Correspondence: María Luisa Gil, Departamento de Microbiología y Ecología, Universitat de València, Facultad de Ciencias Biológicas, Edificio de Investigación, C/Dr Moliner 50, 46100 Burjasot, Valencia, Spain. Tel.: +34 96 354 3410; fax: +34 96 354 4570; e-mail: email@example.com
Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2–3 months old) and aged (18–20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1β, and tumor necrosis factor-α and chemokines (MIP-2) by purified (CD11b+) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.
Candida albicans is the microorganism most frequently causing opportunistic fungal infections in immunocompromised patients. The nature and extent of the impairment of host defense influence the manifestation and severity of infection. The incidence of these infections is increasing as a result of a growing population of immunocompromised individuals due to the use of intensive chemotherapy and immunosuppresive drugs. Systemic candidiasis is associated with high morbidity and mortality, as early diagnosis is difficult and current antifungal therapies are limited by toxicity and resistance (Calderone, 2001; Pfaller & Diekema, 2007).
Resistance to candidiasis requires the coordinated action of innate and adaptive immune defenses. Phagocytes, such as neutrophils and macrophages, are crucial to these processes because they can eliminate the pathogen via phagocytosis. Furthermore, macrophage activation leads to the release of several key mediators such as proinflammatory cytokines, which are important for protecting the host against disseminated candidasis and for inducing a T-helper type 1 (Th1) immune response that activates fungicidal effector mechanisms, and helps in the generation of a protective antibody response (Romani, 2004). Interestingly, although protective immunity to C. albicans is mediated by Th1 cells, some Th2 cytokines (such as IL-10) are required for the maintenance of antifungal immune protection. The balance of proinflammatory and anti-inflammatory signaling is a requisite to allow host survival without excessive proinflammatory pathology (Romani & Puccetti, 2007). Phagocytotic cells recognize the pathogen by a variety of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) (Bellocchio et al., 2004; Poulain & Jouault, 2004; Gil & Gozalbo, 2006; Netea et al., 2006; Zelante et al., 2007). TLRs are essential PRRs and constitute a family of receptors that mediate recognition of microbial challenges, subsequent inflammatory response, and are also regulators of the adaptive responses (West et al., 2006). We have previously shown that TLR2 is the most important TLR involved in interaction with C. albicans, both yeasts and hyphae, triggering cytokine secretion through the MyD88 signaling pathway (Villamón et al., 2004a, b; Gil & Gozalbo, 2006).
Opportunistic yeast infections, such as invasive C. albicans infections, have increasingly become a problem among older adults because they are more likely to be considered for transplantation, receive aggressive regimens of chemotherapy for cancer, and take immunosuppressive drugs for nonmalignant diseases (Kauffman, 2001). Moreover, the immunosenescence process in old adults probably enhances the risk and severity of candidemia (Nucci et al., 1998; Romani, 2005). Recently we have shown that aged C57BL/6 mice develop an altered innate and adaptive immune response to C. albicans and are more susceptible to systemic primary candidiasis (Murciano et al., 2006). In the present work, using young and aged C57BL/6 mice, we have studied the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines [IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and IL-10] and chemokines (MIP-2) by resident peritoneal macrophages in response to yeasts and hyphae of C. albicans, and (3) baseline cell surface TLR2 expression on resident peritoneal macrophages.
Materials and methods
Mice and yeasts strains
Young (2–3 months old) and aged (18–20 months old) C57BL/6 female mice, with an average weight of 18.7±2.9 and 26.9±4.4 g, respectively, were purchased from Harlan Ibérica (Barcelona, Spain). All assays involving mice were approved by the Institutional Animal Care and Use Committee.
Endotoxin-free fixed or heat-killed (inactivated) C. albicansATCC 26555 yeasts or hyphae, and endotoxin-free starved yeast cells of C. albicans PCA2, a low-virulence nongerminative strain, were prepared as described previously (Gil-Navarro et al., 1997; Murciano et al., 2007b). All the assays were performed under conditions designed to minimize endotoxin contamination. Endotoxin-free water and phosphate-buffered saline (PBS) were used; fungal culture media were passed through a detoxi-gel endotoxin removing gel (Pierce, Rockford, IL) and tested for the absence of endotoxin by the E-toxate assay (Sigma, Madrid, Spain).
Isolation and purification of mouse cells
Total resident peritoneal cells from young and aged mice were harvested by instilling and withdrawing 10 mL of complete cell culture medium (RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin stock solution, Gibco, Barcelona, Spain). To obtain recruited peritoneal neutrophils and macrophages, young and aged mice were injected intraperitoneally with 107 heat-killed C. albicansATCC 26555 yeast cells. After 4 or 72 h, peritoneal cells were collected as described above to obtain neutrophils and macrophages, respectively. Bone marrow cells were obtained from femurs and tibias by flushing the bone marrow into a 50-mL conical tube with culture medium using a short 25-G needle and a 10-mL syringe.
CD11b+ macrophages were purified from total resident peritoneal cells and from recruited peritoneal macrophages by immunomagnetic cell sorting using CD11b (Mac-1) MicroBeads (Miltenyi Biotec, Madrid, Spain) according to the manufacturer's instructions. Ly-6G+ cells were purified from recruited peritoneal neutrophils and from bone marrow cells by immunomagnetic cell sorting. For separation, cells were magnetically labeled with anti-Ly-6G-Biotin and anti-Biotin Microbeads, according to the manufacturer's instructions. The purity of the CD11b+ and Ly-6G+ cells was assessed by labeling with PE-labeled anti-CD11b monoclonal antibody (mAb, clone M1/70, eBioscience, San Diego, CA) and PE-labeled anti-Ly-6G mAb (clone RB6-8C5, eBioscience) and flow cytometry analysis with an EPICS XL-MCL flow cytometer (Beckman Coulter).
The percentage of neutrophils and macrophages in the peritoneal cavity was measured by flow cytometry using the phycorythrine-labeled mAb Ly-6G and the PE-labeled mAb F4/80 (clone BM8, eBioscience), respectively. Intraperitoneal cells were also labeled with FITC-labeled mAb anti-major histocompatibility complex (MHC) class II (clone M5/114.15.2, eBioscience).
Determination of C. albicans killing by phagocytotic mouse cells
The ability to kill C. albicans yeasts by phagocytotic cells from young and aged mice was assessed in total recruited peritoneal neutrophils, Ly-6G+ recruited peritoneal neutrophils, Ly-6G+ bone marrow cells, total recruited peritoneal macrophages, CD11b+ recruited peritoneal macrophages, CD11b+ resident peritoneal cells, and CD11b+ depleted resident peritoneal cells.
Cells of C. albicans strain PCA2 were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 28 °C up to the exponential growth phase (A600 nm 0.6–0.8), collected, and washed with water. Cells were resuspended in water and maintained for 3 h at 28 °C with shaking and afterwards at 4 °C for 24 or 48 h (starved yeast cells). The suspension was finally washed and diluted in complete cell culture medium. Candida albicans cells (0.5 × 106) were added to a final volume of 200 μL of culture medium containing 1 × 106 neutrophils or macrophages and incubated for 2 or 4 h at 37 °C, respectively. As control, 0.5 × 106C. albicans cells were inoculated in 200 μL of culture medium without phagocytotic cells. After incubation, samples were diluted in water, plated on Sabouraud/glucose agar, and incubated overnight at 37 °C. The CFUs were counted and the percentages of killing were determined as follows: % killing=[1−((CFU sample (t=2 or 4 h)/(CFU control (t=2 or 4 h))] × 100. A nongerminative strain (PCA2) was chosen for killing assays, in order to facilitate determination of CFU after the incubation period, as no germ tube (hyphae) aggregates are formed.
Measurement of in vitro cytokine and chemokine production
CD11b+ resident peritoneal cells were plated at a density of 2.3 × 105 cells in 200 μL of complete cell culture medium per well in a 96-well tissue culture plate and cultured for 72 h at 37 °C in a 5% CO2 atmosphere before stimulation. Cells were challenged with the indicated stimuli for 24 h. The stimuli used were the yeast cell wall particle zymosan (Molecular Probes, Eugene, OR) and fixed C. albicansATCC 26555 yeasts or hyphae. Assays in the absence of exogenous stimuli were used as negative controls to check background activation. Supernatants were then harvested and tested with commercial enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, IL-1β, IL-10 (eBioscience), and MIP-2 (Peprotech, Rocky Hill, NJ).
Analysis of TLR2 expression by flow cytometry
The level of TLR2 in total resident peritoneal cells and in CD11b+ resident peritoneal cells was measured by flow cytometry; 0.2 × 106 cells were incubated with 0.2 μg of PE-labeled antimouse TLR2 (clone 6C2, eBioscience) or with the respective PE-labeled isotype control (eBioscience) for 20 min at 4 °C in the dark. After incubation, cells were washed with PBS and the percentage of TLR2-positive cells and the fluorescence mean intensity were measured by flow cytometry using an EPICS XL-MCL flow cytometer (Beckman Coulter).
The two-tailed t-test was used to compare cytokine and chemokine production, the percentage and number of neutrophils and macrophages in the peritoneal cavity, and the percentage of C. albicans killing by phagocytotic cells. Data are expressed as mean±SD. Significance was accepted at the *P<0.05 and **P<0.01 level.
Fungicidal activity of phagocytotic cells
To investigate the effect of aging on the fungicidal activity of neutrophils and macrophages, the survival of C. albicans yeast cells was determined following incubation with phagocytotic cells from young and aged mice. Different phagocytotic cells were assayed: recruited peritoneal neutrophils and macrophages elicited by C. albicans injection, resident peritoneal macrophages, and bone marrow neutrophils.
Groups of young and aged mice were injected intraperitoneally with 107 heat-killed C. albicansATCC 26555 yeast cells and exudate peritoneal cells were harvested at 4 h (when the bulk of neutrophils reaches the peritoneal cavity) and 72 h (when the bulk of macrophages reaches the peritoneal cavity). Total recruited cells at 4 and 72 h from young mice were more potent to kill C. albicans PCA2 yeasts than recruited cells from aged mice (Table 1). However, neutrophils (Ly-6G+) and macrophages (CD11b+), purified from the total recruited population of cells, from both young and aged mice, were equally potent to kill C. albicans cells (Table 1). These results suggest differences in the recruitment of phagocytotic cells between young and aged mice.
Table 1. Killing of Candida albicans yeasts by phagocytotic cells from young and aged mice
% Killing=[1−((CFU sample (t=2 or 4 h)/(CFU control (t=2 or 4 h))] × 100.
In order to investigate whether aged mice have a decreased recruitment of neutrophils and macrophages to the peritoneal cavity, we quantified the percentage and the total amount of cells in the peritoneal cavity of young and aged mice at 0, 4, and 72 h after challenge (Table 2). The results showed that the percentage of recruited neutrophils (at 4 h) and recruited macrophages (at 72 h) were lower in aged mice when compared with young mice; however, as the total amount of cells in the peritoneal cavity is significantly higher in aged mice (four- to fivefold increase), the total number of recruited neutrophils and recruited macrophages is actually higher (about a threefold increase) in aged mice. In addition, the percentage of resident peritoneal macrophages (F4/80+) in aged mice is lower than that in young mice, but as the total number of cells is higher, also the total number of resident macrophages is actually higher in aged mice (threefold increase). Interestingly, the population of F4/80− resident peritoneal cells, which are present in aged mice, were positive for MHC class II (data not shown).
Table 2. Percentage of neutrophils and macrophages in the peritoneal cavity of young and aged mice
Total peritoneal cells
% Ly-6G+ cells
% F4/80+ cells
Time, hours after intraperitoneal injection of 107 heat-killed Candida albicans yeasts; total peritoneal cells, average number of peritoneal cells per mouse; %Ly-6G+ cells, percentage of neutrophils in the peritoneal cavity; % F4/80+ cells, percentage of macrophages in the peritoneal cavity. *P<0.05 ; **P<0.01.
The candidacidal activity of resident peritoneal macrophages (CD11b+) was similar in young and aged mice (Table 1); however, the resident peritoneal cells from aged mice, when depleted of CD11b+ cells, showed a diminished fungicidal activity (48.8±2.8% of killing). Therefore, this CD11b− population present in aged mice may account for the lower fungicidal activity of the peritoneal cells from these mice. Finally, neutrophils purified from the bone marrow of young and aged mice were equally potent to kill C. albicans yeasts (Table 1).
Cytokine production by macrophages in response to C. albicans
We have previously reported that in vitro TNF-α production by adherent resident peritoneal cells, in response to C. albicans, was significantly lower by cells from aged mice compared with cells from young mice (Murciano et al., 2006). Taking into account that aged mice have a high number of cells in the peritoneal cavity that lack membrane molecules (F4/80 and CD11b) and are poorly fungicidal, we decided to analyze cytokine production by CD11b+-purified cells from young and aged mice. As described for total resident peritoneal cells, macrophages (CD11b+) from aged mice showed severely impaired production of TNF-α in response to all stimuli tested: zymosan, yeasts cells, and hyphae (Fig. 1a). The production of other proinflammatory cytokines (IL-6 and IL-1β) was also greatly diminished in aged CD11b+ macrophages in response to all stimuli tested (Fig. 1b and c).
We also analyzed the production of MIP-2, a CXC chemokine that causes marked neutrophil chemotaxis and activation. MIP-2 production by macrophages (CD11b+) from aged mice in response to yeasts and hyphae was significantly impaired, compared with production by macrophages from young animals (Fig. 2). In contrast to proinflammatory cytokines and MIP-2, production of IL-10, an anti-inflammatory cytokine, was unaffected in aged macrophages in response to C. albicans yeasts and hyphae (Fig. 3). The production of IL-10, in response to zymosan, was enhanced in aged macrophages as compared with young macrophages (Fig. 3). In all cases, supernatants from negative controls showed undetectable levels of cytokines.
Analysis of TLR2 expression by flow cytometry
As we previously reported that TLR2 is the main TLR triggering in vitro cytokine production by macrophages in response to C. albicans (Villamón et al., 2004a, c; Gil & Gozalbo, 2006), we determined the baseline surface expression of TLR2 by flow cytometry in total resident peritoneal cells and on CD11b+ resident peritoneal cells from aged and control mice (Fig. 4). The results showed that both the percentage of positive cells and the fluorescence mean intensity were lower in total resident peritoneal cells from aged mice than in cells from young mice. Similarly, when the analysis was performed in CD11b+-purified cells, the TLR2 expression was also lower in cells from aged mice.
We have previously described that aging significantly increases the overall susceptibility of mice to systemic C. albicans infection (Murciano et al., 2006). As phagocytes (neutrophils, monocytes, and tissue macrophages) clearly play a role in host resistance to invasive C. albicans infections (Calderone, 2001; Romani, 2004), in this work we have studied whether there are defects in the ability of aged phagocytotic cells to kill C. albicans in vitro. For this, we performed survival assays of yeasts in the presence of phagocytes from aged and young mice. Our first set of experiments, using as phagocytes the total recruited cells to the peritoneal cavity after C. albicans injection, indicated that young cells were more potent to kill the yeasts than aged cells. However, purified neutrophils (Ly-6G+, from recruited cells to the peritoneal cavity or from the bone marrow) and purified macrophages (CD11b+, from recruited or resident peritoneal cells) were able to kill the fungus equally. Therefore, this experimental approach showed no significant differences between the fungicidal activity of neutrophils and macrophages from aged and young C57BL/6 mice, suggesting that phagocytes from aged individuals can normally kill C. albicans cells. This is in accordance with our previous results showing no significant differences in the fungicidal activity between human blood from old and young donors (Murciano et al., 2007a).
Nevertheless, it should be noted that aged mice accumulate a higher amount of cells (a four- to fivefold increase) in the peritoneal cavity than young mice, and that this increase does not correlate with the weight of the mice (1.4 increase). In the peritoneal cavity of aged mice there is a significant population of cells (43.3%) that are F4/80−, MHC class II+ that showed a poor ability to kill C. albicans cells. Although we cannot determine the origin of these cells, it can be suggested that they are macrophages that have lost some membrane molecules. Interestingly, C. albicans uptake (measured by a flow cytometry assay) by total peritoneal cells from aged mice was not modified as compared with the uptake by cells from young animals (data not shown). Therefore, it can be suggested that when these cells internalize yeasts, probably prevent their internalization by the more potent fungicidal macrophages, and in this way hamper the clearance of the pathogen by the aged host.
Proinflammatory cytokines secreted by activated macrophages are critical in protecting the host against disseminated candidiasis (Calderone, 2001; Romani, 2004). Therefore, we determined the effect of aging on signaling cellular pathways for proinflammatory cytokine production upon C. albicans recognition by purified (CD11b+) macrophages. Our results clearly show a reduction in the in vitro TNF-α, IL-6, and IL-1β production by resident peritoneal macrophages (CD11b+) from aged mice in response to both C. albicans yeasts and hyphae as well as in response to zymosan. The production of MIP-2 was also decreased in aged macrophages, although, as mentioned above, aged mice appear to recruit properly functional neutrophils. By contrast, IL-10 production by aged macrophages was increased in response to zymosan and unaltered in response to C. albicans. Therefore, the balance of proinflammatory and anti-inflammatory signaling in response to C. albicans is clearly altered in aged mice and biased toward the anti-inflammatory profile. The decreased production of proinflammatory cytokines together with a sustained level of anti-inflammatory cytokines may result in defective development of the protective cellular (Th1) and humoral adaptive immune response against C. albicans in aged mice. In fact, we have previously described that aged mice develop a lower specific protective Th1 response, which resulted in lower levels of IFN-γ in a model of experimental candidiasis (Murciano et al., 2006). Because TLR2 has been described as the main TLR triggering TNF-α production by macrophages in response to C. albicans (Villamón et al., 2004b, c; Gil & Gozalbo, 2006), we investigated TLR2 expression at the surface of resident peritoneal macrophages, both in total cells and in CD11b+ cells, from aged and young mice. We found a decrease in the proportion of macrophages expressing TLR2 in aged mice, and similar results were obtained when the mean fluorescence intensity of TLR2 was measured.
Our results are in line with several reports supporting the notion of an age-associated alteration in TLR responses in older mice. It has been described that aged macrophages secrete significantly lower levels of proinflammatory cytokines than young macrophages in response to some ligands of TLRs (Renshaw et al., 2002; Boehmer et al., 2004, 2005). However, different explanations have been reported for this observation. Renshaw et al. (2002) found that macrophages from aged mice express lower levels of all 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 signaling. These changes were further evaluated in a microarray study of genes expressed in macrophages from young and aged mice stimulated with lipopolysaccharide (Chelvarajan et al., 2006). It was found that macrophages from aged mice have a global defect in the TLR signaling pathway and in the production of proinflammatory cytokines and chemokines, whereas the production of anti-inflammatory cytokines is increased. Although downstream signaling components, such as MyD88, and several members of the NF-κB pathway are reduced in aged mice, the expression of several TLR members (TLR4, TLR6, and TLR9) in macrophages from aged mice is similar to that of macrophages from the young mice, with the exception of TLR2 that decreases (Chelvarajan et al., 2006). Although most of the evidence, as cited above, indicates an altered function of TLRs in old mice, a recent study showed that myeloid dentritic cells and macrophages from aged mice have intact TLR signaling as compared with cells from young mice (Tesar et al., 2006).
In summary, we found that macrophages from aged mice have an in vitro defective synthesis of proinflammatory cytokines and chemokines but an unaltered secretion of anti-inflammatory cytokines, in response to yeasts and hyphae of C. albicans, when compared with control young mice. This defective proinflammatory secretion by resident peritoneal macrophages from old mice is correlated with a lower expression of TLR2. The balance of proinflammatory and anti-inflammatory signaling may be therefore biased toward the anti-inflammatory response in old mice. However, the fungicidal activity of different phagocytotic cells is well preserved with aging. These consequences of aging on TLR function may impair activation of the protective immune response and contribute to greater morbidity and mortality from candidiasis in older adults.
This work was supported by grant PI04/1472 (Instituto de Salud Carlos III; Ministerio de Sanidad y Consumo, Spain) and ACOMP06/43 and ACOMP07/073 (Conselleria d'Empresa, Universitat i Ciència, Generalitat Valenciana). C.M. and A.Y. are recipients of fellowships from the Ministerio de Educación y Ciencia.