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Journal of Ethnopharmacology 184 (2016) 72–80

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Cytopiloyne, a polyacetylenic glucoside from Bidens pilosa, acts as anovel anticandidal agent via regulation of macrophages

Chih-Yao Chung a,1, Wen-Chin Yang b,c,d,e,1, Chih-Lung Liang f, Hsien-Yueh Liu a,Shih-Kai Lai a, Cicero Lee-Tian Chang a,n

a Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwanb Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwanc Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwand Institute of Pharmacology, Yang-Ming University, Taipei 112, Taiwane Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwanf Department of Microbiology and Immunology, Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung 402, Taiwan

a r t i c l e i n f o

Article history:Received 11 July 2015Received in revised form26 January 2016Accepted 25 February 2016Available online 26 February 2016

Keywords:Bidens pilosaCandidaCytopiloyneMacrophagePKC

x.doi.org/10.1016/j.jep.2016.02.03641/& 2016 Published by Elsevier Ireland Ltd.

espondence to: Department of Veterinary Meity, 250, KuoKuang Rd., Taichung, Taiwan.ail address: [email protected] (C.-T. Changese authors contributed equally to this work

a b s t r a c t

Ethnopharmacological relevance: Bidens pilosa, a tropical and sub-tropical herbal plant, is used as anethnomedicine for bacterial infection or immune modulation in Asia, America and Africa. It has beendemonstrated that cytopiloyne (CP), a bioactive polyacetylenic glucoside purified from B. pilosa, increasesthe percentage of macrophages in the spleen but the specific effects on macrophages remain unclear.Aim of the study: The aim of this study was to evaluate the effects of CP on macrophage activity and hostdefense in BALB/c mice with Candida parapsilosis infection and investigate the likely mechanisms.Materials and methods: RAW264.7 cells, a mouse macrophage cell line, were used to assess the effects ofCP on macrophage activity by phagocytosis assay, colony forming assay and acridine orange/crystal violetstain. To evaluate the activity of CP against C. parapsilosis, BALB/c mouse infection models were treatedwith/without CP and histopathological examination was performed. The role of macrophages in theinfection model was clarified by treatment with carrageenan, a selective macrophage-toxic agent.RAW264.7 macrophage activities influenced by CP were further investigated by lysosome staining,phagosomal acidification assay, lysosome enzyme activity and PKC inhibitor GF109203X.Results: The results showed that CP in vitro enhances the ability of RAW264.7 macrophages to engulf andclear C. parapsilosis. In the mouse model, CP treatment improved the survival rate of Candida-infectedmice and lowered the severity of microscopic lesions in livers and spleens via a macrophage-dependentmechanism. Furthermore, with CP treatment, the fusion and acidification of phagolysosomes were ac-celerated and the lysosome enzyme activity of RAW264.7 macrophages was elevated. PKC inhibitorGF109203X reversed the increase in phagocytic activity by CP demonstrating that the PKC pathway isinvolved in the macrophage-mediated phagocytosis of C. parapsilosis.Conclusions: Our data suggested that CP, as an immunomodulator, enhances macrophage activity againstC. parapsilosis infections.

& 2016 Published by Elsevier Ireland Ltd.

1. Introduction

Candida species, commensal yeast cells in the human body,have emerged as a frequent threat to patients whose immunity iscompromised by immune deficiencies, transplantation and can-cers (Fidel Jr., 2002; Guinea, 2014). These pathogens cause

dicine, National Chung-Hsing

)., and are the co-first writers.

candidiasis, which is characterized by symptoms ranging fromlocal complications to systemic candidemia (Fidel Jr., 2002; Gui-nea, 2014). Systemic Candida infection has a mortality rate of up to35% (Guinea, 2014; Kontoyiannis and Lewis, 2002). In humans, themost common cause of opportunistic fungal disease is Candidaalbicans, which accounts for 62% of cases of candidemia (Guinea,2014; Kontoyiannis and Lewis, 2002; Silva et al., 2012). But othernonalbicans Candida spp., including C. parapsilosis have alsoemerged as significant pathogens, especially in women with vul-vovaginal infections (Silva et al., 2012). In addition, the incidenceof C. parapsilosis infection has increased drastically over the past

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decade and is reported to be the second most frequently isolatedCandida species from blood cultures (Silva et al., 2012). The pro-pensity of C. parapsilosis to colonize intravascular devices andprosthetic materials contributes to a significant problem in low-birth-weight neonates, transplant recipients and patients receiv-ing parenteral nutrition, whom require prolonged used of a centralvenous catheter or indwelling devices (Silva et al., 2012).

In host defense mechanisms, the immune system is required tolimit and eliminate Candida dissemination. Macrophages, an es-sential immune cell subset in microbial clearing, function pre-dominantly in defense and trigger adaptive immunity againstCandida (Fidel Jr., 2002; Vazquez-Torres and Balish, 1997). Thecrucial role of macrophages in resistance to systemic Candida in-fection has been confirmed in animal models (Fidel Jr., 2002;Redmond et al., 1993). Phagocytosis is a central process of patho-gen degradation in host defense. Macrophages engulf invadingpathogens into phagosomes (Steinberg et al., 2007). The phago-somes undergo a complex maturation process, fusing with endo-somes and lysosomes to form phagolysosomes, which acidify theirluminal contents and enhance activity of lysosome enzymes (Diet al., 2006; Steinberg et al., 2007). Various intracellular micro-organisms, however, can escape from the phagosomal pathway todevelop persistent infections (Birmingham et al., 2008; Di et al.,2006; Seider et al., 2010). C. krusei can survive and replicate inmacrophages by damaging phagolysosome maturation (Garcia-Rodas et al., 2011). C. albicans shows diverse intracellular survivalmechanisms in macrophages with inhibition of phagosomal acid-ification and nitric oxide production (Fernandez-Arenas et al.,2009; Seider et al., 2010). A secreted lipase deletion of C. para-psilosis impairs its survival in macrophages (Gacser et al., 2007).Therefore, Candida spp. has acquired strategies that permit in-tracellular survival and escape from macrophages.

Anti-fungal drugs are commonly used to treat candidiasis.However, due to the limited efficacy of existing anti-fungal drugsand the increase in drug-resistant Candida mutants, new strategiesare urgently needed to combat such a challenging fungal disease,including increased antifungal dose intensity, combination anti-fungal therapy, investigational antifungals, and immunomodula-tion (Kontoyiannis and Lewis, 2002). Clinical resistance, a persis-tent infection of a laboratory susceptible fungal isolate, is observedin immunodeficient patients (Kontoyiannis and Lewis, 2002). Im-munomodulators which can improve subtle cell functions are seento be a means to overcome this phenomenon (Kontoyiannis andLewis, 2002; Masihi, 2000). Thus, a new therapeutic strategy usinga combination treatment of antifungal drugs and edible im-munomodulatory herbs, might improve the efficacy of antifungaldrug therapy in patients treated with immunosuppressors underhigh incidence of opportunistic fungal infection (Kontoyiannis andLewis, 2002; Masihi, 2000).

Bidens pilosa, an edible Asteraceae plant widely found in tro-pical and subtropical areas of the world, is traditionally eaten as avegetable, and used in teas and herbal medicines in Africa,America and Asia (leaves, stems, flowers, roots and whole plantsare all used), for wounds, bacterial and malarial infection, in-flammation, etc. (Bartolome et al., 2013; Geissberger and Sequin,1991; Lans, 2007; Rabe and van Staden, 1997). Compounds ex-tracted from B. pilosa have been shown to have im-munomodulatory (Chang et al., 2007b,c; Chiang et al., 2007) andantimicrobial (Chang et al., 2007c; Nakama et al., 2012; Tobinagaet al., 2009; Yang et al., 2015) activity. Among these compounds,polyacetylenes, which have multiple bioactivities, could be can-didates for Candida treatment. Cytopiloyne (CP), a novel poly-acetylene compound extracted from B. pilosa (Chiang et al., 2007),was able to prevent type 1 diabetes via regulation of T helper (Th)cell differentiation (Chang et al., 2007a; Chiang et al., 2007; Yang,2014). This compound also increased the percentage of

macrophages in spleens (Chang et al., 2007a). DNA microarrayanalysis of CP on LPS-stimulated THP-1 monocytes showed thatthe extracellular signal-regulated kinase (ERK) 1/2 pathway is akey target of CP (Chiu et al., 2010). In addition, in a type 2 diabetesmouse model, CP was able to increase insulin expression andprotect pancreatic β cells via a PKC-dependent mechanism (Changet al., 2013; Chien et al., 2009; Yang, 2014). The data clearly sug-gest the role of CP in pancreatic β cells, T cells and macrophages.However, the effect of CP on macrophage function and macro-phage-mediated clearance of Candida species remains unclear.

In this study, the anti-fungal action and mechanism of CP invitro and in vivo was examined. The in vitro anti-fungal effect of CPon Candida species was detected and the in vivo effect of CP againstC. parapsilosis, the second most common causative agent of can-didiasis worldwide (Silva et al., 2012), was evaluated in mice.Furthermore, the mechanisms by which CP helped macrophagesclear Candida in mice was investigated.

2. Materials and methods

2.1. Chemicals, cells and mice

B. pilosa plants were collected from the campus of AcademiaSinica, Taiwan and authenticated by the Biodiversity Center, Aca-demia Sinica, Taiwan. CP was prepared to 98% purity from wholeplant of B. pilosa as previously described (Chang et al., 2007a).Briefly, CP was isolated on an RP-18 HPLC column by methanolextraction and ethyl acetate partition of whole B. pilosa plants.Structure and purity were confirmed by NMR spectra using aBruker DMX-500 spectrometer and nuclear magnetic resonancedetermination, respectively. Ketoconazole (KTC), carrageenan (CA),dimethyl sulfoxide (DMSO), thioglycollate (TG), acridine orange(AO), crystal violet, DX40-FITC, Triton X-100, phosphate-bufferedsaline (PBS), ρ-nitrophenyl phosphate and GF109203X (PKC in-hibitor) were obtained from Sigma-Aldrich (St. Louis, MO, USA).Tryptic soy broth (TSB) and, sabouraud dextrose agar (SDA) wereobtained from Neogen (Lesher PI, Lansing, MI, USA). The periodicacid Schiff (PAS) staining kit came from Merck (Darmstadt, Ger-many). Fluorescence-labeled zymosan particles, fetal calf serum(FBS) and Dulbecco's modified Eagle's medium (DMEM) were or-dered from Gibco (Grand island, NY, USA). Candida parapsilosis(BCRC 20515, Biosource Collection and Research Center, Hsinchu,Taiwan) was stored at �20 °C and cultured on SDA at 25 °C. In-ocula were prepared by suspending one colony of �1 mm indiameter in 5 ml of TSB and incubating at 37 °C for 24 h. Thecolony forming unit (CFU) of the resulting suspension was de-termined using a hemocytometer and confirmed by colony countson an SDA plate. The RAW264.7 cell line (BCRC 60001) was ob-tained from the Bioresource Collection and Research Center (FoodIndustry Research and Development Institute, Hsinchu, Taiwan). Itwas cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% FBS, and 1% penicillin/streptomycin/glu-tamine and maintained at 37 °C in a CO2 humidified incubator.Female 6–8 week-old BALB/c mice (19.2–21.0 g) used in this studywere obtained from the National Laboratory Animal Center (Taipei,Taiwan), and handled according to the guidelines of the Institu-tional Animal Care and Utilization Committee of National ChungHsing University (affidavit of approval of animal use protocol,National Chung Hsing University: 99-77). Mice in this study werekilled by CO2 inhalation.

2.2. Phagocytosis assay, colony forming assay, and acridine orange/crystal violet staining

According to our previous study (Chiang et al., 2007),

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RAW264.7 cells (0.1�106 cells/ml) were pre-treated with DMSOand CP (1, 2.5 and 5 μg/ml) for 24 h at 37 °C. For phagocytosisassay, the cells were incubated with fluorescence-labeled zymosanparticles at 37 °C. Thirty minutes later, the cells were washed withFACS buffer 3 times, fixed by adding 200 μl FACS fixation buffer,and analyzed with a flow cytometer (Di et al., 2006; Kim et al.,2001).

For colony forming assay, C. parapsilosis was added to amonolayer of RAW264.7 cells at a cell/yeast ratio of 1:15. Phago-cyte-free incubation of yeasts was used as a control of yeast via-bility. After 30 min, the non-phagocytized yeast in the super-natants and washings were serially diluted and plated on SDA.Two hundred microliters of 50% SDA in culture mediumwas addedto the monolayer of the cells. After incubation for 3 h at 37 °C, thecells were detached and collected. The intracellular yeasts werereleased by adding 200 μl distilled H2O to lyse phagocytes threetimes and quantified by spreading tenfold dilutions of each sampleon SDA agar plates. The numbers of CFU were counted after 48 h ofincubation at 37 °C. The percentage of yeast killed by the phago-cytes was defined as follows: 100%� [1�(CFU after incubation)/(CFU at time 0�control CFU)] (Gacser et al., 2007).

For acridine orange/crystal violet staining, RAW264.7 cells wereplaced on a cover slide in the presence or absence of C. parapsilosis.The cells were stained with 0.01% acridine orange for 45 s (Pru-zanski and Saito, 1988). After gentle washing with HBSS, the cellswere stained for 30–45 s with 0.05% crystal violet. After 3 runs ofthe rinse, the cells were mounted with the medium and examinedby fluorescent microscopy (Leica DMI3000B, Major, Taipei, Tai-wan). For each experiment, 3 fields in each wells were counted,and at least 200 macrophages were analyzed in each well. Yeastviability was determined by the color of the fluorescence. Dead(ingested) C. parapsilosis displayed bright red/yellow metachro-matic fluorescence, while live yeasts displayed green fluorescence(Gacser et al., 2007).

2.3. Candida infections, CFU determination and histopathology

According to our previous study (Chang et al., 2007a), six tofourteen female (6 to 8-week-old, 19.2–21.0 g) BALB/c mice pergroup received intraperitoneal injection of PBS (control) or PBSsolution of CP (5, 12.5 and 25 μg/kg BW) three times per week for2 weeks. As a positive control, ketoconazole (KTC, 50 mg/kg BW),an antifungal drug, was orally administered to another group ofmice by gavage at 4 h post C. parapsilosis infection and once daily(Cacciapuoti et al., 1992). For survival assay, C. parapsilosis at alethal dose (2.5�108 CFU/300 μl PBS) was intraperitoneally in-jected into BALB/c mice one day after the final injection of PBS orCP.

To clarify the role of macrophages in host defense against C.parapsilosis infection, carrageenan (1 mg, 200 μl), which selec-tively depletes macrophages, was administered by intraperitonealinjection 2 days before infection (Barnes et al., 2008; Wong andHerscowitz, 1979). The animals were examined every 6 h formortality.

To determine the level of yeast dissemination, BALB/c micewere challenged with a dose of C. parapsilosis (5�106 CFU) aspublished (Gacser et al., 2007). The animals were killed at 12 and72 h post challenge (Conchon-Costa et al., 2007). Their liver,spleen, and kidneys were weighed and cut into 2 parts for CFUdetermination and histopathological examination. For CFU count,one part of the liver, spleen, and kidney were homogenized, di-luted and plated on SDA agar incubated at 37 °C for 48 h (Con-chon-Costa et al., 2007; Gacser et al., 2007). The rest of the organswere fixed with 10% formalin. Twenty random fields (200� , H&E)of livers and spleens per mouse were examined for the numberand area of inflammatory foci (Dai et al., 1997). Inflammatory foci

were graded as follows: 1, o1000 mm2; 2, 1000–2000 mm2; 3,2000–3000 mm2; 4, 3000–4000 mm2; 5, 44000 mm2. The in-flammatory score in the liver was obtained based on the formulamodified from the insulitis index (Shen et al., 2011). The degree ofliver inflammation (%)¼100%� (total scores of inflammatory fociin liver/the number of inflammatory foci in liver). Yeasts in theorgans were identified by the deparaffinized section stained withPAS kit (Merck, Darmstadt, Germany).

2.4. Lysosome staining, phagosomal acidification and lysosome en-zyme activity

Pre-treated RAW264.7 cells (0.1�106 cells/ml) were incubatedwith DX40-FITC (1 mg/ml) for 30 and 60 min for lysosome staininganalysis. After extensive washing, the cells were fixed and un-derwent FACS analysis (Di et al., 2006).

For phagosomal acidification assay, RAW274.7 cells were in-cubated for 30 min at 37 °C with the fluorescence-labeled zymo-san. The cells were excited with 485 nm and 535 nm lasers andvisualized with TRIAD multimode reader (Magellan Bioscience,Tampa, FL, USA). Measuring the fluorescence against different pHbuffers performed titrations of the fluorescein-labeled zymosanfluorescence versus pH. The standard curve was determined invitro using a series of buffered solutions at the appropriated pH aspH range tested in Fig. 4B. The data were fit with linear regressionanalysis (Di et al., 2006).

Cellular lysosome enzyme activity was detected using the fol-lowing procedure. RAW264.7 cells were solubilized in 25 μl of 0.1%Triton X-100. The lysates were incubated with 150 μl of 10 mM ρ-nitrophenyl phosphate for 1 h at 37 °C. Fifty microliters of 0.2 mborate buffer (pH 9.8) were added to stop the reaction. The mix-ture underwent spectrophotometry at 405 nm. Relative lysosomeenzyme activity (%) of the mixture was obtained from the ratio ofthe absorbance at 405 nm of CP-treated cells to that of control cellsmultiplied by 100% using spectrophotometry (Kim et al., 2001).

2.5. PKC activation and inhibition assay

RAW264.7 cells (0.1�106 cells/ml) were pre-treated withDMSO and CP (1 μg/ml) for 24 h at 37 °C. The PKC activator andinhibitor, PMA (1 μM) and GF109203X (1 μM), respectively, wereadded 30 min prior to the assays (Chang et al., 2013). RAW264.7cells were then incubated with zymosan particles (1�106, 2 μl)for 30 min at 37 °C. After washing, cells were scattered on slices bycytospin and went through microscopic examination with LiuStain (Chang et al., 2007c; Loke et al., 2007). Phagocytic Activitywas calculated as phagocytic cell number divided by total cellnumber.

2.6. Statistical analysis

Data from three independent experiments or more are pre-sented as mean7SEM. Two-tailed Student's t test, Kruskal-Wallistest and ANOVA test were used for statistical analysis of differ-ences between groups according to the data type, and a P value ofless than 0.05 was considered to be statistically significant.

3. Results

3.1. CP enhances macrophage phagocytosis activity and intracellularkilling

Macrophages play key roles in host defense by recognizing,engulfing, and killing microbes. In this study, we intended to testwhether the CP (Fig. 1A), a plant compound, enhances killing of C.

Fig. 1. Cytopiloyne (CP) enhances phagocytosis and intracellular killing of Candida by macrophages. (A) Chemical structure of CP. (B) Flow cytometry analysis of RAW 264.7cells incubated with fluorescein-labeled zymosan. (C) Number of yeasts in the supernatants of RAW 264.7 cells incubated with C. parapsilosis. (D) The number of yeasts insideRAW 264.7 cells was determined. Data of three or more experiments are presented as mean7SD. ANOVA was used to assess differences between the groups.*Po0.05,**Po0.01, ***Po0.001. (E) RAW 264.7 cells pre-treated with PBS and CP (5 μg/ml) were incubated with C. parapsilosis. Dead C. parapsilosis (arrowheads) display bright red/yellow fluorescence, while live yeasts display green fluorescence (arrows). Representative images are shown. Scale bar¼10 mm.

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parapsilosis by macrophages. Phagocytosis assay with zymosan, aligand of yeast cell walls, showed that the count of fluorescence-stained macrophages increased 1.8–2.2 fold with CP 1–5 μg/ml,but the peaks of fluorescence intensity did not shift (Fig. 1B). Drugsusceptibility and MTT assays were also performed to make surethat CP did not have a direct cytotoxic effect on C. parapsilosis(Supplementary Fig. S1A) and influence the growth of RAW264.7cells (Supplementary Fig. 1B). To further assess the overall effectsof CP on phagocytosis and clearance abilities of macrophages,colony forming assay with C. parapsilosis was performed. The re-sults showed that CP, increased phagocytosis of macrophages 1.6–2.1 fold in a dose dependent manner (Fig. 1C), and enhanced the

intracellular killing to C. parapsilosis 2.1–5.4 fold (Fig. 1D). Ob-servation of the living (green) and dead (bright red/yellow) yeastcells inside macrophages by using acridine orange/crystal violetstaining under fluorescent microscopy also showed that CP en-hanced the phagocytosis activity and intracellular killing to C.parapsilosis (Fig. 1E).

3.2. CP elevates the resistance of mice challenged with C. parapsilosisin a macrophage-dependent fashion

The mouse infection model was used to assess the in vivo effectof CP. In the experiment, the survival rates of CP-treated mice

Fig. 2. Cytopiloyne (CP) protects C. parapsilosis-infected mice from death involvingmacrophages. (A) Survival rate of 6- to 8-week-old BALB/c females that receivedPBS, ketoconazole (KTC, 50 mg/kg BW), or CP (5, 12.5 and 25 μg/kg BW) and werechallenged with a lethal dose of C. parapsilosis (2.5�108 CFU). (B) Survival rate ofPBS or CP (25 μg/kg BW) pre-treated mice injected with carrageenan (CA; 1 mg in200 μl) 2 days before an intraperitoneal challenge with Candida (2.5�108 CFU). Thenumber (n) of mice is indicated in the parentheses. Long rank test was used tocompare the survival rate between control and experimental groups. *Po0.05,**Po0.01, ***Po0.001.

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ranged from 66.7% to 100% at 168 h post Candida infection (CP5,12.5 and 25, Fig. 2A), while the survival rates of PBS-treated andKTC-treated mice were 21.4% and 88.9% (PBS and KTC, Fig. 2A).KTC, an antifungal drug, significantly promoted the survival ratefrom 21.4% to 88.9%. Meanwhile, the survival rate of CP-treatedmice was significantly higher than that of the PBS-treated miceand the survival rate of the mice treated with CP 25 μg/kg BW waseven better than that of KTC-treated mice, although not at a sta-tistically significant level. Carrageenan administration significantlyreduced the survival rate of the mice pre-treated with CP 25 μg/kgBW by 50% (CPþCA, Fig. 2B), but did not change the survival rateof PBS treated group at 168 h post infection (PBSþCA, Fig. 2B).However, the carrageenan treated mice died 24 h earlier than non-treated carrageenan mice (Fig. 2B). Mouse macrophages werecounted to make sure the effect of carrageenan, and the treatmentwas found to have decreased macrophages to 22.6–27.4% of theoriginal amount (Supplementary Fig. S2). Similarly, CP (1.5–25 μg/kg BW) also affected the survival rate of Listeria monocytogenes-infected mice (Fig. 1 in Chung et al., submitted for publication).Therefore, administration with CP higher than 6.25 μg/kg BWcould prevent mice from death after lethal L. monocytogenesinfection.

3.3. CP restrained the dissemination of Candida and ameliorated thehepatic and splenic lesions of mice challenged with Candida

Liver, spleen and kidneys were taken for CFU counting to de-termine the dissemination of yeast. The number of Candida in eachgroup decreased with time (Fig. 3A). In the liver and kidneys, KTCand CP-treated mice had at least 1 order lower number of Candidathan the PBS-treated control mice at 72 h post infection (Fig. 3A).In the spleen, KTC and CP-treated mice had a lower number ofCandida than the PBS-treated control mice at 12 h post infection

but only CP-treated mice had a one-order lower number of Can-dida than those of PBS-treated mice at 72 h post infection (Fig. 3A).Slices of liver and spleen were histopathologically examined tofurther investigate the effect of CP in mice. Due to the inflamma-tion in the liver, the number of inflammatory foci and the per-centage of inflammation increased 2.9 fold and 1.5 fold, respec-tively, in the PBS-treated control mice over time (Fig. 3B and C). Incontrast, the CP-treated mice had lower numbers of inflammatoryfoci than those of PBS-treated control mice at 12 and 72 h postinfection, 40% and 33.3%, respectively, although, over time, thenumber of inflammatory foci increased 2.4 fold (Fig. 3B), The CP-treated mice also had a lower percentage of inflammation than thePBS-treated mice at 72 h post infection and all the areas of in-flammatory foci of CP-treated mice were less than 3000 mm2. Inaddition, the percentage of inflammation in CP-treated miceshowed no difference over time (Fig. 3C). Over all, microscopyshowed that CP decreased size of necrotic and inflammatory le-sions in the livers compared to those of PBS control at 72 h postinfection (Fig. 3D; upper panel). Moreover, lower PAS-positiveCandida were observed in splenic sections of CP-treated mice at 12and 72 h post infection (Fig. 3D; lower panel). Similarly, CP low-ered the CFU counts and lesion severity of Listeria monocytogenes-infected mice (Fig. 2 in Chung et al., submitted for publication).

3.4. CP promotes phagolysosomal fusion, phagosomal acidificationand lysosome enzyme activity of macrophages

To investigate the effect of CP on phagosome maturation, aseries of experiments were performed. Incubation of DX40-FITC(1 mg/ml) with RAW264.7 cells for 30 and 60 min showed that CPenhanced phagolysosomal fusion 1.4–2.8 fold and 1.5–2.4 fold inRAW264.7 cells in a dose-dependent manner (Fig. 4A). The acid-ification assay indicated that CP dose-dependently decreased pHvalues, ranging from 5.5 to 4.3 inside macrophages (Fig. 4B). Fur-ther, CP dose-dependently increased the lysosome enzyme activity1.5–2.4 fold (Fig. 4C).

3.5. CP promotes phagocytosis in a PKC-dependent manner

Our previous study indicated that CP regulated insulin pro-duction by a mechanism that involved the calcium/DAG/PKCαcascade in beta islet cells (Chang et al., 2013). In order to in-vestigate the mechanism by which CP augmented macrophagephagocytosis, PKC inhibitor, GF109203X, was used. The resultshowed that CP, like PMA, increased phagocytic uptake of zymosanparticles. In the meantime, the addition of GF109203X obviouslydiminished phagocytosis (Fig. 5A). The data shown in Fig. 5A werere-plotted into histograms. PMA and CP significantly increasedphagocytic activity 2.6 fold over the control group. The PKC in-hibitor, GF109203X, reduced the phagocytic activity of PMA- andCP-treated macrophages to 35.6% and 26.9% of the control group;and 13.5% of PMA and 10.1% of the CP group (Fig. 5B), respectively.

4. Discussion

Macrophages play a central role in host defense against in-tracellular pathogens such as Candida (Fidel Jr., 2002; Redmondet al., 1993; Vazquez-Torres and Balish, 1997). Although the anti-microbial activity of some polyacetylenes has been reported (To-binaga et al., 2009), the growth of C. parapsilosis showed no dif-ference between the sample that received CP (2.5, 5 and 10 μg/ml)or vehicle DMSO (Supplementary Fig. S1A). On the other hand, thegrowth of RAW264.7 macrophages was not affected by CP (Sup-plementary Fig. S1B), which is consistent with a previous study onTHP-1 cells (Chiu et al., 2010). Moreover, lipase deletion of C.

Fig. 3. Cytopiloyne (CP) diminishes fungal load in different organs of mice challenged with C. parapsilosis. (A) CFU in livers, spleens and kidneys at 12 and 72 h post-infection(n¼5, for each group at each time point). (B) The number of inflammatory foci and (C) the percentage of foci score in livers at 12 and 72 h post-infection. (D) Representativeimages of livers (upper panel; arrowheads: necrotic and inflammatory lesions; inserted numbers: sizes of these lesions) and spleens (lower panel; arrowheads: PAS-positive,C. parapsilosis). Data of 3 or more experiments are presented as mean7SD. *Po0.05, **Po0.01, ***Po0.001. Kruskal-Wallis test were used for the analysis of the CFU dataand the foci score in livers. ANOVA was used to assess the other data. Lesion sizes were measured using ImageJ.

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parapsilosis has been shown to increase the clearance of yeasts byphagocytosis and intracellular killing of macrophages (Gacseret al., 2007). Nevertheless, the consistency of results of phagocy-tosis assays with zymosan and C. parapsilosis suggested that CP hasno effect on the yeast metabolic pathways which do not affectgrowth rate, but rather affected the phagocytosis activity of mac-rophages directly.

In addition, the peaks of fluorescence intensity of phagocytosisassay with zymosan (Fig. 1B) did not shift suggesting that CP dose-dependently increased the number of phagocytosis-active mac-rophages but did not increase the ability of each macrophage to

engulf more pathogens. Although the colony forming assay cannotdifferentiate between these two conditions, the phagocytosis in-dex (the average number of yeasts in phagocytosis-active macro-phages) calculated from acridine orange/crystal violet staining andPKC activation and inhibition assay supported the previous ob-servation. The phagocytosis index ranged from 1.3–1.5 and 1.5–2.6,respectively, and there was no significant statistical differenceamong the groups.

Macrophages are equipped with two intracellular killing me-chanisms, oxygen-dependent and -independent (Fidel Jr., 2002;Vazquez-Torres and Balish, 1997). The result of the colony forming

Fig. 4. Cytopiloyne (CP) promotes phagolysosomal fusion, phagolysosomal acid-ification and lysosomal enzyme activity during phagocytosis in macrophages.(A) Lysosome fusion analysis for RAW 264.7 cells incubated with DX40-FITC.(B) The pH inside phagolysosomes of RAW 264.7 cells (n¼10) incubated withfluorescein-labeled zymosan. (C) Lysosome enzyme activity of RAW 264.7 cells.Data of three or more independent experiments are expressed as mean7SD.ANOVA was used to assess differences between the groups. *Po0.05, **Po0.01,***Po0.001.

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assay suggested that the increase in intracellular killing to Candidawas not simply because of the increase in phagocytosis (Fig. 1C andD). The increase in intracellular killing appears to be a synergisticeffect of the increase in phagocytosis and the activation of lyso-some enzymes or phagolysosomal acidification (Figs. 1 and 4). Onthe other hand, the NO production of macrophages, an oxygen-dependent mechanism, was not affected by CP (data not shown).That is, CP treatment promoted intracellular killing via an oxygen-independent mechanism. Previous studies have shown that CPpromotes IL-4 secretion in mice (Chang et al., 2007a; Chiang et al.,2007); IL-4 and IL-13 has also been demonstrated to enhance ly-sosome fusion (Gordon and Martinez, 2010). Although the pha-golysosomal fusion assay in this study was performed in vitro(Fig. 4A), the effect of CP on phagolysosomal fusion might relate to

its effect on the IL-4 secretion. However, the relationship betweenCP-elevated phagolysosomal fusion and acidification remainsunclear.

We also demonstrated that the macrophage-mediated Candidaeradication involved the PKC pathway (Fig. 5). As CP can regulateinsulin secretion through the calcium/DAG/PKCα cascade in betacells (Chang et al., 2013), it is possible that CP-mediated clearanceof Candida species is via the PKC signaling pathway. PKC is knownto activate macrophages, enhance phagocytosis and affect bacteriainternalization/escape from the phagosome (Schwegmann et al.,2007; Shaughnessy et al., 2007). Interestingly, treatment for 15 hwith PMA has been shown to deplete PKC (von Knethen et al.,2007; von Knethen et al., 2005), while treatment for 24 h with CPincreased the phagocytosis mediated by PKC. This difference im-plies that the molecular mechanism of CP is different from PMA.Despite significant progress in understanding CP made since itsdiscovery, the molecular cascade linking CP to the PKC pathwayand, particularly, macrophage function is still unclear and needs tobe further mapped. In the future, identification of the cellulartarget(s) of CP will be important to decipher the molecular basis ofCP in macrophages and other cells, and explain the mechanismbehind the anti-candidal function of CP.

In the mouse infection model, the use of carrageenan, which isselectively toxic to macrophages, depleted macrophages in mice,leading to a reduction in survival rate in CP-treated mice (Fig. 2B).The effect of CA on macrophages (Supplementary Fig. S2) wasconsistent with another report of a 38% decrease in mice splenicmacrophages with 1 mg carrageenan treatment (Wong and Hers-cowitz, 1979) and a 21% cell survival rate of in vitro 48 h culture ofnormal peritoneal macrophages in the presence of carrageenan(500 μg/mL) (Barnes et al., 2008). Further, it also suggests that thedecreases in Candida load, CFU inflammatory foci, and score inlivers might relate to macrophage functions of CP-treated mice(Fig. 3). However, the effect of CP on Th cell differentiation, in favorof the promotion differentiation into Th2 cells and IL-4 secretion(Chang et al., 2007a; Chiang et al., 2007), might also contribute tothe reduction of death and the inflammatory foci area caused byacute inflammation.The in vivo data illustrate the important role ofmacrophages in CP-mediated Candida clearance although otherimmune cells can also be affected by CP. Since CP treatment in-creased the number of macrophages in mouse spleens, its effect onphagocyte function adds another reason to consider it as a possiblecomplimentary compound to control Candida infection.

We also found that CP protected mice against Listeria infection(Figs. 1 and 2 in Chung et al., submitted for publication). Th1-typeresponses, IFN-γ and TNF-α, are associated with protection againstCandida and Listeria infection (Chang et al., 2007c; Conchon-Costaet al., 2007; Dai et al., 1997; Fidel Jr., 2002; Redmond et al., 1993;Vazquez-Torres and Balish, 1997). This seems contradictory to theresults from a previous study that reported that CP promoted Th2cell differentiation and increased IL-4 secretion (Chang et al.,2007a; Chiang et al., 2007). However, the requirement of sufficientIL-4 in macrophage proliferation has been demonstrated (Jenkinset al., 2011). Also, IL-13, a Th2-type cytokine, has been shown toincrease the phagocytosis of macrophages against C. albicans in-fection (Coste et al., 2003, 2008). IL-4 and IL-13 induce CD36 andmannose receptor expression, respectively, in monocytes/macro-phages via a PPAR-γ signaling pathway (Coste et al., 2003, 2008).Curcumin, a natural plant product, also enhances phagocytosis viaPPAR-γ activation and CD36 expression (Mimche et al., 2012). ThePPAR-γ signaling pathway is also associated with PKC activation(von Knethen et al., 2007). A previous study also showed that CPdoes not inhibit early inflammation expression on LPS-stimulatedTHP-1 (Chiu et al., 2010). Therefore, it seems that the effects of CPon host defense and macrophages cannot simply be explained byTh1/Th2 paradigm.

Fig. 5. Cytopiloyne (CP)-mediated phagocytosis involves the PKC pathway. (A) Liu's staining of RAW 264.7 cell incubation with zymosan particles. (B) Phagocytic activity ofRAW 264.7 cells. Data of three or more experiments are presented as mean7SD. ANOVA was used to assess differences between the groups. *Po0.05, **Po0.01,***Po0.001.

Fig. 6. Schematic diagram indicating the likely mechanism underlying the effect ofcytopiloyne (CP) against Candida. CP can clear C. parapsilosis in mice via an up-regulation of phagocytosis in macrophages involving PKC pathway. CP also canpromote phagosomal acidification, phagolysosome fusion, and lysozyme activity.

C.-Y. Chung et al. / Journal of Ethnopharmacology 184 (2016) 72–80 79

Various intracellular pathogens overturn normal phagosomefunction; for example, inhibit phagolysosome fusion, interferewith scavenger receptor expression, and decrease phagolysosomepH value and increase lysosome activity, to sustain their in-tracellular infection and escape from macrophages (Birminghamet al., 2008; Di et al., 2006; Seider et al., 2010). Here we demon-strated that CP promotes the survival rate of mice with two in-tracellular pathogen infections, C. parapsilosis and L. mono-cytogenes (Figs. 2 and 1 in Chung et al., submitted for publication).The results suggested that CP targeted the same or a similar me-chanism involving in the intracellular survival of C. parapsilosis andL. monocytogenes. Listeriolysin O of L. monocytogenes (Birminghamet al., 2008) and secreted lipase of Candida species (Gacser et al.,2007; Paraje et al., 2008) have been shown to be important viru-lence factors involved in intracellular survival. Therefore, the me-chanism of reversal of intracellular survival by CP might be asso-ciated with lipid metabolism, which seems to be critical in thephagocytic process (Steinberg and Grinstein, 2008).

The increased incidence of oral infection of non-albicans Can-dida species due to inherent resistance to fluconazole, a first-lineantifungal drug has been recognized in the clinic (Kontoyiannisand Lewis, 2002; Silva et al., 2012). Traditional medicines or theirphytochemicals are being researched and developed for use asantifungal agents that limit drug-resistance and residual toxicity(Kontoyiannis and Lewis, 2002; Masihi, 2000). Many extracts andphytocompounds isolated from B. pilosa, for example poly-acetylenes and flavonoids, have been reported to have im-munomodulatory (Chang et al., 2007a,b,c; Chiang et al., 2007),anti-protozoal (Yang et al., 2015) and anti-bacterial (Tobinagaet al., 2009) activity. Several edible Bidens plants have been used asherbal drugs in America, African and Asia for many years (Barto-lome et al., 2013; Yang, 2014). Thus, a new therapeutic strategythat uses a combination treatment of antifungal drugs and edibleimmunomodulatory herbs might improve the efficacy of anti-fungal drug therapy on patients treated with immunosuppressantsunder high incidence of opportunistic fungi infection.

5. Conclusion

In this study, we demonstrated that CP enhances macrophage-mediated Candida eradication via PKC-dependent phagocytosisand intracellular killing to Candida by phagolysosomal fusion andacidification, and lysosome enzyme activation (Fig. 6). Based onour results, it can be concluded that CP from B. pilosa possessesanti-candidal activity in vitro and in vivo. This compound can beconsidered as a novel anti-fungal agent to prevent and treat op-portunistic Candida infection.

C.-Y. Chung et al. / Journal of Ethnopharmacology 184 (2016) 72–8080

Acknowledgements

This work was supported by the Ministry of Science andTechnology of Taiwan (NSC101-2313-B-005-019- and NSC97-2320-B-005-001-MY3).

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jep.2016.02.036.

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