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J. Med. Microbiol. - Vol. 48 (1999), 1095-1 102 0 1999 The Pathological Society of Great Britain and Ireland ISSN 0022-26 15 MYCOLOGY Acute susceptibility of aged mice to infection with Candida albicans ROBERT B. ASHMAN, JOHN M. PAPADIMITRIOU and A L M A FULURIJA School of Dentistry, University of Queensland, Brisbane, Queensland 4072 and Pathology Department, University of Western Australia, Nedlands, WA 6009, Australia The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity. Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice. Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-y and tumour necrosis factor-a cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals. Introduction Immunocompetence declines with advancing age [ 11 and this is associated with a general increase in susceptibility to infection. Epidemiological investigations have shown that the elderly are at higher risk for pneumococcal and other respiratory infections [2], and experimental studies have demonstrated an increased susceptibility of aging mice to organisms as diverse as Leishmania major [3], Trypanosoma musculi [4], Legionella pneumophila [5] and Sendai virus [6]. Candida albicans is an opportunist yeast that is carried as a commensal by the majority of the human population. Mucosal candidosis (oral and vaginal thrush) is common in the general population, although chronic infections usually occur in conjunction with deficiencies or abnormalities in the cell-mediated immune response [7, 81. Systemic, or disseminated candidosis is rare outside the hospital environment and Received 20 Oct. 1998; revised version received 26 April 1999; accepted 30 April 1999. Corresponding author: Professor R. B. Ashman (e-mail: [email protected]). is typically associated with dysfbnctional neutrophils [9] or neutropenia [lo, 111. Oral infections with C. albicans are common in the elderly [ 121, but although advanced age increases susceptibility of mice to intravenous challenge with Cryptococcus neoformans [13], it is not a recognised factor for disseminated candidosis. This paper reports the effect of aging in CBA/CaH mice on susceptibility to systemic challenge with C. albicans and the effect of immunity induced as a consequence of infection with the yeast early in adult life on the severity of infection induced by re-challenge in aged animals. Materials and methods Mice Specific-pathogen-free female CBA/CaH mice were purchased from the Animal Resources Centre, Perth, Australia. Animal experiments were approved by the Animal Experimentation Ethics Committee of the University of Western Australia and performed in accordance with the NH&MRC/CSIRO/Australian Agricultural Council’s Code of Practice for the Care Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 1096 R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA. and Use of Animals for Experimental Purposes in Australia, 1985. Yeast preparation C. albicans isolate 3630 was obtained from the Mycology Reference Laboratory at the Royal North Shore Hospital, Sydney, Australia, and stored at -70°C in Sabouraud's broth with glycerol 15%. Yeasts were grown in Sabouraud's broth for 2 days at room temperature, with constant agitation. Blastospores were washed in phosphate-buffered saline (PBS) and adjusted to the appropriate concentration for inoculation. Infection and immunisation Mice were immunised with 3 X lo5 blastoconidia administered in 0.2 ml of PBS via the lateral tail vein at 6 weeks of age. The same yeast isolate, dose and route of administration were used both for infection of the aged non-immune mice and re-challenge of the aged immune animals. Histology Tissue samples were fixed in formalin, sectioned and stained with haematoxylin and eosin (H&E), or periodic acid-Schiff (PAS). The sections were coded, examined blind and re-evaluated when the code had been broken. Clearance of C. albicans Mice were killed at 1,4, 8 and 14 days after infection. A transverse slice 2-3 mm thick was cut from the central portion of the brain and placed in 1 ml of PBS. Additional brain tissue was stored in liquid nitrogen for mRNA extraction and analysis of cytokine gene expression. Each tissue sampled was weighed and disrupted with an Ultra Turrax T-25 homogeniser (IKA Labortechnik, Staufen, Germany) running at 13 500 rpm at room temperature. The samples were diluted appropriately and 100-pl volumes were plated on Sabouraud's agar containing chloramphenicol. The plates were incubated at 37°C for 2 days and the colonies were counted. Each determination was performed in duplicate in a minimum of five mice. The results were calculated as loglo c h / g of tissue. RNA preparation and reverse transcription Total cellular RNA was prepared from infected brain tissue with 'Ultraspec' RNA Isolation Reagent (Biotecx Laboratories, Houston, TX, USA) according to the manufacturer's instructions. The concentration and purity of the RNA samples were determined by spectrophotometry at 260 and 280nm. cDNA was prepared by reverse transcription of 2 pg of each RNA, with an oligo d(T)15 primer and AMV reverse transcriptase, according to the manufacturer's instruc- tions (Promega Corporation, Madison, WI, USA). Briefly, 5 m~ MgClz, 1 X reverse transcription buffer, 1 m~ each dNTP, 0.5 URNAasin, 15 UAMV reverse transcriptase and 0.5 pg oligo (dT)15 primer were incubated in a 20-pl reaction mix at 42°C for 1 h, heated to 99°C for 5 min, then cooled on ice. The cDNA was stored at -20°C until used. PCR Primer sequences for interferon-y (IFN-y), interleukin4 (IL-4), interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) were obtained from published data [14], and the PCR was performed as described previously [ 151. The amplification mix consisted of 50 ng DNA, 200 m d N T P s , 0.5 U Taq polymerase (Biotech International, Perth, Western Australia), 1X reaction buffer, either 1 m~ or 2 m~MgC12and either 1 ml or 2 ml of primers in a total volume of 25 pl. The mixture was overlaid with paraffin oil, then amplified with a PTC-100 thermal cycler (MJ Research, Waltham, MA, USA). The amplification protocol was 94"C, 1 min; 60"C, 2 min; 72"C, 2 min; for 35-40 cycles. Following amplification, 10 pl of product were analysed by electrophoresis through agarose 3% gels. The gels were stained with ethidium bromide and the bands were visualised with a W transilluminator. Competitive PCR for comparison of cytokine cDNA Plasmid MCQ (pMCQ) was generously donated by Dr Cornelia Platzer, Humboldt University, Berlin, Germany. This plasmid (control fragment) contains the primer sequences for interleukins 1-6, IFN-y, TNF, lymphotoxin and p-actin, arranged so that the products amplified by cytokine-specific primers differ in size from those amplified from the target DNA, and thus can be distinguished by electrophoresis through an agarose gel [ 161. Samples showing reactivity with cytokine primers were subjected to semi-quantitative analysis as described previously [ 151. Briefly, the cDNA was adjusted to equal concentrations by coamplification of a fixed amount of the cDNA with 10fold, and then two-fold, dilutions of the control fragment @MCQ), with primers for P-actin. For quantification and comparison of cytokine mRNA expression, equal amounts of cDNA were amplified in the presence of 10-fold and then three-fold dilutions of the control fragment. The linearised plasmid was used at dilutions ranging from lo3 to lo5 for the standardisation of the cDNA and from lo5 to lo8 for comparison of cytokine concentrations. The experiments were repeated three times. The end-points for the aged mice, expressed in logarithms, were subtracted from those for the young controls, and the resultant value gave a factor by which the cDNA concentrations in the aged mice were greater or less than those in the controls. A positive value indicated that the concentra- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 CANDIDIASIS IN AGING MICE tions in the aged mice were greater than in the young animals and vice versa. Statistical analysis The statistical significance of differences between groups in the number of yeast cells recovered from infected brains was determined by Scheffe’s test on the one-way analysis of variance. Cytokines were compared as follows. The mean and standard deviation of the factors by which the aged mice differed fiom the controls were calculated for each data set, and the hypothesis that the cDNA concentrations in young and aged mice were not significantly different was tested by determining whether the 95% confidence intervals included zero. Results Long-term effects of systemic infection Female CBA/CaH mice infected with 3 X lo5 C. albicans yeast cells at 6-8 weeks of age and a group of uninfected, age-matched control mice were maintained in conventional animal facilities for 16 months. The mean weight (30.2 SD 6.1 g) of the aged immune mice was significantly less (p<O.Ol) than that of the uninfected controls (35.2 SD 3.9 g), but they were otherwise active and healthy. Within 3 days after intravenous challenge with 3 X lo5 C. albicans yeast cells, the aged non-immune mice displayed visible signs of distress and this entire group was killed on day 4 for ethical reasons. In contrast, the old immune mice and a group of 6-week-old control mice given a similar challenge remained outwardly healthy and showed no unusual symptomatology at any time during the 14-day period of observation. Histology of the lesions The histological features of disseminated C. albicans infection in 6-8-week-old CBA/CaH mice were as described previously [ 171. Briefly, mycelia were readily detected in the brains of the young control mice on the first day after infection. They were present to a lesser extent in the kidney, but were rarely found in the heart, although a focal myocarditis had already developed. By day 4, the encephalitis, pyelonephritis and myocarditis had increased in severity. The brain exhibited abscesses (Fig. 1A) containing predominantly yeasts, with some mycelial growth forms, the latter being present in greater numbers than on day 1. At day 14, mycelia were difficult to find. The myocarditis had abated, but the encephalitis and pyelonephritis had increased in severity. On day 1 after infection of the aged non-immune mice, mycelia were seen in brain, kidney and heart, in numbers similar to those in the young controls. In the heart, scattered foci of myocardial necrosis were 1097 surrounded by a few inflammatory cells. By the fourth day of infection, the number of mycelia in the brain and kidney had increased markedly in comparison with those in the young mice, the brain being the most severely affected organ (Fig. 1B). Necrotic debris was seen in the vicinity of the mycelia, but only a few inflammatory cells were detected. Mycelia were not seen in the heart, but foci of myocardial necrosis, including calcified cardiac myocytes, were readily found (Fig. 2). Again, inflammatory cells were sparse. The aged immune mice were similar to the other two groups in terms of the numbers of mycelia in the brain and kidney at 24 h after infection. By the fourth day, the mycelial population in the brain (Fig. 1C) and kidney had increased, but to a lesser extent than in the aged non-immune mice. Polymorphonuclear leucocytes (PMNLs) were easily detected at the periphery of the mycelial aggregates in the brain (Fig. 3), while in the heart they were present as focal infiltrates in the interstitium. Abscesses had formed in both brain and kidney at this time point, but the myocarditis remained unchanged. Calcified myocytes were detected, but at a much lower fiequency than in the non-immune aged mice. By day 14, mycelia were only detectable in the kidney. The focal encephalitis, pyelonephritis and myocarditis were reduced in intensity compared with the aged non-immune mice, but were more severe than lesions in the young controls at the same time point. Kinetics of fungal clearance The fungal burden in the brain of both the aged nonimmune and the aged immune mice was significantly less than in the young mice on the first day after infection ( p < O . O l ) , but by day 4 the number of colonies recovered from the brains of aged nonimmune mice was significantly greater (p < 0.01) than that in the other two groups (Fig. 4). After this time, the number of colonies recovered from the brains of the aged immune mice declined to levels similar to those in the young controls. Comparison of cytokine cDNA proJiles It was of interest to determine whether the increased susceptibility of the aged mice was associated with a change in the relative gene expression in the infected brain of cytokines that are known to play a part in host defence against C. albicans infection. The cytokine profiles were assessed by comparison of the end-points of competitive PCR assays. The relative amounts of IFN-y and TNF-a cDNA in the aged non-immune mice were not different fiom those in the young controls (Fig. 5 ) , but IL-6 cDNA levels were significantly higher in the aged infected mice. However, comparison of IL6 gene expression in young and aged uninfected mice showed a 3-5-fold increase in aged animals (data not shown). There were no significant differences between aged immune mice and the young controls in the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 1098 R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA. Fig. 1. (A) Brain of a 6-8-week-old CBA/CaH mouse 4 days after i.v. challenge with 3 X lo5 C. albicans yeast cells. A large abscess contains fungal elements and inflammatory infiltrates. (B) Brain of aged non-immune CBA/CaH mouse 4 days after challenge as above. There is extensive fungal proliferation, with prominent mycelial elements. Few inflammatory cells are present in the lesion. (C) Brain of aged immune CBA/CaH mouse, 4 days after challenge as above. An abscess shows yeast and mycelial forms, and a dense inflammatory infiltrate. All sections stained with periodic acid-Schiff (X380). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 CANDIDIASIS IN AGING MICE 1099 Fig. 2. Aggregates of calcified myocytes (arrows) in the heart of an aging CBA/CaH mouse, 4 days after intravenous challenge with 3 X lo5 C. albicans yeast cells. (X160). Fig. 3. Brain of aged immune mouse 4 days after intravenous challenge with 3 X lo5 C. albicans yeast cells. Inflammatory cells can be seen scattered around the focus of fungal infection. H&E (X400). relative expression of IL-6, IFN-y or TNF-a (data not shown). IL-4 was detected in the brains of all three control mice on day 4 after infection, but was not found in either the aged non-immune (Fig. 6) or aged immune mice (data not shown) at any time point. Discussion Systemic infection with C. albicans in aged mice causes acute disease and early death. The rapid onset of morbidity, within 2-3 days after challenge, suggested that the animals may have suffered some kind of septic shock, a condition that has also been associated with candidaemia in elderly patients [ 181. Although there was a quantitative increase in the fungal burden in the brains of the aged non-immune mice at 4 days after challenge, the striking feature of the infection in these animals was the widespread appearance of mycelial growth forms, which are potent stimulators of TNF-a production by both macrophage cell lines [ 191 and macrophages obtained from different anatomical regions throughout the body POI * Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 1100 R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA. Fig.4.Number of C. albicans in the brains of normal (young; o), aged non-immune (FA),and aged immune (m) CBA/CaH mice, at various times after intravenous challenge with 3 X lo5 yeast cells. Each bar shows the mean and SEM of loglo cfu/gram of tissue. Fig. 5. Relative amounts of cytokine cDNA in the brain of aged non-immune CBA/CaH mice: IFN-y (day 1, 0; day 4, o), IL-6 (day 1, 0;day 4, m), TNF-a (day 1, A; day 4, A). Each point represents the mean and 95% confidence intervals of the difference in cytokine cDNA concentrations between old and young animals, as estimated by competitive PCR with equivalent amounts of cDNA. mRNA was prepared separately from each of three mice per group and each determination was carried out at least twice. Fig. 6. IL-4 gene expression in brain tissue of young (lanes 1-3) and aged non-immune (4-6) mice 4 days after intravenous challenge with 3 x 105 c. albicans yeast cells. TNF-a is a known mediator of septic shock syndromes [21] and accumulates to high levels in the serum of infected mice [22,23]. Although aged mice are more susceptible than younger mice to the lethal toxicity of TNF-a [24], the relative levels of TNF-a cDNA in the brains of the aging non-immune mice were not significantly different from those in the Young control group. This suggested that TNF-a was not implicated in the early morbidity and mortality of these animals. A recent study of C. aEbicans septic Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 15 May 2017 09:49:29 CANDIDIASIS IN AGING MICE shock syndrome in rats [25] also excluded TNF-a as a mediator and the authors of the latter study suggested that the syndrome was mediated either by host cytokines other than TNF-a, or by circulating fungal virulence factors. As mice age, the concentration of leucocytes in the blood rises [26]. This could account for the transient reduction in the number of viable yeasts in the brain of both the aged non-immune and aged immune mice, as compared with the controls, on the first day after infection. However, the ability to mobilise PMNLs from the marrow into blood in the elderly is markedly reduced when compared with young subjects [27] and PMNLs from older individuals also exhibit a pronounced impairment of their chemotactic, phagocytic and killing capacities [28]. PMNLs from aging rats show a reduction in bactericidal capacity and in the generation of oxygen radicals, after stimulation with low levels of IFN-y [29]. Hyphal growth forms of the yeast are readily susceptible to killing by neutrophils [30], and the marked increase in mycelial elements seen in the aging nonimmune mice might have reflected a quantitative or qualitative reduction in the contribution of this cell type to resolution of the lesions [31]. The aging mice also displayed foci of myocardial calcification and necrosis, which have otherwise only been observed in mice depleted of neutrophils [32]. There is increasing evidence that neutrophils are the major effector mechanism mediating host responses against the yeast [33-351, so a gradual decline in neutrophil function may account both for the extent of tissue invasion in the aged non-immune animals and the predominance of mycelial forms throughout the later stages of infection in the aged immune mice. Neutrophil activation for killing of C. albicans is mediated predominantly by IFN-y and TNF-a, acting either separately or synergically [36]. However, the relative gene expression of these two cytokines in the brains of aged non-immune mice was similar to that in the young controls. This tended to exclude a failure of cytokine-mediated activation as a basis for the increased susceptibility of the aged non-immune animals. Nevertheless, cytokine production and function in C. albicans infection are subject to complex regulation, particularly by cytokines of the T-helper type 2 (Th2) subset [37]. Of these, IL-4 was detected in the young but not in the aged non-immune or aged immune animals, although the relevance of this host resistance is unknown. In contrast, IL-6 was expressed in significantly higher amounts in the brains of aged non-immune mice when compared with young controls, but levels in aged uninfected mice were also elevated. The cellular origin of the increased IL-6 in the aged mice, and whether its presence is a cause or a consequence of the increased susceptibility, remain to be determined. 1101 The induction of protective immunity consequent upon recovery from an initial sublethal infection is most readily demonstrated in mice of the CBA/CaH strain [38, 391. The present data confirm that protective immunity elicited by systemic infection of young mice persists for at least 12 months, and probably for the life of the mouse. Colony counts in the brains of the aged immune mice were reduced by about 0.5 loglo relative to the aged non-immune group, which was similar to the effect achieved by immunisation of young animals [3 81. Nevertheless, the increased susceptibility demonstrated by the aged non-immune mice was also evident in the aged immune mice when compared with the responses of younger animals. It is unclear whether this reflects an overall decline in the candidacidal potential of phagocytic effector cells, or a reduction in function of some of the antibody-dependent mechanisms that accelerate clearance of the yeast in immune mice. In summary, these studies in the mouse provide evidence that the changes in host defence mechanisms associated with advanced age markedly increase the severity of disseminated candidosis. 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