Tatsushi Katakura
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA
| | | | | | | | | |
Collapse
|
|
39
|
Jiang W, Reich III CF, Pisetsky DS. Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA. Cell Immunol 2004; 229:31-40. [PMID: 15331326 DOI: 10.1016/j.cellimm.2004.06.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2004] [Accepted: 06/10/2004] [Indexed: 11/25/2022]
Abstract
Bacterial DNA can stimulate the production of cytokines and nitric oxide (NO), while mammalian DNA can block these responses. If mammalian DNA is transfected into macrophages, however, it can stimulate NO production, without inducing IL-12. To define further this activity, signaling pathways induced by transfected calf thymus (CT) DNA were studied. Using RAW264.7 cells as a model, CT DNA in the transfection agent FuGENE 6 activated cells through the NF-kappaB and MAPKs pathways, similar to bacterial DNA and LPS. The role of these pathways was further investigated using specific inhibitors, with studies indicating that NO production is blocked by inhibitors of NF-kappaB and p38 but not other MAPKs. These data indicate that the immune activity of DNA is influenced by context or intracellular location and that, when transfected into cells, mammalian DNA can activate cells through signaling pathways similar to those of bacterial DNA.
Collapse
Affiliation(s)
- Weiwen Jiang
- Department of Medicine, Division of Rheumatology and Immunology, Duke University, Durham, NC, USA
| | | | | |
Collapse
|
|
40
|
Klinman DM, Zeuner R, Yamada H, Gursel M, Currie D, Gursel I. Regulation of CpG-induced immune activation by suppressive oligodeoxynucleotides. Ann N Y Acad Sci 2004; 1002:112-23. [PMID: 14751829 DOI: 10.1196/annals.1281.023] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated "CpG motifs" stimulate an innate immune response characterized by the production of cytokines, chemokines, and polyreactive Igs that promote host survival following infectious challenge. Yet CpG-driven immune activation can have deleterious consequences, such as increasing the host's susceptibility to autoimmune disease. The immunomodulatory activity of CpG DNA can be blocked by DNA containing "suppressive" motifs. This work explores the rules governing cellular recognition of stimulatory and suppressive motifs, and the resultant modulation of the immune system. Results suggest that both CpG and suppressive ODN may find use as therapeutic agents.
Collapse
Affiliation(s)
- Dennis M Klinman
- Section of Retroviral Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
| | | | | | | | | | | |
Collapse
|
|
41
|
Anders HJ, Vielhauer V, Eis V, Linde Y, Kretzler M, Perez de Lema G, Strutz F, Bauer S, Rutz M, Wagner H, Gröne HJ, Schlöndorff D. Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice. FASEB J 2004; 18:534-6. [PMID: 14734643 DOI: 10.1096/fj.03-0646fje] [Citation(s) in RCA: 172] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.
Collapse
MESH Headings
- Animals
- Antibodies, Antinuclear/biosynthesis
- Antibodies, Antinuclear/blood
- Cells, Cultured/drug effects
- Cells, Cultured/metabolism
- Chemokines/biosynthesis
- Chemokines/genetics
- DNA/immunology
- DNA, Bacterial/pharmacology
- DNA-Binding Proteins/drug effects
- DNA-Binding Proteins/physiology
- Dendritic Cells/drug effects
- Dendritic Cells/metabolism
- Disease Models, Animal
- Disease Progression
- Escherichia coli/genetics
- Immunoglobulin G/biosynthesis
- Immunoglobulin G/blood
- Kidney Glomerulus/drug effects
- Kidney Glomerulus/immunology
- Kidney Glomerulus/metabolism
- Lupus Nephritis/blood
- Lupus Nephritis/immunology
- Lupus Nephritis/metabolism
- Macrophages/drug effects
- Macrophages/metabolism
- Mice
- Mice, Inbred MRL lpr
- Oligodeoxyribonucleotides/pharmacology
- Receptors, CCR5/biosynthesis
- Receptors, CCR5/genetics
- Receptors, Cell Surface/drug effects
- Receptors, Cell Surface/physiology
- Toll-Like Receptor 9
Collapse
Affiliation(s)
- Hans-Joachim Anders
- Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Germany.
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
42
|
Yeo SJ, Yoon JG, Yi AK. Myeloid differentiation factor 88-dependent post-transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: tumor necrosis factor-alpha receptor-associated factor 6, a diverging point in the Toll-like receptor 9-signaling. J Biol Chem 2003; 278:40590-600. [PMID: 12902324 DOI: 10.1074/jbc.m306280200] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2-3'-UTR-luciferase) was generated by inserting sequences within the 3'-untranslated region (UTR) of cox-2 to the 3' end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2-3'-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2-3'-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2-3'-UTR-luciferase activity induced by CpG DNA. Overexpression of DN-TRAF6 suppressed CpG DNA-mediated activation of p38 and JNK, but not ERK, explaining the partial inhibitory effects of DN-TRAF6 on CpG DNA-induced COX2-3'-UTR-luciferase activity. Co-expression of DN-TRAF6 and N17Ras completely inhibited CpG DNA-induced COX2-3'-UTR-luciferase activity, indicating the involvement of Ras in CpG DNA-mediated ERK and COX2-3'-UTR regulation. Collectively, our results suggest that MyD88 and MAPKs play a key regulatory role in CpG DNA-mediated cox-2 expression at the post-transcriptional level and that TRAF6 is a diverging point in the Toll-like receptor 9-signaling pathway for CpG DNA-mediated MAPK activation.
Collapse
MESH Headings
- 3' Untranslated Regions
- Adaptor Proteins, Signal Transducing
- Animals
- Antigens, Differentiation/genetics
- Antigens, Differentiation/physiology
- Blotting, Western
- Cell Line
- CpG Islands
- Cyclooxygenase 2
- DNA/metabolism
- DNA-Binding Proteins/metabolism
- Genes, Reporter
- Isoenzymes/biosynthesis
- Isoenzymes/genetics
- Luciferases/metabolism
- MAP Kinase Signaling System
- Mice
- Myeloid Differentiation Factor 88
- Plasmids/metabolism
- Prostaglandin-Endoperoxide Synthases/biosynthesis
- Prostaglandin-Endoperoxide Synthases/genetics
- Proteins/metabolism
- Proteins/physiology
- RNA Processing, Post-Transcriptional
- Receptors, Cell Surface/metabolism
- Receptors, Immunologic/genetics
- Receptors, Immunologic/physiology
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction
- TNF Receptor-Associated Factor 6
- Toll-Like Receptor 9
- Transcription, Genetic
- Transfection
Collapse
Affiliation(s)
- Seon-Ju Yeo
- Children's Foundation Research Center at Le Bonheur Children's Medical Center, Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee 38103, USA
| | | | | |
Collapse
|
|
43
|
Xu H, An H, Yu Y, Zhang M, Qi R, Cao X. Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages. J Biol Chem 2003; 278:36334-40. [PMID: 12867418 DOI: 10.1074/jbc.m305698200] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.
Collapse
Affiliation(s)
- Hongmei Xu
- Institute of Immunology, Second Military Medical University, Shanghai 200433, People's Republic of China
| | | | | | | | | | | |
Collapse
|
|
44
|
Mendez S, Tabbara K, Belkaid Y, Bertholet S, Verthelyi D, Klinman D, Seder RA, Sacks DL. Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability. Infect Immun 2003; 71:5121-9. [PMID: 12933855 PMCID: PMC187328 DOI: 10.1128/iai.71.9.5121-5129.2003] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The inoculation of live, nonattenuated Leishmania major to produce a lesion in a selected site that heals, referred to as leishmanization, is to date the only vaccine against leishmaniasis that has proven to be effective in humans. Its use has been restricted or abandoned entirely, however, due to safety concerns. In an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long-term protection, live parasites were coinjected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) alone or in combination with whole-cell lysates of heat-killed L. major promastigotes bound to alum (ALM). C57BL/6 mice infected intradermally by using L. major plus CpG ODN with or without ALM developed few or no dermal lesions and showed an early containment of parasite growth, while mice infected with L. major with or without ALM developed sizable dermal lesions that required up to 10 weeks to heal. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of gamma interferon-producing CD4(+) lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receiving L. major and CpG ODN plus ALM were totally protected against reinfection with L. major for up to 6 months. By comparison, the immunity elicited by two efficient nonlive vaccines began to wane by 6 months. Our results suggest that immune modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.
Collapse
Affiliation(s)
- Susana Mendez
- Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
45
|
Yeo SJ, Gravis D, Yoon JG, Yi AK. Myeloid differentiation factor 88-dependent transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: role of NF-kappaB and p38. J Biol Chem 2003; 278:22563-73. [PMID: 12695520 DOI: 10.1074/jbc.m302076200] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
CpG DNA induces macrophage cyclooxgenase-2 (Cox-2) production. In this study, we have investigated a biochemical signaling pathway and transcription factors responsible for transcriptional regulation of the Cox-2 gene expression induced by CpG DNA. CpG DNA-induced Cox-2 promoter activity was completely inhibited by an endosomal acidification inhibitor (chloroquine), a TLR9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. In contrast, overexpression of DN-IRAK1 or DN-TRAF6 only partially inhibited CpG DNA-induced Cox-2 promoter activity and NF-kappaB activation, indicating the presence of additional signaling modulators downstream of MyD88. CpG DNA-induced Cox-2 promoter activity was substantially suppressed in cells overexpressing super-suppressive IkappaB (IkappaB-arachidonic acid), DN-p38, or DN-CREB. In addition, Cox-2 promoter-luciferase reporters with alterations in predicted cis-acting transcriptional regulatory elements revealed that C/EBP, Ets-1, NF-kappaB, and CREB-binding sites are essential for optimal Cox-2 expression in response to CpG DNA. Conclusively, these results demonstrate that endosomal DNA processing and TLR9/MyD88-dependent activation of NF-kappaB and p38 are required for transcriptional regulation of Cox-2 expression induced by CpG DNA, and suggest that interleukin-1 receptor-associated kinase and/or TRAF6 may be a diverging point for NF-kappaB activation in response to CpG DNA in RAW264.7 cells.
Collapse
Affiliation(s)
- Seon-Ju Yeo
- Children's Foundation Research Center at Le Bonheur Children's Medical Center, Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee 38103, USA
| | | | | | | |
Collapse
|
|
46
|
Zeuner RA, Verthelyi D, Gursel M, Ishii KJ, Klinman DM. Influence of stimulatory and suppressive DNA motifs on host susceptibility to inflammatory arthritis. ARTHRITIS AND RHEUMATISM 2003; 48:1701-7. [PMID: 12794839 DOI: 10.1002/art.11035] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVE To examine whether systemic administration of immunostimulatory and immunosuppressive oligodeoxynucleotides (ODNs) alter host susceptibility to inflammatory arthritis. METHODS Normal BALB/c mice were treated systemically with CpG ODNs or suppressive ODNs, and then challenged intraarticularly with CpG DNA. The onset and magnitude of the resulting inflammatory response was monitored. RESULTS Systemic delivery of CpG ODNs significantly increased susceptibility to local inflammation, whereas systemic treatment with suppressive ODNs reduced this susceptibility. CD11c+ cells played a key role in mediating host sensitivity to arthritis. These cells were the dominant source of tumor necrosis factor alpha production in CpG-stimulated animals and transferred resistance to arthritis from mice treated with suppressive ODNs. CONCLUSION Systemic exposure to immunostimulatory and immunosuppressive DNA influences host susceptibility to local inflammatory challenge. Current findings raise the possibility that suppressive ODNs may be useful in the prevention/treatment of proinflammatory diseases.
Collapse
|
|
47
|
Blackwell SE, Krieg AM. CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2003; 170:4061-8. [PMID: 12682235 DOI: 10.4049/jimmunol.170.8.4061] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.
Collapse
Affiliation(s)
- Sue E Blackwell
- Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
| | | |
Collapse
|
|
48
|
Anders HJ, Banas B, Linde Y, Weller L, Cohen CD, Kretzler M, Martin S, Vielhauer V, Schlöndorff D, Gröne HJ. Bacterial CpG-DNA aggravates immune complex glomerulonephritis: role of TLR9-mediated expression of chemokines and chemokine receptors. J Am Soc Nephrol 2003; 14:317-26. [PMID: 12538732 DOI: 10.1097/01.asn.0000042169.23931.73] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.
Collapse
Affiliation(s)
- Hans-Joachim Anders
- Nephrological Center, Medical Policlinic, University of Munich, Munich, Germany
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
49
|
Mutwiri G, Pontarollo R, Babiuk S, Griebel P, van Drunen Littel-van den Hurk S, Mena A, Tsang C, Alcon V, Nichani A, Ioannou X, Gomis S, Townsend H, Hecker R, Potter A, Babiuk LA. Biological activity of immunostimulatory CpG DNA motifs in domestic animals. Vet Immunol Immunopathol 2003; 91:89-103. [PMID: 12543546 DOI: 10.1016/s0165-2427(02)00246-5] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in domestic animals.
Collapse
Affiliation(s)
- G Mutwiri
- Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3.
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
50
|
Harandi AM, Eriksson K, Holmgren J. A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection. J Virol 2003; 77:953-62. [PMID: 12502811 PMCID: PMC140825 DOI: 10.1128/jvi.77.2.953-962.2003] [Citation(s) in RCA: 126] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.
Collapse
Affiliation(s)
- Ali M Harandi
- Department of Medical Microbiology & Immunology, Göteborg University Vaccine Research Institute, Göteborg University, Sweden.
| | | | | |
Collapse
|