In this study, double-stranded Candida albicans DNA was administered in systemic C. albicans infection in at dose of 20 µg per mouse at 4, 5 and 6 weeks of age. The level of IL-12 in serum was elevated as a result of yeast DNA treatment and correlated with lower mortality and decreased kidney and liver injury. Macrophage activation was demonstrated by an increase of nitric oxide (NO) and IL-12 production. These effects were Janus activation kinases (JAK)/signal transducer and activator of transcription (STAT) dependent as they were inhibited by selective JAK inhibitor tyrphostin AG-490. DNA influenced adaptive immune response through elevation of anti-Candida IgG antibody production in systemic C. albicans infection. Thus, C. albicans DNA augmented innate and adaptive immune responses against the pathogen.
Candida albicans infection
C. albicans DNA
Candida albicans is a polymorphic fungus causing disseminated candidiasis or local inflammation. The incidence of C. albicans infections increases in parallel with the increased number of immunocompromised patients (Bodey, 1988; Dimopoulos et al., 2007). As the fungal pathogen diversity and its molecular basis are still unconfirmed, it is reasonable to consider new possibilities for enhancement of host resistance. Bacterial DNA exerts an immunostimulatory effect on mammalian immune cells, which is mainly due to cytosine–guanine-rich (CpG) motifs (Lipford et al., 1998). Non-CpG sequences may have an immunosuppressive action as they induce Th2 responses (Ho et al., 2003). Nevertheless, the immune action of non-CpG motifs and the existence of stimulatory motifs different from the CpG sequences are not entirely clear. Recent studies have shown that CpG and non-CpG motifs engage Toll-like receptor 9 (TLR9) expressed on macrophages and B cells. As a result, secretion of several Th1 cytokines and enhanced B-cell proliferation have been observed (Akira et al., 2001). Considering the recent data about the immunostimulatory effects of bacterial DNA, one might ask a relevant question as to whether eukaryotic fungal DNA have a similar action. Previously, we have demonstrated that a single administration of C. albicans DNA increased the host resistance against disseminated C. albicans infection best expressed after 2 days (Yordanov et al., 2005). In this study, we aimed to prolong and enhance the protective effect of C. albicans DNA through its administration three times at 4, 5 and 6 weeks of age before lethal systemic infection. As a result of C. albicans DNA treatment, we established increased numbers of survivors in correlation with an elevated level of IL-12 and an augmented anti-infectious antibody response.
Materials and methods
Male and female BALB/c mice were purchased from Iffa-Credo (L'Abrese, France), and maintained in the animal facility under standard conditions of temperature and food. The animal study protocols were approved by the Ethical Animal Commission of the Institute of Microbiology, Sofia.
Candida albicans DNA was prepared as described previously (Yordanov et al., 2005). Escherichia coli DNA, ODN 1826 and control ODN 1826, were purchased from InvivoGen (Cayla, France). Lipopolysaccharide from E. coli (055:B5) and tyrphostin AG-490 were obtained from Sigma-Aldrich (Germany).
Systemic infection and treatment
Candida albicans (clinical isolate 562, Institute of Infectious and Parasitic Diseases, Sofia, Bulgaria; ATCC 18623) was grown for 48 h at 37 °C on Sabouraud dextrose broth (Sigma-Aldrich). After washing with endotoxin-free phosphate-buffered saline (PBS) (Cambrex, UK), a suspension of viable C. albicans cells (5 × 106 mL−1) was prepared. Before the induction of infection, 4-week-old BALB/c mice were intraperitoneally injected three times at 4, 5 and 6 weeks of age with 20 µg of C. albicans DNA (200 µL), 20 µg E. coli DNA (200 µL) or sterile PBS (200 µL). Ten days after the last treatment, C. albicans suspension (5 × 106 cells−1) was intraperitoneally inoculated in mice. The course of infection was followed for 28 days, when the percentage of survivors was calculated. Fungal growth in the kidneys and liver of infected mice was assayed on day 7. Homogenized organs in sterile PBS were plated on Sabouraud dextrose agar (Sigma-Aldrich) containing chloramphenicol (50 µg mL−1). The results are expressed as log CFU adjusted to the weight of organs.
Kidneys were immediately fixed in 4% formaldehyde/PBS solution (pH 7.4). Sections (7 µm) of paraffin-embedded tissues were stained with periodic acid–Schiff reagent (Sigma-Aldrich) and examined using a light microscope (Boeco, Germany).
Enzyme-linked immunosorbent assays (ELISA)
The quantitative ELISA kit (PeproTech, UK) was used to detect total IL-12p40/p70 (Cytolab, Rehovot, Israel) in plasma or in macrophage supernatants. Plasma samples were collected from C. albicans DNA-treated, E. coli DNA-treated or saline-treated mice 6 h postinfection. Culture supernatants were collected after a 24-h incubation of peritoneal macrophage (1 × 106 mL−1) with C. albicans DNA (20 µg mL−1) or lipopolysaccharide (100 ng mL−1) in the presence or absence of tyrphostin AG-490 (10 and 50 µM).
For determination of the anti-Candida IgG level in serum, the ELISA test plates were coated with 1 × 105 mL−1 heat-inactivated yeast cells. Test sera appropriately diluted in PBS/1% bovine serum albumin were dispensed and, after 2 h, at 37 °C, peroxidase-labeled goat anti-mouse IgG (1 : 10 000 diluted) (Cappel, Durham, NC) was added for 2 h. The reaction was developed by o-phenylenediamine and the absorbance was measured at 492 nm using an ELISA reader.
Peritoneal macrophages were harvested as described above. The cells (1 × 106 cells mL−1) were cultured at 37 °C for 18 h in the presence or absence of lipopolysaccharide (100 ng mL−1) or C. albicans DNA (20 µg mL−1) only or along with 10 or 50 µM AG-490. Nitrite concentration was assayed in culture supernatants by a standard Griess reaction as described previously (Dimitrova & Ivanovska, 2006).
Differences between groups were compared by Mann–Whitney U-test for survival and an unpaired t-test for the other assays. Data are presented as mean±SD. A value of P<0.05 was considered to be significant.
Repeated administration of C. albicans DNA increased survival and reduced organ injury in systemic infection
In the present experiments, mice were pretreated with 20 µg C. albicans DNA at 4, 5 and 6 weeks of age, and 10 days thereafter the infection was evoked intraperitoneally. Candida albicans DNA significantly improved the outcome of infection, increasing the percentage of survivors from 40% to 75% (Fig. 1a). Escherichia coli DNA was injected under the same schedule, similar to Candida DNA (20 µg, three times). The rate of survival was higher than that registered for yeast DNA.
Kaplan–Mayer survival curves of BALB/c mice injected with saline (n=15), with 20 µg yeast DNA (n=20) or with 20 µg Escherichia coli DNA (n=15) at 4, 5 and 6 weeks of age. Ten days after the last treatment, mice were inoculated with 5 × 106 CFU. Mice were observed for 28 days. (a) *P<0.05 of groups treated with Candida albicans DNA or E. coli DNA vs. control group. Colonization of the kidney (b) and liver (c) was determined at day 7 of infection. *P<0.01 vs. control group. Kidney sections (d) from control mice and C. albicans DNA-treated mice at day 7 of infection.
Major target organs in generalized candidiasis are the kidneys and liver. The fungal growth in those organs was assayed on day 7 of infection (Fig. 1b and c). A significant reduction of kidney and liver colonization showed groups treated with C. albicans DNA or E. coli DNA (Fig. 1b and c). The observation of tissue sections from kidneys on day 7 of infection showed the dramatic fungal burden and the massive presence of C. albicans hyphae (Fig. 1d).
Candida albicans DNA increased the level of IL-12 in circulation
The initiation of C. albicans infection was related to a high release of IL-12 into the circulation, reaching 2 ng mL−1 at first 3 h. The maximum IL-12 level was determined at 6 h, when E. coli DNA had a more pronounced effect than C. albicans DNA (Fig. 2). Then the cytokine concentration rapidly decreased until 24 h, remaining higher in the E. coli DNA-treated group, compared with the similar levels in the control and Candida DNA-treated groups.
DNA increased the level of IL-12 in vivo. Mice were treated as described in Fig. 1 and, at different time points, sera were collected. *P<0.01; **P<0.001 of DNA-treated groups vs. control group.
Candida albicans DNA action is dependent on Stat-3 signalling
Tyrphostin AG-490 is a specific inhibitor of Janus activation kinase (JAK) kinases. We investigated the influence of tyrphostin on macrophage activation, assayed by IL-12 and nitric oxide (NO) production. In vitro stimulation of peritoneal macrophages with C. albicans DNA induced IL-12 secretion but to a lesser extent than in response to lipopolysaccharide stimulation (Fig. 3a). Tyrphostin AG-490, at a concentration of 50 µM, significantly inhibited IL-12 production, triggered by C. albicans DNA or lipopolysaccharide (Fig. 3a). AG-490 did not have a direct inhibitory effect on unstimulated macrophages because it alone did not change IL-12 release (data not shown). We observed that C. albicans DNA or lipopolysaccharide markedly enhanced the release of NO in supernatants of macrophages stimulated for 18 h (Fig. 3b). Simultaneously, AG-490 was added during the time of cultivation. The substance abrogated DNA and lipopolysaccharide action in a dose-dependent manner.
IL-12 and NO production. Peritoneal macrophages (4 × 106 mL−1) were incubated in the absence (control) or in the presence of 20 µg mL−1Candida albicans DNA or 100 ng mL−1 lipopolysaccharide for 18 h. Two groups were incubated with DNA plus 10 or 50 µM AG-490 and two groups with lipopolysaccharide plus 10 or 50 µM AG-490 (a) Macrophages were treated as described in (a) for 18 h and NO was determined in the supernatants (b). *P<0.01, **P<0.001, DNA+AG-490-treated groups vs. DNA-treated groups; #P<0.01, ##P<0.001 for lipopolysaccharide+AG-490 groups vs. lipopolysaccharide-treated.
Candida albicans DNA increased anti-Candida antibody synthesis in systemic infection
Mice were pretreated with Candida DNA, ODN 1826 and control ODN at 4, 5 and 6 weeks of age and 10 days after they were inoculated. At day 14 of infection, sera were collected and the level of specific antibodies was determined. Yeast DNA administration resulted in an elevation of anti-Candida antibody titer. High activity on antibody production was expressed by ODN 1826, while control ODN 1826 had no effect (Fig. 4).
Serum anti-Candida IgG antibodies at day 14 of infection. Mice were treated with 20 µg ODN 1826, 20 µg control ODN 1826 or 20 µg Candida albicans DNA at 4, 5 and 6 weeks of age. After 10 days, mice were inoculated with 5 × 106 CFU and at day 14 specific antibodies were determined by ELISA. *P<0.05 vs. control group.
Protective immune response to C. albicans is mainly mediated by antigen-specific Th1 cells (Cenci et al., 1999; Romani, 1999). Th1 cells produce IL-2, IFN-γ and IL-12, activate macrophages and provide B cell help. Recent studies demonstrated that Th2 cytokines such as IL-4 are required for the maintenance of Th1-mediated host resistance too (Montagnoli et al., 2002). When the host-commensal relationships are disrupted in immunocompromised patients, C. albicans causes infections and a local immune response is triggered. We hypothesized that yeast DNA, like bacterial DNA, could have immunostimulatory properties and could activate innate immunity to limit the fungal growth and prevent acute infection. Candida albicans DNA improved the outcome of systemic infection; however the effect was weaker compared with E. coli, which ensured almost full protection. The diverse bacterial–host and yeast–host interactions might explain the difference in the protective properties of C. albicans and E. coli DNA. In DNA-treated mice, the infection was limited and the lesions were resolved to a huge extent. Infiltrates of neutrophils and macrophages together with lower numbers of candidal hyphae and blastospores, were seen. Several hypha-specific genes have been described to contribute directly to the virulence of C. albicans (Liu, 2002)
The production of Th1 cytokines is a key factor for the outcome of C. albicans infection. Recently, it has been assumed that the development of protective immunity against C. albicans is linked to the production of endogenous IL-12 (Romani et al., 1994). We suggest that during acute systemic C. albicans infection, DNA released from the ingested pathogen could amplify an innate immune response and could augment the production of proinflammatory cytokine IL-12. The application of DNA caused this effect on IL-12 release. Although, high IL-12 production might cause a deleterious effect in the sites of inflammation, our results indicate that the enhanced IL-12 synthesis can contribute to the improvement of survival rate and to the suppression of organ colonization.
In order to understand better the in vivo effects of C. albicans DNA on IL-12 production and protective immunity, we focused our attention on studying the cytokine-signalling pathway triggered by DNA. Cytokines are recognized by its receptors that subsequently form dimers and activate JAKs. Phosphorylated JAKs are docking sites for a family of transcription factors known as STAT, which regulate the expression of genes for cytokines or inflammatory mediators. Currently, a class of JAK inhibitors termed tyrphostins has been described (Gazit et al., 1989). These data indicated that C. albicans DNA express its action on cytokine production via the JAK/STAT signalling pathway. Further investigations describing precisely this mechanism should be conducted. Recent study has shown that the JAK/STAT pathway is involved in the regulation of iNOS expression in intestinal epithelial cells (Stempelj et al., 2007). Similar observations have been made on macrophages where tyrphostin AG-490 inhibited IFN-γ-induced NO production (Sareila et al., 2006). This effect of yeast DNA can be abrogated by tyrphostin AG-490 indicating the importance of the JAK/STAT signalling pathway for NO synthesis. In conclusion, our in vitro data suggest that C. albicans DNA induced the synthesis of NO and IL-12 in a JAK/STAT-dependent fashion and this effect might contribute to enhanced protective immunity in mice with infections.
The role of Th1 cells and Th1 cytokines in the clearance of pathogens and long-term immune responses in C. albicans infections is well described (Puccetti et al., 1995). The role of antibody immunity in C. albicans infection seems to be controversial. Although the antibodies against certain cell surface antigens of C. albicans can enhance the host resistance in disseminated infection. In the present experiments, C. albicans DNA augmented anti-Candida antibody synthesis significantly in systemic infection. Bernasconi (2003) have shown that the expression of TLR9 and TLR10 is rapidly induced in human naïve B cells following triggering of B-cell-receptor (BCR). The engagement of TLR 9 activates two transcription factors Rel and NF-κB and causes B-cell proliferation (Grumont et al., 1998). When we used ODN 1826 in acute infection, we established an enhancement of the specific antibody response. In order to prove whether CpG motifs in C. albicans DNA affect B cells and antibody production, we further intend to obtain CpG-enriched sequences from yeast DNA and to investigate their in vivo action in animal models of C. albicans infection.
This work was supported by a project Actions Concertees Interpasteuriennes (ACIP) No A/7/2005 and project MU-L-1501/05 (National Scientific Fund, Bulgaria).
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