Research Article

Gene Therapy (2004) 11, 941–948. doi:10.1038/ Published online 26 February 2004

IL-18 gene therapy develops Th1-type immune responses in Leishmania major-infected BALB/c mice: is the effect mediated by the CpG signaling TLR9?

Y Li1, K Ishii1, H Hisaeda1, S Hamano1, M Zhang1, K Nakanishi2, T Yoshimoto2, H Hemmi3, K Takeda3, S Akira3, Y Iwakura4 and K Himeno1

  1. 1Department of Microbiology and Immunology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  2. 2Department of Immunology and Medical Zoology, Hyogo College of Medicine, Hyogo, Japan
  3. 3Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
  4. 4Center for Experimental Medicine, Institute of Medical Science, Tokyo University, Tokyo, Japan

Correspondence: Dr K Himeno, Department of Microbiology and Immunology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3, Higashi-ku, Fukuoka 812-8582, Japan

Received 22 August 2003; Accepted 13 January 2004; Published online 26 February 2004.



IL-18 regulates either Th1 or Th2 responses depending on the cytokine microenvironment. Administration of recombinant IL-18 (rIL-18) alone does not promote Th1 response, but rather induces Th2 response and exacerbates Leishmania major infection in susceptible BALB/c mice. Here, we treated BALB/c mice with an IL-18-expressing plasmid by using a gene gun weekly after L. major infection. This gene therapy resulted in improved pathogenic process and preferential induction of Th1 responses by inducing the expression of IL-12 p40, but treatment with rIL-18 did not. Notably, simultaneous administration of rIL-18 with an empty plasmid vector rendered BALB/c mice resistant to the infection, despite the fact that treatment with either rIL-18 alone or the plasmid vector alone did not influence the susceptibility. The synergistic role of the vector with rIL-18 was found to depend on CpG motifs, which enhanced expression of proinflammatory cytokines, especially IL-12, from APCs through Toll-like receptor (TLR) 9 ligation. Treatment with methylated plasmid vector in which CpG was disrupted could no longer prevent the disease development in coadministration with rIL-18. Taken together, IL-18 gene therapy was shown to develop Th1-type protective immunity in L. major-infected BALB/c mice without the requirement of exogenous IL-12, probably via CpG-TLR9 signaling pathway.


IL-18, Leishmania major, CpG, laboratory mouse



IL-18, primarily produced by macrophages and dendritic cells, was initially described as an IFN-gamma-inducing factor.1 This cytokine serves as a cofactor for IL-12-induced development of Th1 immune response and optimizes IFN-gamma production from effector Th1 cells.2 It plays a critical role in the development of protective immunity against intracellular pathogens such as Leishmania major, Mycobacterium tuberculosis, Cryptococcus neoformans, and Listeria monocytogenes.3, 4, 5, 6 However, administration of IL-18 alone reportedly results in deviation of immune responses to Th2 type in mouse models of helminth infection and allergic asthma with enhanced IgE response.7, 8 NK, T cells and basophils stimulated with IL-18 have been shown to generate Th2-type cytokines such as IL-4 and IL-13 when cultured together with IL-2 and IL-3, respectively.7, 9

BALB/c mice are susceptible to infection with L. major, an intracellular protozoan parasite, with a Th2-type immune response resulting in progressive infection with a fatal outcome.10 According to combined treatment with recombinant IL-18 and IL-12, however, those mice acquire potent protective immunity to ameliorate the infection by inducing and activating Th1-type response. In contrast, treatment with rIL-18 alone does not induce a Th1 response but rather exacerbates the disease by skewing the immune responses toward a Th2 type.3, 11 IL-18-/- mice with a genetically resistant C57BL/6 background are relatively susceptible to infection with L. major, although the disease is finally controlled.12 Thus, IL-18 has been supposed to contribute to the host defense synergistically with IL-12 by accelerating the development of Th1 response through augmentation of IFN-gamma production, but not always to be essential for development of Th1-type protective immunity.

In the present study, we found that gene therapy of susceptible BALB/c mice with an IL-18-expressing plasmid alone using a gene gun directs their immune responses to a Th1 type and prevents development of the disease, at a similar level as that induced by the IL-12 gene against L. major infection. This result is in clear contrast to that obtained using rIL-18 alone. We also investigated the molecular mechanisms underlying the difference between protective potentials of recombinant and plasmid encoded IL-18. The cytokine gene appears to play an important role in enhancement of IL-12 mRNA expression resulting in improving pathogenic process with support of the signaling pathway, especially through TLR9 activated by CpG motifs, which are included in the plasmid vector used in the gene therapy.



Effect of IL-18 gene therapy in L. major-infected BALB/c mice

Previous studies demonstrate that rIL-18 cannot protect BALB/c mice against L. major infection.3 In the present study, experiments were carried out to determine whether treatment of BALB/c mice with an IL-18-expressing plasmid, pCAGGS-IL-18 (pIL-18) (Figure 1a), exerts a therapeutic effect on L. major infection. As shown in Figure 1b, footpad swelling was significantly suppressed in pIL-18-treated mice compared with that in the untreated BALB/c mice (P<0.05 at 2–6 weeks after infection). The lesion size was similar to that in mice delivered with pIL-12 and, surprisingly, also similar to that in mice treated with pIL-12 plus pIL-18. No therapeutic effect was observed in mice that received a control plasmid vector alone. We also examined the parasite burden at 6 weeks after infection. Consistent with the results of footpad swelling, pIL-18-treated mice showed a markedly lower parasite burden in draining lymph nodes (LNs) than did control BALB/c mice (Figure 1c). These results indicate that gene therapy of mice with the IL-18-expressing plasmid induces protective immunity against L. major to a degree similar to that induced by IL-12 gene therapy.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Effect of IL-18 gene therapy on L. major infection in BALB/c mice. (a) Construction of an IL-18-expressing plasmid named pCAGGS IL-18 (pIL-18). The plasmid was designed so that IL-18 is secreted using an INF-beta signal sequence under the control of CMV enhancer/chicken beta-actin promoter. (b) Kinetics of footpad swelling in BALB/c mice treated with pIL-18. After infection of BALB/c mice with 5 times 106 promastigotes, the degree of footpad swelling was measured weekly. Data represent the average plusminuss.d. of seven mice in each group. (c) Parasite burden in draining LNs of treated mice. Six weeks after infection, parasite burden in popliteal LNs was determined. Data represent average plusminuss.d. of triplicate cultures. Asterisks indicate statistical significance compared with values in a group of mice treated with an empty plasmid (P<0.05). (d), Cytokine production by CD4+ T cells in draining LNs. Three weeks after infection, CD4+ T cells purified from popliteal LNs of mice treated with pIL-18 were cultured with L. major antigen, and culture supernatants were assayed for cytokine concentration by ELISA. Results are expressed as the meanplusminuss.d. of three individual mice per group. Asterisks indicate statistical significance compared with values in a group of mice treated with an empty plasmid (P<0.05).

Full figure and legend (202K)

To confirm whether resistance acquired by IL-18 gene therapy is mediated by the induction of a Th1 response, cytokine profiles were measured at 3 weeks after infection (Figure 1d). Production of IFN-gamma and IL-4 by CD4+ T cells from draining LNs was assessed after in vitro stimulation with L. major antigen. CD4+ T cells from mice treated with pIL-18 generated a significantly greater amount of L. major-specific IFN-gamma than did those from mice treated with a control plasmid, which suffered from progressive disease (P<0.05). The amount of IL-4 produced was less in mice treated with pIL-18 than that in mice treated with control plasmid, although rIL-18 is known to promote IL-4 production.7, 8 Similar results were also obtained at the mRNA level (data not shown). Thus, the IL-18 gene therapy appears to skew immune responses in a Th1 direction following L. major infection in BALB/c mice.

Requirement of IFN-bold gamma and TNF-alpha from Th1-type CD4+ T cells for protection conferred by pIL-18

To elucidate the mechanism by which IL-18 gene therapy confers protection against infection, mice were administered neutralizing antibodies specific for each cell type or cytokine after infection. As shown in Figure 2a, mice pretreated with either anti-CD4 or anti-IFNgamma mAb failed to acquire protective immunity by IL-18 gene therapy, and those mice showed greater footpad swelling than did PBS-administered mice (P<0.05 at 3–6 weeks after infection). Cytotoxic activity and IFN-gamma production by NK cells were also significantly increased in pIL-18-treated mice (data not shown), a finding that is consistent with previously reported results.13 However, the protective potential was not cancelled in mice depleted of NK cells by anti-asialo GM1 Ab, suggesting that NK cells are not the main effectors for the protection acquired by IL-18 gene therapy.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Requirement of IFN-gamma and TNF-alpha from Th1-type CD4+ T cells for protection conferred by pIL-18. (a) Footpad swelling in infected BALB/c mice treated with pIL-18. Mice were depleted/neutralized of CD4+ T, NK cells or IFN-gamma prior to infection with 5 times 106 promastigotes. Data represent the average plusminuss.d. of 7 mice. (b, c) Susceptibility of TNF-alpha-deficient mice (TKO) treated with pIL-18. Wild-type BALB/c (WT) or TKO mice (BALB/c background) were infected with 105 promastigotes, and treated with the indicated vectors. Footpad swelling was monitored (b), and production of IFN-gamma specific for L. major antigen was analyzed (c). Details are the same as those given in the legends to Figure 1b and d.

Full figure and legend (182K)

In addition to the increased IFN-gamma production, mice treated with pIL-18 also showed a markedly elevated level of TNF-alpha at 3 weeks after infection (Figure 1d). TNF-alpha is one of the main cytokines involved in production of nitric oxide (NO),14 and we therefore tried to clarify the significance of this cytokine in pIL-18-induced protection. Following IL-18 gene therapy, TNF-alpha-deficient BALB/c mice generated remarkably high levels of IFN-gamma despite the fact that those mice acquired little protection against the infection (Figure 2b and c). Thus, TNF-alpha as well as IFN-gamma is required for protection induced by IL-18 gene therapy.

Essential role of endogenous IL-12 in protection conferred by pIL-18

IL-12 plays a critical role in IFN-gamma production synergistically with IL-18.2.As shown in Figure 3a, quantitative real-time PCR revealed that IL-18 gene therapy increased the level of IL-12 p40 mRNA expression in L. major-infected BALB/c mice. The role of IL-12 was investigated by using IL-12 p40-deficient mice. The KO mice were infected with L. major and treated with pIL-18. All of the KO mice showed susceptible phenotypes (Figure 3b). IFN-gamma secretion in popliteal LN cells of the KO mice was profoundly impaired compared with that in popliteal LN cells of wild-type (WT) mice despite the fact that IFN-gamma production was about 5-fold higher in IL-12 p40-deficient mice treated with pIL-18 than in mice treated with the control plasmid vector (Figure 3c). Quantitative PCR analysis of cytokine mRNA showed similar results (data not shown). These results demonstrated that endogenous IL-12 is necessary for the IL-18 gene therapy, although an alternative IL-12-independent pathway seems to exist in IFN-gamma production.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Essential role of endogenous IL-12 in protection conferred by pIL-18. (a) Level of IL-12 p40 mRNA in mice treated with pIL-18. Two weeks after infection, APCs were separated as plastic-adherent cells from popliteal LNs of BALB/c mice treated with pIL-18, and were analyzed for IL-12 p40 mRNA expression by quantitative real-time PCR. Data are expressed as ratios of each group to the untreated group. (b, c) Wild-type (WT) BALB/c or B6, or IL-12 p40-deficient (12KO)(B6 background) mice were infected with 105 promastigotes and treated with pIL-18. Footpad swelling was monitored (b), and production of IFN-gamma specific for L. major antigen was analyzed (c). Details are the same as those given in the legends to Figure 1b and d.

Full figure and legend (152K)

Contribution of the plasmid backbone to induction of resistance to L. major infection by pIL-18

It is well known that treatment of L. major-infected mice with rIL-18 alone does not lead to control of the infection but, rather, exacerbates the disease.3, 11 As shown in the present study, however, treatment of mice with a plasmid encoding IL-18 gene is markedly protective. We hypothesized that the difference in protective potentials of pIL-18 and rIL-18 is attributed to the plasmid vector contained in pIL-18, since the vector alone induced production of a significant amount of IL-12 after infection with L. major (Figure 3a). To confirm this possibility, we injected rIL-18 into L. major-infected BALB/c mice over a period of 7 days with or without delivery of an empty plasmid vector. Footpad lesions in mice treated with rIL-18 progressed more than did those in untreated BALB/c mice. In contrast, mice treated with rIL-18 plus the plasmid vector acquired a similar degree of resistance as that of mice treated with pIL-18 (Figure 4a). Parasite burden in draining LNs was also significantly decreased by this combined treatment (Figure 4b). Administration of mice with rIL-18 augmented IL-4 production and did not influence IFN-gamma production. Notably, addition of the plasmid vector reversed the Th2 cytokine profile to Th1 (low IL-4 and high IFN-gamma) (Figure 4c). However, the treatment with pIL-18 appears to have more advantage than that with rIL-18 plus vector. That is, a weekly delivery system was enough in the former, while a daily treatment system was required in the latter. It is probably due to the low dose but long duration of cytokine secretion caused by the cytokine gene in vivo.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Contribution of the plasmid backbone to induction of resistance to L. major infection by pIL-18. After infection of BALB/c mice with 106 promastigotes to the hind footpad, the mice were treated with rIL-18 daily for the first 7 days and/or an empty vector pCAGGS weekly from the day of infection (n=7/group). Severity of infection was assessed by monitoring footpad swelling weekly (a) and parasite burden at 6 weeks after infection (b). Cytokine production was analyzed as described in the legend to Figure 1d (c).

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Induction of IL-12 through TLR9 ligation with CpG motifs in the plasmid vector

Thus, resistance conferred by pIL-18 was dependent on both exogenous IL-18 and endogenous IL-12 induced by the plasmid structure. We next analyzed the molecular mechanisms of IL-12 induction. Quantitative PCR analyses again revealed increased levels of IL-12 p40 mRNA expression in the APCs of susceptible BALB/c mice delivered with pIL-18 or an empty vector. However, rIL-18 could not induce IL-12 (Figure 5a). The most likely candidate is CpG motifs because the plasmid used in this study had the structure that stimulates APCs to produce IL-12. Since CpG DNA transduces signals through TLR9,15 we examined the IL-12-inducing ability of CpG motifs in our gene gun-delivery system by using TLR9-/- mice. Further, TLR4-/- mice were employed as the reference since previous study reported that TLR4-/- mice responded normally to CpG.16 PIL-18 was delivered to these KO mice or wild-type C57BL/6 mice, and IL-12 p40 mRNA expression in APCs was examined. Two- to three-fold increased level of IL-12 was detected in WT and TLR4-/- mice but not in TLR9-/- mice (Figure 5b). A similar result was also obtained in mice delivered with an empty plasmid vector (data not shown). Thus, CpG motifs included in pIL-18 appear to play an essential role in induction of a Th1 response in L. major-infected BALB/c mice.

Figure 5.
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Induction of IL-12 through TLR9 ligation with CpG motifs in the plasmid vector. BALB/c mice (a) were treated with rIL-18, an empty vector, or pIL-18. WT of B6, TLR4-/- and TLR9-/- mice (B6 background) (b) were delivered with pIL-18 weekly. Splenic APCs were separated on 17 days after the first treatment, and quantitative real-time PCR was carried out to determine IL-12 p40 mRNA expression. Data are expressed as ratios of each treatment group to the naive WT or KO mice, respectively.

Full figure and legend (69K)

Confirmation of CpG motif contribution to induction of protective immunity

Immunostimulatory activity of bacterial DNA is attributed to the presence of unmethylated CpG motifs, and methylated CpG motifs show an inhibitory effect on immune activation induced by unmethylated CpG.17 Then, we confirmed the function of CpG directly by using methylated plasmid containing CpG motifs. Treatment with rIL-18 plus unmethylated vector, again, improved pathogenic process as evaluated by either footpad swelling or parasite burden in the draining LNs (Figure 6a and b), whereas, in treatment with methylated vector the effect was lost (Figure 6a and b), and the Th1-type cytokine profile was reversed to Th2 type (Figure 6c).

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Confirmation of contribution of CpG motifs to protective immunity. Plasmid vector of pCAGGS was methylated by CpG methylase. BALB/c mice were infected with 106 L. major promastigotes in hind footpad and treated with methylated (mvector) or unmethylated (vector) plasmid vector weekly by using a gene gun in conjunction with a daily injection of rIL-18 for the first 7 days from the day of infection. Data show the footpad swelling (a) and parasite burden at 6 weeks after infection (b). Cytokine production by CD4+ T cells in draining LNs was analyzed at 3 weeks after infection (c). Details are the same as those given in the legends to Figure 1b–d. Asterisks indicate statistical significance compared with values in a group of mice treated with plasmid vector alone (P<0.05).

Full figure and legend (116K)



In murine leishmaniasis models, treatment of genetically susceptible BALB/c mice with rIL-18 is reportedly therapeutic only when rIL-12 is coadministrated, while treatment with rIL-18 alone exacerbates the disease and enhances Th2 responses.3 Interestingly, we found that gene therapy using an IL-18-expressing plasmid alone could render susceptible BALB/c mice resistant to the infection without the requirement of immunomodulating reagents such as IL-12. The efficacy of this therapy depended on the immune stimulatory function triggered by the CpG motifs in plasmid backbone, which contribute to the promotion of expressions of proinflammatory cytokines, especially IL-12, probably via a TLR9-mediated signaling pathway. The present study is the first report that showed the applicability of IL-18 gene alone in the trial of cytokine gene therapy to intracellular protozoan infections.

IL-18 and IL-12 synergistically exert their IFN-gamma-inducing activities in T cells and NK cells. At first, IL-12 induces IL-18Ralpha expression on naive T cells and, in turn, IL-18 subsequently upregulates IL-12Rbeta2.2 Then, IL-12 and IL-18 activate STAT4 and activator protein-1 (AP-1), respectively, resulting in enhanced transcription of IFN-gamma mRNA.18 On the other hand, treatment of mice with rIL-18 alone induces CD40L expression and production of IL-4 and IgE to promote Th2 responses.2, 7, 8 NKT cells might be the major source to secrete IL-4 in response to IL-18 administration as we reported recently.19 We show here that gene therapy with pIL-18 alone is enough to induce Th1-type protective immunity similar to that with IL-12 gene in BALB/c mice infected with L. major (Figure 1), suggesting that an additional factor(s) in the plasmid vector of pIL-18 may promote Th1-dependent protective immunity during L. major infection synergistically with IL-18 in vivo. Notably, delivery of pIL-18 augmented the expression of IL-12 p40 mRNA, but rIL-18 administration did not (Figures 3a and 5a). To confirm the role of IL-12, IL-12 p40-deficient mice were employed (Figure 3). Our results demonstrate that endogenous IL-12 is essential for the development of Th1-type protective immunity initiated by pIL-18.

Next, we addressed the issue of how pIL-18 induces endogenous IL-12. CpG motifs contained in the plasmid vector appear to play a key role in the difference between the therapeutic efficacies with pIL-18 and rIL-18 since CpG motifs have a capacity to elicit IL-12 production from APC via TLR9. CpG motifs are bacterial DNA sequences containing repeats of unmethylated C and G, which is a pathogen-associated molecular pattern recognized by TLR9.20 The recognition of CpG motifs by TLR9 induces activation of signaling cascades such as c-Jun N-terminal kinase (JNK) and NF-kappaB. Expression of TLR9 is essential for CpG to induce NF-kappaB, which finally activates the IL-12 p40 promoter.20, 21, 22 Notably, methylated CpG motifs lose the ability in immune stimulation.20 We confirmed the necessity of the CpG-TLR9 pathway by using TLR9-/- mice (Figure 5) and the methylated plasmid (Figure 6).

Adjuvant activity of CpG DNA has become well established in vaccination with various antigenic proteins and in DNA vaccination.23, 24, 25 A large amount of CpG-ODN is known to confer protection against L. major infection with promotion of IL-12 production and expression of IL-12Rbeta2-chains, even in the absence of a specific antigen.26 Elimination of CpG motifs from plasmid markedly reduces the efficiency of intramuscular DNA vaccination.27 In our gene gun-mediated delivery system, administration of the empty plasmid vector alone did not result in amelioration of the disease, probably owing to the small quantity of CpG DNA delivered by a gene gun. That is, the quantity of DNA administered by the ballistic route is about 100-fold smaller than that in the case of intramuscular injection.28 Interestingly, however, the small amount of CpG motifs was sufficient to trigger Th1 responses when rIL-18 was coadministered.

The amount of TNF-alpha was elevated in mice treated with pIL-18 (Figure 1d). IL-18 induces TNF-alpha production through the activation of NF-kappaB.29 Neutralization of IL-18 results in a decrease of TNF-alpha production in lethal endotoxemia as well as in the cornea infected with Pseudomonas aeruginosa.29, 30 Administration of TNF-alpha at a therapeutic dose leads to an amelioration of symptoms in susceptible mice, whereas resistant C57BL/6 mice lacking TNF rapidly succumb to progressive leishmaniasis.31, 32 TNF-alpha-/- BALB/c mice showed high susceptibility to L. major infection even when a substantial amount of IFN-gamma was generated in those KO mice treated with pIL-18 (Figure 2). Thus, TNF-alpha, in addition to IFN-gamma, was shown to be necessary for the development of protective immunity against L. major infection.

In conclusion, the present study demonstrated that gene therapy with an IL-18-expressing plasmid alone improves pathogenic process in L. major-infected BALB/c mice without the requirement of exogenous IL-12, ameliorating disease-progressive Th2 responses to protective Th1 responses. CpG motifs included in the delivered plasmids appear to play a key role in developing Th1 responses through the production of proinflammatory cytokines, especially IL-12, at the APC level via TLR9 ligation.


Materials and methods

Animals and parasites

Studies using mice were performed in accordance with the institutional guidelines of Kyushu University. Female BALB/cN Sea (BALB/c), DBA/2N Sea (DBA/2) and C57BL/6N Sea (B6) mice were purchased from Seac Yoshitomi (Fukuoka, Japan). TNF-alpha-/- mice were generated as described previously33 and backcrossed onto the BALB/c background. IL-12 p40-deficient mice on the C57BL/6 background were kindly provided by Dr J Magram (Hoffmann-LaRoche, Nutley, USA). Generation of TLR4-/- and TLR9-/- mice has been described previously.15, 34 All mice were housed under specific pathogen-free conditions. Sex- and age-matched (8–12 week old) mice were used in all experiments.

L. major (MHOM/SU/73-5-ASKH) was maintained by serial passages in the footpads of BALB/c mice. Promastigotes harvested from stationary-phase cultures were inoculated into the left hind footpad of each mouse.


Mouse anti-CD4 (GK1.5). and anti-IFN-gamma (R4-6A2) mAbs were purified from ascites. Anti-asialo GM-1 polyclonal serum was purchased from Wako Chemicals (Osaka, Japan). Mouse rIL-18 was obtained from Medical & Biological Laboratories (Nagoya, Japan). ELISA Development Kits for IFN-gamma and IL-4 were purchased from Genzyme Techne (Minneapolis, MN, USA). FITC-conjugated anti-CD4 antibody was purchased from BD Pharmingen (San Diego, CA,USA). Anti-FITC Microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). CpG methylase (M. SssI) was obtained from New England Biolabs (Beverly, MA, USA).

Cytokine-expressing plasmids

An IL-18-expressing plasmid, designated pCAGGS-IL-18, was constructed by inserting the mouse IFN-beta signal sequence and mature mouse IL-18 cDNA (a gift from Dr I Hara, Kobe University, Japan)35 into the unique EcoRI site between the CAG promoter and a 3'-flanking sequence of the rabbit beta-globin gene of the pCAGGS expression vector (a gift from Dr J Miyazaki, Osaka University, Japan) (Figure 1a). An IL-12-expressing plasmid, designated pCAGGS-IL-12, was constructed as described previously.36 The plasmids were replicated in Escherichia coli DH5alpha and purified using the QIAGEN Plasmid Kit Maxi or Mega (QIAGEN, Chatsworth, CA, USA).

Treatment protocol

A Helios Gene Gun (Bio-Rad, Hercules, CA, USA) was used for gene therapy as described previously.36 Briefly, plasmid DNA was precipitated onto gold particles 1.6 mum in diameter and coated onto the inner face of the tubing by a tube loader. The final tubing segment resulted in delivery of 0.125 mg of gold particles and 2 mug of plasmid DNA. Administration of two nonoverlapping shots (total 4 mug of DNA) per mouse into freshly shaven abdominal skin at a helium pressure of 300 psi was started on day 0 after the infection and repeated weekly. In some experiments, in order to deplete CD4+ T cells and NK cells, 0.5 mg of anti-CD4 mAb or 100 mug of anti-asialo GM-1 polyclonal serum was injected intraperitoneally (i.p.) into the mice on days -1, 3, 7, 10, 14, 21 and 28 after infection. Over 95% of the cells of each corresponding subset was invariably found to be deleted. Anti-IFNgamma mAb was administered with the same schedule. In some experiments, mice were injected intraperitoneally each day with 1 mug of rIL-18 for the first 7 days after L. major infection with or without delivery of the plasmid vector.

Determination of lesion size and parasite burden

Footpad lesion size was measured weekly with a dial caliper and expressed as the difference in thickness between the infected footpad and uninfected contralateral footpad. To determine the number of viable L. major parasites in the tissues of infected mice, draining popliteal lymph nodes (LNs) were obtained from mice 6 weeks after infection. The LNs were homogenized by slide glass. Then, cells were washed twice with Medium 199, plated at a concentration of 106 per 2 ml Medium 199 containing 10% FBS in 12-well tissue plates and cultured at 25°C. The number of parasites was counted using a microscope 7 days later. Parasite burden was expressed as the number of parasites per LN.

Cytokine production

Draining LNs were obtained from mice of each group at 3 or 6 weeks after infection. The nodes from a minimum of three mice in each group were pooled and then purified into CD4+ T-cell subpopulations using Magnetic Separation Columns (Miltenyi Biotec, Germany). Flow cytometry confirmed > 95% purity of CD4+ T lymphocytes. The cells were then plated in a 96-well microtiter plate at 2.5 times 105 cells per 200 mul RPMI1640 supplemented with 10% FBS, with or without L. major antigen obtained from freeze-thawed promastigotes, in the presence of irradiated (30 Gy) splenocytes. Culture supernatants were harvested 72 h later, and cytokine concentration was determined by sandwich ELISA.

Analysis of mRNA expression

APCs were collected from spleen or LN as plastic-adherent cells as described.37.Mainly macrophages were separated in this case. Total RNA was prepared from these cells and was reverse-transcribed with SuperScript II (Takara Biomedicals, Tokyo, Japan). Primer sequences for IFN-gamma and IL-4 have been described previously.38 The primer sequences for IL-12 p40 are 5'-AAC CTC ACC TGT ACA CGC C-3' (sense) and 5'-CAA GTC CAT GTT TCT TTG CAC G-3' (antisense). Quantitative real-time PCR was performed using an ABI Prism 7700 Sequence Detector System (P.E. Biosystems, Warrington, GB). Results are expressed as n-fold difference relative to the expression of beta-actin.

DNA methylation

Full CpG methylation of plasmid was performed with SssI methylase39 according to the manufacturer (New England Biolabs). Briefly, methylation of every 100 mug of plasmid DNA was conducted by incubation with 30 U SssI in 500 mul buffer, 640 muM S-adenosylmethionine, for 2 h at 37°C. Complete methylation was verified by extensive digestion with the methylation sensitive restriction enzyme BstUI.

Statistical analysis

Data are expressed as meanplusminuss.d. Differences between experimental groups in each experiment were analyzed by using Student's t-test and were considered significant if P<0.05.



  1. Okamura H et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 1995; 378: 88–91. | Article | PubMed | ISI | ChemPort |
  2. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol 2001; 19: 423–474. | Article | PubMed | ISI | ChemPort |
  3. Ohkusu K et al. Potentiality of interleukin-18 as a useful reagent for the treatment and prevention of Leishmania major infection. Infect Immunity 2000; 68: 2449–2456. | Article |
  4. Kinjo Y et al. Contribution of IL-18 to Th1 response and host defense against infection by Mycobacterium tuberculosis: a comparative study with IL-12p40. J Immunol 2002; 169: 323–329. | PubMed | ISI | ChemPort |
  5. Kawakami K et al. IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells. J Immunol 2000; 165: 941–947. | PubMed | ChemPort |
  6. Neighbors M et al. A critical role of interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on interferon gamma production. J Exp Med 2001; 194: 343–354. | Article | PubMed | ISI | ChemPort |
  7. Yoshimoto T et al. IL-18, although anti-allergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA 1999; 96: 13962–13966. | Article | PubMed | ChemPort |
  8. Wild JS et al. IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma. J Immunol 2000; 164: 2701–2710. | PubMed | ISI | ChemPort |
  9. Hoshino T, Wiltrout RH, Young HA. IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response. J Immunol 1999; 162: 5070–5077. | PubMed | ISI | ChemPort |
  10. Reiner S, Locksley RM. The regulation of immunity to Leishmania major. Annu Rev Immunol 1995; 13: 151–177. | Article | PubMed | ISI | ChemPort |
  11. Xu D et al. IL-18 induces the differentiation of Th1 or Th2 cells depending upon cytokine milieu and genetic background. Eur J Immunol 2000; 30: 3147–3156. | Article | PubMed | ISI | ChemPort |
  12. Monteforte GM et al. Genetically resistant mice lacking IL-18 gene develop Th1 response and control cutaneous Leishmania major infection. J Immunol 2000; 164: 5890–5893. | PubMed | ISI | ChemPort |
  13. Takeda K et al. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 1998; 8: 383–390. | Article | PubMed | ISI | ChemPort |
  14. Liew FY, Li Y, Millott S. Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide. J Immunol 1990; 145: 4306–4310. | PubMed | ChemPort |
  15. Hemmi H et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740–745. | Article | PubMed | ISI | ChemPort |
  16. Hacker H et al. Immune cell activation by bacterial CPG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF) 6. J Exp Med 2000; 192: 595–600. | Article | PubMed | ISI | ChemPort |
  17. Chen Y et al. Identification of methylated CpG motifs as inhibitors of the immune stimulatory CpG motifs. Gene Therapy 2001; 8: 1024–1032. | Article | PubMed |
  18. Nakahira M et al. Synergy of IL-12 and IL-18 for IFN-gamma gene expression: IL-12-induced STAT 4 contributes to IFN-gamma promoter activation by up-regulation of the binding activity of IL-18-induced activator protein 1. J Immunol 2002; 168: 1146–1153. | PubMed | ISI | ChemPort |
  19. Yoshimoto T et al. Nonredundant roles for CD1d-restricted natural killer T cells and conventional CD4+ T cells in the induction of immunoglobulin E antibodies in response to interleukin-18 treatment of mice. J Exp Med 2003; 197: 997–1005. | Article | PubMed |
  20. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20: 709–760. | Article | PubMed | ISI | ChemPort |
  21. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001; 2: 675–680. | Article | PubMed | ISI | ChemPort |
  22. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003; 21: 335–376. | Article | PubMed | ISI | ChemPort |
  23. Klinman DM, Yamshchikov G, Ishigatsubo Y. Contribution of CpG motifs to the immunogenicity of DNA vaccines. J Immunol 1997; 158: 3635–3639. | PubMed | ISI | ChemPort |
  24. Krieg AM, Davis HL. Enhancing vaccines with immune stimulatory CpG DNA. Curr Opin Mol Ther 2001; 3: 15–24. | PubMed | ISI | ChemPort |
  25. Rhee EG et al. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med 2002; 195: 1565–1573. | Article | PubMed | ISI | ChemPort |
  26. Zimmermann S et al. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J Immunol 1998; 160: 3627–3630. | PubMed | ISI | ChemPort |
  27. Sato Y et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996; 273: 352–354. | Article | PubMed | ISI | ChemPort |
  28. Fensterle J, Grode L, Hess J, Kaufmann SHE. Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun. J Immunol 1999; 163: 4510–4518. | PubMed | ISI | ChemPort |
  29. Netea MG et al. Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia. J Immunol 2000; 164: 2644–2649. | PubMed | ISI | ChemPort |
  30. Huang X, McClellan SA, Barrett RP, Hazlett LD. IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production. J Immunol 2002; 168: 5756–5763. | PubMed |
  31. Titus RG, Sherry B, Cerami A. Tumor necrosis factor plays a protective role in experimental murine cutaneous leishmaniasis. J Exp Med 1989; 170: 2097–2104. | PubMed |
  32. Wilhelm P et al. Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF. J Immunol 2001; 166: 4012–4019. | PubMed | ISI | ChemPort |
  33. Tagawa Y, Sekikawa K, Iwakura Y. Suppression of concanavalin A-induced hepatitis in IFN-gamma-/- mice, but not in TNF-alpha-/- mice. J Immunol 1997; 159: 1418–1428. | PubMed | ISI | ChemPort |
  34. Hoshino K et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999; 162: 3749–3752. | PubMed | ISI | ChemPort |
  35. Nagai H et al. Gene transfer of secreted-type modified interleukin-18 gene to B16F10 melanoma cells suppresses in vivo tumor growth through inhibition of tumor vessel formation. J Invest Dermatol 2002; 119: 541–548. | Article | PubMed | ISI | ChemPort |
  36. Sakai T et al. Gene gun-mediated delivery of an interleukin-12 expression plasmid protects against infection with the intracellular protozoan parasite Leishmania major and Trypanosoma cruzi in mice. Immunology 2000; 99: 615–624. | Article | PubMed | ChemPort |
  37. Hamano S et al. Role of macrophages in acute murine cytomegalovirus infection. Microbiol Immunol 1998; 42: 607–616. | PubMed |
  38. Yoshida H et al. WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection. Immunity 2001; 15: 569–578. | Article | PubMed | ISI | ChemPort |
  39. Renbaum P et al. Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI). Nucleic Acids Res 1990; 18: 1145–1152. | PubMed | ISI | ChemPort |


We thank Drs J Miyazaki and I Hara for kindly providing the pCAGGS vector and mouse IL-18 cDNA and Dr J Magram for providing IL-12 p40-deficient mice. We also thank Drs Y Yoshikai and H Yoshida for their constructive discussions regarding the manuscript. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15019075, 15025255, 15390136, 15659265).