Candida albicans Activation of Human Monocytes Toward M2 Profile is Reversed by Amphotericin B and Fluconazole

Research Article

J Bacteriol Mycol. 2021; 8(2): 1168.

Candida albicans Activation of Human Monocytes Toward M2 Profile is Reversed by Amphotericin B and Fluconazole

Icely PA1,2, Vigezzi C1,2, Rodriguez E1,2, Miró MS1,2, Salido Renteria B1 and Sotomayor CE1,2*

1Department of Clinical Biochemistry, National University of Cordoba, Argentina

2Center for Research in Clinical Biochemistry and Immunology, CIBICI-CONICET, Argentina

*Corresponding author: Claudia E Sotomayor, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000, Cordoba, Argentina

Received: February 20, 2021; Accepted: March 22, 2021 Published: March 29, 2021


Phagocytes, including monocytes/macrophages, play an important role in the host defense during Candida albicans infections. In the L-arginine metabolism, the balance between the activation of two enzymes, inducible Nitric Oxide Synthase (iNOS) and arginase, promotes in the macrophages two alternative metabolic states, while M1 profile is related with host protection, M2 favored the fungal growth and evasion. Our aim was to evaluate the effect of Amphotericin B (AMB) and Fluconazole (FLC) on polarization of human monocytes to M2 profile induced by C. albicans. The human monocytic (Mo) cell line U937 was co-cultured with viable yeast of C. albicans, or Lipopolysaccharides (LPS) or Phorbol-12-myristate-13-acetate (PMA). Nitric Oxide (NO), cytokines production and arginase activity were evaluated. The effect of AMB or FLC on these metabolic pathways in immune cells and on fungus intrinsic arginase activity was studied. C. albicans inhibits NO production in human-monocyte and induces strong host arginase activity (p<0.0001). AMB and FLC inhibited C. albicansinduced arginase activity in immune cells (p<0.001), reaching a percentage of inhibition of 90% for AMB and 78% for FLC. Arginase intrinsic activity of the fungus was blocked by nor-NOHA (arginase inhibitor) and AMB (p<0.05). These results show that C. albicans drives human Mo toward M2 profile and that both antifungal drugs evaluated have the ability to revert C. albicans-induced M2 profile. In a relevant manner, it also provides data about additional effect of AMB as inhibitor of C. albicans endogenous arginase activity. Here in we provide new evidence for the effect of these drugs over the immune cells and the yeast.

Keywords: Amphotericin B; Fluconazole; Candida albicans; Monocytes; L-arginine pathway; Candida albicans arginase activity


During the last decades the medical importance of the fungus as pathogens have increased dramatically, as a result of intensive use of antibiotics, immune suppressor therapies and HIV epidemic [1]. Candida albicans is a component of the human oral and gastrointestinal flora and this opportunistic fungus can in certain circumstances switch between the yeast to the mycelial form increasing its invasiveness, entering to host tissues and causing a local or systemic disease [2,3]. The host´s resistance to C. albicans is determined by a coordinated interaction between the innate and the adaptive mechanisms of defense [4].

In the innate immune response, the phagocytic cells such as macrophages (MF) and neutrophils are considered the effector cells [1]. In neutropenic patients, the mononuclear cells (Monocytes/ Macrophages) (Mo/MF) contribute to the protection against the infection, although the molecular mechanisms of the macrophagefungus interaction have not been completely established. In the last years, a relevant role has been assigned to this cell population in the host defense against this opportunistic pathogen [4]. The Mo/ MF have the capacity of killing C. albicans with the production of metabolites such as H2O2, superoxide anion, Nitric Oxide (NO) and peroxidenitrites that have a candidacidal activity more powerful than their progenitors [4-6]. The NO and other intermediaries of oxygen are important effectors molecules that directly act on the yeast and the hyphal form of fungus [6].

The ability to release NO can be modulated by different stimulus, including bacterial products, tumor cells and cytokines [6]. Its production depends on a cytosolic enzyme, the inducible Nitric Oxide Synthase (iNOS) and the availability of the substrate L-arginine [5]. Besides iNOS, the other major L-arginine consuming enzyme, arginase, hydrolyzes the L-arginine producing L-ornithine and urea. Both metabolic pathways depend on the extracellular supply of L-arginine. The competition between arginase and iNOS is more evidently when the availability of L-arginine is compromised as it occurs during sepsis [7,8]. Several lines of investigation suggest that the induction of one of the enzymes undergo with the suppression of the other, indicating two competitive states in murine macrophages [9]. The polarization of the metabolism of the L-arginine is associated with two types of response: the induction of the iNOS linked to MFM1 profile and the T-helper 1 type (Th1) response, denominated “classic pathway”, and the arginase activation associated to MFM2 profile and the Th2 response known as “alternative pathway” [5,9]. While M1 profile is associated with host protection, M2 favored the fungus resistance, making the M1 vs. M2 balance, a relevant event in the development of infection.

Our group showed evidence about the induction of both metabolic pathways in an experimental model of candidemia [10,11]. In the present work we evaluated the activation of the L-arginine pathway in a human monocytic cell line exposed to C. albicans and the effect of two antimicrobial agents frequently used in Latin America, Fluconazole (FLC) and Amphotericin B (AMB). We also provide novel evidence about the effect of these antifungal agents on C. albicans intrinsic arginase activity. Our results support an interesting perspective about the complex relation between pathogen, host and the antifungal drugs, extending the properties of the last ones beyond its well-known classic effects.

Materials and Methods


The C. albicans strain ATCC36801 was used in all the experiments [12,13]. Yeast cells were kept by serial subculture on Sabouraud Dextrose Agar (SDA) (Britania, Argentina) at Room Temperature (RT). For the experiments, yeast cells were grown on SDA for 48h at RT, harvested in sterile Phosphate-Buffered Saline (PBS) and centrifuged at 1000×g; after two washes in PBS, the yeast were counted and diluted to the desired concentration [12,13].


The human monocytic cell line U937 (ATCC-CRL-1593.2, USA) was cultured in RPMI1640 medium (GIBCO, USA) supplemented with 10% SBF (NATOCOR, Argentina) heat inactivated (56°C, 30min), 2mM L-glutamine, 50Uml penicillin, 50μg/ml streptomycin at 37°C in a 5% CO2/95% air atmosphere. Cell viability was determined by using trypan blue exclusion test (≥96%) [11].

Antifungal agents

A stock solution of each antifungal (FLC or AMB) was prepared using sterile distilled water. For FLC the stock solution of 2mg/ml from Lazar Laboratory (Argentina) was diluted with RPMI1640 medium (GIBCO-USA) to a final concentration of 1μg/ml in the well [14]. For AMB the stock solution of 5mg/ml from Fada Laboratory (Argentina) was diluted to a final concentration of 2.5μg/ml with RPMI1640 medium (GIBCO-USA) [14]. Both concentrations were below the Minimum Inhibitory Concentration (MIC) and the fungal viability after incubation with FLC or AMB was checked by the MTT assay [12].

Experimental design

The U937 cells (3x105cell/ml) were co-cultured in 96 multiwell plates with viable yeast of C. albicans, or Lipopolysaccharides (LPS) or Phorbol-12-myristate 13-acetate (PMA) and incubated for 48h at 37°C, 5% CO2/95% air atmosphere; U937 cells cultured alone (without stimuli) were used as control. C. albicans was used in a relation of 5:1 yeast/cell; LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich, USA) was used at a final concentration of 10μg/ml in the well and PMA (Sigma-Aldrich) was used at a final concentration of 1μg/ml.

Also U937 cells were cultured during 48h with AMB or FLC at established concentration, in the absence or presence of C. albicans. In other set of experiments, the specific arginase inhibitor, Nω- Hydroxy-nor-L-arginine (nor-NOHA, Roche- Switzerland) was used at 50μM with the purpose to evaluate it effects on: a) C. albicans alone, b) U937 cells co-cultured with C. abicans and c) U937 cells co-cultured with C. abicans in presence of AMB or FLC. After incubation, multiwell plates were centrifuged 10min at 2000rpm, the supernatants were removed and used to determine NO and cytokines production. The cells monolayer was used to determine arginase activity. Each experiment conditions consisted of three replications and at least three individual experiments were carried out.

Cell viability

Eukaryotic cell viability was determined after 48h of incubation using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma-Aldrich). The MTT assay is a colorimetric method that detects mitochondrial activity present in living cells and the signal generated is directly proportional to the number of viable cells. This method was validated to detect fungus and cell viability [12,15]. MTT solution was added to each well at final concentration of 5mg/ml and incubated 4hat 37°C. After wards 200μl of DMSO was added into each well to dissolve the formed darkblue formazan crystals. Absorbance was measured at 570nm on a microplate reader (Bio-Rad, Hercules, USA).

Nitric oxide production

NO production by U937 cells was determined by measuring the nitrite concentration in the supernatant following a microplate assay method as previously described [16]. Briefly, 100μl of the culture supernatants were collected and mixed with 200μl of Griess reagent (sulphanylamide 1.5% in 1N HCl and N-(1-naphtyl) ethylenediamine-dihydrochloride 0.13% in H2O). Plates were incubated for 15min at RT in the dark and absorbance was measured at 540nm in a microplate reader (Bio-Rad). NO concentration was calculated by means of a calibration curve made between 2 and 125 μM of nitrite using sodium nitrite (Sigma-Aldrich) as standard. The results were expressed as μM of NO.

Arginase activity

To assess arginase activity, cell monolayers were washed with PBS and treated with 0.15ml of 0.1% Triton X-100 containing protease inhibitors [16]. Cell lysate (50μl) was added to 50μl of Tris-HCl (50nM; pH=7.5) containing 10nM MnCl2. Monocytes arginase was then activated by heating this mixture at 55-60°C for 10min. The hydrolysis reaction of L-arginine by arginase was carried out by incubating the mixture containing activated arginase with 50μl of L-arginine (0.5M; pH=9,7) at 37°C for 1h and was stopped by adding 400μl of the acid solution mixture (H2PO4:H3PO4:H2O=1:3:7). For colorimetric determination of urea, a-isonitroso-propiophenone (25μl, 9% in absolute ethanol) was then added, and the mixture was heated at 100°C for 45min. After placing the samples in the dark for 10min at RT, the urea concentration was determined spectrophotometrically by the absorbance at 550nm measured with a microplate reader (Bio-Rad). The amount of the urea produced was referred as arginase activity and expressed in mU/106 cells.

Cytokine immune assays

The level of cytokines in U937 culture supernatants was quantified using IL-1β, IL-8 and TNF (OptEIA BD, USA) ELISA tests as previously described [13]. Absorbance was measured using a microplate reader (Bio Rad), and concentrations extrapolated from standard curves. Data were expressed as pg/ml.

Statistical analyses

Data were expressed as means ± SEM. Differences between group means or ratios were assessed using one-way or two-way ANOVA followed by Bonferroni test for multiple comparisons. A p-value<0.05 was considered statistically significant. GraphPad Prism version 5.0 for Windows (GraphPad Software, USA) was employed. All experiments were repeated at least three times to test the reproducibility of results.


C. albicans upregulates the alternative L-arginine pathway in human monocytes

It was reported that human Mo are able to express the iNOS, the limitant enzyme for the NO production, after the activation by different stimulus [17]. In order to evaluate if C. albicans induces the production of this nitric derivative product in human Mo, the U937 cell line was cultured with yeast, o with different stimulus for 48h and the production of NO was measured in the cell culture supernatants. We observed in Figure 1A, that the fungus induces a significant decrease in the basal production of the toxic nitrogen derivative (p<0.0001). In this system, the human Mo were refractory to the E.coli LPS and PMA activation. We also tested the Mo viability exposed to different stimuli using MTT assay directly in the U937 cell cultured in basal condition and stimulated with LPS, PMA, or C. albicans. The results demonstrate that U937 cells were alive at all different treatments (Figure 1B).