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. 2005 Jun;115(2):223–230. doi: 10.1111/j.1365-2567.2005.02150.x

CpG motif acts as a ‘danger signal’ and provides a T helper type 1-biased microenvironment for DNA vaccination

Ling Liu 1,1, Xiaohui Zhou 1,1,2, Hua Liu 1, Li Xiang 1, Zhenghong Yuan 1
PMCID: PMC1782151  PMID: 15885128

Abstract

The method of delivering a DNA vaccine can influence the type of immune response induced by the vaccine. Application of a DNA vaccine by gene gun typically induces a T helper type 2 (Th2)-type reaction, whereas needle inoculation triggers a Th1 response. In the present study, we found that physical trauma, gold-particle bombardment and the CpG motif can act as ‘danger signals’ that recruit inflammatory cells to damaged tissues. Analysis of the cytokine profiles of draining lymph nodes or lymph-node-derived mononuclear cells from different groups by real-time reverse transcription–polymerase chain reaction revealed that, while gene-gun-bombardment induced a Th2-type cytokine microenvironment with increased interleukin-4 (IL-4) and IL-10 mRNA expression and almost no increase in IL-12 and interferon-γ mRNA expression in draining lymph nodes, intradermal injection as well as subcutaneous injection of muscle induced the opposite. We further studied whether the addition of the CpG motif can switch the Th2-type cytokine microenvironment produced by gene-gun bombardment in draining lymph nodes. Results showed that the addition of the CpG motif can increase IL-12 mRNA expression in draining lymph nodes whether induced by intradermal injection, intramuscular injection, or gene-gun bombardment. These data suggest that delivery of the CpG motif induced a Th1-biased microenvironment in draining lymph nodes. Taken together, the CpG motif can act as a ‘danger signal’ and Th1 immune response enhancer in DNA vaccination. These results may help to explain the mechanism of different types of immune response induced by DNA vaccines delivered by different routes and facilitate the application of DNA vaccines.

Keywords: CpG motif, danger signal, DNA vaccine, gene gun, physical trauma

Introduction

DNA vaccines are usually administered by intradermal or intramuscular injection or are coated with gold particles and propelled into the epidermis using a gene gun.1–4 However, different methods of antigen delivery can affect the type of immune response. Typically, intradermal and intramuscular injections induce a predominantly T helper type 1 (Th1) response, whereas gene-gun-mediated DNA immunization triggers predominantly Th2-type responses.5–8 These differences between needle injection and gene-gun inoculation have been attributed to the amount of plasmid DNA and the associated CpG motifs, as well as the nature of the antigen.9–11

As the activation of antigen-presenting cells (APCs) has a great impact on both the induction and the qualities of the acquired immune response,12,13 it is hypothesized that gene-gun-mediated delivery and intradermal and intramuscular injection of DNA vaccine may deliver different ‘danger signals’ to localized APCs.14,15 The danger signals of intradermal injection of DNA may come from physical trauma, while the danger signals of intramuscular injection of DNA come from the CpG motifs built into the plasmid construct, and those of gene-gun-mediated DNA immunization come from the gold-particle bombardment of the skin.

To test this hypothesis, local skin that had been gene-gun-bombarded or intradermally injected and needle-injected muscle were sectioned and analysed by haematoxylin & eosin (H&E) staining. The cytokine profiles of draining lymph nodes or lymph-node-derived mononuclear cells from the three different sample groups were further studied by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Results showed that remarkable inflammatory infiltration could be seen in skin after intradermal injection and gene-gun-bombardment. Interestingly, although single intramuscular injection with normal saline (NS) cannot induce inflammatory cell infiltration, injection with CpG motif recruits large numbers of inflammatory cells to the muscle. Quantitative RT-PCR experiments revealed that the cytokine profiles of draining lymph nodes or lymph-node-derived mononuclear cells were different among these three groups. Gene-gun-bombardment induced a Th-2 microenvironment, while intradermal injection as well as needle-injection of muscle induced the opposite. Furthermore, adding CpG-oligodeoxynucleotide (ODN) to gold particles could switch the cytokine microenvironment in draining lymph nodes to a Th1 type. Taken together, the CpG motif may act as a ‘danger signal’ and an enhancer of the Th1 immune response in DNA vaccination.

Materials and methods

Animals

Female BALB/c mice were purchased from BK Co. (Shanghai, China) and housed in the Shanghai Medical Center of Fudan University with free access to food and water. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.

Plasmids and reagents

Sodium chloride injections (NS) were purchased from Chanzheng Fumin Co., Ltd (Shanghai, China). The plasmid pYQF-2CpG/s used for DNA-based immunization was constructed in our laboratory.9 Briefly, this plasmid contains CpG motifs (AACGTT) at the Sac II and SphI sites, and the gene encoding hepatitis B virus surface protein in the multiple-cloning site. DNA plasmids were extracted from transformed Escherichia coli by alkaline sodium dodecyl sulphate (SDS) lysis and were column-purified (Qiagen Plasmid Maxi Kit) using the standard protocol. The plasmids were then dissolved in NS and stored at −20°. The phosphorothioate-modified CpG-ODN) 1668 (5′-TCCATGACGTTCCTGATGCT-3′), which contains an immunostimulatory sequence and phosphorothioate-modified GpC-ODN 1668gc (5′-TCCATGAGCTTCCTGATGCT-3′) as a negative control sequence were synthesized by SBS Gentech Co., Ltd (Shanghai, China) and dissolved in NS.

Formulation of plasmid DNA or plasmid DNA-ODN onto cartridges

The plasmid pYQF-2CpG/s or ODN was precipitated onto the surface of 1-μm gold beads (Bio-Rad, Hercules, CA) in the presence of calcium chloride and spermidine as previously described.16 Briefly, each cartridge contains 0·5 mg gold particles, together with 1 μg plasmid DNA or 25 μg ODN.

Animal treatment protocol

For gene-gun-mediated immunization, freshly shaven mouse abdominal skin was bombarded with DNA-coated gold particles at 2·5 MPa pressure, resulting in a target area of 2 cm2. Intramuscular and intradermal injections of DNA administered in saline were carried out as specified elsewhere.17,18 Mice were killed at different time-points after injection, and the draining lymph nodes were removed for RNA isolation and real-time RT-PCR assay. The mononuclear cells of draining lymph nodes were separated and adjusted to a final concentration of 1 × 106 cells/ml in 10% fetal calf serum and RPMI-1640. The mononuclear cell suspensions were plated on 24-well tissue culture plates (1 ml per well) and stimulated with 5 μg/ml concanavalin A (Con A). Relative copies of RNA of each cytokine were determined by real-time RT-PCR at the optimized time [interferon-γ (IFN-γ), Con A for 48 hr; interleukin-12 (IL-12), Con A for 24 hr; IL-4 and IL-10, Con A for 72 hr) after stimulation.

Quantitative RT-PCR analysis of cytokine mRNA expression

The total RNA of draining lymph nodes was extracted with TRIzol reagent (Gibco BRL, Rockville, MD) following the manufacturer's instructions. After treatment with DNaseI, reverse transcription was performed with random hexamers (Promega, Fitchburg, WI) for priming and 200 U of SuperScript™ II (Gibco). Real-time PCR (LightCycler; Roche, Switzerland) amplifications were performed using the CYBR I reagents (Roche) according to the manufacturer's protocol. Hypoxanthineguanine phosphoribosyl transferase (HPRT) mRNA was used as an internal control for cell number and metabolic status. The oligonucleotide primers used for PCR were as follows: IL-12p40, 5′-TGCTGGTGTCTCCACTCATGGC (sense) and 5′-TTTCAGTGGACCAAATTCCATT (antisense); IFN-γ, 5′-AACGCTACACACTGCATCTTGG (sense) and 5′-CAAGACTTCAAAGAGTCTGAGG (antisense); IL-4, 5′-GAATGTACCAGGAGCCATATC (sense) and 5′-CTCAGTACTACGAGTAATCCA (antisense); IL-10, 5′-CGGGAAGACAATAACTG (sense) and 5′-CATTTCCGATAAGGCTTG (antisense); and HPRT, 5′-GTTGGATACAGGCCAGACTTTGTTG (sense) and 5′-GATTCAACTTGCGCTCATCTTAGGC (antisense). Forty cycles of PCR were performed with cycling conditions of 15 seconds at 95°, 20 seconds at 55°, 25 seconds at 72°. The real-time PCR signals were analysed using lightcycler 3 software. Each experiment was repeated at least three times.

H&E staining

Three hours after gene-gun administration or intramuscular injection, or 6 hr after intradermal injection, target skin or muscle was excised, fixed with 10% formalin, and embedded in paraffin. Sections (6 mm in thickness) were sliced and stained with H&E. Photographs were taken under × 400 magnification.

Statistical analysis

All quantitative data were presented as mean ± standard error of three independent experiments. Statistical significance was assessed using an unpaired Student's t-test.

Results

Cytokine mRNA profiles of draining lymph nodes after intradermal injection and the effects of CpG motif

We have previously demonstrated that physical trauma induced by intradermal injection with a certain volume of NS leads to the accumulation of TLR9-positive cells in mouse skin.18 H&E staining further showed that these TLR9-positive cells may be derived from APC recruited from peripheral blood (Fig. 1a). To investigate whether activation of these TLR9-positive cells that are induced by physical trauma with CpG motif could alter the immune response microenvironment in draining lymph nodes, cytokine profiles of IFN-γ, IL-12, IL-10 and IL-4 were studied after mice had been intradermally injected with 20 μl NS, GpC-ODN or CpG-ODN using quantitative RT-PCR analysis. As shown in Fig. 1(b), IL-12p40 mRNA was transiently detected in all three groups within 24 hr postinjection, and was significantly increased in the CpG-ODN group compared with the NS and GpC groups at 6–9 hr after injection (P < 0·05). Unlike IL-12p40, expression of IFN-γ, IL-10 and IL-4 mRNA was weakly detected and showed no differences in all three groups within 24 hr postinjection. Results suggested that physical trauma from the intradermal injection, which acted as a ‘danger signal’,14,15 could switch the cytokine microenvironment of draining lymph nodes to Th1 type, and the effect could be enhanced by CpG-ODN. These results may help to explain the mechanism of inducing a Th1-type-biased response by intradermal injection of plasmid DNA.

Figure 1.

Figure 1

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(a) H&E staining of mouse ear skin after intradermal injection of NS. Mouse ears were intradermally injected with 20 μl NS (II) or left untreated (I) for 6 hr followed by H&E staining as described in the Materials and methods section. Arrows show a cluster of inflammatory cells including neutrophils and monocytes/macrophages accumulated in the ear of the NS-treated group, but not in vehicle mice. Magnifications were × 400.(b) Cytokine profile in draining lymph nodes induced by intradermal injection of CpG-ODN. Mouse ears were intradermally injected with 20 μl NS, GpC-ODN, or CpG-ODN and relative copies of mRNA in auricular lymph nodes were determined by real-time RT-PCR after the indicated time period. Results are presented as mean ± standard error of three independent experiments.

Cytokine mRNA profiles of draining lymph nodes after intramuscular injection and the effects of CpG motif

Intramuscular injection of plasmid DNA is also known to induce a Th1-type immune response.5,6 To detect whether the CpG motif or physical trauma derived from intramuscular injection could also act as a ‘danger signal’, mice were intramuscularly injected with 100 μg pYQF-2CpG/s and 25 μg CpG-ODN in a volume of 100 μl NS, with 100 μl NS alone, or were left untreated. Three hours after injection, the target muscle was sectioned and stained with H&E. As shown in Fig. 2(a), clusters of inflammatory cells occurred in the pYQF-2CpG/s + CpG-ODN-treated group, but not in the NS-treated and control groups. The results suggested that it was the CpG motif but not the physical trauma derived from intramuscular injection that acted as a ‘danger signal’ to recruit inflammatory cells to the injection area. To detect the effect of intramuscular injection on the cytokine microenvironment of draining lymph nodes, mice were intramuscularly injected with 100 μg pYQF-2CpG/s, 25 μg GpC-ODN or 25 μg CpG-ODN in a volume of 100 μl NS, and the cytokine profiles of IFN-γ, IL-12, IL-10 and IL-4 were analysed by quantitative RT-PCR after the indicated time period. As shown in Fig. 2(b), IL-12p40 mRNA was significantly increased in the CpG-ODN-treated and plasmid groups compared with the GpC-ODN-treated group 3–12 hr after intramuscular injection but decreased to basic level in all three groups 24 hr postinjection. Similarly, IFN-γ mRNA was markedly increased only in the plasmid-treated group compared to the GpC-ODN and CpG-ODN groups. IL-10 mRNA and IL-4 mRNA showed no significant difference in the three groups.

Figure 2.

Figure 2

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(a) H&E staining of target muscle after intramuscular injection of CpG motif. Mice were intramuscularly injected with 100 μg pYQF-2CpG/s (IV), 25 μg CpG ODN (III) in a volume of 100 μl NS, 100 μl NS alone (II), or left untreated (I). Three hours after injection, the muscles were sectioned and stained with H&E. Arrows shows clusters of inflammatory cells in the pYQF-2CpG/s-treated and CpG-ODN-treated groups, but not in the NS-treated and vehicle groups. Magnifications were × 400. (b) Cytokine profile in draining lymph nodes after intramuscular injection of CpG motifs. Mice were intramuscularly injected with 100 μg pYQF-2CpG/s, 25 μg GpC-ODN, or 25 μg CpG-ODN in a volume of 100 μl NS, and the popliteal and inguinal lymph nodes were collected and pooled at each time-point. Cells separated from them were cultured and stimulated with Con A. Relative copies of mRNA of each cytokine were determined by real-time RT-PCR at the optimized time after stimulation. Results are presented as mean ± standard error of three independent experiments.

Intramuscular injection of CpG motif can act as a ‘danger signal’ to recruit inflammatory cells from blood and facilitate a more Th1-type cytokine secretion profile in draining lymph nodes.

Cytokine mRNA profiles of draining lymph nodes after gene gun bombardment of skin and the effects of CpG motif

Compared to a more Th1-type-biased response induced by intramuscular or intradermal injections of plasmid DNA with syringe and needle, a predominant Th2-type response has been observed after gene-gun-mediated DNA immunization.7,8 It has been reported that the gold-particle bombardment of the skin by a gene gun represents a type of Th2 adjuvant.15 To verify whether gold-particle bombardment of the skin by gene gun has any effect on the recruitment of inflammatory cells to the target skin, mice were challenged with gene-gun bombardment with gold particles or were left untreated. Three hours after bombardment, the abdominal skin was sectioned and stained with H&E. As shown in Fig. 3(a), clusters of inflammatory cells had invaded the skin 3 hr post-gold-particle bombardment, but were not seen in the control group. Furthermore, to analyse the cytokine mRNA profiles of draining lymph nodes after gene-gun bombardment of skin and the effects of CpG motif on the cytokine microenvironment of draining lymph nodes after gene-gun bombardment, mice were inoculated by gene-gun bombardment of 1 μg plasmid DNA coated on gold particles, 25 μg CpG-ODN coated onto gold particles, or with gold particles alone. As shown in Fig. 3(b), gene-gun bombardment of 1 μg plasmid DNA coated on gold particles or gold particles alone resulted in a Th2-type cytokine microenvironment with increased IL-4 and IL-10 mRNA expression and almost no increase in IL-12 and IFN-γ mRNA expression in draining lymph nodes. However, compared with the 1 μg plasmid DNA and gold particle group at 6–12 hr after gene-gun bombardment, the IL-12p40 mRNA was significantly increased in the CpG-ODN-treated group and the mRNA level of IL-4 and IL-10 were significantly decreased in the same conditions (P < 0·05). Thus, gene-gun bombardment with gold particles alone or 1 μg plasmid DNA led to a Th2-type response; coating 25 μg CpG-ODN onto gold particles could change the Th2-type cytokine microenvironment induced in draining lymph nodes by gene-gun bombardment to a Th1-type response.

Figure 3.

Figure 3

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(a) H&E staining of mouse abdomen skin after gold-particle bombardment. Mice were inoculated by gene-gun bombardment of gold particles (II) or were left untreated (I). Three hours after bombardment, the abdomen skin was sectioned and stained with H&E. Gold particles appear as small dark dots. Arrow shows clusters of inflammatory cells. Magnifications were × 400.(b) Cytokine profile in draining lymph nodes after gene-gun bombardment. Mice were inoculated by gene-gun bombardment of 1 μg plasmid DNA coated on gold particles, or 25 μg CpG-ODN coated onto gold particles, or gold particle alone. Inguinal and axillary–brachial lymph nodes were collected and pooled at each time-point. Cells separated from them were cultured and stimulated with Con A. Relative copies of mRNA of each cytokine were determined by real-time RT-PCR at the optimized time after stimulation. Results are presented as mean ± standard error of three independent experiments.

Discussion

DNA vaccine was regarded as one of the most promising approaches for future vaccine development in light of its capability to overcome the disadvantages in conventional vaccines (such as the capability to induce both humoral and cellular immunity) and of its stability as well as simplicity of production.19–21 Gene-gun-mediated DNA vaccination, which developed in the last decade, requires only minute quantities of DNA to induce a strong immune response, and so has great potential in its application to humans.5,22,23 However, compared to the more Th1-type biased response induced by intramuscular or intradermal injections of plasmid DNA with syringe and needle, a predominant Th2-type response has been observed after gene-gun-mediated DNA immunization.5–8 The exact mechanism for this difference is yet to be completely understood. The tendency to preferentially prime a Th2 response by gene-gun-mediated DNA immunization makes it less attractive because it is Th1 immunity, which is characterized by strong cellular immunity and moderate humoral immunity, that plays a vital role in host defence against viral, intracellular bacterial infections and tumours.24,25

Considerable efforts are now being focused on understanding the molecular mechanisms of the induction of different types of immune response by gene-gun-mediated DNA immunization or syringe-needle-injection DNA vaccination, and on developing ways of shifting the Th2-type immune response induced by gene-gun-mediated DNA immunization to a Th1-type response.7,9,26 In this study, the mechanism of Th2 immune response induction by gene-gun-mediated DNA immunization was investigated and the adjuvant roles of CpG motif in the different routes of DNA vaccine inoculation were compared.

In this study, H&E staining showed that remarkable inflammatory infiltration could be seen in skin intradermally injected with NS, in CpG-motif-injected muscle and in gold-particle bombarded skin, which suggests that the physical trauma of intradermal injection or gold-particle bombardment may provide a danger signal that may promote inflammatory cytokine or chemokine reactions in the target area of DNA vaccine inoculation. Unlike intradermal injection or gold-particle bombardment, intramuscular injection of NS did not recruit apparent inflammatory cells, while intramuscular injection of CpG motif did. We speculate that for the loose structure of muscle, intramuscular injection of NS to muscle cannot cause inflammatory cells to infiltrate.

The possible impacts of these ‘danger stimuli’ on the polarity (Th1/Th2) of the adaptive immune response were further studied by analysing the cytokine profile from draining lymph nodes. Results showed that intradermal injection of NS resulted in a Th1-type cytokine microenvironment in the draining lymph nodes, and the effect could be enhanced by intradermal injection of CpG motif. Intramuscular injection of 100 μg plasmid DNA or 25 μg CpG-ODN was able to induce a Th1-type cytokine microenvironment in draining lymph nodes. Gold-particle bombardment resulted in a Th2-type cytokine microenvironment, which was consistent with the finding of Weiss and colleagues.15 Gene-gun bombardment of skin with gold particles coated with 1 μg plasmid DNA still resulted in a Th2-type cytokine microenvironment in draining lymph nodes. However, coating 25 μg CpG-ODN onto gold particles could change the Th2-type cytokine microenvironment induced in the draining lymph nodes by gene-gun bombardment to a Th1 type. Based on the above results, it can be inferred that the bombardment of gold particles coated with plasmid DNA, which results in Th2 biased ‘danger signals’, may be the result of a small amount of CpG motif. Because of the limitation of the gold beads' capacity, it is difficult to increase the amount of plasmid DNA used in gene-gun bombardment.16 However, as we have described previously, co-delivery of 10 μg CpG-ODN with 1 μg plasmid DNA by gene gun can shift the DNA immune response from Th2 towards Th1.9

Danger signals have been considered to be a model of immunity that suggests that the immune system responds to substances that cause damage. Danger signals, such as molecules or molecular structures derived from cells undergoing stress or abnormal cell death, are perceived by resting APCs followed by activation of those APCs to initiate immune responses.27–29 In our present study, we showed that physical trauma and gold-particle bombardment can act as ‘danger signals’ causing recruitment of inflammatory cells to damaged tissues and administration of certain amounts of CpG motif could increase the Th1 immune response microenvironment in related draining lymph nodes. It could be the result of the local interaction between CpG motif and TLR9-positive cells as studies have demonstrated that some inflammatory cells, such as B cells, macrophages and dendritic cells, express TLR9 physically.30–32 In summary, CpG motif may act as a ‘danger signal’ and Th1 immune response enhancer in DNA vaccination. These results may help to explain the mechanism of different types of immune response induced by different DNA vaccination delivery routes and to facilitate the process of application of DNA vaccine into practice.

Acknowledgments

We thank Drs Ying Lin, Chen Yang, Aimin Xue and Jianying Shi for helpful discussion and technical help. This work was supported by the State Key Basic Research Program (Grant No: G1999054105) and High Technology Project (Grant No:2001AA215011) from the Ministry of Science and Technology of China.

Abbreviations

APC

antigen-processing cell

Con A

concanavalin A

H&E

haematoxylin and eosin

HPRT

hypoxanthineguanine phosphoribosyl transferase

IFN-γ

interferon-γ

IL-12

interleukin-12

NS

normal saline

ODN

oligodeoxynucleotide

RT-PCR

reverse transcription–polymerase chain reaction

SDS

sodium dodecyl sulphate

Th1

T helper type 1

TLR

Toll-like receptor

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