An immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif protects senescence-accelerated mice from lethal influenza virus by augmenting the T helper type 1 response

Age-related deterioration of immune functions is designated immunosenescence. The traits and mechanisms of immunosenescence are multifactorial, especially being related to reduction in or alteration of lymphocyte functions (Pawelec et al., 1995; Miller, 1996). Dysfunction of T-cell repertoires appears to play a critical part in the process of age-related immune deficiency in both humoral and cellular responses. It is well documented that dysregulation of the T helper type 1 (Th1)/Th2 balance in immunosenescence increases the incidence of infectious diseases, autoimmune disorders and cancers, and raises morbidity and mortality in the geriatric population (Rink et al., 1998). In the case of virus infection, human peripheral blood mononuclear cells stimulated with influenza virus antigen in vitro tend to show an age-associated decrease in type 1 cytokine production (Gardner & Murasko, 2002). The role of the Th1 immune response definitely differs from that of the Th2 response: cellular immunity, including cytotoxic T-lymphocytes (CTLs) and Th1 inflammatory cytokines, contributes to the protection of the host from infectious agents (Ramshaw et al., 1997), while IL-4, which is typical of Th2 cytokines, causes severe and prolonged infection (Moran et al., 1996). Therefore, the crucial point in prevention and cure of infectious virus diseases in the elderly relies on the amelioration of immunosenescence (Minutello et al., 1999).

Recently, an oligodeoxynucleotide (ODN) containing unmethylated cytidine-guanosine (CpG) motifs has been demonstrated to be an excellent immune stimulator to induce a strong Th1 immune response (Chu et al., 1997; Davis, 2000). It is compelling to investigate whether the CpG ODN is capable of improving immunosenescence in the elderly through energizing the Th1 response. In the present study, we adopted a newly developed senescence-accelerated mouse (SAM) system (Takeda et al., 1997a). The senescence-prone strain of SAM-P1 mice shows various signs of rapid ageing, such as a shortened lifespan of about half that of ordinary control mice, wrinkled skin and age-dependent geriatric disorders (Takeda et al., 1997b). The storage and function of CD4⁺ T-cells of SAM-P1 mice are altered, displaying the Th2-biased status of immune responses and the consequently increased susceptibility to influenza virus infection (Dong et al., 2000). Prolonged infection with influenza virus in immunocompromised, aged mice has also been observed (Bender et al., 1991). Herein, we show that treatment of SAM-P1 mice with CpG ODN repairs age-associated immune dysfunction and thereby the mice are saved from lethal influenza virus infection.

Experimental infection of animals.
The senescence-prone SAM-P1 strain of mice was obtained from the Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan. Specific-pathogen-free mice of this strain were established and maintained in the Laboratory Animal Centre of Fukui Medical University, Fukui, Japan. Mice had fresh water and autoclaved food and were kept at 23 °C under bioclean conditions throughout all experiments. SAM-P1 mice (3-month-old) showing genetically stable impairment of Th1-associated immune functions (Dong et al., 2000) were adopted for all experiments. Mice were anaesthetized mildly by intraperitoneal administration of pentobarbital solution (0·025 mg g^-1 body weight) and inoculated in the right nostril with the mouse-adapted strain of human influenza A/PR/8/34 (H1N1) virus in 20 µl sterile PBS. Virus growth in the lung was assayed by plaque titration on MDCK cell monolayers, as described elsewhere (Mori et al., 1995). This work was approved by the Committee of Institutional Animal Care and Use in Fukui Medical University.

CpG ODN.
Nuclease-resistant phosphorothioate-modified CpG ODNs, such as ODN 1826, ODN IL-12p40, control non-CpG ODN (Hokkaido System Science), ODN g10 and ODN CG-30 (Nisshinbo), were dissolved in endotoxin/pyrogen-free PBS. Sequence data of these ODNs are listed in Table 1 (CpG motifs are underlined).

Table 1. Cytokine production by SAM-P1 mouse spleen cells stimulated in vitro with CpG ODN and concanavalin A SAM-P1 spleen cells (3·0x106) were incubated in medium containing 1 µM ODN or 1 µg concanavalin A ml-1 for 24 h. Each cytokine in the culture supernatant was assayed by ELISA. Data are the mean±SD of results for each group of five mice tested.

Cytokine assay.
For induction of cytokines in vitro, spleen cells were infected with influenza virus at an input m.o.i. of 1 p.f.u. per cell and then cultured in medium containing 1 µM ODN. The amounts of cytokines such as IL-12, IL-4, IFN-γ and TNF-α were assayed using a mouse cytokine detection ELISA kit (BioSource), in accordance with the manufacturer's instructions.

Antibody assay.
Virus-specific immunoglobulins (Igs) were measured with an ELISA Ig Quantitative kit (Bethyl Laboratories). Briefly, microtitre plates were coated with 20 µg potassium tartrate-purified influenza virus at 4 °C overnight. After blocking with 1 % BSA for 30 min, serum samples were added into the well and incubated for 1 h. Bound antibodies were reacted with goat horseradish peroxidase-labelled anti-mouse IgG, IgG1, IgG2a or IgA. Plates were read at 490 nm after the addition of O-phenylenediamine. Antibody titres were calculated through a standard curve made from the reference serum using the calculation software SPECTRA MAX 250 (Molecular Devices).

Assay of CTL and natural killer (NK) cell activities.
Spleen cells were obtained from mice that had been primed with influenza virus 3 weeks before. Lymphocytes were collected through density-gradient centrifugation with lymphocyte-separation solution (Antibody Institute) and re-stimulated in vitro by co-cultivation for 5 days with syngeneic spleen cells that had been infected with influenza virus 1 h before. Target cells were prepared using mouse L929 cells infected with influenza virus at an input m.o.i. of 10 p.f.u. per cell. Effector lymphocytes and target cells were mixed and incubated for 6·5 h at 37 °C. Specific lysis of target cells was determined by lactate dehydrogenase-release assay (Decker & Lohmann-Matthes, 1988) using a Cytotoxicity Detection kit (Roche). Data were expressed as the percentage of specific release: 100x[(target with effector -effector spontaneous) - target spontaneous]/(target maximum -target spontaneous). NK cell activity was measured by specific lysis of Yac-1 cells.

Flow cytometry analysis.
B-cells were doubly stained using the FITC-labelled monoclonal antibody (mAb) for CD19 (Beckman Coulter) and the phycoerythrin (PE)-labelled mAb for CD40. T-cells were stained using the PE-labelled antibodies for CD3, CD4 and CD8 (Caltag) and the FITC-labelled mAb for CD154 (Serotec). The fluorescence intensity of cell samples was assayed on a fluorescence-activated cell sorter (EPICS XL, Beckman Coulter), acquiring 10 000 events per sample. Data were analysed using the computer program SYSTEM 2, version 1.0.

Statistical analysis.
Data represent the mean±SD. Fisher's exact test, the Student t-test or the MannWhitney U-test were used to determine whether a significant difference (P<0·05) existed between the groups tested. Statistical analyses were performed using STATVIEW J4.11 (SAS Institute).

Shift from Th2- to Th1-type cytokine production following CpG ODN treatment
(a) In vitro
SAM-P1 mice are genetically burdened with Th2-biased senescence immune responses (Dong et al., 2000). When spleen cells of SAM-P1 mice were cultured with CpG ODN in vitro, an increased production of Th1-type cytokines such as IFN-γ and IL-12 were detected (Table 1). No such immunostimulatory effect could be induced by an ODN lacking a CpG motif. Among the stimulatory CpG ODNs tested, ODN 1826 was capable of inducing the most efficient Th1-type response and was, therefore, selected for use in all following experiments.

In response to influenza virus infection, spleen cells of SAM-P1 mice displayed the Th2-type dominant cytokine production profile when compared with that of normal mice (Dong et al., 2000). An additional treatment of SAM-P1 spleen cells with ODN 1826 increased the ratio of the titre of IFN-γ to that of IL-4, showing a shift to the Th1-type response (Table 2).

Table 2. Effect of CpG ODN on cytokine production of influenza virus-infected spleen cells in vitro Spleen cells (2·5x106) were infected with influenza virus at an input m.o.i. of 1 p.f.u. per cell and incubated with or without 1 µM ODN 1826. Culture fluids were harvested at the indicated time and assayed for cytokines by ELISA. Data are the mean±SD of results for each group of five mice tested.

(b) In vivo
SAM-P1 mice were administered intraperitoneally with 1 µg ODN 1826 ml^-1 and infected with influenza virus 1 week later. Virus-specific CTL activity was enhanced by ODN 1826 treatment. Specific lysis was elevated to 65·3±6·1 %, about fourfold higher than that of mock-treated control mice (15·4±3·4 %) (Fig. 1). No appreciable lysis was detected when uninfected cells were used as the control target cells. Furthermore, NK cell activity of spleen cells was increased from 5·4±1·2 to 23·1±2·2 % after in vitro stimulation with ODN 1826 (Fig. 2).

(14K): Fig. 1. Augmentation of influenza virus-specific CTL activity of SAM-P1 mice following CpG ODN treatment. Mice were administered 1 µg ODN 1826 ml-1 intraperitoneally and then immunized with influenza virus. Data are the mean±SD of results for each group of five mice tested. Significant difference compared with non-treated mice is shown (*, P<0·01; †, P<0·05).

(10K): Fig. 2. Augmentation of NK cell activity by immunostimulatory CpG ODN in vitro. Spleen cells from 3-month-old SAM-P1 mice were cultured in medium containing 1 µM ODN 1826 for 20 h. NK cell activity against Yac-1 target cells was assayed at an effector : target cell ratio of 100 : 1. Data are the mean±SD of results for each group of five mice tested. Significant difference compared with non-treated mice (P<0·01).

Production of virus-specific IgG and IgA antibodies increased significantly in the ODN 1826-treated mice (Table 3). In particular, an increase of IgG2a, a typical Th1 IgG isotype, was observed, while no significant changes in the IgG1 level, which is related to the Th2-type response, were detected.

Table 3. Influenza virus-specific antibody production in SAM-P1 mice primed with CpG ODN Data are the mean±SD of results for each group of five mice tested.

Enhanced expression of CD154 (CD40 ligand) molecules following CpG ODN treatment in vitro
Treatment of SAM-P1 mouse spleen cells with ODN 1826 increased the number of CD154⁺ (CD40 ligand) CD4⁺ T-cells, with more intensified expression after virus infection (Fig. 3). ODN 1826 treatment also increased the intensity of CD40 expression on B-cells. It should be noted that expression of CD154 (CD40 ligand) occurs quickly, as early as 3 h after CpG ODN stimulation, peaking at 6 h, while CD40 expression on B-cells occurs 24 h later. Thus, it can be inferred that CpG ODN, by activating the CD40CD154 (CD40 ligand) interaction, contributes to elevating the Th1 immune response in the immunosenescence system of SAM-P1 mice.

(37K): Fig. 3. Increased expression of CD154 (CD40 ligand) and CD40 molecules on spleen lymphocytes of SAM-P1 mice following CpG ODN treatment in vitro. Spleen cells were infected with influenza virus at an input m.o.i. of 10 p.f.u. per cell (A, B, E and F) and incubated in 1 µM ODN 1826 (C, D, E and F). Cells were doubly stained either for CD154 (CD40 ligand) and CD4 at 6 h after stimulation (A, C and E) or for CD40 and CD19 at 24 h after stimulation (B, D and F). Blocked lines indicate non-treated spleen cells. The representative result of three independent experiments is shown with the percentage of double-positive cells and the mean fluorescence intensity.

Protection from lethal influenza virus following CpG ODN treatment
To investigate the protective efficacy of ODN 1826 in vivo, SAM-P1 mice were administered ODN 1826 and then challenged with influenza virus. Virus growth in the lung was inhibited remarkably on day 2 after infection and the virus was eliminated as early as day 7, while in non-treated mice, virus shedding still persisted on day 9 (Table 4). Of the SAM-P1 mice pre-treated with ODN 1826, 90 % survived the lethal influenza virus infection (Fig. 4).

Table 4. Elimination of influenza virus from the lungs of SAM-P1 mice primed with CpG ODN SAM-P1 mice were administered 1 µg ODN 1826 ml-1 intraperitoneally on days -3 and -1 and then infected intranasally with 103 p.f.u. influenza virus per mouse. Data are the mean±SD of results for each group of five mice tested. The ratio of the number of mice with virus detected to the number of mice tested is shown in parentheses.

(18K): Fig. 4. Protection of SAM-P1 mice from lethal influenza following CpG ODN treatment. SAM-P1 mice were pre-treated with 1 µg ODN 1826 ml-1 on days -7, -3 and -1 before infection. A lethal dose of influenza virus (105 p.f.u. per mouse) was used for challenge experiments. Data were obtained from 10 mice of each group.

Most of the previous works dealing with CpG ODNs were carried out in ordinary hosts (Brazolot Millan et al., 1998; Oxenius et al., 1999). In the present study, we found that immunostimulatory CpG ODN also has the capacity to act as a Th1 immune stimulator, even in senescence-immunocompromised SAM-P1 mice, which remain bound to Th2-biased immune conditions and have decreased innate immunities (Dong et al., 2000). SAM-P1 mice that were treated with CpG ODN displayed a remarkably increased NK cell activity accompanied with an elevated release of antiviral cytokines such as IL-12, IFN-γ and TNF-α. Such innate immune responses play an important part in the defence system at an early stage of infection by killing virus-infected cells and consequently, by inhibiting virus growth and its spread in the respiratory tract (Monteiro et al., 1998). Rapid clearance of the virus from infected organs results in a reduction in symptoms and shortening of the disease period (Gibbons, 1992). Furthermore, the CpG ODN-stimulated virus-specific immune responses in SAM-P1 mice, including enhanced CTL activity and the preponderance of Th1-type IgG2a antibody, made it possible to achieve a quick recovery and satisfactory protection from infection (Fig. 4).

Unmethylated ODNs with certain flanking bases of CpG motifs are virtually absent in the vertebrate DNA. This fact suggests that the immunostimulatory actions of CpG ODN is an evolutionary phenomenon whereby the CpG ODN acts as a danger signal that the innate defence system of the vertebrate can recognize and respond to (Krieg et al., 1996). Immunostimulatory CpG ODN exerts a number of stimulatory effects on NK cells (Yamamoto et al., 1992) and antigen-presenting cells (APCs) such as macrophages, dendritic cells and B-cells (Sparwasser et al., 1998; Hartmann & Krieg, 2000). The cellular and molecular mechanisms of the CpG ODN action are evidenced as follows: (1) upregulation of proinflammatory cytokines like IL-6, IL-12 and IFN-γ; (2) upregulation of cell surface molecules of MHC, CD40, CD80 (B7-1), CD86 (B7-2) and intracellular adhesion molecule (ICAM)-1; and (3) activation of classic signal transduction pathways such as the stress kinase pathway and the NF-κB pathway (Hacker et al., 1998). CpG ODN has little capacity for inducing direct effects on either CD4⁺ or CD8⁺ T-cells and functions through a Th cell-independent pathway (Cho et al., 2000). However, a current report has demonstrated that phosphorothioate CpG ODN stimulates thymocytes directly (Mannon et al., 2000). The expression of MHC class II molecule and adhesion molecule ICAM-1 on dendritic cells and B-cells is impaired in SAM-P1 mice, resulting in the age-related dysfunction of APCs (Haruna et al., 1995). Furthermore, SAM-P1 mice show a unique deficiency in the cell number of CD4⁺ T-cells (Dong et al., 2000) and in the expression of the CD154 (CD40 ligand) molecule (Fig. 3). Because the CD40CD40 ligand interaction is crucial for the IL-12-dependent priming of Th1 cells (Cella et al., 1996), it is conceivable that the age-associated impairment of CD40CD40 ligand expression is a causative factor of immunosenescence. CpG ODN treatment of SAM-P1 mice induced a quick response of the increased expression of the CD40 ligand on CD4⁺ T-cells (Fig. 3), thus establishing protective Th1 immunity.

This finding suggests another novel mechanism involved in T-cell activation by CpG ODN. Although there appear to be multiple defects in immune function of the elderly and the mechanisms are complicated, restoration of the defective Th1 immune response is the most important strategy for protecting the elderly from infection. CpG ODN has been found to be a potent stimulator of a Th1 response that is effective against many types of pathogens. Thus, CpG ODN treatment, especially of elderly individuals, becomes a promising remedy for infectious diseases and age-related immune disorders.

Li Dong was the recipient of an Encouragement of Young Scientist Award from the Nakanihon Infectious Diseases Foundation, Japan.