Itraconazole Promotes Macrophage M1 Polarization and Phagocytic Capacity of Macrophage to Candida Albicans

Xiao-Li Zheng1,2,Guan-Zhao Liang2,Dong-Mei Shi3,Hui-Ping Yao1,Lu Zhang1,Wei-Da Liu2,?,

Guan-Zhi Chen1,?

1Department of Dermatology,The Affiliated Hospital of Qingdao University,Qingdao,Shandong 266003,China,2Department of Mycology,Hospital for Skin Diseases(Institute of Dermatology),Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing,Jiangsu 210042,China,3Department of Dermatology,Jining No.1 People’s Hospital,Jining,Shandong 272011,China.

Abstract

Keywords:itraconazole,macrophage,immunomodulatory,macrophage polarization,Candida albicans

Introduction

Itraconazole (ITZ), an anti-fungal triazole drug, is commonly used for the treatment of a broad spectrum of fungal infections and also in severe cases of candidiasis and aspergillosis.1The major anti-fungal effect of ITZ is based on inhibition of 14-α-sterol demethylase,which is a microsomal cytochrome P450 protein enzyme,and can interrupt the synthesis of ergosterol,a key component of fungal cell membranes,2consequently leading to an accumulation of lanosterol and thereby inhibit the cholesterol biosynthesis pathway.3

Moreover,recently anti-tumor effects of ITZ have been reported.4It has been identified as a potential anti-tumor agent to non-small-cell lung cancer,basal cell carcinoma,melanoma,and other tumors,4whereas other anti-fungal azole drugs such as fluconazole, voriconazole, and miconazole were inactive toward tumors. This and recently discovered immunomodulatory effects of ITZ,5indicates different biological activity of ITZ in contrast to other anti-fungal azole drugs.The differences might be related to its effects on macrophages.6

Generally,macrophages can be divided into two groups:classically activated macrophages(M1),characterized by high expression of the major histocompatibility complex class II,nitric oxide,reactive oxygen species,and proinflammatory cytokines,such as tumor-necrosisfactor(TNF),interleukin(IL)-1,and IL-6.7Secondly,the alternative activated macrophages(M2)are having immunosuppressive activity and tumor-promoting functions.In response to a variety of stimuli,for instance IL-4,M2 can produce more IL-10 and arginase(Arg),leading to cell proliferation and enhanced collagen production.Therefore,the polarization of M1 cells have positive effects to infectious diseases and cancer.However,the effects of ITZ on the polarization of macrophages are mainly unknown.

In the current study,we used a LPS-induced in vitro model to investigate whether ITZ possess direct immunomodulatory properties in response to M1 polarization of macrophage-like RAW264.7 cells, and used an IL-4-induced in vitro model to observe whether ITZ modulate M2 polarization of RAW264.7 cells.To imitate Candida albicans(C.albicans)infection,we used non-hazardous yeast cells killed by UV radiation and observed the immunoregulatory effects of ITZ on RAW264.7 cells in interaction with C.albicans.We investigated the effects of ITZ on the production of inducible nitric oxide synthase(iNOS)and Arg-1 and analyzed the cytokine profiles in lipopolysaccharide(LPS),IL-4 and C.albicans activated mouse macrophage-like RAW264.7 cells. Our data pointed to an increased polarization of M1 macrophage in the in vitro experiments and improved phagocytosis.

Materials and methods

Cells type and cultivation

Mouse macrophage-like RAW264.7 cells obtained from American Type Culture Collection were maintained in Roswell Park Memorial Institute(RPMI)1640 medium(Gibco,Carlsbad,CA,USA)supplemented with 10%fetal bovine serum(Gibco)and incubated at 37°C supplied with 5% CO2and humidified atmosphere. ITZ (purity＞98.0%; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethylsulfoxide and stored in -20°C.RPMI 1640 supplemented with lipid-depleted serum medium was used for all experiments.

Cell counting kit-8(CCK-8)assay

RAW264.7 cells were seeded in 96-well plates at the density of 6×103cells/well and ITZ was added to the final concentrations(～16μmol/L)in lipid-depleted fetal bovine serum medium for 12,24,and 48h.And then 200μL CCK-8 solution (Yeasen, China) were added to each well and the plates were incubated for another 3hours in dark.Spectra MAX 190 absorbance microplate reader (Molecular Devices, San Jose, CA USA)was used to detect the light absorbency at the wavelength of 450nm.Experiments were repeated for three times.

Detection of cytokines secreted by RAW264.7 cells under ITZ treatment and co-stimulated with LPS,IL-4 or C.albicans

RAW264.7 cells were collected,washed and seeded in 6-well plates at density of 1.0×106in.One hour later,ITZ was added to the plates to the final concentrations of 0,0.25,0.5,and 1μmol/L and incubated for 1hour.Then LPS(Sigma,St.Louis,MO,USA)or IL-4(Peprotech,Rocky Hill,NJ,USA)was added to the final concentration of 1μg/mL or 20ng/mL,respectively.All the plates were incubated for 12hours.

For C.albicans treated groups,first,RAW264.7 cells were seeded in plates at density of 1.0×106.One hour later,ITZ was added to the RAW264.7 cells with the indicated concentrations(0,0.25,0.5,and 1μmol/L)and incubated for 12hours.Then ultraviolet(UV)-killed C.albicans were added to the indicated groups and incubated for another 1.5hours.The group treated without ITZ and C.albicans was used to detect the basic secretion of cytokines. All the supernatants were collected and cytokines were measured by Luminex or Cytometric Bead Array(BD Bioscience San Diego,CA,USA)following the manufacturer’s protocol.

Western blot assay

RAW264.7 cells were stimulated by LPS,IL-4,or C.albicans as described above and the cell lysates were collected after treatment.Aliquots lysates of each group were boiled at 100°C for 5minutes.Electrophoresis was performed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels,then it was transferred to polyvinylidene difluoride membranes. The membranes were probed with primary rabbit anti-iNOS antibody(1:1,000,Santa Cruz Biotechnology,Santa Cruz,CA,USA),rabbit anti-Arg-1 antibody(1:1,000,Santa Cruz),and mouse anti-β-actin antibody(1:200;Santa Cruz).Chemiluminescence detection system was used to visualize the secondary anti-rabbit IgG antibody (Santa Cruz).Adobe Photoshop CS5 software(Adobe Systems Incorporated, CA, USA) was used to perform densitometric analysis.For the quantification,the expression level had been normalized against the β-actin control,whereby the β-actin level has been set to 1.

Phagocytosis assay

Cells were seeded in plates with a density of 5×105.One hour later,ITZ was added in final concentrations of 0,0.25,0.5,and 1μmol/L and incubated for additional 12 hours.And then,UV-killed C.albicans cells,which were labeled with fluorescein isothiocyanate (Sigma), were mixed with RAW264.7 cells at a ratio of cells:yeast=1:4 or 1:8.The plates were incubated for 1.5hours and washed twice with phosphate-buffered saline(PBS)to remove unbound yeast cells.After cell collection 100μL trypan blue(250μg/mL in PBS)was added and incubated at room temperature for 1minute to quench the fluorescence of bounded yeast cells,which had not been internalized.The cells were washed twice with PBS and resuspended in 2%paraformaldehyde and analyzed by flow cytometry.Parallel,PKH 26 Red Fluorescent Cell Linker Kits(Sigma)was used to stain the membrane of RAW264.7 cells.Image of internalized fluorescent yeasts was visualized and analyzed by fluorescence microscope(OLIMPUS,Japan).

Statistical analyses

GraphPad Prism ver.6.02 software(GraphPad Software,San Diego,CA,USA)was used to analyze the data,and data are presented as mean±standard deviation.The Student’s t test and one-way analysis of variance were used to calculate the differences,whereby the threshold of significance was set to P＜0.05.

Results

Cell toxicity of ITZ

As shown in Fig. 1, ITZ significantly inhibited the growth of the cells in both a time and dose-dependent manner.

Figure 1.Itraconazole inhibited the growth of the cells is time and dose-dependent.Results are expressed as percentage of cell viability compared with control.All values represented means±SD.

A concentration of 1μmol/L ITZ for 12h had no significant cytotoxic effects on the RAW264.7 cells compared to the untreated control group, and cell toxicity has appeared and been increased with the concentration increasing and time prolongation. So,1μmol/L for 12hours was evaluated as optimal concentration to examine the effects of ITZ on macrophages in further experiments.

Effects of ITZ on polarization of RAW264.7 cells induced by LPS or IL-4

When stimulated by LPS,compared with the control(370.43±33.98pg/mL),ITZ increased the production of IL-6 in the group treated with 0.25μmol/L(538.03±60.23pg/mL, P＜0.05), 0.5μmol/L (550.32±47.87 pg/mL,P＜0.05),and 1μmol/L(626.95±75.24pg/mL,P＜0.01) ITZ (Fig. 2A). The production of TNF-α of the group treated with 1μmol/L ITZ(2,521.51±444.06pg/mL,P＜0.05)was also significant higher than that in the control group (1,617.85±94.57pg/mL;Fig.2B).

When induced by IL-4,the production of IL-6 and TNFα was increased in the high dose group(1μmol/L),with the detected concentration of 528.33±11.60pg/mL(P＜0.05)and 4.85±0.32pg/mL(P＜0.05),respectively,compared to the control(466.99±28.32pg/mL,4.30±0.19pg/mL,respectively;Fig.2C and 2D).Also,vary concentrations of ITZ(0.25μmol/L,0.5μmol/L,and 1μmol/L enhanced the production of IL-1β in IL-4 induced([325.95±13.97 pg/mL,P＜0.05],[332.38±11.97pg/mL,P＜0.05],and[334.35±16.23pg/mL, P＜0.05] respectively) (control group,291.62±17.03pg/mL;Fig.2E).Moreover,ITZ decreased the production of IL-10 at 1μmol/L(7.21±0.68 pg/mL vs 9.11±0.14pg/mL,P＜0.05;Fig.2F).

As shown by Fig.3,ITZ significantly improved the expression of iNOS in protein levels in the LPS and IL-4-induced groups.The expression of Arg-1 was slightly downregulated by ITZ at the concentration of 1μmol/L in both LPS and IL-4 groups.

Effects of ITZ on polarization of RAW264.7 cells induced by C.albicans

While stimulated with C.albicans,ITZ at 1μmol/L(38.34±1.36pg/mL)obviously improved the production of IL-6 of cells compared to that of the control group(32.32±0.84pg/mL, P＜0.05, Fig. 4A). Also, a similar result was observed in the secretion of TNF-α (1060.17±80.16pg/mL vs.890.84±52.82pg/mL,P＜0.01,Fig.4B).A decrease of IL-4 was identified in ITZ-treated group,0.5μmol/L(2.86±0.20pg/mL,P＜0.05)and 1μmol/L(2.24±0.33pg/mL,P＜0.001),compared to the control(3.91±0.23pg/mL;Fig.4C).Further,the production of IL-10 was evidently reduced in the group treated with 1μmol/L ITZ(19.46±2.05pg/mL vs.25.67±1.95pg/mL,P＜0.05;Fig.4D).

Figure 2.The effects of ITZ on the production of cytokines in LPS or IL-4 induced RAW264.7 cells.As is shown from A to F,ITZ increased the production of IL-6 and TNF-α in LPS-and IL-4-induced RAW264.7 cells,increased IL-1β and decreased IL-10 in IL-4 induced RAW264.7 cells,while IL-4 and IL-10 were undetected in the cells treated with LPS.All values represented means±standard deviation of three independent experiments.?P＜0.05,??P＜0.01 vs.LPS or IL-4 treated but itraconazole untreated group(in white).ITZ:itraconazole;IL:interleukin;LPS:lipopolysaccharide;TNF-α:tumor-necrosis factor-α.

As shown by Fig.5,ITZ significantly improved the protein level of iNOS after the induction with C.albicans.The expression of Arg-1 was slightly downregulated at the concentration of 1μmol/L ITZ.

ITZ increased phagocytosis of RAW264.7 cells

From the photos taken by fluorescence microscope we found that the merged color of yellow was much more noticeable in the 1μmol/L ITZ group than that in the control group(Fig. 6).The detection of phagocytosis measured by flow cytometry was shown in Fig.7.The phagocytic activity of RAW264.7 cells were increased at 1μmol/L of ITZ by 7.53%±2.21%and 9.73%±2.03%when co-inoculation of a 1:4 and 1:8 rate in comparison to the control group(Fig.8,P＜0.01).While in the 0.5μmol/L ITZ group,phagocyticactivityofcellswasincreasedonlyby 6.13%±0.55%and 5.54%±2.80%,respectively,compared to the control group(Fig.8,P＜0.05).

Discussion

The synergistic effects of the phagocytes of C.albicans and the immuoregulatory effects of ITZ on macrophages polarization were not clear.We demonstrated for the first time that ITZ has the potential to promote M1 over the M2 polarization of macrophages.

Macrophages, with their essential role in defending against infection and cancer, are involved in many pathogenesis.8-10In response to different microenvironmental signals,macrophages can polarize into M1 or M2 phenotype.11 It has been widely accepted that M1 macrophages is a class of effective fungicidal cells,whereas M2 macrophages is in contrast.Previous study indicated the pro-inflammatory effects of ITZ by increasing the TNF-α and IL-6 protein levels in mouse macrophage-like cells in vitro in the presence of LPS.6Our results confirm these results with significantly higher levels of TNF-α and IL-6 in mouse macrophage-like RAW264.7 cells during ITZ and LPS co-stimulation,and evidences of M1 polarization.11M1 macrophages play an important role in anti-microbial activity which connected to the secretion of enzymes that generate microbicidal factors,including iNOS.11-13Our data showed that ITZ improved the expression of iNOS in LPS-induced macrophages and downregulated the expression of Arg-1,which indicates a functional effects on modulating macrophage polarization.

Figure 3.The effects of ITZ on the protein expression of iNOS and Arg-1 in RAW264.7 cells induced by LPS or IL-4.ITZ significantly improved the protein expression of iNOS in protein levels after the induction with LPS(A and B)or IL-4(C and D),and slightly inhibited the expression of Arg-1 after 12hours treatment.All values represented means±standard deviation of three independent experiments.?P＜0.05,??P＜0.01 vs.ITZ-untreated control(in white),respectively.Arg:arginase;ITZ:itraconazole;iNOS:inducible nitric oxide synthase;IL:interleukin;LPS:lipopolysaccharide.

M2 macrophages are mainly found in Th2-dominated responses,as the Th2-driving IL-4 is a strong inducer of M2 polarization.14Stimulation with IL-4 in vitro resulted in M2 polarization,as evidenced by uniform induction of the applicable marker protein expression in RAW264.7 cells(Fig.3).Our data demonstrated that macrophages stimulated together with IL-4 and ITZ slightly inhibited the M2 polarization.This statistically significant trend is supported by the decreased IL-10,but increased TNF-α and IL-6 expression(Figs.2 and 4).Macrophages and their polarization state are crucial factors in anti-microbial defenses.Our data showed that ITZ downregulated Arg-1,but upregulated iNOS levels.This is the first published evidence,that M2 polarization by IL-4 was slightly, at least in part, reversed after stimulation of ITZ(Figs.2-5).

Figure 4.The effects of ITZ on the production of cytokines in RAW264.7 cells induced by C.albicans.As is shown from A to D,ITZ increased the production of IL-6 and TNF-α and decreased IL-4 and IL-10 in C.albicans-stimulated RAW264.7 cells.All values represented means±standard deviation of three independent experiments.?P＜0.05,???P＜0.001 vs.C.albicans treated but itraconazole untreated group(in white).Arg:arginase;C.albicans:Candida albicans;IL:interleukin;ITZ:itraconazole;TNF-α:tumor necrosis factor-alpha.

Figure 5.The effects of ITZ on the expression of iNOS and Arg-1 in RAW264.7 cells stimulated by C.albicans.ITZ significantly improved the expression of iNOS in protein levels after the stimulation of C.albicans,and slightly inhibited the expression of Arg-1.All values represented means±standard deviation of three independent experiments.?P＜0.05,??P＜0.01 vs.itraconazole-untreated control(in white),respectively.Agr:arginase;C.albicans:Candida albicans;iNOS:inducible nitric oxide synthase;ITZ:itraconazole.

Figure 6.Phagocytosis of C.albicans in ITZ-treated RAW264.7 cells measured by fluorescence microscope.ITZ enhances the phagocytosis of RAW264.7 cells in vitro.RAW264.7 cells ingested FITC-stained C.albicans is observed(Green:FITC-stained C.albicans;Red:PKH26-stained RAW264.7 cells;Yellow:merged color,RAW264.7 cells ingested C.albicans).C.albicans:Candida albicans;FITC:fluorescein isothiocyanate;ITZ:itraconazole.

Figure 7.Phagocytosis of C.albicans in ITZ-treated RAW264.7 cells measured by flow cytometry.Phagocytosis of C.albicans is both increased under ITZ in an indicated ratio of cells:yeast at 1:4 and 1:8.C.albicans:Candida albicans;ITZ:itraconazole.

Although clearance of C.albicans infection depends on the development of protective T-cell-mediated immunity,macrophages are the distal effectors,which either perform fungicidal function or improve intracellular persistence of C.albicans.10Phagocytosis is a key mechanism in the innate immune response to both bacterial and fungal clearance.We found that treatment of macrophages with ITZ increased phagocytosis of C.albicans.Moreover,ITZ upregulated the expression of TNF-α,IL-6,and iNOS,but downregulated the expression of IL-10,IL-4,and Arg-1,hence enhanced M1 polarization and inhibited M2 polarization.1μmol/L ITZ and a treatment for 12hours turned out to be optimal,which is similar to the pharmic concentration in human blood,when ITZ was orally taken 200mg,twice a day.15We emphasize the necessity to adjust the concentration and incubation time,when using other conditions or cell types.Lacking this preparatory step could explain some contradictory results of previous studies.

The study also has limits that we identified a M1 macrophage induced effects of ITZ in vitro LPS or IL-4 induced patterns,and in C.albicans induced macrophages,it not only enhanced M1 polarization,but also increased the phagocytosis of C.albicans.Also it may not completely parallel with the in vivo changes, for polarization is dynamic across time and involves the tissue microenvironment.Evaluating such dynamism is complex,but this study only taking a“snapshot”at some point in the inflammatory process.Besides,the immunoregulation of ITZ may be dependent on the microenvironment and then functionally regulated the phenotype and action of macrophages.As a result,the analysis of macrophage polarization status should be performed in vivo in mice models.

In conclusion,in the LPS and IL-4 induced macrophage polarization model,ITZ enhanced M1 polarization and slightly inhibited M2 polarization.When using C.albicans to imitate infection conditions, ITZ may have the potential to enhanced M1 polarization(enhanced the release of pro-inflammatory cytokines and the expression of iNOS),but also increased the phagocytosis of C.albicans yeast cells,which suggests that ITZ not only has its own bacteriostasis effect, but also promotes the elimination of fungi by promoting the release of proinflammatory factors and enhancing the phagocytosis of fungi.Consequently,ITZ may also improve the immunological responses by promoting M1 polarization against causative microorganism in vivo.Further studies will give more insight in the effects of ITZ as medical drug against fungal infection.It turns out that not only the fungicidal propriety of ITZ is effective,but also immunomodulatory proprieties.

Figure 8.Inhibitory effects of ITZ on the phagocytosis of RAW264.7 cells by flow cytometric method.The phagocytic activity of RAW264.7 cells is showed that 1μmol/L ITZ increases the phagocytic ability of cells by 7.53%±2.21%and 9.73%±2.03%at a ratio of 1:4(A)and 1:8(B)in comparison to the control group(P＜0.01).While at the 0.5μmol/L ITZ,phagocytic activity was increased by 6.13%±0.55%and 5.54%±2.80%(P＜0.05),respectively.Mean±standard deviation of data from three independent experiments is shown.ITZ-untreated group served as the control,?P＜0.01.ITZ:itraconazole.

Acknowledgement

This work was supported by the Chinese Academy Medical Sciences Initiative for Innovative Medicine(No.2016-I2M-3-021).