Suppression of anti-candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stress

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1 ORIGINAL ARTICLE Microbiol Immunol 2009; 53: doi: /j x Suppression of anti-candida activity of macrophages by a quorum-sensing molecule, farnesol, through induction of oxidative stress Shigeru Abe 1,2, Rumi Tsunashima 1,3, Ryosuke Iijima 4, Tsuyoshi Yamada 1, Naho Maruyama 1, Tatsuya Hisajima 1, Yoshie Abe 1, Haruyuki Oshima 3 and Masatoshi Yamazaki 4 1 Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo , Japan; 2 Faculty of Medical Technology, Teikyo University, 359 Otsuka, Hachioji, Tokyo , Japan; 3 Department of Bioscience, School of Science and Engineering, Teikyo University, 1-1, Toyosatodai, Utsunomiya-shi, Tochigi , Japan; and 4 Faculty of Pharmaceutical Sciences, Teikyo University, Suarashi Sagamiko, Sagamihara Kanagawa , Japan ABSTRACT Farnesol is well known as a quorum-sensing molecule of Candida albicans. To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro. Murine macrophages, when cultured in the presence of μm of farnesol for 1 2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti-oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol-treated macrophages was shown by fluorescence of DCFH-DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti-candida activity, perhaps through induction of ROS. Key words anti-oxidants, pathogenic factor, terpenoid alcohol. Farnesol is known as a quorum-sensing molecule produced by Candida albicans (1). The wide range of concentration (2 100 μm) of this sesquiterpene alcohol in culture medium of C. albicans inhibits the morphological change of the fungus from yeasts to hyphae without influencing growth rate (2). The effects of farnesol on the morphological signaling pathway of C. albicans have been studied: increased expression of the hyphal formationassociated gene, TUP1 (3), and regulation of the twocomponent signal transduction pathway (4). Roles of this molecule in physiological or pathological interactions between Candida and hosts have been only partially elucidated. Navaranthna et al. (5) reported that C. albicans with increased ability of farnesol produc- tion enhanced its pathogenicity for systemic candidiasis in mice. They also reported that farnesol had pathogenic activity to systemic candidiasis in mice (6), and that i.p. administration of farnesol to mice suppressed lymphoid cell functions, such as cytokine production and its activities (7). We know that the first-line defense against Candida invasion in mucus and various organs mainly consists of phagocytes such as neutrophils and macrophages (8). In particular, macrophages with a relatively long lifespan around infected lesions work not only as effectors but also as modulators in host defense mechanisms. The first purpose of the present study was to determine whether farnesol affects effector activities of macrophages Correspondence Shigeru Abe, Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo , Japan. Tel: ; fax: ; sabe@main.teikyo-u.ac.jp Received 4 November 2008; revised 23 January 2009; accepted 8 February List of Abbreviations: CV, crystal violet; DCF, 2,7 -dichlorofluorescein; DCFH-DA, 2,7 -dichlorodihydrofluorescein diacetate; DMSO, dimethylsulfoxide; LDH, L-lactate dehydrogenase; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SDS, sodium dodecylsulfate. c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 323

2 S. Abe et al. against C. albicans. The second purpose was to examine the cytotoxicity of farnesol to macrophages. Here we observed that farnesol suppressed anti-candida activity of murine macrophages in vitro and that the suppression might be caused by the cytotoxicity of farnesol to macrophages, which was coupled with apoptosis and production of active oxygens. On the basis of these results, the roles of farnesol in the interaction between host macrophages and C. albicans were discussed. MATERIALS AND METHODS Agents Trans,trans-farnesol (Sigma-Aldrich, St Louis, MO, USA) was solubilized to 10% solution by DMSO, then further diluted by RPMI-1640 medium containing 1% fetal calf serum (complete medium). Probucol (Wako Pure Chemical, Osaka, Japan) and a semi-hydrophilic derivative of tocopherol, trolox (Sigma-Aldrich) were solubilized to 100 mm by DMSO and further diluted by the complete medium. Animals All animal experiments were carried out according to the guidelines for the care and use of animals approved by Teikyo University. Six-week-old male ICR mice (Charles River Japan, Inc., Kanagawa, Japan) were used for macrophagepreparation.thephotoperiodswereadjusted to 12 hr of light and 12 hr darkness daily, and the environmental temperature was constantly maintained at 21 C. The mice were kept in cages housing four to six animals and were given ad libitum access to food and water. Murine macrophage preparation Murine macrophages were prepared from peritoneal exudate as described previously (9). ICR mice were injected i.p.with0.2mlof0.5%glycogen(wakopurechemical)in saline. Twenty hours later, peritoneal cells were collected. After centrifugation at 430 g at 4 C for 5 min, the precipitate was suspended in the complete medium and seeded at cells/0.2 ml into 96-well flat-bottomed microplates. For measurement of cellular peroxide and DNA fragmentation, the macrophage suspension was seeded at and cells/3 ml into a Petri dish (35 mm diameter), and for phagocytosis assay, cells/0.4 ml onto a Lab-Tek R Chamber Slide TM (Nalge Nunc International Corp., Naperville, IL, USA). The plates were incubated for 2 hr at 37 C, 5% CO 2, and were then washed with warm PBS to remove non-adherent cells. The resulting monolayer consisted of macrophages (>95%) as described previously (9). Candida albicans The clinically isolated strain of C. albicans, TIMM1768, was maintained in our laboratory and was stored at 80 C in Sabouraud dextrose broth (Becton Dickinson, Sparks, MD, USA) containing 0.5% yeast extract (Becton Dickinson) and 10% glycerol until the experiment was carried out. TIMM1768 was washed with the complete medium by centrifugation (430 g, 3 min, 4 C) and suspended in cells/ml. For phagocytosis assay, 2 ml heat-killed C. albicans ( cells/ml) was opsonized by pre-incubation with 0.8 ml of fresh mouse serum for 15 min. Assay of anti-candida activity of macrophages A CV staining assay (10) was carried out to determine the macrophage-mediated inhibition of the mycelia growth of C. albicans. Murine macrophage monolayer was pre-incubated with 100 μl of various concentrations of farnesol (final concentrations: 28, 56, 90, 112 μm) with 100 μl ofcomplete medium containing anti-oxidants at 37 C, 5% CO 2 for 1 hr. After the pre-incubation, the macrophages were washed three times by the complete medium. Then, 100 μl of the complete medium and 100 μl of cells/ml of C. albicans cells were poured into these macrophage wells, and the cells were incubated at 37 C, 5% CO 2 for 16 hr. The adherent mycelial Candida cells were sterilized by immersion of the plates in 70% ethanol, and the macrophages were washed out with 100 μl of % SDS. Each well was washed with water three times and the mycelia were stained with 100 μl of 0.02% CV for 15 min. After washing with distilled water, mycelia-binding CV was solubilized by the addition of 150 μl isopropanol including 0.04 N HCl and 50 μl of 0.25% SDS, and the optical density at 620 nm of the samples was measured photometrically. The relative mycelial growth percent of Candida was calculated as follows: {[absorbance (Candida withmacrophages) absorbance (macrophages alone)]/absorbance (Candida alone) 100} (%). Thevalues of relative growth percent of Candida have been confirmed to correspond to the percent of Candida estimated by the [ 3 H]-glucose incorporation method (10). Assay of phagocytosis activity of macrophages Murine macrophage monolayer was incubated with 100 μl of various concentrations of farnesol (final concentrations; 28, 56, 90 μm), 140 μl opsonized C. albicans (final concentration: cells/ml) and 160 μl ofcomplete medium at 37 C for 1 hr. After the incubation, the macrophages were washed by the complete medium. The 324 c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

3 Suppression of macrophage by farnesol phagocytosis of macrophages was observed after staining by the addition of a drop of 0.4% Trypan blue in 0.2% eosin yellow solution. Assay of farnesol cytotoxicity to macrophages Cytotoxicity was assessed by LDH release assay (11). Murine macrophage monolayer was cultured with 100 μl farnesol (final concentrations: 28, 56, 90, 112 μm) with or without 100 μl probucol (final concentrations: 25, 50 μm) and trolox (final concentrations: 1, 5, 25 mm) at 37 Cfor 10 min to 4 hr. The activities of LDH in the culture supernatants, which were released from the cytosol of the damaged macrophages, were measured using a Cytotox 96 non-radioactive cytotoxicity assay kit (Promega Corporation, Madison, WI, USA). Cytotoxic action of farnesol was observed microscopically using a Lab-Tek R Chamber Slide TM System (Nalge Nunc International). The macrophages cultured in the slide chamber were stained by Diff-Quick (International Reagents Corp., Hyogo, Japan). DNA fragmentation assay DNA fragmentation of macrophages was checked as described previously (12). Macrophage monolayers were cultured in the presence or absence of farnesol (final concentrations: 56 μm) and probucol (final concentrations: 50, 100 μm) at 37 C, 5% CO 2 for 2 hr in Petri dishes. After the culture, adherent macrophages were recovered by scraping and washed twice with PBS and collected by centrifugation (430 g, 5 min, 4 C). These cells were suspended in 200 μl PBS, and then subjected to total DNA extraction with a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer s instructions. Aliquots of 2 μg of the obtained DNA were separated by electrophoresis on 0.8% (w/v) agarose gels, stained with ethidium bromide and visualized under UV irradiation. Detection of intracellular ROS generation As a parameter of ROS generation, production of intracellular peroxide after farnesol treatment was analyzed using a fluorescent dye, DCFH-DA (Invitrogen Corporation, Carlsbad, CA, USA) as described previously (13). DCFH-DA is a non-polar compound that readily diffuses into cells and is then converted into a non-fluorescent polarderivativedcfhbyesterases.dcfhismembrane impermeable and is easily oxidized to the highly fluorescent compound DCF by the presence of intracellular ROS, especially hydrogen peroxide. Before farnesol treatment, the medium was replaced with Hanks solution containing 5 μm DCFH-DA for 5 min. After DCFH-DA incubation, cells were washed with PBS and then cultured for 2 hr with complete medium containing farnesol and other agents. The DCF fluorescence was observed by a Leica DMR-HC fluorescence microscope (Leica Microsystems, Heidelberg, Germany) excitation at 488 nm. Measurement of cellular peroxide The peroxide accumulation in the farnesol-treated cells was measured with a PeroxiDetect TM KIT (Sigma- Aldrich). Peroxides convert Fe 2+ to Fe 3+ ions under acidic conditions, and these Fe 3+ ions form a blue-colored adduct of xylenol orange (14). Macrophage monolayers were cultured in the presence of farnesol and other reagents for 2 hr in Petri dishes. After the culture, adherent macrophages were recovered by scraping and washed twice with PBS. The packed cells obtained by centrifugation (1710 g, 5 min) were extracted with 95% methanol 5% water. Nine volumes of organic peroxide color reagent were then added, the mixture was incubated at room temperature for 30 min and the optical density at 570 nm was measured. Tert - butylhydroperoxidewasusedasastandardcompoundof hydroperoxides. Statistical analysis The results were expressed by the mean ± standard deviation. The data were statistically compared using the Students t-test. RESULTS Suppression of macrophage function by farnesol in vitro We previously (10) reported that macrophages effectively inhibited the mycelial growth of C. albicans. The effects of farnesol on anti-candida activities of murine macrophages were examined in vitro. Murine macrophages inhibited mycelial growth of C. albicans to less than 40% (Fig. 1a). When the macrophages were preincubated with more than 56 μm farnesol for 1 hr, their growth inhibitory activity against Candida was suppressed significantly. As farnesol was washed out before addition of C. albicans in this experimental condition, this result means that farnesol suppressed the anti-candida activity of the macrophages. In order to determine the role of ROS in this suppressive action, effects of several anti-oxidants were examined. Figure 1a also shows that the addition of the anti-oxidant, probucol (50 μm), to the medium attenuated the suppressive effect of farnesol on the macrophage function. c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 325

4 S. Abe et al. These results were confirmed microscopically. As shown in Figure 1b, macrophages inhibited the mycelial growth of C. albicans, which was clearly blocked by pretreatment of macrophages with farnesol. This farnesol action seemed to be neutralized by coexistence with 50 μmprobucol. We also checked the effects of farnesol on the phagocytosis activity of macrophages. Macrophages revealed phagocytosis of Candida in 1 hr incubation. When macrophages were incubated with more than 28 μm farnesol and Candida simultaneously, the phagocytosis activity was suppressed dose dependently and the macrophages seemed to be killed by approximately 90 μm farnesol (data not shown). Cytotoxicity of farnesol to macrophages Cytotoxic action of farnesol against murine macrophages was examined by LDH release test. Figure 2a clearly shows that 2 hr culture in the presence of 90 μm farnesol caused the release of LDH from macrophages. This release was dose-dependently blocked by the anti-oxidant probucol or trolox. The effects of concentration and incubation time of farnesol on the cytotoxicity were also examined. As shown in Figure 2b, when macrophages were incubated with Fig. 1. Inhibition of Candida mycelial growth by macrophages and the effects of farnesol and/or probucol pretreatment on the anti-candida activity of macrophages. Murine macrophage monolayers were preincubated with farnesol and/or probucol for 2 hr, then washed out. Cell suspensions of C. albicans were added to the monolayer and cultured for 16 hr. (A) Relative mycelial growth of C. albicans measured by crystal violet staining as described in Materials and Methods., control;, without probucol;,with50μmprobucol; P < (B) Microscopic observation of mycelial growth of C. albicans just after 16 hr incubation. Colonies with spikes (black arrows) represent mycelial growth of C. albicans and small dots dispersed in the whole area (white arrows) are adherent macrophages. (a) Non-treated, (b) control, (c) pre-incubation with farnesol (112 μm), (d) pre-incubation with farnesol (112 μm) and probucol (50 μm). Bar = 0.2 mm. Fig. 2. Cytotoxicity of farnesol to macrophages. Murine macrophage monolayers were cultured in the presence or absence of farnesol for various periods within 4 hr. Cytotoxicity of macrophages was measured by the LDH release test. LDH release (%) was calculated as relative value to 100% release of detergent-treated cells as indicated by the manufacturer. P < (a) Macrophages were cultured in the presence of farnesol and/or probucol or trolox for 2 hr. (b) Time course., detergent treated;, control;,withfarnesol28μm;,withfarnesol56μm;, farnesol 90 μm. 326 c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

5 Suppression of macrophage by farnesol Fig. 3. Microscopic feature of macrophages cultured in the presence of farnesol with or without probucol or trolox. (a) Control, (b) farnesol (90 μm), (c) farnesol (90 μm) with probucol (50 μm), (d) farnesol (90 μm) with trolox (25 mm). Bar = 10 μm. Arrows indicate blebbing cell surface. 90 μm farnesol, their release of LDH began to increase from 30 min and was extremely enhanced after 1.5 hr of culture. In the case of 56 μm farnesol, the release increased from 1.5 hr, and 4 hr was required to elicit a strong cytotoxic reaction. This result indicated that suppression of anti-candida activity of macrophages by farnesol may be explained by its cytotoxic action to macrophages. This cytotoxic action of farnesol was observed microscopically. As shown in Figure 3, the macrophages cultured with 90 μm farnesol for 2 hr lost their spindle shape. Their major parts showed obscure round outlines and the others displayed blebbing on the cell surface. The nucleus of these cells revealed an abnormal figure suggesting disassembly of nuclei. These suggest apoptotic change. Co-presence of 50 μm probucol or 25 mm trolox partially protected them from decay of the cell structure. These results mean that farnesol had a cytotoxic effect against macrophages, perhaps through induction of apoptosis and that this effect could be blocked by anti-oxidants. Fig. 4. DNA fragmentation of macrophages induced by farnesol. Murine macrophages were incubated at 37 C for 2 hr with or without farnesol and/or probucol. Cells were then collected, and total DNA was extracted. Aliquots of 2 μg of the obtained DNA were separated by electrophoresis on 0.8% (w/v) agarose gels, stained with ethidium bromide and visualized under UV irradiation. DNA standard fragment sizes are shown on the left. ROS production in farnesol-treated macrophages The results of the experiments using anti-oxidants suggest the possibility that macrophages might be damaged through oxidative stress induced by farnesol. We microscopically examined the level of intra-macrophage ROS. The macrophages were pre-stained with a fluorescent dye, DCFH-DA, and then incubated with or without 90 μm farnesol for 2 hr. In the farnesol-treated macrophages, a rise of DCF fluorescent intensity was observed in comparison with control macrophages, and this fluorescence appeared to be partially suppressed when the cells were treated with farnesol and probucol (Fig. 5). DNA fragmentation of farnesol-treated macrophages In order to check apoptotic change of macrophages, degree of DNA fragmentation of the cells was examined by the gelelectrophoresis method. Figure 4 shows that the farnesol treatment of macrophages for 2 hr caused fragmentation of their DNA. This fragmentation seemed to be partially inhibited by cell culture with the addition of 100 μm probucol. Fig. 5. DCF fluorescence intensity of macrophages cultured in the presence of farnesol with or without probucol. (a) Control, (b) farnesol (90 μm), (c) farnesol (90 μm) with probucol (50 μm). Bar = 40 μm. c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 327

6 S. Abe et al. Fig. 6. Reactive oxygen production by the macrophages cultured in the presence of farnesol. Macrophages ( cells) were cultured in the presence of farnesol and/or anti-oxidants for 2 hr. They were obtained by scraping and washed. Peroxide production of the cells was measured by PeroxiDetect TM KIT. P < We assessed the oxidative stress of the macrophages by another ROS detection method, xylenol orange colorimetric quantitation. Figure 6 shows that 56 μm farnesol significantly increased peroxide production. It was drastically increased in the cells treated with 90 μm farnesol. This increase of peroxides was also inhibited significantly by the addition of 50 μmprobucolor25mmtrolox.the clear correlation between accumulation of cellular peroxides and cytotoxic responses suggests that the former reaction is coupled with cytotoxicity. DISCUSSION Macrophages are well recognized to play major effector and regulatory roles in host-fungus interaction around C. albicans-infected lesions (8). Biological activities of farnesol on macrophage functions have not been investigated, although this molecule is attracting our attention as a pathogenic regulator in not only mucocutaneous but also in systemic candidiasis (2, 15). Here, we showed that the pretreatment of murine macrophages by 56 μm farnesol clearly suppressed the inhibitory activity of mycelial growth of C. albicans and induced cell death as estimated by LDH release. As far as we know, this is the first report providing evidence that farnesol suppresses macrophage function and induces cell death of macrophages. In parallel with macrophage cytotoxicity, farnesol treatment rapidly induced DNA fragmentation of macrophages within 2 hr. These findings, including our observation of nuclear disassembly and membrane blebbing in the farnesol-treated macrophages as shown in Figure 3, suggest that farnesol induces apoptosis of macrophages. The apoptosis-inducing activity of farnesol to macrophages is not so unusual, as farnesol is known to cause apoptosis of various types of tumor cells (16 18). Rioja et al. (18) reported that 30 μm farnesol caused apoptosis of leukemic cells but not of primary monocytes. Although this finding seems contradictory to our own, we speculate that 30 μm farnesol might be just below the active concentration or that murine macrophages induced by glycogen injection were relatively sensitive to farnesol. We examined the effects of ROS-absorbants on farnesol actions for macrophages to learn the mechanism of farnesol cytotoxicity. The most important finding about these phenomena is that all activity of farnesol to macrophages can be blocked by the co-presence of the semi-hydrophilic anti-oxidants, probucol and trolox. In this context, we confirmed induction of ROS generation in farnesol-treated macrophages by elicitation of DCFH-DA fluorescence and increase of intracellular peroxides. The increase in these parameters of ROS generation was also inhibited by the anti-oxidants. We have measured ROS production in the medium of farnesolpretreated macrophage culture. It was below the detection sensitivity by the PeroxiDetect TM KIT (data not shown), so we think that ROS was produced mainly intracellularly and it rapidly diminished with a short half-life period. Therefore, we can propose the following hypothesis: farnesol may suppress macrophage functions and cause cell apoptosis through induction of intracellular oxidative stress. This hypothesis can also be supported by the finding by Machida et al. that farnesol accelerates ROS generation in the cell of Saccharomyces cerevisiae and that the growth inhibition by farnesol is blocked by semi-hydrophilic anti-oxidants (19). They also mentioned that farnesol produced ROS by inhibiting electron transformation in mitochondria (19), and Joo et al. reported that apoptosis of lung carcinoma cells was induced by farnesol which was coupled with endoplasmic reticulum stress (17). Farnesol might produce ROS in macrophages the same as in S. cerevisiae, but the precise mechanism of ROS production and macrophage apoptosis remains to be clarified. We have no information about the distribution of farnesol in Candida-infected tissues in vivo.hornby etal.reported that clinical strains of C. albicans produce farnesol at μg/g (dry weight of Candida cells), which is equivalent to a concentration of 2 to 4 μm farnesolata yeast cell density of 10 8 cells/ml. This level of production can be enhanced approximately 8 45-fold by the subinhibitory concentration of several antifungal agents with 328 c 2009 The Societies and Blackwell Publishing Asia Pty Ltd

7 Suppression of macrophage by farnesol inhibitory activity to sterol biosynthesis (20). Therefore, we can speculate that local concentration of this compound in boundary areas between Candida focus and host tissues may reach more than 56 μm, which showed suppressive activities to macrophages and cytotoxic activity to them in vivo. To date, the relationships of macrophages to the suppression of lymphocyte functions by farnesol reported by Navarathna et al. (7) have not yet been investigated. It was reported that the growth of C. albicans was not inhibited even in the presence of more than 200 μm farnesol (1). We speculate that farnesol may have a pathogenic role through inactivating macrophages around Candida-infected tissues without affecting the viability of C. albicans. In a preliminary experiment, we histologically observed the morphological changes of inflammatory phagocytes located near C. albicans foci in experimental murine oral candidiasis. In further experiments, we wish to determine whether or not macrophages around Candida-infected lesions induced apoptotic cell death. From another point of view, we wish to discuss the role of farnesol in host-fungus interaction, as we recently reported that giving oral farnesol alleviated local symptoms of murine oral candidiasis without a clear decrease of the viable cell number of C. albicans (15). In the seemingly protective activity of farnesol, disappearance of an inflammatory response in Candida-infected tongues was quite remarkable. This observation and the suppression of macrophage functions by farnesol reported here suggest that farnesol may show anti-inflammatory actions, which maintain a non-inflammatory condition of mucocutaneous membranes even when infected with C. albicans. Furthermore, we can speculate that this anti-inflammatory action of farnesol may contribute to the mechanisms of stable and long-term colonization of C. albicans as a normal microflora member in vertebrate intestinal mucocutaneous membranes. Our preliminary experimental results suggest that farnesol suppressed neutrophil functions in a similar manner to that in macrophages (unpublished data). This means that farnesol can locally suppress phagocyte functions, which can be blocked by the administration of antioxidants. However, this speculation should be checked by further study on the level of farnesol production in Candida-infected lesions and their cytotoxic activities for macrophages. Pharmacological actions of farnesol and/or anti-oxidants to mucocutaneous candidiasis are under investigation. REFERENCES 1. Hornby J.M., Jensen E.C., Lisec A.D., Tasto J.J., Jahnke B., Shoemaker R., Dussault P., Nickerson K.W. (2001) Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol 67: Nickerson K.W., Atkin A.L., Hornby J.M. (2006) Quorum sensing in dimorphic fungi: Farnesol and beyond. Appl Environ Microbiol 72: Cao Y.Y., Cao Y.B., Xu Z., Ying K., Li Y., Xie Y., Zhu Z.Y., Chen W.S., Jiang Y.Y. (2005) cdna microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol. Antimicrob Agents Chemother 49(2): Yamada-Okabe T., Mio T., Ono N., Kashima Y., Matsui M., Arisawa M., Yamada-Okabe H. (1999) Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans. JBacteriol181(23): Navarathna D.H., Hornby J.M., Hoerrmann N., Parkhurst A.M., Duhamel G.E., Nickerson K.W. (2005) Enhanced pathogenicity of Candida albicans pre-treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis. J Antimicrob Chemother 56: Navarathna D.H., Hornby J.M., Krishnan N., Parkhurst A., Duhamel G.E., Nickerson K.W. (2007) Effect of farnesol on a mouse model of systemic candidiasis, determined by use of a DPP3 knockout mutant of Candida albicans. Infect Immun 75: Navarathna D.H., Nickerson K.W., Duhamel G.E., Jerrels T.R., Petro T.M. (2007) Exogenous farnesol interferes with the normal progression of cytokine expression during candidiasis in a mouse model. Infect Immun 75: Vartivarian S., Smith C.B. (1993) Pathogenesis, host resistance, and predisposing factors. In:Bodey G.P., eds. Candidiasis, 2nd edn. New York: Raven Press, pp Tokuda Y., Tsuji M., Yamazaki M., Kimura S., Abe S., Yamaguchi H. (1993) Augmentation of murine tumor necrosis factor production by amphotericin B in vitro and in vivo. Antimicrob Agents Chemother 37(10): Abe S., Satoh Y., Tokuda Y., Tansho S., Yamaguchi H. (1994) A rapid colorimetric assay for determination of leukocyte-mediated inhibition of mycelial growth of Candida albicans. Microbiol Immunol 38: Yui S., Kudo T., Hodono K., Mimaki Y., Kuroda M., Sashida Y., Yamazaki M. (2003) Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin, securioside B against BAC1.2F5 macrophages. Mediators Inflamm 12(3): Morofuji S., Abe S., Okinaga K. (2005) Characterization of effector cells with anti-candida activity obtained from murine bone marrow cells cultured in the presence of rhg-csf: comparison between normal and CY-treated mice. Microbiol Immunol 49(9): Ohba M., Shibanuma M., Kuroki T., Nose K. (1994) Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. JCell Biol 126(4): Jiang Z.Y., Woollard A.C., Wolff S.P. (1990) Hydrogen peroxide production during experimental protein glycation. FEBS Lett 268(1): Hisajima T., Maruyama N., Tanabe Y., Ishibashi H., Yamada T., Makimura K., Nishiyama Y., Funakoshi K., Oshima H., Abe S. (2008) Protective effects of farnesol against oral candidiasis in mice. Microbiol Immunol 52: Haug J.S., Goldner C.M., Yazlovitskaya E.M., Voziyan P.A., Melnykovych G. (1994) Directed cell killing (apoptosis) in human lymphoblastoid cells incubated in the presence of farnesol: effect of phosphatidylcholine. Biochim Biophys Acta 1223(1): c 2009 The Societies and Blackwell Publishing Asia Pty Ltd 329

8 S. Abe et al. 17. Joo J.H., Liao G., Collins J.B., Grissom S.F., Jetten A.M. (2007) Farnesol-induced apoptosis in human lung carcinoma cells is coupled to the endoplasmic reticulum stress response. Cancer Res 67(16): Rioja A., Pizzey A.R., Marson C.M., Thomas N.S Preferential induction of apoptosis of leukaemic cells by farnesol. FEBS Lett 467(2 3): Machida K., Tanaka T., Fujita K., Taniguchi M. (1998) Farnesol-induced generation of reactive oxygen species via indirect inhibition of the mitochondrial electron transport chainintheyeastsaccharomyces cerevisiae. JBacteriol180: Hornby J.M., Kebaara B.W., Nickerson K.W. (2003) Farnesol biosynthesis in Candida albicans: cellularresponsetosterol inhibition by zaragozic acid B. Antimicrob Agents Chemother 47: c 2009 The Societies and Blackwell Publishing Asia Pty Ltd