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1 Departamento de Microbiología y Ecología, Universitat de València, Facultad de Ciencias Biológicas, Edificio de Investigación, C/Dr. Moliner 50, 46100 Burjasot, Valencia, Spain
2 Laboratorio de Citómica, Unidad Mixta CIPF-UVEG, Centro de Investigación ‘Principe Felipe’, Valencia, Spain
Correspondence
M. Luisa Gil
m.luisa.gil{at}uv.es
Received 25 May 2006
Accepted 14 August 2006
by macrophages from aged mice in response to yeast cells and hyphae of C. albicans was significantly lower than production by macrophages from young mice. In vitro production of cytokines, such as TNF- and gamma interferon (IFN-
), by antigen-stimulated splenocytes from mice intravenously infected with C. albicans cells was also diminished in old mice. This decrease in production of T helper 1 cytokines in old mice correlated with a diminished frequency of IFN--producing CD4+ T lymphocytes, although the ability to develop an acquired resistance upon vaccination (primary sublethal infection) of mice with the low-virulence PCA2 strain was not affected in aged mice. The diversity of antigens recognized by C. albicans-specific antibodies in sera from infected aged mice was clearly diminished when compared with that from infected young mice. Taken together, these data show that aged mice develop an altered innate and adaptive immune response to C. albicans and are more susceptible to systemic primary candidiasis.
Abbreviations: IFN-, gamma interferon; IL, interleukin; i.v., intravenous(ly); Th1, T helper 1; TLR, Toll-like receptor; TNF, tumour necrosis factor.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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Resistance to candidiasis requires the coordinated action of innate and adaptive immune defences. Neutrophils and macrophages can clear the pathogen via phagocytosis, and macrophage activation also leads to the release of several key mediators such as pro-inflammatory cytokines, which are important for protecting the host against disseminated candidiasis. It is accepted that antifungal CD4+ T helper 1 (Th1)-mediated responses play a central role in anti-C. albicans defences, providing control of fungal infectivity through the production of gamma interferon (IFN-), which is required for optimal activation of phagocytes and for helping in the generation of a protective antibody response. A special feature of C. albicans is the morphological transition from the yeast to the hyphal form, which is strictly associated with virulence, as several agerminative mutants invariably have low systemic pathogenicity. Phagocytosis of the yeast form induces murine dendritic cells to produce interleukin (IL)-12 and to prime Th1 lymphocytes, whereas ingestion of the hyphal form results in IL-4 production, which favours Th2 cell priming (Calderone, 2001; Romani, 2004).
In the elderly, many alterations of both innate and adaptive immunity have been described, generally viewed as a deterioration of immunity, leading to the use of the term immunosenescence (Effros, 2001; Ginaldi et al., 2001; McGlauchlen & Vogel, 2003; Plackett et al., 2004). Most research concerning immunosenescence has been carried out on T and B cells, which show an altered cytokine pattern, a reduction in clonal expansion and function of antigen-specific T and B cells, and a decline in antigen-presenting-cell function (Effros, 2001; McGlauchlen & Vogel, 2003). Similarly, the functions of macrophages, neutrophils and natural killer cells, components of the innate immunity, are also decreased (Plackett et al., 2004). The decline in immune function leads to increased susceptibility of aged individuals to viral, bacterial and fungal infections, reactivation of latent viruses and a decreased response to vaccines (Effros, 2001; Ginaldi et al., 2001). Invasive infections with opportunistic fungi, such as Candida species, have become an increasing problem in old adults because they are more likely to be considered for transplantation, to receive aggressive regimens of chemotherapy for cancer and to take immunosuppressive drugs for non-malignant diseases (Kauffman, 2001). In addition, the immunosenescence process in older adults probably enhances the risk and severity of candidaemia (Nucci et al., 1998).
In the present study, using young and aged mice, we studied the influence of ageing on (i) susceptibility in vivo to experimental candidiasis, (ii) in vitro tumour necrosis factor (TNF)- release by macrophages in response to C. albicans, (iii) the production of Th1 cytokines (IFN- and TNF-) by splenocytes, (iv) the development of a Th1 response, (v) the ability to develop an acquired resistance upon vaccination (primary sublethal infection) and (vi) the development of an acquired humoral response.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Endotoxin-free starved and heat-inactivated C. albicans ATCC 26555 yeast cells or hyphae and endotoxin-free starved yeast cells of C. albicans PCA2, a low-virulence non-germinative strain, were prepared as described previously (Villamón et al., 2004a, b, c). All of the assays were performed under conditions designed to minimize endotoxin contamination. Endotoxin-free water and PBS were used, and fungal culture media were passed through a Detoxi-Gel endotoxin-removing gel (Pierce) and tested for the absence of endotoxin by the E-Toxate assay (Sigma).
Infection model and survival curves. Young and aged mice were intravenously infected with starved yeast cells of the high-virulence C. albicans strain ATCC 26555. Mice were challenged with 8x105 yeast cells in a volume of 0.2 ml PBS and survival was checked daily for 21 days.
In vaccination assays, aged mice were infected with the low-virulence C. albicans strain PCA2 by intravenous (i.v.) injection of 106 yeast cells in a volume of 0.2 ml (primary candidiasis). Fifteen days later, immunized mice were i.v. infected (secondary infection) with C. albicans ATCC 26555 (106 yeast cells in a volume of 0.2 ml) and survival was checked daily for 14 days. Control aged mice were intravenously infected with 106 yeast cells of C. albicans ATCC 26555 (primary candidiasis) and survival was checked for 14 days.
To assess the tissue outgrowth of the micro-organism, young and aged mice were i.v. infected with 8x105 C. albicans ATCC 26555 yeast cells. At 3 and 5 days post-infection (p.i.), mice were killed and the kidneys were removed aseptically, weighed and homogenized in 1 ml PBS; dilutions of the homogenates were plated on Sabouraud/glucose agar. The c.f.u. were counted after 24 h of incubation at 37 °C and expressed as c.f.u. (g tissue)–1. The amount of TNF- in the supernatants of kidney homogenates was determined using a commercial ELISA kit (eBioscience). Mice in a moribund condition were sacrificed irrespective of the planned schedule and were not evaluated for c.f.u. or TNF-.
Isolation of mouse peritoneal macrophages and splenocytes. Resident peritoneal macrophages were harvested by injecting and withdrawing 10 ml complete cell-culture medium (RPMI 1640 supplemented with 5 % heat-inactivated fetal bovine serum and 1 % penicillin/streptomycin; Gibco). Cells were washed once with the same medium and plated at a density of 2.3x105 cells in 200 µl medium per well in a 96-well tissue culture plate. Peritoneal macrophages were allowed to adhere for 2 h at 37 °C in a 5 % CO2 atmosphere, the non-adherent cells were removed and the adherent macrophages were cultured for 72 h prior to challenge, as described previously (Villamón et al., 2004b).
Splenocytes were obtained as described elsewhere (Villamón et al., 2004a, c). Mice were i.v. infected (4x105 yeast cells per mouse) with either C. albicans PCA2 or ATCC 26555 yeast cells. Three days after infection, total spleen cells were obtained by collagenase D treatment of the spleens, washed once with complete cell-culture medium and plated at a density of 107 cells in 1 ml medium per well in a 24-well tissue culture plate, in the presence of 2.5 µg amphotericin B (Gibco) ml–1.
Measurement of in vitro cytokine production.
Cells (peritoneal macrophages or splenocytes) were challenged with the indicated stimuli for 24 or 48 h, respectively, at 37 °C. Samples with no stimuli were used as controls. Supernatants were harvested and tested using commercial ELISA kits for TNF- and IFN- (eBioscience).
The stimuli used were LPS from Escherichia coli O111 : B4 (Sigma), the yeast cell-wall particle zymosan (Molecular Probes) and heat-inactivated C. albicans ATCC 26555 yeast cells or hyphae, obtained as reported elsewhere (Gil-Navarro et al., 1997; Gozalbo et al., 1998). Briefly, starved yeast cells were inoculated [200 µg cells (dry wt) ml–1] in a minimal synthetic medium and incubated for 3 h at 28 °C to obtain yeast cells or at 37 °C to obtain hyphae. For heat inactivation, yeast cells and hyphae were resuspended in PBS and treated at 100 °C for 1 h. After inactivation, fungal cells were washed in PBS and brought to the desired cell density in complete cell-culture medium.
IFN--secretion assay.
After i.v. infection of mice with 4x105 yeast cells of either C. albicans PCA2 or ATCC 26555, total spleen cells were obtained as described above, challenged in vitro with heat-killed C. albicans ATCC 26555 yeast cells or hyphae for 18 h, and analysed for IFN- secretion using the mouse IFN--secretion assay detection kit (Miltenyi Biotec) according to the manufacturer's instructions. Cells were also incubated with a saturating amount of FITC-conjugated CD4 mAb (RM4-5; Pharmingen) and phycoerythrin (PE)–Cy5-conjugated CD3 mAb (145-2C11; Pharmingen). After washing twice with PBS, the stained cells were analysed on an EPICS XL-MCL flow cytometer (Beckman Coulter).
Western immunoblot. Soluble cell extracts, obtained from yeast cells or hyphae by boiling cells in PBS supplemented with 1 % SDS and 1 % DTT, were separated by SDS-PAGE and transferred to PVDF membranes (Amersham Pharmacia Biotech). Blots were incubated with a 1 : 10 dilution of mouse serum obtained before primary infection with strain PCA2 (uninfected sera) and 14 days after secondary infection with strain ATCC 26555 (see above). Reactive bands were developed using goat anti-mouse IgG horseradish peroxidase-conjugated antibody (1 : 1000 dilution; Sigma), and hydrogen peroxide and 4-chloro-1-naphthol as the chromogenic reagent.
Statistical analysis. Survival curves were analysed using the Kaplan–Meyer log rank test. Student's two-tailed t test was used to compare cytokine production and numbers of c.f.u. Data were expressed as mean±SD. Significance was accepted at levels of P<0.05 and P<0.01.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This dose was selected following preliminary studies evaluating the mortality rates in C57BL/6 young mice according to the number of C. albicans cells injected. Under these conditions, the percentage of dead animals was 83 % in aged mice, whilst only 44 % of the young mice had died at day 21; this statistically significant difference (P=0.0031) indicated that old mice are more susceptible to invasive candidiasis in this experimental model. To investigate further the effect of ageing on disseminated candidiasis, mice were infected using the same conditions (8x105 yeast cells per mouse), killed at random on days 3 and 5 p.i. and the number of c.f.u. (g kidney)–1 was determined (Fig. 1b). The actual fungal growth in this target organ was significantly higher in aged mice compared with young mice on days 3 and 5, further supporting the high susceptibility of aged mice to invasive candidiasis. In spite of the higher fungal burden in the kidneys of aged infected mice, the level of TNF- in the kidney homogenates was similar to that of young mice (Fig. 1c). Therefore, when TNF- levels in kidney homogenates, expressed as levels of TNF- (106 c.f.u.)–1, were compared, a significant difference (P<0.05) was observed at day 3 between young (3500±1100) and aged (550±240) mice. Similar qualitative differences between young and aged mice concerning fungal burden and levels of TNF- (106 c.f.u.)–1 in kidneys were obtained at day 3 following i.v. infection with a lower (sublethal) dose of C. albicans ATCC 26555 (not shown).
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production by macrophages production by macrophages in response to C. albicans (Gil & Gozalbo, 2006; Villamón et al., 2004b) and it has been described that ageing negatively skews macrophage TLR2- and TLR4-mediated pro-inflammatory responses (Boehmer et al., 2004, 2005; Renshaw et al., 2002). Therefore, we checked the in vitro production of TNF- by macrophages from young and aged mice in response to C. albicans (Fig. 2). Zymosan (a known TLR2 agonist) and LPS (a TLR4 agonist) were included as control stimuli. The synthesis of TNF- in response to LPS and zymosan was significantly lower by macrophages from aged mice compared with the synthesis by macrophages from young mice. Similarly, production of TNF- by macrophages from aged mice in response to yeast cells and hyphae of C. albicans was also significantly impaired at all doses tested.
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and IFN- production by C. albicans-stimulated splenocytes were assessed 3 days after infection. Splenocytes stimulated with LPS and zymosan served as controls. As described above for resident peritoneal macrophages, cells from aged mice showed severely impaired production of TNF- in response to all stimuli tested: LPS (55 % inhibition), zymosan (70 % inhibition), yeast cells (50–90 % inhibition, depending on the dose) and hyphae (47–87 % inhibition, depending on the dose). Also, as early as 3 days p.i., IFN- production was dramatically impaired in aged mice compared with young mice, in response to LPS, zymosan, yeast cells and hyphae (Fig. 3a).
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by splenocytes was also greatly diminished in aged mice in response to all stimuli tested (Fig. 3b
). As described previously, mice produced more IFN- upon exposure to yeast cells than upon exposure to hyphae (Fig. 3a, b) (Romani, 2004; Romani et al., 1991; Villamón et al., 2004a).
Effect of ageing on development of the Th1 response in mice
To define the effect of ageing on development of the Th1 anticandidal response, we determined the frequency of IFN--producing CD4+ T lymphocytes in mice infected with C. albicans. To this purpose, mice were infected i.v. with the low-virulence strain PCA2; 3 days after infection, total spleen cells were challenged in vitro with heat-killed C. albicans ATCC 26555 yeast cells or hyphae for 18 h and analysed for IFN- secretion using the mouse IFN--secretion flow cytometric assay. The cells were simultaneously labelled with FITC-conjugated CD4 mAb and PE–Cy5-conjugated CD3 mAb. As shown in Fig. 4(a), the number of IFN--producing CD4+ T lymphocytes in C. albicans-infected mice was significantly reduced in aged mice compared with young mice in response to both yeast cells and hyphae. A similar result was obtained when the frequency of IFN--producing CD4+ T lymphocytes was determined following infection of mice with a sublethal dose of the high-virulence C. albicans strain ATCC 26555 (Fig. 4b). As expected, the frequency of IFN--producing CD4+ T lymphocytes was higher in response to yeast cells than in response to hyphae, both in aged and young mice infected with C. albicans PCA2 or ATCC 26555. These results were in accordance with the in vitro levels of IFN- produced by splenocytes (see Fig. 3).
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). Naive aged mice i.v. injected with 106 cells of the high-virulence strain ATCC 26555 (primary candidiasis in naive mice) were used as controls (Fig. 5). Aged mice surviving PCA2 infection (vaccinated) were resistant to C. albicans ATCC 26555 infection (90 % survivors at day 14), whereas 100 % of control aged mice primarily infected with the high-virulence strain (non-vaccinated) died within 8 days. As a control, an identical immunization protocol was performed in parallel assays with young mice and, as expected, 100 % survival was observed at day 14 after secondary infection (not shown). These results suggest that although effector mechanisms of resistance to primary C. albicans infection are impaired in aged mice, they are still able to develop a significant acquired resistance upon vaccination (primary sublethal infection).
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). Control sera from uninfected aged mice showed a higher background than control sera from uninfected young mice tested at the same dilution. The diversity of antigens, from both yeast cells and hyphae, recognized by sera from aged infected mice was clearly diminished when compared with that recognized by sera from young infected mice. The number of bands and the intensity of the staining were lower with sera from aged mice than with sera from young mice.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Our model of disseminated candidiasis demonstrated that ageing significantly increased the overall susceptibility of mice to systemic C. albicans infection. Decreased survival of aged mice compared with young mice was evident and a higher fungal burden in the kidneys of aged infected mice was detected. However, the level of TNF- measured in homogenates from the kidneys of aged mice was similar to that of young mice, in spite of their higher fungal burden. These results suggested a lower TNF- production by cells from aged mice in response to C. albicans infection, as deduced from a comparison of the levels of TNF- (106 c.f.u.)–1.
It has been described that aged macrophages secrete significantly lower levels of pro-inflammatory cytokines than young macrophages in response to different ligands of TLRs (Boehmer et al., 2004, 2005; Renshaw et al., 2002), although different explanations have been reported for this observation. Renshaw et al. (2002) found that macrophages from aged mice expressed lower levels of TLRs, whereas Boehmer et al. (2004, 2005) concluded that decreased expression of mitogen-activated protein kinases could be the mechanism responsible for age-related deterioration of TLR-mediated signalling. We have previously described that TLR2 is the main receptor triggering TNF- in macrophages in response to C. albicans (Gil & Gozalbo, 2006; Villamón et al., 2004b) and that pro-inflammatory cytokines, such as TNF-, secreted by activated macrophages are critical in protecting the host against disseminated candidiasis (Netea et al., 1999; Steinshamn et al., 1996). Therefore, we determined the effect of ageing on signalling cellular pathways for TNF- production upon C. albicans recognition by macrophages. Our results clearly showed a reduction in TNF- production in vitro by resident peritoneal macrophages from aged mice in response to both yeast cells and hyphae of C. albicans, supporting decreased production of TNF- in vivo during primary infection, as mentioned above. Therefore, this impaired TNF- production may be partly responsible for the increased susceptibility of aged mice to candidiasis.
In addition, levels of TNF- and IFN- production by C. albicans-stimulated splenocytes after infection with strain PCA2 were dramatically impaired in aged mice compared with young mice. This result indicated that, early in infection, production of pro-inflammatory cytokines, including the Th1 cytokine IFN-, is impaired in aged mice. To correlate these findings with the acquired immunity to C. albicans, we measured the frequency of IFN--producing CD4+ T lymphocytes in mice i.v. infected with the low-virulence strain PCA2, which induces a Th1-acquired immune response (Romani et al., 1991). Our results showed a greatly diminished frequency of IFN--producing CD4+ T cells in aged mice compared with young mice, indicating that aged mice developed an impaired protective Th1 cellular response against C. albicans infection. In order to determine whether this impaired Th1 response may help explain why aged mice were more susceptible to primary infection with a high-virulence strain, we investigated the development of a Th1 response following infection with a sublethal dose of the virulent C. albicans strain ATCC 26555. In this case, aged mice also showed an impaired specific Th1 response and lower levels of IFN-, indicating that this defect may be involved directly in the susceptibility of aged mice to primary candidal infection.
We also studied the effect of ageing in the development of the acquired humoral response against C. albicans during secondary invasive infection. Sera from uninfected aged mice showed a higher background than sera from uninfected young mice, probably as a consequence of the increase in the levels of serum immunoglobulins that has been described in elderly subjects (Ginaldi et al., 2001). At day 15 after secondary infection, IgG titres were similar in sera from both aged and young mice (data not shown); however, the pattern of C. albicans antigens recognized by Western blot analysis was different. Sera from infected aged mice recognized fewer antigenic proteins than sera from infected young mice, in both yeast and hyphal extracts. This result may be explained by a decreased helper T-cell function and also by an intrinsic primary B-cell deficit (the ageing-associated decrease in the number of total B lymphocytes and in the high-affinity protective antibody response) (Ginaldi et al., 2001; McGlauchlen & Vogel, 2003). Therefore, the acquired humoral response to C. albicans was modified in aged mice compared with young mice; however, there was no evidence indicating whether this might account for the high susceptibility of aged mice to primary candidiasis, as antibody responses to C. albicans are complex, with the presence in immune sera of protective, non-protective and deleterious antibodies (Bromuro et al., 2002). Although it is accepted that the humoral response is not critical for host defence against C. albicans during primary systemic infection, administration of antibodies against certain C. albicans antigens confers protection in naive mice against infection, and also other C. albicans epitopes have been used for antibody-mediated protection against systemic candidiasis (Bromuro et al., 2002; Matthews et al., 2003; Montagnoli et al., 2004; Torosantucci et al., 2005; Vilanova et al., 2004; Zhang et al., 2006). In spite of the impaired acquired Th1 immune response and modified humoral response in aged mice, they were still capable of mounting an acquired resistance upon vaccination by the primary sublethal model of infection with a low-virulence strain, indicating that, although ageing is associated with a decline in immune function, a protective response may still be conserved. We have previously described a similar result using TLR2–/– mice, which showed a diminished Th1 response, although it was still sufficient to confer acquired protection against secondary infection (Villamón et al., 2004c). As mentioned above, the role of antibodies in the development of a protective response upon vaccination is complex and therefore the role of altered antibody production by aged mice in protection remains to be established.
In summary, we found that aged mice were more susceptible to in vivo experimental primary infection by C. albicans. This increased susceptibility was associated with defective synthesis of TNF-, a lower specific protective Th1 response, which resulted in lower levels of IFN-, and altered development of specific antibodies. However, aged mice were still capable of mounting an acquired resistance upon vaccination. Our results suggest that the ageing-associated decreased immune function may result in an increased susceptibility to fungal infections. Identification of the multiple molecular mechanisms underlying the age-associated decline of the immune function is required for the development of interventions designed to prevent or reverse immunosenescence, in order to decrease the incidence of these infections in elderly people.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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lymphotoxin- double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans. J Immunol 163, 1498–1505.This article has been cited by other articles:
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  A. Marcil, C. Gadoury, J. Ash, J. Zhang, A. Nantel, and M. Whiteway Analysis of PRA1 and Its Relationship to Candida albicans- Macrophage Interactions Infect. Immun., September 1, 2008; 76(9): 4345 - 4358. [Abstract] [Full Text] [PDF]
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