CLINICAL INVESTIGATION
Immunomodulatory Effects of Voriconazole and Caspofungin on Human Peripheral Blood Mononuclear Cells Stimulated by Candida albicans and Candida krusei Isil Fidan, MD, Emine Yesilyurt, PhD, Ayse Kalkanci, MD, PhD, Sevgi O. Aslan, MD, Nur Sahin, MSc, Mustafa Chihat Ogan, MD and Murat Dizbay, MD
Abstract: Background: Candida infections are frequently associated with high morbidity and mortality rates in immunosuppressed patients. T cell–mediated and phagocytic immunity are the primary protective immune responses against fungal infections. Antifungal agents such as voriconazole and caspofungin enter phagocytic cells and lead to various intracellular activities. In this study, the authors aimed to investigate the immunomodulatory effects of voriconazole and caspofungin on human peripheral blood mononuclear cells (PBMC) stimulated by Candida albicans and Candida krusei. Methods: Human PBMC isolation was performed by Ficoll-hypaque density-gradient centrifugation method. Cell proliferation was assessed by colorimetric method using MTT. The cytokine levels in the human PBMC culture supernatants stimulated by C. albicans and C. krusei were determined by enzyme-linked immunosorbent assay. Results: The addition of voriconazole and caspofungin lead to proliferation of PBMC. In the presence of voriconazole and caspofungin, the levels of IL-2, IFN-g and IL-6 remarkably increased in PBMC stimulated by C. albicans and C. krusei. However, the combination of antifungal drugs and PBMC stimulated by Candida species did not increase the levels of TGF-b and IL-10. Conclusions: The results indicate that voriconazole and caspofungin have immunomodulatory effects on human PBMC stimulated by Candida species. The interaction between antifungal drugs and PBMC stimulates Th1-type cytokine secretion. Cytokine stimulation from immune cells can assist in the elimination of fungal pathogens. Therefore, during the treatment of fungal infection, putative immunomodulatory effects of antifungal agents should be taken into account. Key Indexing Terms: Immunomodulatory effects; Voriconazole; Caspofungin; Candida. [Am J Med Sci 2014;348(3):219–223.]
C
andida infections are associated with high morbidity and mortality rates in immunosuppressed patients.1 Although Candida albicans is the most frequently isolated species in patients with candidemia, infection rates caused by other Candida species such as Candida krusei is increasing. The development of resistance against most of the earlier antifungal agents among Candida species has made the treatment of these infections more difficult.2 The development of echinocandins such as caspofungin and newer triazole agents such as vorico-
From the Department of Medical Microbiology, Department of Infectious Disease (IF, EY, AK, NS), Gazi University, Ankara, Turkey; Ankara Oncology Hospital (MCO), Cancer Research Institute, Ankara, Turkey; and Department of Infectious Disease (MD), Gazi University, Ankara, Turkey. Submitted April 26, 2013; accepted in revised form November 22, 2013. This study was supported by the Gazi University Research Fund (no: 01/ 2011-87), Ankara, Turkey. The authors have no financial or other conflicts of interest to disclose. Correspondence: Isil Fidan, MD, Department of Medical Microbiology, Gazi University, Dekanlik Binasi 2.Kat, Bes¸evler, Ankara 06500, Turkey (E-mail: isilfi[email protected]).
The American Journal of the Medical Sciences
nazole has become important in the treatment of infections caused by resistant Candida species. Voriconazole (VRC) is an antifungal triazole with activity against medically important fungal infections in immunocompromised patients.3 VRC inhibits the synthesis of ergosterol by inhibiting the lanosterol 14a-demethylase enzyme.4 Caspofungin, the 1st clinically used echinocandin, is a member of a new class of antifungal antibiotics.5 Caspofungin inhibits fungal b-1,3 glucan synthesis and is effective in the treatment of mucosal and invasive fungal infections.6 T cell–mediated immunity and phagocytic immunity are the primary protective immune responses against fungal infections. Phagocytic cells such as monocytes and monocytederived macrophages are known to be important in clearing fungal infections. Phagocytes are activated by pattern recognition receptors that recognize fungus and trigger expression of cytokines that modulate the antifungal host defense.7 Peripheral blood mononuclear cells (PBMCs) are important cells in host defense against fungal infections, especially in invasive infections. Interactions with these cells may influence the efficacy of antifungal agents.8 The modulatory effects of antifungal agents against on the host immune response are not well understood. Recently, the immunomodulatory effects of antifungal agents, especially newer antifungals such as azoles and echinocandins, have been described.9 Antifungal agents either alone or combined with phagocytes are effective in inhibiting fungal growth.7 Antifungal agents such as VRC and caspofungin enter phagocytic cells and cause intracellular activities.10 Therefore, it is suggested that antifungal agents have also direct immunomodulatory effects. A better understanding of the immunomodulatory effects of antifungal agents may provide new strategies to improve the host immune response in invasive fungal infections. The aim of this study was to evaluate the immunomodulatory effects of VRC and caspofungin on human PBMC stimulated by C. albicans and C. krusei.
METHODS Preparation of Human PBMC Heparinized blood was collected from 3 healthy male blood donors. The age range of the donors was between 20 and 40. All samples were negative for HBsAg, HCV antibody and HIV antibody enzyme-linked immunosorbent assay (ELISA) tests. PBMC isolation was performed by Ficoll-hypaque densitygradient centrifugation method. After centrifugation, buffy coats were collected and washed in phosphate-buffered saline (PBS; Gibco, Darmstadt, Germany) for 3 times and resuspended at a concentration of 2 3 106 cells per milliliter with complete RPMI 1640 medium containing 2 mM l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.05 mM 2-mercaptoethanol and supplemented with 10% fetal calf serum (Gibco, Germany). Cell viability was evaluated as 95% by the trypan blue exclusion
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Yeast Strains C. albicans ATCC 10231 and C. krusei ATCC 6258 were used in this study. Candida suspensions were adjusted at the concentration of 106 CFU/mL. Afterward, Candida cells were inactivated by heating at 60°C for 30 minutes.
Cytokine Secretion Levels of cytokines such as IFN-g, IL-2, IL-6, IL-10 and TGF-b were determined by specific ELISA techniques according to the manufacturer’s instructions (Biosource, California). The concentration of cytokines was determined spectrophotometrically. The absorbance was read at 450 nm. A standard curve was created using cytokine standards. The cytokine concentrations for unknown samples were calculated according to the standard curve.
Drug Preparation The lyophilized powders of Vfend (Pfizer, Sandwich, UK) and Cancidas (Merck, Darmstadt, Germany) were reconstituted in dimethyl sulfoxide, diluted with sterile water.
Statistical Methods The results were analyzed using the 1-way analysis of variance. The Bonferroni test was used as post hoc analysis. P , 0.05 was considered as statically significant.
test. Antifungal susceptibility of Candida isolates were performed using the broth microdilution test as recommended by the CLSI, document M27-A2.
The MIC Values of Antifungal Agents The MICs of VRC and caspofungin were measured by the broth microdilution test as recommended by the CLSI, document M27-A2.11 MICs of VRC/caspofungin for C. albicans were 1 mg/mL and 2 mg/mL, respectively. For C. krusei, MICs of VRC/caspofungin were 0.25 mg/mL and 0.5 mg/mL, respectively. Incubation of PBMC With Candida Species and Antifungal Agents PBMCs (2 3 106) were delivered in culture flasks. PBMCs were incubated with heat-inactivated C. albicans or C. krusei (106 CFU/mL) at an effector:target ratio of 1:1 in the presence or absence of VRC or caspofungin. Concentrations of 13 MIC of VRC and caspofungin were added to the wells. Control culture flasks contained only PBMC or only antifungals. All flasks were studied for 3 times. Culture flasks were incubated in 5% CO2 for 24 and 48 hours at 37°C. At the end of each selected time period, the culture supernatants were removed and stored at 230°C until used. Cell Proliferation The proliferation of PBMCs was assessed by colorimetric method using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma, Steinheim, Germany). PBMCs were washed and incubated with MTT for 24, and after 48 hours, they were treated with C. albicans or C. krusei in the presence of VRC or caspofungin. Then, 0.5 mg/mL MTTmedia solution were added to each well for 4 hours at 37°C. After centrifugation, DMSO was added. The absorbance was read at 560 nm with a spectrophotometer.
RESULTS The addition of VRC and caspofungin leads to the proliferation of PBMC stimulated by Candida (Figure 1). Both of the antifungal agents lead to remarkable proliferation in PBMCs stimulated by C. albicans and C. krusei. The PBMC proliferation produced by caspofungin was determined at much higher levels after 48 hours. In the presence of VRC and caspofungin, the levels of IL-2 increased remarkably in PBMC stimulated by C. albicans and C. krusei (Figure 2) (P , 0.05). Moreover, the addition of VRC and caspofungin to PBMCs stimulated by C. albicans resulted in more significant increase in the IL-2 levels compared with the PBMCs stimulated by C. krusei (P , 0.001). VRC and caspofungin caused a statistically significant increase in the release of IFN-g from PBMCs stimulated by C. albicans and C. krusei at the 24th and 48th hours compared with the wells without any antifungal agent (Figure 3) (P , 0.05). Caspofungin produced the most remarkable increase in the IFN-g levels at the 48th hour in the presence of C. albicans (P , 0.001). The addition of VRC and caspofungin to PBMCs stimulated by C. albicans and C. krusei caused a statistically significant increase in the IL-6 levels compared with the PBMC wells without any antifungal agents at the 24th and 48th hours (Figure 4) (P , 0.05). C. krusei caused much more increase in the IL-6 levels in PBMCs in the presence of antifungal agents at the 24th and 48th hours compared with the wells with C. albicans. However, the increase in the wells with VRC after 24 hours and in the wells with VRC and caspofungin after 48 hours was found to be statistically significant in the presence of C. krusei compared with the wells with C. albicans (P 5 0.001, P 5 0.001, P 5 0.027, respectively).
FIGURE 1. analysis.
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FIGURE 2. IL-2 production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
The addition of VRC and caspofungin to PBMCs led to a statistically significant decrease in TGF-b release in PBMCs stimulated by Candida species without antifungal agents (Figure 5) (P , 0.05). However, the VRC and caspofungin did not any change the levels of IL-10 in PBMCs stimulated by Candida species.
DISCUSSION Invasive fungal infections constitute a major threat for immunocompromised patients. Even after the invention of new therapeutic antifungal agents, the treatment of invasive fungal infections is still unsatisfactory.12 In addition, the mortality due to fungal infections is still increasing.13 This situation entails further research for new treatment alternatives that can essentially regulate the immune system during the treatment of fungal infections in addition to antifungal agents. In some studies, it is reported that there is a synergy between phagocytic cells and antifungals for enhanced killing of Candida species.14,15 Vora et al16 reported that VRC and fluconazole can collaborate with phagocytic cells such as polymorphonuclear neutrophils or monocytes for enhanced candidacidal activity. Bopp et al2 also shown that the intracellular phagocytic capacity of these cells against Candida species increases when VRC enters human monocytes. In recent studies, it is also observed that newer antifungals, such as second-generation triazoles and the echinocandins may modificate the host immune response.17 The antifungal agent effect on host immune cells may be relevant to patients receiving
the agents for either prophylaxis or therapeutic purposes.9 Antifungal agents may also directly affect the immune system. This finding may help the use of immunotherapy as an adjunctive approach against invasive fungal infections. Depending on this fact, we evaluated the immunomodulatory effects of 2 different classes (azole and echinocandin) of antifungal agents on the human PBMCs, which is in vitro stimulated by Candida species. It is demonstrated that Th (T helper) cells play the major role in regulating immune responses to the fungal infection. Th1-type response is responsible for resistance against Candida infections, whereas Th2-type response is associated with susceptibility to infection.18 Th1 response is characterized by the production of proinflammatory cytokines such as IL-1, IL-2, IL-6, IL-12, TNF-a, IFN-g and IL-6. Th2 responses are associated with increased production of IL-4, IL-10 and TGF-b.18–20 Proinflammatory cytokines are produced at very early phase of response developed against infections and are important in being involved in both innate and acquired immunity.21 In our study, it was observed that 2 antifungal agents from triazole and echinocandin classes caused proliferation in human PBMCs. Furthermore, in the presence of VRC and caspofungin, IL-2, IFN-g and IL-6 proinflammatory cytokines, which is associated with Th1-type response, was found remarkably increased in PBMCs stimulated by Candida species. In the presence of antifungal agents, a decrease was detected in the levels of TGF-b, which causes Th2-type response, whereas there was no change in IL-10 levels, another Th2-type response-associated cytokine. We can assert that in
FIGURE 3. IFN-g production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
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FIGURE 4. IL-6 production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
the presence of antifungal agents, there were some changes both in the cytokine secretion and cell proliferation in PBMCs stimulated by Candida species. This situation suggests that antifungal agents have effects on immune cells and can create immunomodulatory effects by causing various alterations both in the proliferation of these cells and in the levels of cytokines that those cells produce. Simitsopoulou et al7 observed that the combination of VRC and Aspergillus fumigatus hypae increased the expression of TLR2 and TNF-a compared with the samples that did not include VRC. According their results, they suggested that VRC has an immunomodulatory effect leading to more efficient to A. fumigatus. In another study, the same researchers found that VRC significantly increased the expression of proinflammatory cytokines such as TNF-a in human monocytes stimulated by A. fumigatus.22 In previous studies, it was demonstrated that there was a synergism between IFN-g and antifungal agents against fungal pathogens such as Candida species and A. fumigatus.23 IFN-g is a potent activator of macrophage function that can enhance the antifungal activity of macrophages. The finding in our study showing that IFN-g increases in the presence of antifungal agents suggests that antifungal agents have immunomodulatory effects. According to the results of our study, antifungal agents increase the release of the cytokines that cause Th1-type responses in PBMCs. Th1 response enhances cell-mediated immunity, phagocyte activation and resistance against reinfection.24 In addition, the progression of infection is associated
with predominance of Th2 responses.20 Therefore, we suggest that the interactions of antifungals with immune cells may help the induction of protective immunity. Furthermore, decreased TGF-b response (associated with Th2-type response) in the presence of antifungal agents shows that although antifungal agents produce fungicidal activities, they also create some immunomodulatory effects by decreasing Th2-type cytokine release from immune cells. Our results suggest that VRC and caspofungin have immunomodulatory effects on human PBMC stimulated with Candida species. It is also suggests that antifungal agents and PBMC may show an additive antifungal effect against Candida species. The interaction between antifungal drugs and PBMCstimulated Th1-type cytokine secretion is associated with cellmediated immunity. Cytokine stimulation from immune cells can assist in the elimination of fungal pathogens. Baltch et al1 also reported that cytokines increase the intracellular anticandidal effect of VRC. We believe that our study will shed light on further studies because it is one of the first studies investigating the immunomodulatory effects of 2 different classes of antifungal agents in Candida infection. Antifungal treatment is essential against causative agents. Immunomodulatory properties of antifungal agents may provide beneficial effects, depending on factors to the host. So, during the treatment of fungal infection, putative immunomodulatory effects of antifungal agents should be taken into account. However, clinical evidence supporting
FIGURE 5. TGF-b production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
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immunomodulatory properties of antifungal agents in patients with fungal infection too limited to allow conclude treatment recommendations.
11. National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard M27–A2, 2nd ed. Wayne, PA: NCCLS; 2002.
REFERENCES
12. Antachopoulos C, Roilides E. Cytokines and fungal infections. Br J Haematol 2005;129:583–96.
1. Baltch AL, Bopp LH, Smith RP, et al. Effects of voriconazole, granulocyte-macrophage colony-stimulating factor, and interferon g on intracellular fluconazole-resistant Candida glabrata and Candida krusei in human manocyte-derived macrophages. Diagn Microbiol Infect Dis 2005;52:299–304. 2. Bopp LH, Baltch AL, Ritz WJ, et al. Antifungal effect of voriconazole on intracellular Candida glabrata, Candida krusei and Candida parapsilosis in human monocyte-derived macrophages. J Med Microbiol 2006;55:865–70. 3. Simitsopoulou M, Roilides E, Likartsis C, et al. Expression of immunomodulatory genes in human monocytes induced by voriconazole in the presence of Aspergillus fumigatus. Antimicrob Agents Chemother 2007;51:1048–54. 4. Kiraz N, Dag I, Yamac M, et al. Synergistic activities of three triazoles with caspofungin against Candida glabrata isolates determined by timekill, Etest, and disk diffusion methods. Antimicrob Agents Chemother 2010;54:2244–7. 5. Bowman JC, Scott Hicks P, Kurtz MB, et al. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 2002;46:3001–12. 6. Deresinski SC, Stevens DA. Caspofungin. Clin Infect Dis 2003;36: 1445–57. 7. Simitsopoulou M, Roilides E, Paliogianni F, et al. Immunomodulatory effects of voriconazole on monocytes challenged with Aspergillus fumigatus: differential role of Toll-like receptors. Antimicrob Agents Chemother 2008;52:3301–6. 8. Farowski F, Cornely OA, Vehreschild JJ, et al. Intracellular concentrations of posaconazole in different compartments of peripheral blood. Antimicrob Agents Chemother 2010;54:2928–31. 9. Choi JH, Kwon EY, Park CM, et al. Immunomodulatory effects of antifungal agents on the response of human monocytic cells to Aspergillus fumigatus conidia. Med Mycol 2010;48:704–9. 10. Baltch AL, Bopp LH, Smith RP, et al. Anticandidal effects of voriconazole and caspofungin, singly and in combination, against Candida glabrata, extracellularly and intracellularly in granulocyte-macrophage colony stimulating factor (GM-CSF)-activated human monocytes. J Antimicrob Chemother 2008;62:1285–90.
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13. Eschenauer G, Depestel DD, Carver PL. Comparison of echinocandin antifungals. Ther Clin Risk Manag 2007;3:71–97. 14. Natarajan U, Randhawa N, Brummer E, et al. Effect of granulocytemacrophage colony-stimulating factor on candidacidal activity of neutrophils, monocytes or monocyte-derived macrophages and synergy with fluconazole. J Med Microbiol 1998;47:359–63. 15. Gujral S, Brummer E, Stevens DA. Role of extended culture time on synergy of fluconazole and human monocyte-derived macrophages in clearing Candida albicans. J Infect Dis 1996;174:888–90. 16. Vora S, Purimetla N, Brummer E, et al. Activity of voriconazole a new triazole, combined with neutrophils or monocytes against Candida albicans: effect of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Antimicrob Agents Chemother 1998;42:907–10. 17. Steinbach WJ, Stevens DA. Review of newer antifungal and immunomodulatory strategies for invasive aspergillosis. Clin Infect Dis 2003; 37(suppl 3):S157–87. 18. Mencacci A, Perruccio K, Bacci A, et al. Defective antifungal T-helper 1 (TH1) immunity in a murine model of allogeneic T-cell– depleted bone marrow transplantation and its restoration by treatment with TH2 cytokine antagonists. Blood 2001;97:1483–90. 19. Stevens DA. Th1/Th2 in aspergillosis. Med Mycol 2006;44:229–35. 20. Shoham S, Levitz SM. The immune response to fungal infections. Br J Haematol 2005;129:569–82. 21. Feldmann M, Saklatvala J. Proinflammatory cytokines. In: Oppenheim JJ, Feldman M, editors. Cytokine Reference. New York, NY: Academic Press; 2001. p. 291–305. doi:10.1006/rwcy.2000.0204. 22. Simitsopoulou M, Roilides E, Likartsis C, et al. Expression of immunomodulatory genes in human monocytes induced by voriconazole in the presence of Aspergillus fumigatus. Antimicrob Agents Chemother 2007;5:1048–54. 23. Rodriguez-Adrian LJ, Grazziutti ML, Rex JH, et al. The potential role of cytokine therapy for fungal infections in patients with cancer: is recovery from neutropenia all that is needed? Clin Infect Dis 1998;26:1270–8. 24. Blanco JL, Garcia ME. Immune response to fungal infections. Vet Immunol Immunopathol 2008;15:47–70.
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Immunomodulatory Effects of Voriconazole and Caspofungin on Human Peripheral Blood Mononuclear Cells Stimulated by Candida albicans and Candida krusei Isil Fidan, MD, Emine Yesilyurt, PhD, Ayse Kalkanci, MD, PhD, Sevgi O. Aslan, MD, Nur Sahin, MSc, Mustafa Chihat Ogan, MD and Murat Dizbay, MD
Abstract: Background: Candida infections are frequently associated with high morbidity and mortality rates in immunosuppressed patients. T cell–mediated and phagocytic immunity are the primary protective immune responses against fungal infections. Antifungal agents such as voriconazole and caspofungin enter phagocytic cells and lead to various intracellular activities. In this study, the authors aimed to investigate the immunomodulatory effects of voriconazole and caspofungin on human peripheral blood mononuclear cells (PBMC) stimulated by Candida albicans and Candida krusei. Methods: Human PBMC isolation was performed by Ficoll-hypaque density-gradient centrifugation method. Cell proliferation was assessed by colorimetric method using MTT. The cytokine levels in the human PBMC culture supernatants stimulated by C. albicans and C. krusei were determined by enzyme-linked immunosorbent assay. Results: The addition of voriconazole and caspofungin lead to proliferation of PBMC. In the presence of voriconazole and caspofungin, the levels of IL-2, IFN-g and IL-6 remarkably increased in PBMC stimulated by C. albicans and C. krusei. However, the combination of antifungal drugs and PBMC stimulated by Candida species did not increase the levels of TGF-b and IL-10. Conclusions: The results indicate that voriconazole and caspofungin have immunomodulatory effects on human PBMC stimulated by Candida species. The interaction between antifungal drugs and PBMC stimulates Th1-type cytokine secretion. Cytokine stimulation from immune cells can assist in the elimination of fungal pathogens. Therefore, during the treatment of fungal infection, putative immunomodulatory effects of antifungal agents should be taken into account. Key Indexing Terms: Immunomodulatory effects; Voriconazole; Caspofungin; Candida. [Am J Med Sci 2014;348(3):219–223.]
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andida infections are associated with high morbidity and mortality rates in immunosuppressed patients.1 Although Candida albicans is the most frequently isolated species in patients with candidemia, infection rates caused by other Candida species such as Candida krusei is increasing. The development of resistance against most of the earlier antifungal agents among Candida species has made the treatment of these infections more difficult.2 The development of echinocandins such as caspofungin and newer triazole agents such as vorico-
From the Department of Medical Microbiology, Department of Infectious Disease (IF, EY, AK, NS), Gazi University, Ankara, Turkey; Ankara Oncology Hospital (MCO), Cancer Research Institute, Ankara, Turkey; and Department of Infectious Disease (MD), Gazi University, Ankara, Turkey. Submitted April 26, 2013; accepted in revised form November 22, 2013. This study was supported by the Gazi University Research Fund (no: 01/ 2011-87), Ankara, Turkey. The authors have no financial or other conflicts of interest to disclose. Correspondence: Isil Fidan, MD, Department of Medical Microbiology, Gazi University, Dekanlik Binasi 2.Kat, Bes¸evler, Ankara 06500, Turkey (E-mail: isilfi[email protected]).
The American Journal of the Medical Sciences
nazole has become important in the treatment of infections caused by resistant Candida species. Voriconazole (VRC) is an antifungal triazole with activity against medically important fungal infections in immunocompromised patients.3 VRC inhibits the synthesis of ergosterol by inhibiting the lanosterol 14a-demethylase enzyme.4 Caspofungin, the 1st clinically used echinocandin, is a member of a new class of antifungal antibiotics.5 Caspofungin inhibits fungal b-1,3 glucan synthesis and is effective in the treatment of mucosal and invasive fungal infections.6 T cell–mediated immunity and phagocytic immunity are the primary protective immune responses against fungal infections. Phagocytic cells such as monocytes and monocytederived macrophages are known to be important in clearing fungal infections. Phagocytes are activated by pattern recognition receptors that recognize fungus and trigger expression of cytokines that modulate the antifungal host defense.7 Peripheral blood mononuclear cells (PBMCs) are important cells in host defense against fungal infections, especially in invasive infections. Interactions with these cells may influence the efficacy of antifungal agents.8 The modulatory effects of antifungal agents against on the host immune response are not well understood. Recently, the immunomodulatory effects of antifungal agents, especially newer antifungals such as azoles and echinocandins, have been described.9 Antifungal agents either alone or combined with phagocytes are effective in inhibiting fungal growth.7 Antifungal agents such as VRC and caspofungin enter phagocytic cells and cause intracellular activities.10 Therefore, it is suggested that antifungal agents have also direct immunomodulatory effects. A better understanding of the immunomodulatory effects of antifungal agents may provide new strategies to improve the host immune response in invasive fungal infections. The aim of this study was to evaluate the immunomodulatory effects of VRC and caspofungin on human PBMC stimulated by C. albicans and C. krusei.
METHODS Preparation of Human PBMC Heparinized blood was collected from 3 healthy male blood donors. The age range of the donors was between 20 and 40. All samples were negative for HBsAg, HCV antibody and HIV antibody enzyme-linked immunosorbent assay (ELISA) tests. PBMC isolation was performed by Ficoll-hypaque densitygradient centrifugation method. After centrifugation, buffy coats were collected and washed in phosphate-buffered saline (PBS; Gibco, Darmstadt, Germany) for 3 times and resuspended at a concentration of 2 3 106 cells per milliliter with complete RPMI 1640 medium containing 2 mM l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.05 mM 2-mercaptoethanol and supplemented with 10% fetal calf serum (Gibco, Germany). Cell viability was evaluated as 95% by the trypan blue exclusion
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Yeast Strains C. albicans ATCC 10231 and C. krusei ATCC 6258 were used in this study. Candida suspensions were adjusted at the concentration of 106 CFU/mL. Afterward, Candida cells were inactivated by heating at 60°C for 30 minutes.
Cytokine Secretion Levels of cytokines such as IFN-g, IL-2, IL-6, IL-10 and TGF-b were determined by specific ELISA techniques according to the manufacturer’s instructions (Biosource, California). The concentration of cytokines was determined spectrophotometrically. The absorbance was read at 450 nm. A standard curve was created using cytokine standards. The cytokine concentrations for unknown samples were calculated according to the standard curve.
Drug Preparation The lyophilized powders of Vfend (Pfizer, Sandwich, UK) and Cancidas (Merck, Darmstadt, Germany) were reconstituted in dimethyl sulfoxide, diluted with sterile water.
Statistical Methods The results were analyzed using the 1-way analysis of variance. The Bonferroni test was used as post hoc analysis. P , 0.05 was considered as statically significant.
test. Antifungal susceptibility of Candida isolates were performed using the broth microdilution test as recommended by the CLSI, document M27-A2.
The MIC Values of Antifungal Agents The MICs of VRC and caspofungin were measured by the broth microdilution test as recommended by the CLSI, document M27-A2.11 MICs of VRC/caspofungin for C. albicans were 1 mg/mL and 2 mg/mL, respectively. For C. krusei, MICs of VRC/caspofungin were 0.25 mg/mL and 0.5 mg/mL, respectively. Incubation of PBMC With Candida Species and Antifungal Agents PBMCs (2 3 106) were delivered in culture flasks. PBMCs were incubated with heat-inactivated C. albicans or C. krusei (106 CFU/mL) at an effector:target ratio of 1:1 in the presence or absence of VRC or caspofungin. Concentrations of 13 MIC of VRC and caspofungin were added to the wells. Control culture flasks contained only PBMC or only antifungals. All flasks were studied for 3 times. Culture flasks were incubated in 5% CO2 for 24 and 48 hours at 37°C. At the end of each selected time period, the culture supernatants were removed and stored at 230°C until used. Cell Proliferation The proliferation of PBMCs was assessed by colorimetric method using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma, Steinheim, Germany). PBMCs were washed and incubated with MTT for 24, and after 48 hours, they were treated with C. albicans or C. krusei in the presence of VRC or caspofungin. Then, 0.5 mg/mL MTTmedia solution were added to each well for 4 hours at 37°C. After centrifugation, DMSO was added. The absorbance was read at 560 nm with a spectrophotometer.
RESULTS The addition of VRC and caspofungin leads to the proliferation of PBMC stimulated by Candida (Figure 1). Both of the antifungal agents lead to remarkable proliferation in PBMCs stimulated by C. albicans and C. krusei. The PBMC proliferation produced by caspofungin was determined at much higher levels after 48 hours. In the presence of VRC and caspofungin, the levels of IL-2 increased remarkably in PBMC stimulated by C. albicans and C. krusei (Figure 2) (P , 0.05). Moreover, the addition of VRC and caspofungin to PBMCs stimulated by C. albicans resulted in more significant increase in the IL-2 levels compared with the PBMCs stimulated by C. krusei (P , 0.001). VRC and caspofungin caused a statistically significant increase in the release of IFN-g from PBMCs stimulated by C. albicans and C. krusei at the 24th and 48th hours compared with the wells without any antifungal agent (Figure 3) (P , 0.05). Caspofungin produced the most remarkable increase in the IFN-g levels at the 48th hour in the presence of C. albicans (P , 0.001). The addition of VRC and caspofungin to PBMCs stimulated by C. albicans and C. krusei caused a statistically significant increase in the IL-6 levels compared with the PBMC wells without any antifungal agents at the 24th and 48th hours (Figure 4) (P , 0.05). C. krusei caused much more increase in the IL-6 levels in PBMCs in the presence of antifungal agents at the 24th and 48th hours compared with the wells with C. albicans. However, the increase in the wells with VRC after 24 hours and in the wells with VRC and caspofungin after 48 hours was found to be statistically significant in the presence of C. krusei compared with the wells with C. albicans (P 5 0.001, P 5 0.001, P 5 0.027, respectively).
FIGURE 1. analysis.
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FIGURE 2. IL-2 production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
The addition of VRC and caspofungin to PBMCs led to a statistically significant decrease in TGF-b release in PBMCs stimulated by Candida species without antifungal agents (Figure 5) (P , 0.05). However, the VRC and caspofungin did not any change the levels of IL-10 in PBMCs stimulated by Candida species.
DISCUSSION Invasive fungal infections constitute a major threat for immunocompromised patients. Even after the invention of new therapeutic antifungal agents, the treatment of invasive fungal infections is still unsatisfactory.12 In addition, the mortality due to fungal infections is still increasing.13 This situation entails further research for new treatment alternatives that can essentially regulate the immune system during the treatment of fungal infections in addition to antifungal agents. In some studies, it is reported that there is a synergy between phagocytic cells and antifungals for enhanced killing of Candida species.14,15 Vora et al16 reported that VRC and fluconazole can collaborate with phagocytic cells such as polymorphonuclear neutrophils or monocytes for enhanced candidacidal activity. Bopp et al2 also shown that the intracellular phagocytic capacity of these cells against Candida species increases when VRC enters human monocytes. In recent studies, it is also observed that newer antifungals, such as second-generation triazoles and the echinocandins may modificate the host immune response.17 The antifungal agent effect on host immune cells may be relevant to patients receiving
the agents for either prophylaxis or therapeutic purposes.9 Antifungal agents may also directly affect the immune system. This finding may help the use of immunotherapy as an adjunctive approach against invasive fungal infections. Depending on this fact, we evaluated the immunomodulatory effects of 2 different classes (azole and echinocandin) of antifungal agents on the human PBMCs, which is in vitro stimulated by Candida species. It is demonstrated that Th (T helper) cells play the major role in regulating immune responses to the fungal infection. Th1-type response is responsible for resistance against Candida infections, whereas Th2-type response is associated with susceptibility to infection.18 Th1 response is characterized by the production of proinflammatory cytokines such as IL-1, IL-2, IL-6, IL-12, TNF-a, IFN-g and IL-6. Th2 responses are associated with increased production of IL-4, IL-10 and TGF-b.18–20 Proinflammatory cytokines are produced at very early phase of response developed against infections and are important in being involved in both innate and acquired immunity.21 In our study, it was observed that 2 antifungal agents from triazole and echinocandin classes caused proliferation in human PBMCs. Furthermore, in the presence of VRC and caspofungin, IL-2, IFN-g and IL-6 proinflammatory cytokines, which is associated with Th1-type response, was found remarkably increased in PBMCs stimulated by Candida species. In the presence of antifungal agents, a decrease was detected in the levels of TGF-b, which causes Th2-type response, whereas there was no change in IL-10 levels, another Th2-type response-associated cytokine. We can assert that in
FIGURE 3. IFN-g production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
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FIGURE 4. IL-6 production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
the presence of antifungal agents, there were some changes both in the cytokine secretion and cell proliferation in PBMCs stimulated by Candida species. This situation suggests that antifungal agents have effects on immune cells and can create immunomodulatory effects by causing various alterations both in the proliferation of these cells and in the levels of cytokines that those cells produce. Simitsopoulou et al7 observed that the combination of VRC and Aspergillus fumigatus hypae increased the expression of TLR2 and TNF-a compared with the samples that did not include VRC. According their results, they suggested that VRC has an immunomodulatory effect leading to more efficient to A. fumigatus. In another study, the same researchers found that VRC significantly increased the expression of proinflammatory cytokines such as TNF-a in human monocytes stimulated by A. fumigatus.22 In previous studies, it was demonstrated that there was a synergism between IFN-g and antifungal agents against fungal pathogens such as Candida species and A. fumigatus.23 IFN-g is a potent activator of macrophage function that can enhance the antifungal activity of macrophages. The finding in our study showing that IFN-g increases in the presence of antifungal agents suggests that antifungal agents have immunomodulatory effects. According to the results of our study, antifungal agents increase the release of the cytokines that cause Th1-type responses in PBMCs. Th1 response enhances cell-mediated immunity, phagocyte activation and resistance against reinfection.24 In addition, the progression of infection is associated
with predominance of Th2 responses.20 Therefore, we suggest that the interactions of antifungals with immune cells may help the induction of protective immunity. Furthermore, decreased TGF-b response (associated with Th2-type response) in the presence of antifungal agents shows that although antifungal agents produce fungicidal activities, they also create some immunomodulatory effects by decreasing Th2-type cytokine release from immune cells. Our results suggest that VRC and caspofungin have immunomodulatory effects on human PBMC stimulated with Candida species. It is also suggests that antifungal agents and PBMC may show an additive antifungal effect against Candida species. The interaction between antifungal drugs and PBMCstimulated Th1-type cytokine secretion is associated with cellmediated immunity. Cytokine stimulation from immune cells can assist in the elimination of fungal pathogens. Baltch et al1 also reported that cytokines increase the intracellular anticandidal effect of VRC. We believe that our study will shed light on further studies because it is one of the first studies investigating the immunomodulatory effects of 2 different classes of antifungal agents in Candida infection. Antifungal treatment is essential against causative agents. Immunomodulatory properties of antifungal agents may provide beneficial effects, depending on factors to the host. So, during the treatment of fungal infection, putative immunomodulatory effects of antifungal agents should be taken into account. However, clinical evidence supporting
FIGURE 5. TGF-b production from PBMCs incubated with Candida species and antifungals. Bars indicate standard errors of the mean.
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immunomodulatory properties of antifungal agents in patients with fungal infection too limited to allow conclude treatment recommendations.
11. National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, Approved Standard M27–A2, 2nd ed. Wayne, PA: NCCLS; 2002.
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